MOLECULAR BIOLOGY OF THE CELL - ascb.org
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MOLECULAR BIOLOGY OF THE CELL - ascb.org
MBoC MOLECULAR BIOLOGY OF THE CELL ASCB AWARD ESSAYS, BIG DATA AND THE FBI, AND THE 2015 PAPER OF THE YEAR MBoC MOLECULAR BIOLOGY OF THE CELL Published by the American Society for Cell Biology 2015 ASCB Award Essays, Selected Perspective, and MBoC Paper of the Year Contents EDITORIAL Great science inspires us to tackle the issue of data reproducibility D. G. Drubin 1–2 ASCB AWARD ESSAYS Advice to a young scientist (by someone who doesn’t know how to give it) V. Denic What does it take to get the job done? M. Serpe How nontraditional model systems can save us A. S. Gladfelter A case for more curiosity-driven basic research A. Amon Surviving as an underrepresented minority scientist in a majority environment E. D. Jarvis An unconventional route to becoming a cell biologist E. Fuchs 3–5 6–8 9–11 12–13 14–18 19–21 PERSPECTIVE Biosecurity in the age of Big Data: a conversation with the FBI K. G. Kozminski 22–25 MBoC PAPER OF THE YEAR Subcellular optogenetic inhibition of G proteins generates signaling gradients and cell migration P. R. O’Neill and N. Gautam 27–36 MBoC MOLECULAR BIOLOGY OF THE CELL Published by the American Society for Cell Biology ASCB Award Essays, BIg Data and the FBI, and the 2015 Paper of the Year A cell activated by spatially uniform chemoattractant responds by increasing the level of a signaling lipid all over the cell, shown as translocation of green fluorescence from the cytosol to the plasma membrane. After this transient response subsides, optical activation (white box) is applied to one side of the cell to recruit an inhibitor (red) of an intracellular signaling protein (heterotrimeric G protein) that is known to be activated downstream of the receptor. This causes a subsequent increase in the level of the signaling lipid and initiation of migration, both directed toward the opposite side of the cell. See the 2015 MBoC Paper of the Year (O’Neill and Gautam, Mol. Biol. Cell 25, 2305–2314; reprinted on p. 27). The MBoC Paper of the Year is selected by the Editorial Board from among papers published in the journal each year that have a postdoc or student as the first author. (Image: Patrick O’Neill, Washington University School of Medicine, St. Louis) The Philosophy of Molecular Biology of the Cell Molecular Biology of the Cell (MBoC) is published by the nonprofit American Society for Cell Biology (ASCB) and is free from commercial oversight and influence. We believe that the reporting of science is an integral part of research itself and that scientific journals should be instruments in which scientists are at the controls. Hence, MBoC serves as an instrument of the ASCB membership and as such advocates the interests of both contributors and readers through fair, prompt, and thorough review coupled with responsible editorial adjudication and thoughtful suggestions for revision and clarification. Our most essential review criterion is that the work significantly advances our knowledge and/or provides new concepts or approaches that extend our understanding. At MBoC, active working scientists—true peers of the contributors—render every editorial decision. The Society and MBoC are committed to promoting the concept of open access to the scientific literature. MBoC seeks to facilitate communication among scientists by • publishing original papers that include full documentation of Methods and Results, with Introductions and Discussions that frame questions and interpret findings clearly (even for those outside an immediate circle of experts); • exploiting technical advances to enable rapid dissemination of articles prior to print publication and transmission and archiving of videos, large datasets, and other materials that enhance understanding; and • making all content freely accessible via the Internet only 2 months after publication. Statement of Scope MBoC publishes studies presenting conceptual advances of broad interest and significance within all areas of cell biology, genetics, and developmental biology. Studies whose scope bridges several areas of cell and developmental biology are particularly encouraged. MBoC aims to publish papers describing substantial research progress in full: Papers should include all previously unpublished data and methods essential to support the conclusions drawn. MBoC will not, in general, publish papers that are narrow in scope and therefore better suited to more specialized journals, merely confirmatory, preliminary reports of partially completed or incompletely documented research, findings of as yet uncertain significance, or reports simply documenting well-known processes in organisms or cell types not previously studied. Submissions that report novel methodologies are encouraged, particularly when the technology will be widely useful, when it will significantly accelerate progress within the field, or when it reveals a new result of biological significance. Given the scope of MBoC, relevant methodologies include (but are not limited to) those based on imaging, biochemistry, computational biology, and recombinant DNA technology. Note that MBoC places a premium on research articles that present conceptual advances of wide interest or deep mechanistic understanding of important cellular processes. As such, articles dealing principally with describing behavior or modification of specific transcription factors, or analysis of the promoter elements through which they interact, will not generally be considered unless accompanied by information supporting in vivo relevance or broad significance. MBoC MOLECULAR BIOLOGY OF THE CELL Published by the American Society for Cell Biology Editor-in-Chief David G. Drubin University of California, Berkeley Editors Jennifer Lippincott-Schwartz National Institutes of Health W. James Nelson Stanford University Thomas D. Pollard Yale University Jean E. Schwarzbauer Princeton University Features Editors William Bement University of Wisconsin Paul Forscher Yale University Thomas D. Fox Cornell University Margaret Gardel University of Chicago Reid Gilmore University of Massachusetts Mark H. Ginsberg University of California, San Diego Benjamin S. Glick University of Chicago Robert D. Goldman Northwestern University Jean E. Gruenberg University of Geneva Doug Kellogg University of California, Santa Cruz J. Silvio Gutkind University of California, San Diego Keith G. Kozminski University of Virginia Jeffrey D. Hardin University of Wisconsin Associate Editors Francis A. Barr University of Oxford Patricía Bassereau Institut Curie Laurent Blanchoin CEA Grenoble Kerry S. Bloom University of North Carolina Charles Boone University of Toronto Patrick J. Brennwald University of North Carolina Julie Brill The Hospital for Sick Children Marianne Bronner California Institute of Technology Fred Chang Columbia University Jonathan Chernoff Fox Chase Cancer Center Carl-Henrik Heldin Ludwig Institute for Cancer Research Erika Holzbaur University of Pennsylvania Kozo Kaibuchi Nagoya University Sandra Lemmon University of Miami Daniel J. Lew Duke University Rong Li Johns Hopkins University Diane Lidke University of New Mexico Adam Linstedt Carnegie Mellon University Kunxin Luo University of California, Berkeley Thomas M. Magin University of Leipzig Orna Cohen-Fix National Institutes of Health Wallace Marshall University of California, San Francisco Stephen Doxsey University of Massachusetts Thomas F. J. Martin University of Wisconsin Leah Edelstein-Keshet University of British Columbia A. Gregory Matera University of North Carolina Richard Fehon University of Chicago Alex Mogilner University of California, Davis Denise Montell University of California, Santa Barbara Keith E. Mostov University of California, San Francisco Akihiko Nakano RIKEN Donald D. Newmeyer La Jolla Institute for Allergy and Immunology Asma Nusrat Emory University Carole Parent National Institutes of Health Robert G. Parton University of Queensland Samara Reck-Peterson Harvard Medical School Howard Riezman University of Geneva Mark J. Solomon Yale University Thomas Sommer Max Delbrück Center for Molecular Medicine Anne Spang University of Basel Gero Steinberg University of Exeter Susan Strome University of California, Santa Cruz Suresh Subramani University of California, San Diego Thomas Surrey The Francis Crick Institute William P. Tansey Vanderbilt University Manuel Théry CEA, Hopital Saint Louis Peter Van Haastert University of Groningen Gia Voeltz University of Colorado, Boulder Yu-Li Wang Carnegie Mellon University Valerie Marie Weaver University of California, San Francisco Karsten Weis ETH Zurich Marvin P. Wickens University of Wisconsin Sandra Wolin Yale University Yukiko Yamashita University of Michigan Alpha Yap University of Queensland John York Vanderbilt University Tamotsu Yoshimori Osaka University Molecular Biology of the Cell (ISSN 1059-1524) is published online twice per month by The American Society for Cell Biology, 8120 Woodmont Avenue, Suite 750, Bethesda, MD 20814-2762. Correspondence: Molecular Biology of the Cell, ASCB, 8120 Woodmont Avenue, Suite 750, Bethesda, MD 20814-2762. Email: mbc@ascb.org. Subscriptions: Institutional subscriptions are available according to a tiered rate structure; please visit www.ascb.org/files/2016_MBoC_INST_RATES.pdf for additional information. 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Yixian Zheng Carnegie Institution Xueliang Zhu Chinese Academy of Sciences Board of Reviewing Editors Richard Anderson Ken-ichi Arai Mark Ashe Kathryn Ayscough William Balch Georjana Barnes Diana Bautista Arnold Berk Magdalena Bezanilla Sue Biggins Robert Boswell David Burgess James Casanova David Chan Richard Chi Melanie Cobb Charles Cole Ruth Collins Duane Compton Pierre Coulombe Rik Derynck William Dunphy William Earnshaw Gregor Eichele Harold Erickson John Eriksson Marilyn Farquhar Victor Faundez James Feramisco Christine Field Alison Frand Stanley Froehner Joseph Gall Michael Glotzer David Glover Bob Goldstein Bruce Goode Kathleen Gould Barth Grant Wei Guo Rosine Haguenauer-Tsapis Nissm Hay Rebecca Heald Daniel Hebert Martin Hemler Mark Hochstrasser David Hockenbery Thomas Hope Sui Huang Anna Huttenlocher Ken Inoki Andrei Ivanov Catherine Jackson Leanne Jones Kenneth Kemphues Mary Kennedy Daniel Klionsky David Kovar Helmut Kramer Hua Lou Alberto Luini Eugene Marcantonio Michael Marks Satyajit Mayor Tom Misteli David Mitchell Andrea Munsterberg Coleen Murphy Karla Neugebauer Davis Ng Patrick O’Farrell Thoru Pederson Craig Peterson Rob Piper Kornelia Polyak Maureen Powers Michael Rape Karin Romisch Sarita Sastry Dorothy Schafer Danny Schnell Jonathan Scholey Nava Segev Jeff Settleman Shu-ou Shan Alexander Sorkin Harald Stenmark Alex Strongin Woan-Yuh Tarn Peter ten Dijke Mary Tierney Margaret Titus Linton Traub Claire Walczak Paul Wassarman Orion Weiner Matthew Welch Beverly Wendland Zena Werb Mark Winey Howard Worman Michael Yaffe Tadashi Yamamoto Jennifer Zallen Founding Editors Erkki Ruoslahti Bumham lnstitute (Founding Editor, Cell Regulation) David Botstein Princeton University Keith R. Yamamoto University of California, San Francisco Publisher Executive Director Stefano Bertuzzi Publications Director W. Mark Leader Journal Production Manager Eric T. Baker © 2015 by The American Society for Cell Biology. Molecular Biology of the Cell is available online at www.molbiolcell.org and through PubMed Central at www.pubmedcentral.nih.gov. Two months after being published at www.molbiolcell.org, the material in Molecular Biology of the Cell is available for non-commercial use by the general public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0). Under this license, the content may be used at no charge for noncommercial purposes by the general public, provided that: the authorship of the materials is attributed to the author(s) (in a way that does not suggest that the authors endorse the users or any user’s use); users include the terms of this license in any use or distribution they engage in; users respect the fair use rights, moral rights, and rights that the Authors and any others have in the content. ASCB®, The American Society for Cell Biology®, and Molecular Biology of the Cell® are registered trademarks of The American Society for Cell Biology. MBoC | EDITORIAL adequate independent replicates and report: 1) how many independent replicates were performed and 2) the variance in the results. Appropriate statistical practices are important but are not a panacea. This is because there is a difference between precision and accuracy. An experimental setup with a systematic design flaw can produce data that are precise but inaccurate. Second, it is vital that the variables in each procedure be altered systematically to deterDavid G. Drubin mine which parameters are critical for making results reproducible. Department of Molecular and Cell Biology, University of California, Having done these two things, it is next essential that the study be Berkeley, Berkeley, CA 94720 communicated in sufficient detail to allow others to reproduce the key findings. Finally, one of the best ways to insure that a result is correct is to get the same answer using at least two independent approaches. In their classic study, Jamieson and Palade (1967) used This special MBoC edition celebrates the American Society for Cell both cell fractionation and radioautography to discover the intracelBiology’s 2015 award winners by featuring essays describing their lular trafficking route. inspiring scientific journeys and sharing their impressive wisdom. Journals are part of this reproducibilThese articles remind us that, while much ity issue in another way. Many scientists attention has recently been focused on feel that it is necessary to publish in highconcerns about research practices and profile journals to secure funding, emdata reproducibility, we live in an era of unployment, and career advancement. precedented achievements in biomedical Attempting to publish in such journals, research discovery, such as immune checkhowever, may make authors feel the point therapy for cancer, which arose dineed to oversimplify their results and rectly out of basic research (Sharma and omit inconvenient data, both of which Allison, 2015). compromise the integrity of the reported As the successful careers of these ASCB results. Moreover, higher-profile journals awardees serve to remind us, it is importend to have severe constraints on article tant that all scientists promote research length, compounding these problems. practices and standards that result in highVital to making research reproducible is quality, reproducible research. If the scienbeing able to report ALL of the nuances tific community cannot convince the pubof the experimental procedures and relic that we have control of this issue, we sults, including “inconvenient facts” that risk reduced funding and imposition of might not fit perfectly with the major guidelines developed and enforced by findings of a study. One back-to-basics government legislators. David G. Drubin solution to this problem is to publish reBecause peer-reviewed publications Editor-in-Chief search articles in professional society are both the product of research and the journals like MBoC that are concerned vehicles for communicating scientific disonly with results being new and true and not with their popular coveries, journals have a critical role to play, promoting the practices appeal or flashiness. MBoC and some other professional society that make research reproducible. It is appropriate that scientist-run journals do not have artificial limits on the number of figures or journals like MBoC take a lead in this effort. MBoC has always emthe length of the text. Such limits impair the ability to provide braced a “back-to-basics” approach to promote research integrity. sufficient detail so others can reproduce published work. In other words, major innovations are not needed to promote reproA promising way to address reproducibility issues is through deducibility, just an emphasis on sound fundamentals. velopment of field-specific, community standards. Achieving reproReproducibility must begin with those individuals performing the ducibility can be challenging, because scientific research is difficult initial study. First and foremost, it is vital that investigators perform and protocols are often complex (Aschwanden and King, 2015). However, this is not an excuse for publication of results that cannot DOI:10.1091/mbc.E15-09-0643. Mol Biol Cell 26, 3679–3680. be reproduced. Rather, it is an important acknowledgment of the David G. Drubin is Editor-in-Chief of Molecular Biology of the Cell. reality that there are a lot of variables in performing experiments Address correspondence to: David G. Drubin (drubin@berkeley.edu). and in collecting and analyzing data. Seemingly insignificant © 2015 Drubin. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to changes in execution or analysis can have profound impacts on rethe public under an Attribution–Noncommercial–Share Alike 3.0 Unported Cresults. Because the ways in which complex phenomena are observed, ative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0). classified, and reported are often research area specific, one-size® ® “ASCB ,” “The American Society for Cell Biology ,” and “Molecular Biology of fits-all solutions for the reproducibility issue are unattainable. The the Cell®” are registered trademarks of The American Society for Cell Biology. Great science inspires us to tackle the issue of data reproducibility 2015 ASCB Award Essays, Selected Perspective, and MBoC Paper of the Year 1 autophagy field has developed its own standards by consensus (Klionsky et al., 2012), providing a powerful example for others to follow. Many of these approaches to the data-reproducibility problem are discussed in a report by the ASCB Data Reproducibility Task Force (American Society for Cell Biology, 2015). In the near future, MBoC will be developing strategies to implement the task force’s recommendations in ways that do not place excessive administrative burdens on authors. In closing, I offer congratulations to the 2015 ASCB award winners! You inspire us all with your creativity and passion, exemplify sound science practices, and remind us that great scientific achievements result when all of these elements are combined. 2 | D. G. Drubin REFERENCES American Society for Cell Biology (2015). How can scientists enhance rigor in conducting basic research and reporting research results? A white paper from the American Society for Cell Biology. www.ascb.org/files/ How-can-scientist-enhance-rigor.pdf. Aschwanden C, King R (2015). Science isn’t broken. FiveThirtyEight. http:// fivethirtyeight.com/features/science-isnt-broken. Jamieson JD, Palade GE (1967). Intracellular transport of secretory proteins in the pancreatic exocrine cell. II. Transport to condensing vacuoles and zymogen granules. J Cell Biol 34, 597–615. Klionsky DJ, Abdalla FC, Abeliovich H, Abraham RT, Acevedo-Arozena A, Adeli K, Agholme L, Agnello M, Agostinis P, Aguirre-Ghiso JA, et al. (2012). Guidelines for the use and interpretation of assays for monitoring autophagy. Autophagy 8, 445–544. Sharma P, Allison JP (2015). The future of immune checkpoint therapy. Science 348, 56–61. Molecular Biology of the Cell MBoC | ASCB AWARD ESSAY Advice to a young scientist (by someone who doesn’t know how to give it) Vladimir Denic Department of Molecular and Cellular Biology, Harvard University, Cambridge, MA 02138 ABSTRACT While trying to extract original and general advice from the details of my career, I realized this might not be possible. My path, like those of so many others, had too many idiosyncratic twists and turns that had to work out just the way they did to be mined for generally useful strategies. So I abandon the conceit of advice and simply give you my story. There are many like it, but this one is mine. Take what you wish from it. AND YET IT COLLAPSES that Mr. Patterson refused to confirm or In Belgrade, where I grew up, I was a merefute my explanation. Instead, he chaldiocre science student, unlikely to sponlenged me to devise an experiment that taneously improve. I have to believe this could falsify my working model. I returned was because the subject was taught by the challenge: “Doesn’t this way of thinkrote memorization, but regardless, I was ing call into question all the other stuff in more interested in the indolent pursuits the textbook?” Smiling mischievously, he of disaffected youth in latter-day Yugoslaretorted, “What do you think?” I didn’t via, like stealing car radios (easier than have an answer, but what I should have you might think) and pilfering supermarsaid is “I think, therefore I am … a workket baguettes (harder). In an attempt to ing model.” alter my steady course toward juvenile Learning that I, rather than the authoridelinquency, my mom sent me to live with ties (textbooks, Mr. Patterson himself) my dad, then in the throes of his second could be both originator and verifier of hymarriage, in the mythic land of affluent potheses was one of the most empowerhigh school kids I had been watching on ing revelations of my life, a quiet and meltelevision: the United States. For the next ancholic form of resistance against my year, despite being in rural Pennsylvania, I Vladimir Denic parents’ divorce, against the authoritarian lived a new life that seemed as glamorous system back home, and, I realized as I got and as far from post-Tito Belgrade as the older, against the dying day itself. As summer began, Steven Spielone Brandon and Brenda Walsh were living in Beverly Hills. berg fed my growing interest in science by genetically resurrecting One day, in Mr. Patterson’s chemistry lab, I finally took notice of the dinosaurs. Soon thereafter, I was sent back to Belgrade, just in science. The task: explain why a soda can containing a dollop of time for the war in Bosnia. I dodged the draft by immigrating to boiling water collapsed when inverted in a beaker of ice water. New Zealand, where I attended college. Perhaps inspired by the What was remarkable to me was not that the can collapsed, but velociraptors—clever girls—I majored in biochemistry. DOI:10.1091/mbc.E15-06-0341. Mol Biol Cell 26, 3681–3683. Vladimir Denic is the recipient of the 2015 Early Career Life Scientist Award from the American Society for Cell Biology. Address correspondence to: Vladimir Denic (vdenic@mcb.harvard.edu). Abbreviations used: ER, endoplasmic reticulum; VLCFAs, very-long-chain fatty acids. © 2015 Denic. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0). “ASCB®,” “The American Society for Cell Biology®,” and “Molecular Biology of the Cell®” are registered trademarks of The American Society for Cell Biology. 2015 ASCB Award Essays, Selected Perspective, and MBoC Paper of the Year “DEFINITELY. IT TAKES ANOTHER 3 HOURS BY PLANE FROM SYDNEY” Near the end of college, I started reading recent papers in major journals. One study that caught my eye described how unfolded proteins in the endoplasmic reticulum (ER) send a signal to the nucleus to activate genes encoding ER chaperones (Cox and Walter, 1996). This feedback loop required the ER transmembrane protein kinase Ire1 and a new transcription factor, Hac1. Instead of activating Hac1 by yet another kinase cascade, Ire1 splices an intron from 3 the HAC1 mRNA to relieve a block in Hac1 synthesis by ribosomes. The work was done at UCSF (closer to Beverly Hills) in the lab of Peter Walter. This was not the first molecular biology paper that I read, but it was the first that made me dream. I cold-called Peter from a phone booth in Auckland and asked him to let me work in his lab. He initially demurred, suggesting half-jokingly (or, knowing Peter, not jokingly at all) that a competing lab that had recently relocated to Australia was sending me as an infiltrator. Ultimately, he assented, but only after I convinced him that New Zealand and Australia are different countries. When I left New Zealand after my third year of college, I planned to return at the end of the summer break, but I never did. Instead, I worked as an intern in Peter’s lab for little over a year before joining the graduate program at UCSF. Living in San Francisco over the next 10 years, I came of age both personally and scientifically. THE LONELINESS OF THE LONG-DISTANCE GRADUAL STUDENT I pursued my PhD in Jonathan Weissman’s lab, a scientific paradise that I managed to turn into a personal scientific hell—but I’m getting ahead of myself. I was attracted to the Weissman lab partly because it was new and relatively small, so Jonathan was often available to hang out in the lab and discuss science. However, discussing science with Jonathan meant always being a few steps behind. My brilliant solution was to insist that I work, essentially in isolation, on a problem that was at best tangential to Jonathan’s main research interests. I had also somehow gotten the idea that a mentee should be petulant and jokingly dismissive of his mentor’s scientific ideas. Despite my recalcitrance, Jonathan offered me several projects that were guaranteed to work, but I turned them down in favor of pursuing my own ideas. Cut to four years, several “clever” genetic screens, and zero publications later. Jeffery Cox, one of the students who revealed Ire1 and Hac1’s unique relationship in Peter’s lab, once said (to someone else), “If you can’t clone the gene you love, love the gene you clone.” What he didn’t say is what to do if you don’t know what love is. In my case, that meant not knowing how to explore the other worlds of cell biology that lay in the direction my cloned genes were trying to take me. In part, my resistance was based on fear that pursuing the obvious questions would require me to master biochemistry, which at the time I considered to be both less elegant and more laborious than genetics. By my sixth year of graduate school, the dream of crushing my own can of science was slipping away. But then something unremarkable happened: existing projects in the lab needed an extra pair of hands to get finished. My hands, idled by disillusionment, were available. I got some results. Results became figures. Figures became papers. Year 7. Some of the aforementioned results had suggested that the uncharacterized gene YJL097w was involved in sphingolipid metabolism. In a previous clever (but fruitless) genetic screen, I had cloned two other genes involved in sphingolipid metabolism. As that project collapsed into irrelevance, I had occupied myself by accumulating an absurdly disproportionate familiarity with the sphingolipid literature. On the basis of that knowledge (heretofore useless to me), I intuited that YJL097w might be the missing biosynthetic enzyme for very-long-chain fatty acids (VLCFAs), the building blocks of sphingolipids. Contemporaneously, a couple of publications from another lab had argued that the plant homologue of YJL097w was a protein phosphatase involved in the cell cycle. I was unconvinced by these data and felt that all of the phenotypes associated with mutations in 4 | V. Denic the plant homologue could be explained by a defect in VLCFA synthesis. Thus, finally, I hit my stride: from my first tenuous baby steps in Mr. Patterson’s chemistry lab, to a few Bambi-on-ice moments while finishing other people’s projects, to making what was by far the coolest science prediction I had ever made, which—cherry on top!—was at odds with the accepted view. The exhilarating thought of testing (and possibly even confirming) this hypothesis motivated the next 6 months of labor—at the end of which a peak on a chromatogram showed me that purified Yjl097w had made a dehydrated VLCFA product. My working model had worked! We submitted our paper to a major journal, where it was rejected on the grounds that it lacked general interest. Still intoxicated by my discovery that Yjl097w was the missing dehydratase, I decided that the general reader would be generally interested in total VLCFA synthesis in vitro using Yjl097w and three other enzymes. Unfortunately, all of these enzymes were integral membrane proteins sensitive to detergent. Groping for a path forward, I was inspired by a paper written by Görlich and Rapoport on an unrelated topic (Görlich and Rapoport, 1993). In their approach, one places several pure membrane proteins in detergent, mixes them with detergent-solubilized synthetic phospholipids, and then removes the detergent (with something called “biobeads”) to yield proteoliposomes containing the desired proteins. Despite the strategy’s straightforward logic, the remarkably detailed methods section suggested that there might still be some magic involved (for example, only lot number 810017 of Big CHAP worked), so Jonathan put me in touch with a former UCSF student, Manu Hegde, who was making proteoliposomes regularly in his own lab at the National Institutes of Health. Manu and I spent hours on the phone, like teenagers (“Did you know how much humidity in Bethesda affects my biobeads?” “Tell me about it. No, seriously, tell me ALL about it.”), and a few weeks later, I was making proteoliposomes that were making VLCFAs. We submitted our work to another major journal, where it was rejected on the grounds that it didn’t demonstrate anything new. Meanwhile, I had figured out how two different versions of yeast VLCFA enzymes synthesize VLCFA products of different lengths. In a “natural experiment,” I noticed that evolution had changed the distance between the active site on the cytosolic end of the synthase (where carbon building blocks feed the growing end of the fatty acid–chain substrate) and a lysine near the luminal end of a transmembrane alpha helix. Remarkably, I could make new VLCFA products of predictable lengths by “sliding” the position of the lysine, like molecular calipers, up or down the helix. Several months later, Jonathan and I compiled the data for the molecular caliper story and sent it to the journal that issued our first rejection. (This felt a bit like trying to convince your ex-girlfriend to take you back because you spent a year in the gym.) A few weeks after the submission, as I waited in line in my favorite San Francisco bakery, Jonathan called to tell me that the paper had been accepted without revisions. The moment was ecstatic, but also sentimental, because it meant that our mentor–mentee relationship was finally coming to an end. It was the culmination of nine years of Jonathan’s patience with me, during which he cheered me on, just as loudly every time I fell down as when I finally won the race. GO EAST(?), YOUNG MAN I spent my last six months in the Weissman lab helping another project in the lab get finished, lining up a postdoc in Japan, and hedging my career bets by applying for a job to a few departments that expressed interest in me after the caliper work was published. In the end, I bailed on Japan and started my lab at Harvard University. At Molecular Biology of the Cell the time that I was contemplating taking the Harvard job, the word on the street was not good (“They eat their young”). Why did I still choose to go there? First, I really enjoyed my interview interactions with several senior members of the department (I know what you are thinking: senior, not junior; red flag), who convinced me that they could be decent Jonathan substitutes for this phase of my career. Second, I believed that bad reputations are often the disproportionately long shadows of atypical events (I know what you are thinking: shadows grow long when it is too late in the day for change to occur). And admittedly, my own hubris came into play: even if the place was bad for junior faculty, I thought I would be somehow different (I don’t even wanna know what you’re thinkin’). After a year at Harvard, however, progress was not swift. Only two students had rotated with me, and they both joined other labs (run by senior faculty). Self-doubt and fear spread through my veins like poison. As an antidote, I considered an offer from another department with a better reputation for cultivating junior faculty. Why did I stay, in the end? An old saying: “wherever you go, there you are.” So, rather than entertaining Borgian fantasies about my senior lab “competitors,” I tried to improve my own contributions to the process of attracting talented students. I got myself on the student radar by spearheading a journal club for first-year students and faculty, modeled on one I had enjoyed at UCSF, and started pitching projects with the unabashed verve of a used car salesman. Over the next five years, our group figured out how tail-anchored proteins are inserted into the ER membrane by the GET pathway. Before I left the Weissman lab, I had developed a cell-free system for studying this pathway, which my group stripped down to its purified components. These were exhilarating times, because we were racing against several fantastic labs to answer the same mechanistic questions. Even though I was a newcomer to the membrane protein insertion field, I was encouraged by more senior figures—especially Manu Hegde, who taught me that scientific competition and criticism are not mutually exclusive with scientific openness. As the lab established itself, we started parallel work on autophagy. My interest in this field arose during grad school, when I read a paper from Yoshinori Ohsumi’s lab (where, incidentally, I had planned to do a postdoc). Autophagy is a half century–old puzzle in cell biology: How do cells wrap targets with a membrane to make a vesicle that then delivers targets to the lysosome? Many imaging methods have been used to track the formation of this membrane, but few biochemical approaches had been attempted. After a couple of years, we built a cell-free system that allowed us to initiate autophagosome 2015 ASCB Award Essays, Selected Perspective, and MBoC Paper of the Year membrane formation in situ using a purified autophagy target. Other protein-targeting fields have been transformed following the development and rapid adoption of cell-free systems. Our work adds selective autophagy to this list and will hopefully accelerate the elucidation of key mechanisms underlying this process A TALE TOLD BY AN IDIOT, FULL OF SOUND AND FURY, SIGNIFYING NOTHING Here is where I intended to summarize my tale, with the implicit purpose of inspiring younger scientists to do as I did. But what would that advice be? “Here’s what you need to do, kids: Fail repeatedly for years, alone, but then get serendipitously lucky and pick a winning horse years in advance of a final payoff. Then sit back and wait by the phone for a job offer from Harvard, which despite everything you’ve heard will give you exactly the kind of support you need to succeed as junior faculty. You’re welcome [mic drop].” But one person’s rose-tinted view of their own idiosyncratic story does not constitute “advice,” especially not in an endeavor where we value reproducibility; I’m not sure I could reproduce my own good fortune, much less expect someone else to reproduce it from the same set of initial conditions. The only thing I know for sure is that the support I was repeatedly given at every stage of my career was critical to what success I did have throughout my career. That support enabled me to stay with it through failures and to do something productive at those times when I needed, more than anything else, to produce something. Not everyone who had the support with which I was privileged would have reached the same result, but I know that I wouldn’t have succeeded without it. And so, for that, I am truly grateful. ACKNOWLEDGMENTS I thank Chris Patil for helping me write this essay and for helping me finish my college education in San Francisco. I dedicate this essay to my mother for giving me actually useful advice my whole life. REFERENCES Cox JS, Walter P (1996). A novel mechanism for regulating activity of a transcription factor that controls the unfolded protein response. Cell 87, 391–404. Görlich D, Rapoport TA (1993). Protein translocation into proteoliposomes reconstituted from purified components of the endoplasmic reticulum membrane. Cell 75, 615–630. No advice to a young scientist | 5 MBoC | ASCB AWARD ESSAY What does it take to get the job done? Mihaela Serpe Unit on Cellular Communication, Program in Cellular Regulation and Metabolism, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892 ABSTRACT I am extremely honored to be the recipient of the 2015 Women in Cell Biology Junior Award. When I reflect on my journey in science, many great people and memorable experiences come to mind. Some of these encounters were truly career-defining moments. Others provided priceless lessons. In this essay, I recount some of the moments and experiences that influenced my scientific trajectory with the hope that they may inspire others. THE BIG QUESTION multidisciplinary approaches for solving It was a tense day in late fall of 2011. I was problems. This scientific strength was going through my first site visit at the Nashaped by my diverse background and tional Institutes of Health (NIH), a compreexperiences and is fueled by a need to hensive review that would define my scienunderstand the molecular mechanisms tific career. My three-year-old lab had underlying biological phenomena. I am published a significant paper on mechadrawn to long-standing mysteries in the nisms that shape bone morphogenetic field, and my first impulse is to imagine protein (BMP) morphogen gradients and what kind of molecule/function(s) could control early patterning (Peluso et al., fill the missing link. What does it take to 2011), and we had discovered Drosophila get the job done? In my search for anNeto, an obligatory auxiliary subunit for swers, I reach across disciplines and glutamate receptors. The question filled communicate with diverse experts, from the room: “The BMP/transforming growth cell biologists to neuroscientists and factor β (TGF-β) field has been tackled for computational biologists. I gather comquite some time by many laboratories. You prehensive knowledge of the system and propose to work on these pathways. What the phenomena to understand the “job” are you hoping to bring that is new to the Mihaela Serpe and to describe it in molecular terms. field and how?” It was a very fair and simThen I use biochemistry and structure– ple question coming from one of the most function insights to envision possibilities and formulate a testable accomplished and clear thinkers in cellular and developmental biolhypothesis. ogy today, Eric Wieschaus. In a few words he crystallized the key “What does it take to get the job done?” not only describes issue any junior principal investigator should think hard about: What the thinking process in my lab but also captures our mode of do I bring that is new to science? operation. We will do everything possible to answer the next In my lab, I address fundamental issues of cellular communicaquestion, whether it means perfecting our skills, inventing tools, tion using a genetic system and a unique set of powerful, bringing new technologies to the lab, or establishing relevant collaborations. DOI:10.1091/mbc.E15-06-0428. Mol Biol Cell 26, 3684–3686. Mihaela Serpe is the recipient of the 2015 ASCB Women in Cell Biology Junior Award for Excellence in Research. Address correspondence to: Mihaela Serpe (mihaela.serpe@nih.gov). Abbreviations used: BMP, bone morphogenetic protein; iGluRs, glutamate-gated ion channels; NIH, National Institutes of Health; NMJ, neuromuscular junction; pMad, phosphorylated Smad; TGF-β, transforming growth factor β. © 2015 Serpe. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0). “ASCB®,” “The American Society for Cell Biology®,” and “Molecular Biology of the Cell®” are registered trademarks of The American Society for Cell Biology. 6 | M. Serpe FINDING ONE’S PASSION It takes scholarly work and courage to break new ground, but first one must find his/her passion. I knew early on that I wanted to be a scientist. At first I thought I would be a mathematician. But, because I was a girl, I was persuaded to look elsewhere. I chose chemistry and genetics and decided to study biochemistry. This was one of the best decisions in my life and was entirely mine. I was 17 years old, and I literally took three days to think hard and consider everything that I was passionate about: logical thinking, Molecular Biology of the Cell genetics, molecules, living creatures, and solving puzzles. Within a year I moved to Bucharest to study biochemistry. My first encounter with cell biology was self-driven. The biochemistry curriculum at the University of Bucharest was very heavy on chemistry, especially in the first years. I was searching for ways to put things in perspective. A friend recommended a cell biology textbook adapted from Molecular Biology of the Cell by Bruce Alberts and colleagues (Alberts et al., 2014). I bought it and started to read. I ended up immersed in it, losing track of time. After graduation, I was drawn toward the Institute for Cellular Biology and Pathology in Bucharest, the place with the best cell biology research in Romania. Many scientists have turning points in their careers, when they find out what they really want to do in science. Mine was when I met George Emil Palade, who came to give a seminar at the institute. He made a comment that intrigued me: he considered his work on protein regulation by phosphorylation even more important than the discovery of ribosomes. From the perspective of a freshly graduated scientist, the body of work that brought Palade the Nobel Prize was simply monumental. How could anything be more important than that? What was so significant about protein phosphorylation, and why was this a key finding in biology? I was enthralled. This was my introduction to cellular signaling. This moment triggered a fascination with signaling and macromolecular complexes that I have to this day. THE JOURNEY To work on signaling, I joined Dan Kosman’s laboratory at the State University of New York at Buffalo, which focused on cellular mechanisms to acquire and metabolize iron. The first winter in Buffalo was unforgettable: braving my way through record snowfall, I was taking terrific courses, such enzyme kinetics taught by Cecile Pickart and molecular biology taught by Ed Niles. For my PhD thesis, I studied how a simple eukaryotic cell, the budding yeast, senses and responds to the levels of copper and iron in its environment. It was a time of intense discoveries in the field. Dan Kosman encouraged us to go to meetings and grasp the latest news. I learned a lot from seeing how he discussed our findings and interacted with his competitors. He is a rigorous scientist and a master strategist. For postdoctoral training, I joined Mike O’Connor’s laboratory at the University of Minnesota to work on signaling by BMP/TGF-β factors during Drosophila development. Up to that point, I did not know anything about development, but it seemed to be the best place to probe for the biological relevance of signaling. It was a daunting yet exhilarating experience to dive into a model system with such prominent history in genetics and development. I went back to take courses, including Drosophila genetics at the Cold Spring Harbor Laboratory and developmental biology taught by Ann Rougvie at the University of Minnesota. It was a steep learning curve, but Mike O’Connor’s laboratory and the Developmental Biology Center offered an interactive and stimulating environment. The lab was extremely productive and full of fantastic people. New discoveries were coming in big leaps rather than in a linear progression. We all had different individual projects, and it seemed that each story tackled another field. I was always mesmerized by Mike O’Connor’s ease at entering a new field and making an important discovery. The lab was in the middle of a vibrant fly community, including Tom Hays, Jeff Simon, Tom Newfeld, and Hiroshi Nakato. Fly stocks and good ideas were moving freely. I learned to do microinjections in Xenopus embryos in Jamie Lohr’s lab. And I tested the axon guidance defects of a Caenorhabditis elegans mutant in Lishia Chen’s lab. During these years, I described several molecular mechanisms that shape morphogen gradients during patterning (Serpe et al., 2005, 2008; Umulis et al., 2006). I formed a long-term 2015 ASCB Award Essays, Selected Perspective, and MBoC Paper of the Year collaboration with David Umulis (now at Purdue University), who introduced me to computational biology. And I characterized a TGF-β pathway required for motor neuron axon guidance and formation of neural circuitry (Serpe and O’Connor, 2006). This last bit was the beginning of a new chapter, neural development, which was about to captivate me for the rest of my career. THE UNIT ON CELLULAR COMMUNICATION The big surprise in starting a new lab is the loneliness that comes with its beginning. Fresh from a lab buzzing with people and experiments, you are faced with an empty space. And it is up to you to bring it to life and make things happen. The way I looked at it was that it another new ground to break, which takes scholarly work and courage. This time I bought the book At the Helm by Kathy Barker (Barker, 2010), and I took management courses. Courage was asking for help. My corner of the NIH is full of excellent scientists who challenge themselves and push the boundaries. In this environment, I found many answers and important role models. I learned from Alan Hinnebusch to set high standards and from Mary Dasso to become an effective mentor and woman scientist. I learned to distill questions in neurobiology from the late Howard Nash, who initiated and mentored the Drosophila neurobiology interest group. We regularly brainstorm on neurons and circuits in joint lab meetings with Chi-Hon Lee and Ed Giniger. The fly neuromuscular junction (NMJ) has been a powerful genetic system to study synapse development. The easily accessible synaptic structures were well described, the subunits that form the glutamate-gated ion channels (iGluRs) were known and relatively well characterized, and dynamic studies have captured growing synapses (DiAntonio, 2006; Thomas and Sigrist, 2012). But a nagging problem was holding up the field: the mechanisms controlling the synaptic recruitment of iGluRs remained unknown for decades. Studies on iGluR C-tails, which provide rich regulation for these receptors in other systems, brought marginal progress in flies. We found an interesting transmembrane molecule, Neto (Neuropillin and Tolloid-like protein), with a spectacular phenotype: netonull embryos are completely paralyzed and cannot hatch into the larval stages. This is reminiscent of the phenotype seen with the loss of iGluRs and lack of functional NMJ. In fact, we found that Neto and iGluRs form complexes and depend on each other for trafficking and clustering at synaptic sites (Kim et al., 2012). Neto is an obligatory auxiliary protein for the fly NMJ iGluRs. The discovery of Neto provides an entry point to address key questions in iGluR cell biology and to start dissecting the individual steps of iGluR assembly, surface delivery, trafficking, and stabilization at synaptic locations; function; and postsynaptic composition. We have already found that Neto engages in intracellular and extracellular interactions that recruit postsynaptic components and stabilize iGluRs at synaptic locations (Kim et al., 2015; Ramos et al., 2015). In collaboration with Mark Mayer, we have achieved the long-sought functional reconstitution of NMJ iGluRs in heterologous systems and showed that Neto modulates the functioning of iGluRs but not their assembly or surface delivery (Han et al., 2015). Neto also appears to be at the center of transsynaptic complexes that monitor the synapse activity status and relay this information to the presynaptic BMP signaling pathway (Sulkowski et al., 2014). At the fly NMJ, BMP signaling triggers accumulation of phosphorylated Smad (pMad) in motor neuron nuclei and at synaptic terminals. Nuclear pMad controls gene expression and promotes NMJ growth; the role of synaptic pMad eluded the field for more than a decade. We found that synaptic pMad is part of a completely new BMP pathway that is genetically distinguishable from all known What does it take to get the job done? | 7 BMP signaling cascades. This novel pathway does not contribute to the NMJ growth and instead appears to set up a positive-feedback loop that modulates synapse maturation as a function of synapse activity. Thus, BMPs may monitor synapse activity and coordinate it with synapse growth and maturation. THE PEOPLE When I reflect on how this all came together, many great people and mentors come to mind. First are the people in my lab, a team of talented, budding scientists whom I have been privileged to guide in exciting endeavors. Their success is critical to me from the moment they join my lab to, I suspect, long after they leave. Second are my colleagues—and here I hit the jackpot. Ours can be a stressful profession, particularly at the beginning of a new lab. But if you have colleagues like I do, incredibly accomplished scientists with big hearts and a deep commitment to mentoring their juniors, you could survive even when the building is falling on you. I am especially grateful to Alan Hinnebusch, Mary Dasso, and Chi-Hon Lee for their continuous guidance and support. They believed in me from the beginning, showed confidence in my decisions, and helped me keep the course. I owe a great deal to our collaborators, who taught us exciting new things and enriched our science. Bing Zhang completed the first physiology recordings for our mutants, and then helped set up our own rig. Without Mark Mayer, we could not have done the iGluR functional reconstitution and structural studies. We learned to harness the power of superresolution imaging in Jennifer Lippincott-Schwartz’s laboratory. In science, as in life, one could never return the help and support received along the way. All we can hope is to do the same in the future. When my trainees go on the job market, I paraphrase for them the advice I received: In this profession there will be many worries, good and bad. What BMPs do in a particular cell is a “good worry.” Try to find the place where your worries will be mostly good. And figure out fast “What does it take to get the job done?” 8 | M. Serpe REFERENCES Alberts B, Johnson A, Lewis J, Morgan D, Raff M, Roberts K, Walter P (2014). Molecular Biology of the Cell, 6th ed., Garland Science. Barker K (2010). At the Helm: Leading Your Laboratory, 2nd ed., Cold Spring Harbor, NY: Cold Spring Harbor Laboratory. DiAntonio A (2006). Glutamate receptors at the Drosophila neuromuscular junction. Int Rev Neurobiol 75, 165–179. Han TH, Dharkar P, Mayer ML, Serpe M (2015). Functional reconstitution of Drosophila melanogaster NMJ glutamate receptors. Proc Natl Acad Sci USA 112, 6182–6187. Kim YJ, Bao H, Bonanno L, Zhang B, Serpe M (2012). Drosophila Neto is essential for clustering glutamate receptors at the neuromuscular junction. Genes Dev 26, 974–987. Kim YJ, Igiesuorobo O, Ramos CI, Bao H, Zhang B, Serpe M (2015). Prodomain removal enables Neto to stabilize glutamate receptors at the Drosophila neuromuscular junction. PLoS Genet 11, e1004988. Peluso CE, Umulis D, Kim YJ, O’Connor MB, Serpe M, Shaping BMP (2011). Morphogen gradients through enzyme-substrate interactions. Dev Cell 21, 375–383. Ramos CI, Igiesuorobo O, Wang Q, Serpe M (2015). Neto-mediated intracellular interactions shape postsynaptic composition at the Drosophila neuromuscular junction. PLoS Genet 11, e1005191. Serpe M, O’Connor MB (2006). The metalloprotease tolloid-related and its TGF-β-like substrate Dawdle regulate Drosophila motoneuron axon guidance. Development 133, 4969–4979. Serpe M, Ralston A, Blair SS, O’Connor MB (2005). Matching catalytic activity to developmental function: tolloid-related processes Sog in order to help specify the posterior crossvein in the Drosophila wing. Development 132, 2645–2656. Serpe M, Umulis D, Ralston A, Chen J, Olson DJ, Avanesov A, Othmer H, O’Connor MB, Blair SS (2008). The BMP-binding protein Crossveinless 2 is a short-range, concentration-dependent, biphasic modulator of BMP signaling in Drosophila. Dev Cell 14, 940–953. Sulkowski M, Kim YJ, Serpe M (2014). Postsynaptic glutamate receptors regulate local BMP signaling at the Drosophila neuromuscular junction. Development 141, 436–447. Thomas U, Sigrist SJ (2012). Glutamate receptors in synaptic assembly and plasticity: case studies on fly NMJs. Adv Exp Med Biol 970, 3–28. Umulis DM, Serpe M, O’Connor MB, Othmer HG (2006). Robust, bistable patterning of the dorsal surface of the Drosophila embryo. Proc Natl Acad Sci USA 103, 11613–11618. Molecular Biology of the Cell MBoC | ASCB AWARD ESSAY How nontraditional model systems can save us Amy S. Gladfelter Department of Biological Sciences, Dartmouth College, Hanover, NH 03755; Marine Biological Laboratory, Woods Hole, MA 02543 ABSTRACT In this essay I would like to highlight how work in nontraditional model systems is an imperative for our society to prepare for problems we do not even know exist. I present examples of how discovery in nontraditional systems has been critical for fundamental advancement in cell biology. I also discuss how as a collective we might harvest both new questions and new solutions to old problems from the underexplored reservoir of diversity in the biosphere. With advancements in genomics, proteomics, and genome editing, it is now technically feasible for even a single research group to introduce a new model system. I aim here to inspire people to think beyond their familiar model systems and to press funding agencies to support the establishment of new model systems. My career as a biologist began in the orange groves and lake waters of central Florida. An unstructured childhood was spent learning to observe and wonder. Without realizing it at the time, my training began with the mantra, “Study nature, not books,” the familiar entreaty of Louis Agassiz, a founder of what would become the Marine Biological Lab (MBL) in Woods Hole, Massachusetts. In that humid air, listening to cicadas click, I subconsciously practiced asking basic questions about the structure of the natural world. I suspect many of us began our careers this way, even though we ended up thinking about systems of molecules from behind the black curtains of the microscope room, immersed in the frosty air of the cold room, or bathed in the glow of a computer screen. Before the grown-up challenges of funding, publishing, and progressing in a career, we easily marveled at the complexity and surprises of the living world. How can we recapture the joy that comes from curiosity-driven inquiry? This being an essay for the midcareer award, it seems appropriate to blend material for a midlife with a plan for how we as a cell biology community can identify big new questions. So instead of a fancy car, a new microscope, or a dangerous (professional) liaison, try developing a new model system as an outlet for your midlife crisis! Photo Credit: Rob Strong Amy S. Gladfelter DOI:10.1091/mbc.E15-06-0429. Mol Biol Cell 26, 3687–3689. Amy S. Gladfelter is the recipient of the 2015 ASCB Women in Cell Biology Mid-Career Award for Excellence in Research. Address correspondence to: Amy S. Gladfelter (Amy.Gladfelter@Dartmouth .edu). Abbreviations used: MBL, Marine Biological Lab; NIH, National Institutes of Health; NSF, National Science Foundation. © 2015 Gladfelter. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0). “ASCB®,” “The American Society for Cell Biology®,” and “Molecular Biology of the Cell®” are registered trademarks of The American Society for Cell Biology. 2015 ASCB Award Essays, Selected Perspective, and MBoC Paper of the Year NONTRADITIONAL SYSTEMS PRODUCE PUZZLES AND PARADOXES I begin with my own experiences in working with a nontraditional model fungus called Ashbya gossypii. As a postdoc in Peter Philippsen’s group in UniBasel, Switzerland, I started work with Ashbya, which is relatively closely related on the genome scale to the budding yeast Saccharomyces cerevisiae but has some strikingly different cell biology (Dietrich et al., 2004). As a postdoc, I began studying the perplexing ability of nuclei in Ashbya’s multinucleate cells to divide asynchronously despite being in a common cytoplasm (Gladfelter et al., 2006). This was a paradox, because decades of classic work demonstrated that cytosolic factors (CDK/cyclins) control the cell cycle and should synchronize nuclei in a syncytium (Johnson and Rao, 1970). This observation has fueled many experiments in my lab for the past decade, and we have been led to new models for compartmentalizing cytosol, have gained insight 9 into how ploidy can vary in syncytia, and have been continually surprised and inspired (Anderson et al., 2013, 2015; Lee et al., 2013, 2015). For example, one set of experiments led us to realize functional uses for polyQ-tract proteins in localizing the mRNAs encoding cyclins (Lee et al., 2013). This discovery was an early example of a now growing list of functional uses for protein aggregation outside pathologies. Importantly, as a junior faculty member, I had enough of an autonomous niche that my students and I could ask big questions without concerns about being scooped by bigger labs. FIGURE 1: Images of nontraditional model systems discussed in this essay. (A) Ashbya (fungus), image provided by Hanspeter Helfer. (B) Tardigrade (water bear), image provided by Bob Goldstein (University of North Carolina, Chapel Hill). (C) Aiptasia (sea anemone), image provided by John Pringle (Stanford University). (D) Sepioteuthis sepioidea (reef squid), image provided by Roger Hanlon (Marine Biological Laboratory). WHAT ARE THE CHALLENGES OF WORKING IN A NONTRADITIONAL SYSTEM? While there is a tremendous amount of stimulation and freedom that comes from work on a less-studied system, there are also challenges. For example, there is not a large community of users, so I sought out the yeast, filamentous fungus, and cell biology communities—all of whom have been open to Ashbya as an alternative system. It also means most reagents are not simply an email away but instead typically have to be developed in the lab. This is slow, but again, if you are not in a race to publish, it is less of a concern. A molecular geneticist extraordinaire, such as Patricia Occhipinti, a long-term technician in my lab, makes it possible to generate any kind of new strain. Investing in a person like this as a lab constant is critical for establishing a new system. Also, if a system is somewhat related to an established model system, some tools may be transportable or at least adaptable, and so we have borrowed many tricks from the yeast genetics world. Finally, one has to convince funding agencies of the value of the problems that can be addressed by this system. (More on this later.) If the system is conducive to studying a fundamental problem, brings contrasting mechanisms into our current thinking about a key process, and is tractable, it should be possible to make a compelling case for support. There are truly unknown problems in biology waiting in the wings. SUCCESS STORIES OF ESTABLISHING NONTRADITIONAL MODEL SYSTEMS In the most successful new system launches, there is either new biology or a new handle on an old problem that make the systems especially suited for investment. The entry point to a new experimental system tends to be either through old descriptive literature that captures some fascinating phenomena or through a genomic approach that reveals a surprising manifestation of, or absence of, genes thought to be critical for a process. For example, Bungo Akiyoshi, who recently established his own lab at Oxford, comes from a PhD working on kinetochores in yeast with Sue Biggins. Akiyoshi noticed a complete absence of typical kinetochore proteins in the genome of Trypanosoma brucei, a protozoan parasite (Akiyoshi and Gull, 2013, 2014; Figure 1). He went on to identify a novel mechanism of chromosome segregation for eukaryotes, and, because of the unique identity of the proteins, they are likely to be drug-able targets for treatment of African sleeping sickness disease, which is caused by infection with T. brucei. In seeking out basic biology in an understudied system, Akiyoshi’s lab is finding new mechanisms to old cell biological problems that could lead to a potential disease cure. 10 | A. S. Gladfelter Adoptions of alternative systems can also stem from a lab with a principal investigator familiar with established systems and willing to take risks. Wallace Marshall at the University of California, San Francisco, for example, long a student of flagella in Chlamydomonas, has recently established work in Stentor, a ciliate among the largest single-celled organisms known. Marshall’s lab has established genetic techniques, proteomics, and transcriptomics to study cell polarity, cytoplasmic flow, and regeneration in these simple and intriguing pond dwellers (Slabodnick et al., 2013, 2014; Slabodnick and Marshall, 2014). Similarly, Bob Goldstein at the University of North Carolina, Chapel Hill, an established leader in Caenorhabditis elegans developmental cell biology, invested in establishing another microscopic animal—the Tardigrade or aptly named “water bear” (Figure 1). The water bear is fascinating in terms of evolutionary development but also in its ability to withstand prolonged desiccation (Gabriel and Goldstein, 2007; Gabriel et al., 2007; Tenlen et al., 2013). The water bear has already journeyed into space for experiments with the aim of understanding how these tiny animals that normally reside in moss are so resilient. Both water bears and Stentor are proving to be tractable, rich sources of new biology and may provide important insight into stress responses and regeneration. There are also many “forgotten” systems that were once widely studied but fell out of favor in the recombinant DNA revolution at the end of the 20th century. Many of these creatures happen to reside in the sea or other aquatic habitats and boast deep literatures of physiology, behavior, and, in some cases, cell biology (Figure 1). The squid is a great example, showing us microtubule motors and the action potential, yet the squid research community has shrunk dramatically, in part because squid cannot be readily cultured, thus requiring a steady supply from a marine lab such as the MBL in Woods Hole. Yet there is still tremendous biology being discovered in squid and other cephalopods. These organisms widely use RNA editing to generate variation in protein sequences posttranscriptionally, as is being studied intensively by Josh Rosenthal at the University of Puerto Rico and the MBL (Alon et al., 2015). RNA editing is dependent on temperature and, likely, other environmental conditions (Garrett and Rosenthal, 2012). Investment in cephalopodomics is promising to bring these traditional systems back in vogue. Finally, a hero of yeast geneticists, John Pringle, almost completely switched model systems to a simple sea anemone about 10 years ago. He has rejuvenated his program with new questions relevant to coral ecology and symbiosis (Lehnert et al., 2012, 2014). The sea is vast, and we have tapped into an extremely small amount of the biodiversity, especially at the cell and molecular level. Molecular Biology of the Cell HOW CAN WE MAKE IT MORE FEASIBLE FOR LABS TO ESTABLISH NEW SYSTEMS? Millions of years of evolution has done the hard work of creating vast biodiversity on the planet, so how can we start feasting intellectually on this biology at the level of molecules, cells, and tissues? There is no question that, in this century, we will face problems arising out of climate change, habitat destruction, and expanding and aging human populations that are highly mobile, allowing rapid spread of infectious diseases. How can we best prepare to solve the challenges that will arise from these monumental changes on the planet? I would argue that the track record of basic science solving problems relevant to human health and society is sparkling. Why should we stop here when the technology is in our reach to readily tame many systems? The question is who can pay for the risk of attempting to establish a new system. For Ashbya, Peter Philippsen had the drive and ingenuity to fund sequencing the Ashbya genome more than 15 years ago. He was willing to take the risk and had the creativity to invest in a completely new system after decades of work in budding yeast. He had enough funding flexibility at the time, being in a European institution with industry support, to see it through to complete establishment. What could be done to make it more feasible for even small and new labs to find a parallel alternative model? It could be argued that this is for the realm of the National Science Foundation (NSF), as the work will likely initially result in new science but may not immediately be translated to human health. However, the National Institute of General Medical Sciences at the National Institutes of Health (NIH) has a stellar history in funding basic science that leads to Nobel Prizes and cures for diseases with time. What if everyone who had an R01 and the interest could apply for a “model organism development” supplement to perform foundational experiments to establish a new system in parallel with their conventional system? The advantages and joys of work in an unchartered system revolve around the intellectual challenges, the breathing room of broad problems, which is especially useful for junior scientists, and the potential to discover truly new solutions in biology and for society. Each summer I move part of my lab to the MBL in Woods Hole, and one future for this hallowed place may well be to facilitate the development of new systems—if we can convince funding agencies and our deans to allow us this freedom. My children, ages seven and nine, eagerly attend the Children’s School of Science in Woods Hole while we are there, and each day they head to the ponds, the surf, and the fields to observe nature. For their generation, who are currently learning to gaze at diverse creatures, I hope they have a menagerie of choices of systems in which to study biology. ACKNOWLEDGMENTS I apologize to any nonconventional model systems that I did not have space to discuss. I acknowledge my intellectual family of formal 2015 ASCB Award Essays, Selected Perspective, and MBoC Paper of the Year advisors, mentors, students, lab managers, and postdocs for making this such a rewarding profession. I am also grateful to the U.S. government for generous support of our work through the NIH and the NSF. Finally, I would not be in this position without the encouragement of my parents and sister or support of my husband and children. REFERENCE Akiyoshi B, Gull K (2013). Evolutionary cell biology of chromosome segregation: insights from trypanosomes. Open Biol 3, 130023. Akiyoshi B, Gull K (2014). Discovery of unconventional kinetochores in kinetoplastids. Cell 156, 1247–1258. 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Nontraditional model systems | 11 MBoC | ASCB AWARD ESSAY A case for more curiosity-driven basic research Angelika Amon Koch Institute for Integrative Cancer Research, Department of Biology, Howard Hughes Medical Institute, Massachusetts Institute of Technology, Cambridge, MA 02139 ABSTRACT Having been selected to be among the exquisitely talented scientists who won the Sandra K. Masur Senior Leadership Award is a tremendous honor. I would like to take this opportunity to make the case for a conviction of mine that I think many will consider outdated. I am convinced that we need more curiosity-driven basic research aimed at understanding the principles governing life. The reasons are simple: 1) we need to learn more about the world around us; and 2) a robust and diverse basic research enterprise will bring ideas and approaches essential for developing new medicines and improving the lives of humankind. are lists, some better than others. We now When I was a graduate student, curiosity-driven know how many coding genes define a basic research ruled. Studying mating-type given species and how many protein kiswitching in budding yeast, for example, was nases, GTPases, and so forth there are in exciting because it was an interesting problem: the various genomes we sequenced. This How can you make two different cells from a sinknowledge, however, does not even gle cell in the absence of any external cues? We scratch the surface of understanding their did not have to justify why it is important to study function. When I browse the Saccharomywhat many would now consider a baroque quesces cerevisiae genome database (my section. Scientists and funding agencies alike agreed ond-favorite website), I am still amazed that this was an exciting biological problem that how many genes there are that have not needed to be solved. I am certain that all scieneven been given a name. tists of my generation can come up with similar To me the most important achieveexamples. ment the new genome-sequencing and Since the time I was a graduate student, the genome-editing technologies brought us field of biological research has experienced a is that nearly every organism can be a revolution. We can now determine the genetic model organism now. We can study and makeup of every species in a week or so and have Angelika Amon manipulate the processes that most fascian unprecedented ability to manipulate any genate us in the organisms in which they ocnome. This revolution has led to a sense that we cur, with the exception, of course, of humans. Thus, I believe that understand the principles governing life and that it is now time to the golden era of basic biological research is not behind us but in apply this knowledge to cure diseases and make the world a better front of us, and we need more people who will take advantage of place. While applying knowledge to improve lives and treat disthe tools that have been developed in the past three decades. I eases is certainly a worthwhile endeavor, it is important to realize am therefore hoping that many young people will chose a career that we are far from having a mechanistic understanding of even the in basic research and find an exciting question to study. The more basic principles of biology. What the genomic revolution brought us of us there are, the more knowledge we will acquire, and the higher the likelihood we will discover something amazing and DOI:10.1091/mbc.E15-06-0430. Mol Biol Cell 26, 3690–3691. important. There is so much interesting biology out there that Angelika Amon is the recipient of the 2015 ASCB Women in Cell Biology Sandra we should strive to understand. Some of my favorite unanswered K. Masur Senior Leadership Award. questions are: What are the biological principles underlying symAddress correspondence to: Angelika Amon (angelika@mit.edu). biosis and how did it evolve? Why is sleep essential? Why do © 2015 Amon. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to plants, despite an enormous regenerative potential, never die of the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Crecancer? Why do brown bears, despite inactivity, obesity, and high ative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0). levels of cholesterol, exhibit no signs of atherosclerosis? How do “ASCB®,” “The American Society for Cell Biology®,” and “Molecular Biology of sharks continuously produce teeth? the Cell®” are registered trademarks of The American Society for Cell Biology. 12 | A. Amon Molecular Biology of the Cell One could, of course, argue that the knowledge we have accumulated over the past 50 years provides a reasonable framework, and it is now time to leave basic science and model organisms behind and focus on what matters—curing diseases, developing methods to produce energy, cleaning up the oceans, preventing global warming, building biological computers, designing organisms, or engineering whatever the current buzz is about. Like David Botstein, who eloquently discussed the importance of basic research in these pages in 2012 (Botstein, 2012), I believe that the notion that we already know enough is wrong and the current application-centric view of biology is misguided. Experience has taught us over and over that we cannot predict where the next important breakthrough will be emerge. Many of the discoveries that we consider groundbreaking and that have brought us new medicines or improved our lives in other ways are the result of curiosity-driven basic research. My favorite example is the discovery of penicillin. Alexander Fleming, through the careful study of his (contaminated) bacterial plates, enabled humankind to escape natural selection. More recent success stories such as new cures for hepatitis C, the human papillomavirus vaccine, the HIV-containment regimens, or treatments for BCR-ABL induced chronic myelogenous leukemia have also only been possible because of decades of basic research in model organisms that taught us the principles of life and enabled us to acquire the methodologies critical to develop these treatments. Although work from my own lab on the causes and consequences of chromosome mis-segregation in budding yeast has not led to the development of new treatments, it has taught us a lot about how an imbalanced karyotype, a hallmark of cancer, affects the physiology of cancer cells and creates vulnerabilities in cancer cells that could represent new therapeutic targets. These are but a few examples for why it is important that we scientists must dedicate ourselves to the pursuit of basic knowledge and why we as a society must make funding basic research a priority. Achieving the latter requires that we scientists tell the public about the importance of what we are doing and explain the potential implications of basic research for human health. At the same time, it 2015 ASCB Award Essays, Selected Perspective, and MBoC Paper of the Year will be important to manage expectations. We must explain that not every research project will lead to the development of new medicines and that we cannot predict where the next big breakthroughs will materialize. We must further make it clear that this means we have to fund a broad range of basic research at a healthy level. Perhaps a website that collects examples of how basic research has led to breakthroughs in medicine could serve as a showcase for such success stories, bringing the importance of what we do to the public. While conducting research to improve the lives of others is certainly a worthy motivation, it is not the main reason why I get up very early every morning to go to the lab. To me, gaining an understanding of a basic principle in the purest Faustian terms is what I find most rewarding and exciting. Designing and conducting experiments, pondering the results, and developing hypotheses as to how something may work is most exciting, the idea that I, or nowadays the people in my lab, may be (hopefully) the first to discover a new aspect of biology is the best feeling. It is these rare eureka moments, when you first realize how a process works or when you discover something that opens up a new research direction, that make up for all the woes and frustrations that come with being an experimental scientist in an expensive discipline. For me, having a career in curiosity-driven basic research has been immensely rewarding. It is my hope that basic research remains one of the pillars of the American scientific enterprise, attracting the brightest young minds for generations to come. We as a community can help to make this a reality by telling people what we do and highlighting the importance of our work to their lives. ACKNOWLEDGMENTS I am grateful to my friend and colleague Frank Solomon for his thoughts and discussions. REFERENCE Botstein D (2012). Why we need more basic biology research, not less. Mol Biol Cell 23, 4160–4161. Why we need more basic research | 13 MBoC | ASCB AWARD ESSAY Surviving as an underrepresented minority scientist in a majority environment Erich D. Jarvis Department of Neurobiology, Duke University Medical Center, Durham, NC 27710; Howard Hughes Medical Institute, Chevy Chase, MD 20815 ABSTRACT I believe the evidence will show that the science we conduct and discoveries we make are influenced by our cultural experience, whether they be positive, negative, or neutral. I grew up as a person of color in the United States of America, faced with challenges that many had as members of an underrepresented minority group. I write here about some of the lessons I have learned that have allowed me to survive as an underrepresented minority scientist in a majority environment. THE DIRECT INFLUENCE OF E. E. JUST ON MY CAREER heard gunshots several nights a week. Most of those gunshots were not meant I am honored to be the recipient of the for target practice, including the one that 2015 Ernest Everett Just Award from the killed my father. One of my mentors at American Society for Cell Biology and to Rockefeller at the time, Peter MacLeish, write this associated essay. Just had an iman African-American assistant professor in pact on my scientific life well after his the lab of Nobel laureate Torsten Wiesel death in 1941. I was a beginning graduate and professor next door at Weill Cornell student at the Rockefeller University in Medical College, sat me down in his office New York City at the end of the 1980s, to talk about my life. At the end of our struggling to get a grip on the drama that conversation, MacLeish gave me his copy was unfolding in my life. I had graduated of a book published five years earlier from Hunter College in New York City with (1983) titled The Black Apollo of Science: a double major in biology and mathematThe Life of Ernest Everett Just by Kenneth ics, published several papers from my unR. Manning. MacLeish said, “I want you to dergraduate research, was accepted into read the book, and then come back and top-tier graduate programs, and then my talk to me.” grandfather, who helped support me, died Erich D. Jarvis I read the book and identified with of a heart attack, his brothers died soon Just’s experience. Like Just, my mother after, my homeless father was shot and was single, raising multiple (four) children. I had been ill on and off killed, my first child was on the way, and although technically being at a young age (6–8 years old), intermittently taken out of school, a middle-class person of color with a house I inherited from my and partly as a result was delayed in my education, being below grandfather, I lived in a Bronx ghetto neighborhood where we often grade level in reading and writing. One physician even told my mother that I could become mentally retarded due to a blow my father had made to my back. He did it in a state of anger for me goDOI:10.1091/mbc.E15-06-0451. Mol Biol Cell 26, 3692–3696. ing into his pants pockets and eating some of his drugs (which he Erich D. Jarvis is the 2015 recipient of the E. E. Just Award from the American had synthesized as a student of chemistry) and also for my brother Society for Cell Biology. and me throwing toys and clothes out the window in our Harlem Address correspondence to: Erich D. Jarvis (jarvis@neuro.duke.edu) apartment. Most importantly, Just was an African American who was © 2015 Jarvis. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to struggling to survive as a scientist in a Caucasian majority environthe public under an Attribution–Noncommercial–Share Alike 3.0 Unported Crement (I say environment instead of world, because the majority of ative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0). the world was and still is not Caucasian). Just had been considered “ASCB®,” “The American Society for Cell Biology®,” and “Molecular Biology of the Cell®” are registered trademarks of The American Society for Cell Biology. for a faculty position at Rockefeller in the 1930s for his genius in cell 14 | E. D. Jarvis Molecular Biology of the Cell biology but was rejected in part due to a scientific dispute with a former mentor and collaborator, Jacques Loeb at Rockefeller, and in part due to his race. I came back to MacLeish, had that discussion with the resolve that if Just could make it through his hardships, so could I. And further, Just’s hardships were orders of magnitude more difficult than mine, due in part to greater discrimination and lower expectations for him. It was at that time I began to appreciate how much of a profound impact ethnicity, culture, and gender can have on an individual’s career. Before then, I was surrounded by persons of diverse backgrounds, many of whom looked like me. However, now I was at a mostly Caucasian institution, clearly one of the world’s best in biomedical science, faced with culture shock of wondering why did most of the students have a shared experience different from me? Although they had their struggles, why were mine different and, in many cases, tougher? In this essay, I discuss some of the answers to these questions and lessons learned that helped me survive as an underrepresented minority scientist, all of which I hope will be helpful for all scientists and all people as they navigate their careers. THEY KILLED HIM BECAUSE OF THE COLOR OF HIS SKIN I was born in New York City in 1965, at a high point of the Civil Rights movement, including the signing of the Voting Rights Act. My maternal grandparents and paternal great-grandparents were from North Carolina and Virginia, having moved north to New York either in the late 1800s or during the Great Depression of the 1930s, most being descendants of slaves. I remember the day when I was watching a black-and-white television at my maternal grandparents’ house in Queens, New York, where we lived after my mother divorced in the early 1970s, about a news story of the anniversary of Martin Luther King Jr.’s death. Being between 6 and 7 years old and an inquisitive child, I asked my mother, “Why did they kill him?” She seemed to have a hard time explaining it me, and finally came out with it was because the color of his skin, he was black, a Negro, and wanted to bring peace to all. I remember looking at my skin, feeling afraid, and wondering, “Are they going to kill me one day?” From that time onward as a child, I remember wanting my skin to become white, my hair straighter, and my nose and lips thinner. My family was diverse, and I envied those who looked more European than me. I internalized a feeling of “less than.” It now makes me wonder how young African-American children feel today, when they see on the news stories about young black men killed by police with the reason partly linked to ethnicity. BEING TRAINED TO PERSEVERE IN THE FACE OF DAUNTING OBSTACLES Following my father’s conversion to a Japanese sect of Buddhism (Soka Gakkai), my mother converted as well and taught us (her children) its philosophies. Although her mother, a Baptist Christian, secretly told us kids that she thought it was the devil’s religion, many of the views of Buddhism made sense to me. I applied them to my life, including that I am responsible for my own destiny, must pursue my most ambitious dreams, dream the impossible, and that, no matter what, all obstacles can be overcome as long as you work at it. Although I stopped practicing the faith-based part of the religion in my teens, I still followed the philosophical views, which I found I needed to get through the “less than” internalized feelings. I first applied them to pursue a career in dancing. I was accepted into the High School of Performing Arts, was on scholarships at the Joffrey Ballet and Alvin Ailey Dance schools in New York, and performed with the Westchester Ballet Company. But at the end my senior year, 2015 ASCB Award Essays, Selected Perspective, and MBoC Paper of the Year I was at a junction between choosing opportunities for a career in dance or something else I fell in love with, science. I chose science, following my mother’s training of doing something that has a positive impact on society. I thought I could accomplish that better as a scientist than as a dancer. So again, I applied my training in Buddhism and now in the arts, into the sciences as an undergraduate student at Hunter College of the City University of New York. I found that the transition between dance and science was natural, as both required discipline, creativity, hard work, and, often, acceptance of failure before something works. Hunter was and still is an ethnically diverse school, although I did not realize that at the time, because it looked like the rest of New York City’s melting pot. There I was taken under the wing of Rivka Rudner, a molecular microbiologist studying ribosomal genes, which synthesize proteins. I invested many hours in the lab, sometimes staying overnight to finish my experiments, and pursued projects that eventually led me to publish six research papers with her, including two as first author and one as senior corresponding author (three of which were published by the time I graduated). We developed molecular tools to map the chromosomal organization of the protein synthesis genes in bacteria and determined how their organization affected their genome evolution and function. Making such discoveries, working with collaborators, and publishing these papers gave me the confidence that I could be a scientist. At this juncture, I was considering the choices of applying to medical school, graduate school, or both to pursue a MD/PhD. This was a struggle many from economically challenged backgrounds face, where there is a drive in the family to have my “son/daughter the doctor.” Although I prepared for and performed well on entrance exams for both kinds of degrees, I decided to focus on the basic science PhD route because this was where my passion was, and it felt more closely connected to my artistic side. THE ONLY WAY I FOUND I WAS ABLE TO OVERCOME THE FEELING OF LESS THAN WAS BY BEING SUCCESSFUL I applied to and was accepted into many of the top graduate programs in the country in addition to Rockefeller, including MIT, Harvard, Yale, Princeton, Johns Hopkins, Berkeley, and the University of California, San Francisco. During my interviews, some of the faculty members I interviewed with cautiously informed me that they were surprised that an African-American kid from Harlem had achieved what I did and were wondering how I did it. I did not have an answer and was wondering, “What did I do?” I never thought that I could not. More ethnically sensitive questions and statements came from students. At one place, in a group conversation, a student told me that I should be careful of going to a specific neighborhood, because there are “blacks and Puerto Ricans there and it is dangerous.” I stared back at the person speechless, with a half-smile, wondering to whom does he think he is talking? That is the kind of neighborhood I come from; my wife at the time was Puerto Rican. Were she and I dangerous? Another place had two of their students, one African American and the other Caucasian, contact me on separate occasions to say that if I did not accept their offer for graduate school, there would be no more African-American students in their PhD program. I wondered whether I needed their program, or they needed me. Why was this happening? I accepted the offer to join the graduate student program at Rockefeller but found a different experience from my previous schools. At Hunter, as at many institutions with a diverse population, there was a hold-your-hand support system practiced by the faculty. At Rockefeller, it was sink or swim, as it is at many Research 1 institutions. I was swimming and sinking in my first four years as a graduate Underrepresented scientist survival | 15 student. I had never heard of parents being able to purchase a car for their children after they graduate college; instead, I was helping my parents keep out of financial troubles. There was an unspoken feeling among a very small number of students that I was there to fill a quota, and although tiny, it was enough to contribute to me feeling that way too. At the same time, my father was killed, elders in my family were dying of natural but still more long-term preventable causes, I was taking care of a family, I had failed my first prelim examination (which I passed the second time), and I was struggling to get my experiments to work. I began to again internalize feelings of less than. I had felt that I did not belong in this sink-or-swim world, despite that fact that I had by then seven publications with my undergraduate advisor. But I did not give up. I worked hard, trying different ways of overcoming these obstacles, and in my last two years of graduate school, my earlier life experience training kicked in. Once the small amount of negative influence around me moved on, I found my groove and quickly progressed in my research, publishing three papers from my graduate research (including one as first author). I decided to break the rules and stay on as a postdoctoral fellow in the same lab where I did my PhD, that of Fernando Nottebohm at Rockefeller, because I felt I was just beginning to really swim. My litmus test for making decisions was asking: “What are people going to remember me for after I die?” Whether I followed standard rules or made a significant impact in science? From the three years of my postdoctoral research, I published three papers on the molecular biology of vocal learning, eventually 10 altogether, in high-impact journals, including Nature. I had finally overcome the feeling of less than. I found that the only way I could overcome the feeling of less than was to be successful in what I set out do to. THE COLOR OF MY SKIN AND MY GENDER IS EITHER A DISADVANTAGE OR AN ADVANTAGE, BUT RARELY NEUTRAL My postdoctoral years were the mid-1990s, a period during which funding in science had been looking grim. Very good people were applying for academic jobs and not getting them. Politicians were being blamed for decreasing funding to science and making it harder for us to make new discoveries and contribute to society. I made a vow to myself that I was going to figure out a way to succeed and survive by doing the best science I could do. I worked very hard, sometimes too hard. I applied for a small number of professorship research positions and received offers from all. Once again, similar to my experience as a graduate student, faculty members at these institutions appeared to be surprised that a person of color, an African American, born in the United States, had accomplished what he did. Suddenly, was I heavily recruited, like an up-and-coming basketball star. I felt that the color of my skin and the drive to increase diversity among the academic ranks combined with my scientific successes suddenly made me a commodity. This was the first time in my life, at 32, when I felt the color of my skin was an advantage. Then I was told about a Duke University tuition benefit program in which, if I were a faculty member for more than five years, Duke University would pay for 100% of up to two of my children’s tuition at Duke or up to 75% of Duke’s rate at any other university in the world. Finding this out floored me. It washed away generations of oppression for me in an instant. African Americans were not allowed to be students at Duke before the early 1960s, soon before I was born, and still had a hard time being accepted. My parents and grandparents would not have been able to get into Duke, regardless of their talents. And now, my children (I had two) would later be able to get a high-quality, expensive college tuition 16 | E. D. Jarvis paid for, anywhere in the world. It hit me that this was an affirmative action program for Caucasians that had been around for generations. I cried, and accepted Duke’s offer. I was now in one of the world’s leading neuroscience programs with the resources I needed to accomplish the science I wanted to achieve and make sure my children had an opportunity for a high-quality education. From this experience, I learned that the color of my skin or my gender or that of anyone else is either an advantage or a disadvantage, but rarely neutral. For most of my life it had been a disadvantage, but for once and at that moment, it was an advantage. I wanted it to be neutral, but this was beyond my immediate control. It also made me realize that the affirmative action programs I had benefited from, such as the National Institute of General Medical Sciences Minority Biomedical Research and Minority Access to Research Careers programs as an undergraduate and graduate student were affirmative action programs that offset a disadvantage that many underrepresented students and others do not realize that they have. I HAVE TWO JOBS: BE THE BEST SCIENTIST I CAN BE AND HELP CURE SOCIETY’S RACIAL DISEASE After I arrived at Duke in 1998, I was inducted into many initiatives to help diversify the scientific workforce, including the push for women in science. I wondered, as a man, what did I know about women? But there was an assumption that a person of an underrepresented minority background knew more of what was needed for any minority to succeed, including women in science, relative to white males. There may be some truth to this, but certainly not an absolute truth. I began to realize that as a young professor at Duke University, and within the scientific community generally, I was being unintentionally asked to take on two jobs: 1) be the best scientist I could be, as expected of everyone else; and 2) help cure society’s racial disease, unlike everyone else. After two years, I made the conscious decision that I could not do both jobs well at the same time. I decided on job 1, to pursue being the best scientist I could be, and only taking on those few tasks for job 2 in which I felt I could make the biggest impact. However, I felt that job 1 could indirectly help cure society’s disease. There were many people before me who had taken on activist roles in bringing down obstacles and opening up opportunities for others like me to become scientists. I felt what we needed more of now was underrepresented minority scientists leading by example, so that others would not say they are there because of a quota, that they are less than, and that they have an advantage because of the color of their skin. This is what I set out to do as a scientist and professor at Duke University. To do so, I had to learn how to say no to many requests, balanced with yes for those that had the biggest impacts. Before doing so, I would often consult with others to get a second opinion. I also would also encourage others who were not underrepresented minorities to help with job 2, as I felt the cure to this disease required participation and education of everyone and not just underrepresented minorities. OVERCOMING CULTURE SHOCK AND LEARNING HOW TO REACH OUT When I first arrived at Duke, I had a similar but more stark experience than at Rockefeller, where I felt I was in the middle of Europe in terms of ethnicity. This was despite the fact that, relative to the rest of the country, there was a high proportion of African Americans in the surrounding population. I recall interviewing a female Hispanic student our department brought in as a candidate for graduate school; during the interview, she opened a booklet that included the Duke student demographics. African Americans were in the low Molecular Biology of the Cell single digits, and Hispanics even lower. She actually cried in my office, wondering whether she had any chance of getting in, and if she did, would she really be accepted culturally? Pertaining to faculty, years later in 2006, the dean called a meeting of African-American faculty members of the medical school to discuss our views of the now infamous Duke lacrosse case, in which an African-American female stripper accused team members of rape (later turned out to be false). At the meeting, I learned that I was only the second basic science African-American faculty member in the medical school, out of more than 200, in more than 28 years. The hiring of clinical faculty fared better in absolute numbers, although not proportionally, as there were more than 1800 clinical faculty members. Basically, some others and I, including students, were feeling culture shock. The ethnic diversity at Duke and some other Research 1 schools has changed quite a bit, particularly, at the student level, since I became a professor in the late 1990s. The change was ushered in by purposeful efforts made by leadership, including the Duke President’s Council on Black Affairs chaired by President Nannerl Keohane, the Dean’s Council on Diversity led by Nancy Andrews, admissions offices of the medical schools and graduate schools led by Brenda Armstrong and Jacklynn Looney, respectively, and now the Office of Biomedical Graduate Diversity started by Dona Chickaraishi; this last office is now led by one of the students I mentored at Duke and the second African-American student to graduate from our neurobiology department, Sherilynn Black. What I have learned from these experiences about surviving as an underrepresented minority in the sciences is that many such students do not take proactive steps to reach out, seek help, and get answers to concerns they have. They wait for someone to come to them. But they must learn how to reach out in order to survive. For institutions, creating a culture of inclusion and a space for these students to air their thoughts is a great help. ACCEPT ALL APPROACHES AND BY ANY MEANS NECESSARY I have seen from firsthand experience how cultural background plays a strong role in the way we go about conducting our science. Growing up as a child in the 1960s and 1970s, my perception was that you were either a “Martin Luther King family” or a “Malcolm X family.” The Martin Luther King family adopted the belief of loving and accepting everyone to bring about world peace; the Malcolm X family adopted the belief of achieving equality by any means necessary. We were a Martin Luther King family. In this regard, in my laboratory, I brought together people of diverse ethnicities and cultures with diverse ways of thinking, and found that this diversity led to more rapid advances in our science relative to a mono-ethnic or mono-gender group. But I have stolen from Malcolm X’s thinking as well and have taken the approach to addressing scientific questions by any means necessary, as long as it does not harm anyone. In this manner, I have incorporated molecular biology, anatomy, physiology, evolutionary biology, genetics, and computational biology into our research program to address questions on the brain mechanisms and evolution of vocal learning and spoken language. An analogy is the marriage between physics and biology that led to the discovery of the structure and genetic code of DNA. If I had not been trained to think in this way as a child from a diverse background, it might have been harder for me to learn how to do so as an adult. ACCEPTING ALL OF ME Growing up in the United States, we are often trained to think in a black-and-white world. Further, black was considered bad, white good, and any drop of blackness meant that you were considered 2015 ASCB Award Essays, Selected Perspective, and MBoC Paper of the Year black. Although the definition of mulatto existed, it or admixture was not a recognizable category in the check box of many forms. You had to pick and choose, which became the title of a popular song by one my cousins, a Native American singer, Pura-Fe. My perception of growing up and how I was treated was mainly (∼80%) African, with a mixture of (∼10% each) Native American and European. However, my family had debates about exactly what we were. I decided at a young age to investigate the discrepancies, which I still continue to do to this day. In contrast to perception, based on the oral history of my elders and many others (and even in contrast to some of their own perceptions), my ancestors have been admixed over and over again between these three ethnic groups for several hundred years. My oral history calculations are 48% African, 37% European, and 14% Native American, each of multiple ethnic groups from these ancestral populations. But my genetic ancestry from ancestry.com, 23andMe, and whole-genome sequencing shows 49% African, 49% European, and less than 1% Native American. There are various reasons for the oral and genetic discrepancies, including that either the oral tradition is wrong or there is not sufficient Native American DNA diversity in the databases. One thing that genetics taught me is that my African and European ancestries are heavily admixed from seven different ethnic groups each, and this is not because they were already admixed before arrival in the Americas—most of the admixtures occurred after arrival in the Americas. The biggest lesson I learned from taking a cultural and scientific perspective in trying to figure out who I am, was that I had to learn to accept all of me in order to help propel me along my scientific journey more successfully. Neither I nor anyone else is really living in a black-and-white world. Our current president, Barack Obama, is considered the first “African-American” president of the United States. But like me, he is as much European as he is African. So what happened to the European part in the minds of Americans? It is buried, not thought about, because we still live with this social disease of racism. If you can transcend that thought, accept all of who you are, all of who we are, then I think you have a much greater ability to communicate and interact with the broader world and advance in your science or whatever path in life you choose. In closing, the challenges that Ernest Everett Just faced externally and internally and the approach he took to try to overcome them has influenced how I handled trying to overcome the challenges I faced. Some of my challenges were easier, and some different, due in part to others making it more possible for me to succeed than was possible in Just’s time. I suspect and hope that my brief story here will help the next generation to overcome their challenges, which will invariably have overlap and differences. One such difference I predict is that the countries and thus the environments in which science is highly valued will have less of a black-and-white view of the world, in part due to a greater understanding of human and thus species genetics, and due in part to greater numbers of admixed peoples according to current racial categories. I hope that my story can be of comparable help to underrepresented minorities and the majority. Finally, I note that not all formulas work for all people. My PhD advisor, Fernando Nottebohm, said he would tell his son to “understand 100% of what I say, but only believe in 50% of it.” This meant to me that not all formulas work for all, but there is always room for improvement and change, and sometimes we get things wrong, need to recognize when we do, correct them, and then move on. For further reading on my life story in the sciences written by others see publications by Rimer (1989), Dreifus (2003), Adler (2006), Blakeslee (2005), NOVA (2005), and Berstein (2015a, 2015b). For Underrepresented scientist survival | 17 reading about some of the past and recent scientific discoveries I have contributed to that I feel are broadly important see publications by Jarvis (2004), Jarvis et al. (2005, 2014), Petkov and Jarvis (2012), Pfenning et al. (2014), Whitney et al. (2014), and Zhang et al. (2014a,b). ACKNOWLEDGMENTS I acknowledge the support of my mother, father, stepfather, grandparents, other family members, and friends, as well as my undergraduate advisor Rivka Rudner and graduate advisor Fernando Nottebohm, who helped me get to where I am now. I also acknowledge the opportunities and funding provided to me by the National Institutes of Health (particularly the National Institute of General Medical Sciences), the National Science Foundation, the Howard Hughes Medical Institute, the Society for Neuroscience Scholars Program, Hunter College, The Rockefeller University, Duke University, and many others, without which it would not have been possible for me to progress as a scientist. REFERENCES Adler J (2006). Song and dance man. Smithsonian November. www .smithsonianmag.com/making-a-difference/song-and-dance -man-135440722. Berstein R (2015a). Following the birdsong of science. Science Careers January 19. http://sciencecareers.sciencemag.org/career_magazine/ previous_issues/articles/2015_01_19/caredit.a1500015. Berstein R (2015b). Science by any means necessary. Science 347, 686. www .sciencemag.org/content/347/6222/686.short. Blakeslee S (2005). Minds of their own: birds gain respect. New York Times February 1. www.nytimes.com/2005/02/01/science/minds-of-their-own -birds-gain-respect.html. 18 | E. D. Jarvis Dreifus C (2003). A conversation with: Erich Jarvis: a biologist that explores the minds of birds that learn to sing. New York Times, January 7. www.nytimes.com/2003/01/07/science/conversation-with-erich-jarvis -biologist-explores-minds-birds-that-learn-sing.html. Jarvis ED (2004). Learned birdsong and the neurobiology of human language. Ann NY Acad Sci 1016, 746–777. Jarvis ED, Güntürkün O, Bruce L, Csillag A, Karten H, Kuenzel W, Medina L, Paxinos G, Perkel DJ, Shimizu T, et al. (2005). Avian brains and a new understanding of vertebrate brain evolution. Nat Rev Neurosci 6, 151–159. Jarvis ED, Mirarab S, Aberer AJ, Li B, Houde P, Li C, Ho SYW, Faircloth BC, Nabholz B, Howard JT, et al. (2014). Whole genome analyses resolve the early branches to the Tree of Life of modern birds. Science 346, 1320–1331. Manning KR (1983). The Black Apollo of Science: The Life of Ernest Everett Just, New York: Oxford University Press. NOVA (2005). ScienceNOW profile of Jarvis. www.pbs.org/wgbh/nova/ nature/erich-jarvis.html. Petkov CI, Jarvis ED (2012). Birds, primates, and spoken language origins: behavioral phenotypes and neurobiological substrates. Front Evol Neurosci 4, 121–24. Pfenning A, Hara E, Whitney O, Rivas MV, Wang R, Roulhac PL, Howard JT, Wirthlin M, Lovell PV, Ganapathy G, et al. (2014). Convergent transcriptional specializations in the brains of humans and song learning birds. Science 346, 1256846. Rimer S (1989). Random death claims a man who struggled to regain life. New York Times May 27. www.nytimes.com/1989/05/27/nyregion/ random-death-claims-a-man-who-struggled-to-regain-life.html. Whitney O, Pfenning AR, Howard JT, Blatti CA, Liu F, Ward JM, Wang R, Audet JN, Kellis M, Mukherjee S, et al. (2014). Core and region enriched gene expression networks of behaviorally regulated genes and the singing genome. Science 346, 1256780. Zhang G, Jarvis ED, Gilbert MTP (2014a). A flock of genomes. Science 346, 1308. Zhang G, Li C, Li Q, Li B, Larkin DM, Lee C, Storz JF, Antunes A, Greenwold MJ, Meredith RW, et al. (2014b). Comparative genomics reveals insights into avian genome evolution and adaptation. Science 346, 1311–1320. Molecular Biology of the Cell MBoC | ASCB AWARD ESSAY An unconventional route to becoming a cell biologist Elaine Fuchs Howard Hughes Medical Institute, Rockefeller University, New York, NY 10065 ABSTRACT I am honored to be the E. B. Wilson Award recipient for 2015. As we know, it was E. B. Wilson who popularized the concept of a “stem cell” in his book The Cell in Development and Inheritance (1896, London: Macmillan & Co.). Given that stem cell research is my field and that E. B. Wilson is so revered within the cell biology community, I am a bit humbled by how long it took me to truly grasp his vision and imaginative thinking. I appreciate it deeply now, and on this meaningful occasion, I will sketch my rather circuitous road to cell biology. I could hardly wait until I was in junior I grew up in a suburb of Chicago. My father high school, when I could enter science was a geochemist, and for everyone whose fairs. You would think that my scienceparents worked at Argonne National Labominded family might help me choose and ratories, Downers Grove was the place to develop a research project. True to their live. My father’s sister was a radiobiologist mentoring ethos, they left these decisions and my uncle was a nuclear chemist, both to me. My first project was on crayfish beat Argonne; they lived in the house next havior. I recorded the response of the craydoor. Across the street from their house fish I had caught to “various external stimwas the Schmidtke’s Popcorn Farm—a uli.” At the end of this assault, I dissected great door to knock on at Halloween. The the crayfish and, using “comparative anatcornfields were also super for playing hideomy,” attempted to identify all the parts. and-seek, particularly when you happened The second project was no gentler. I foto be shorter than those Illinois cornstalks. cused on tadpole metamorphosis and the I remember when the first road in the effects of thyroid hormone in accelerating area was paved. It made biking and rollerdevelopment at low concentrations and skating an absolute delight. Fields of butdeath at elevated concentrations. Someterflies were everywhere, and with develElaine Fuchs how, I ended up going all the way to the opment came swamps and ponds filled state fair, where it became clear that I had with pollywogs and local creeks with crayserious competition. That experience, however, whetted my appefish. It was natural to become a biologist. When coupled with a famtite to gain more lab experience and to learn to read the literature ily of scientists and a mother active in the Girl Scouts, all the remore carefully. sources were there to make it a perfect path to becoming a My experience with high school biology prompted me to gravscientist. itate toward chemistry, physics, and math. When it came to college, my father told me that if there was a $2000/year (translated in 2015 to be $30,000/year) reason why I should go anywhere DOI:10.1091/mbc.E15-06-0333. Mol Biol Cell 26, 3697–3699. besides the University of Chicago (where Argonne scientists reElaine Fuchs is the recipient of the 2015 E. B. Wilson Medal awarded by the American Society for Cell Biology. ceived a 50% tuition cut for their children) or the University of IlAddress correspondence to: Elaine Fuchs (fuchslb@rockefeller.edu). linois (then $200/year tuition), we could “discuss” it further. HavAbbreviations used: EBS, epidermolysis bullosa simplex; EH, epidermolytic ing a sister, father, aunt, and uncle who went to the University of hyperkeratosis; Ifs, intermediate filaments. Chicago, I chose the University of Illinois and saved my Dad a © 2015 Fuchs. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to bundle of money. At Illinois, I thought I might revisit biology, but the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Cremy choices for a major were “biology for teachers” or “honors ative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0). biology.” The first did not interest me; the second seemed ® ® “ASCB ,” “The American Society for Cell Biology ,” and “Molecular Biology of intimidating. the Cell®” are registered trademarks of The American Society for Cell Biology. 2015 ASCB Award Essays, Selected Perspective, and MBoC Paper of the Year 19 I enrolled as a chemistry major. Four years went by, during which time I never took a biology class. I enjoyed quantum mechanics, physics, and differential equations, and problem solving became one of my strengths. In the midst of the Vietnam War era, however, Illinois was a hotbed of activity. I was inspired to apply to the Peace Corps, with a backup plan to pursue science that would be more biomedically relevant than quantum mechanics. I was accepted to go to Uganda with the Peace Corps, but with Idi Amin in office, my path to science was clear. Fortunately, the schools I applied to accepted me, even though, in lieu of GRE scores, I had submitted a three-page essay on why I did not think another exam was going to prove anything. I chose Princeton’s biochemistry program. This turned out to be a great, if naïve choice, as only after accepting their offer did I take a biochemistry class to find out what I was getting into. I chose to carry out my PhD with a terrific teacher of intermediary metabolism, Charles Gilvarg, who worked on bacterial cell walls. My thesis project was to tackle how spores break down one cell wall and build another as they transition from quiescence to vegetative growth. By my fourth year of graduate school, I was trained as a chemist and biochemist and was becoming increasingly hooked on biomedical science. I listened to a seminar given by Howard Green, who had developed a method to culture cells from healthy human skin under conditions in which they could be maintained and propagated for hundreds of generations without losing their ability to make tissue. At the time, Howard referred to them as epidermal keratinocytes, but in retrospect, these were the first stem cells ever to be successfully cultured. I was profoundly taken by the system, and Howard’s strength in cell biology inspired me. It was the perfect match for pursuing my postdoctoral research. The time happened to be at the cusp of DNA recombinant technology. At MIT, I learned how to culture these cells. I wanted to determine their program of gene expression and how this changed when epidermal progenitors embark on their terminal differentiation program. While the problem in essence was not so different from that of my graduate work at Princeton, I had miraculously managed to receive my PhD without ever having isolated protein, RNA, or DNA. Working in a quintessential cell biology lab and tackling a molecular biology question necessitated venturing outside the confines of the Green lab and beyond the boundaries of my expertise. Fortunately, this was easy at MIT. Richard Hynes, Bob Horvitz, Bob Weinberg, and Graham Walker were all assistant professors, and their labs were very helpful, as were those of David Baltimore and Phil Sharp, a mere walk across the street. On the floor of my building, Steve Farmer, Avri Ben Ze’ev, Gideon Dreyfuss, and Ihor Lemischka were in Sheldon Penman’s lab just down the hall, and they were equally interested in mRNA biology, providing daily fuel for discussions. Uttam Rhajbandary’s and Gobind Khorana’s labs were also on the same floor, making it easy to learn how to make oligo(dT)-Sepharose to purify my mRNAs. Vernon Ingram’s lab was also on the same floor, so learning to make rabbit reticulocyte lysates to translate my mRNAs was also possible. Howard bought a cryostat, so I could section human skin and separate the layers. And as he was already working with clinicians at Harvard to apply his ability to create sheets of epidermal cells for the treatment of burn patients, I had access to the leftover scraps of human tissue that were also being used in these operations. The three years of my postdoc were accompanied by three Fuchs and Green papers. The first showed that epidermal keratinocytes spend most of their time expressing a group of keratin proteins with distinct sequences. The second showed that these keratins were each encoded by distinct mRNAs. The third showed that, 20 | E. Fuchs as epidermal keratinocytes commit to terminally differentiate, they switch off expression of basal keratins (K5 and K14) and switch on the expression of suprabasal keratins (K1 and K10). That paper also revealed that different stratified tissues express the same basal keratins but distinct sets of suprabasal keratins. I am still very proud of these accomplishments, and my MIT experience made me thirst to discover more about the epidermis and its stem cells. My first and only real job interview came during my second year of postdoc, at a time when I was not looking for a job. I viewed the opportunity, initiated by my graduate advisor, as a free trip home to visit my parents and my trial run to prepare me for future searching. I was thrilled when this interview materialized into an offer to join the faculty, for which the University of Chicago extended my start time to allow me to complete my three years with Howard. Times have clearly changed, and it is painful to see talented young scientists struggle so much more today. That said, I have never looked ahead very far, and having a lack of expectations or worry is likely to be as helpful today as it was then. I am sure it is easier said than done, but this has also been the same for my science. I have always enjoyed the experiments and the joy of discovery. There was no means to an end other than to contemplate what the data meant in a broader scope. I arrived at the University of Chicago with a well-charted route. My aim was to make a cDNA library and clone and characterize the sequences and genes for the differentially expressed keratins I had identified when I was at MIT. It was three months into my being at Chicago when my chair lined up some interviews for me to hire a technician. I was so immersed in my science that I did not want to take time to hire anyone. I hired the first technician I interviewed. Fortunately, it worked out. However, I turned graduate students away the first year, preferring to carry out the experiments with my technician and get results. After publishing two more papers—one on the existence of two types of keratins that were differentially expressed as pairs and the other on signals that impacted the differential expression of these keratin pairs, I decided to accept a student, who analyzed the human keratin genes. My first postdoc was a fellow grad student with me at Princeton; she studied signaling and keratin gene expression. My second postdoc was initiated by my father, who chatted with him at the elevator when I was moving into my apartment. He set up DNA sequencing and secondary-structure prediction methods, and the lab stayed small, focused, and productive. I was fascinated by keratins, how they assembled into a network of intermediate filaments (Ifs). When thalassemias and sickle cell anemia turned out to be due to defects in globin genes, I began to wonder whether there might be human skin disorders with defective keratin genes. I had no formal training in genetics, and there were no hints of what diseases to focus on. Thus, rather than using positional cloning to identify a gene mutation associated with a particular disease, we took a reverse approach: we first identified the key residues for keratin filament assembly. After discovering that mutations at these sites acted dominant negatively, we engineered transgenic mice harboring our mutant keratin genes and then diagnosed the mouse pathology. Our diagnoses, first for our K14 mutations and then for our K10 mutations, turned out to be correct: on sequencing the keratins from humans with epidermolysis bullosa simplex (EBS), we found K14 or K5 mutations; similarly, we found K1 or K10 mutations in affected, but not in unaffected, members of families with epidermolytic hyperkeratosis (EH). Both are autosomaldominant disorders in which patients have skin blistering or degeneration upon mechanical stress. Without a proper keratin network, the basal (EBS) or suprabasal (EH) cells could not withstand pressure. Ironically, family sizes of all but the mildest forms of these disorders Molecular Biology of the Cell were small, meaning that the disorders were not amenable to positional cloning. But the beauty of this approach is that once we had made the connection to the diseases, we understood their underlying biology. In addition, the IF genes are a superfamily of more than 100 genes differentially expressed in nearly all tissues of the body. Once we had established EBS as the first IF gene disorder, the pathology and biology set a paradigm for a number of diseases of other tissues that turned out to be due to defects in other IF genes. Fortunately, I had students, Bob Vassar (professor, Northwestern University) and Tony Letai (associate professor, Harvard Medical School), and a postdoc, Pierre Coulombe (chair, Biochemistry and Molecular Biology, Johns Hopkins University), who jumped into this fearless venture with me. We had to go off campus to learn transgenic technology. I had never worked with mice before. When Bob returned to campus with transgenic expertise, we hired and trained Linda Degenstein, whose love for animal science was unparalleled. Pierre’s prior training in electron microscopy was instrumental in multiple ways. Additionally, I was not a dermatologist and had no access to human patients. Fortunately Amy Paller, MD, at Northwestern volunteered to work with us. The success of this project attests to an important recipe: 1) Pursue a question you are passionate about. 2) In carrying out rigorous, well-controlled experiments, each new finding should build upon the previous ones. 3) If you have learned to be comfortable with being uncomfortable, then you will not be afraid to chart new territory when the questions you are excited to answer take an unanticipated turn. 4) Science does not operate in a vacuum. Interact well with your lab mates and take an interest in their science as well as your own. And wherever you embark upon a pathway in which the lab’s expertise is limited, do not hesitate to reach out broadly to other labs and universities. I have followed this recipe now for more than three decades, and it seems to work pretty well. A lab works only when its students and postdocs are interactive, naturally inquisitive, and freely share their ideas and findings. I have been blessed to have a number of such individuals in my lab over the years. When push comes to shove, I am always inclined first to shave from the “brilliant” category and settle for smart, nice people who are passionate and interactive about science and original and unconventional in their thinking. So what questions have I been most passionate about? I have always been fascinated with how tissues form during development, how they are maintained in the adult, and how tissue biology goes awry in human disorders, particularly cancers. I first began to think about this problem during my days at Princeton. I also developed a 2015 ASCB Award Essays, Selected Perspective, and MBoC Paper of the Year dogma back then that I still hold: to understand malignancies, one must understand what is normal before one can appreciate what is abnormal. I think this is why I have spent so much of my life focusing on normal tissue morphogenesis, despite my passion for being at the interface with medicine. And because skin has so many amazingly interesting complexities, and because it is a great system to transition seamlessly between a culture dish and an animal, I have never found a reason to choose any other tissue over the one I chose many years back. I will not dwell on the various facets of skin biology we have tackled over the years. Our initial work on keratins was to obtain markers for progenitors and their differentiating lineages. This interest broadened to understanding how proliferative progenitors form cytoskeletal networks and how the cytoskeleton makes dynamic rearrangements during tissue morphogenesis. From the beginning, the lab has also been fascinated by how tissue remodeling occurs in response to environmental signals. Indeed, signals from the microenvironment trigger changes in chromatin dynamics and gene expression within tissue stem cells. Ultimately, this leads to changes in proteins and factors that impact on cell polarity, spindle orientation, asymmetric versus symmetric fate specifications, and ultimately, the balance between proliferation and differentiation. The overarching theme of my lab over these decades is clear, namely, to understand the signals that unspecified progenitors receive that instruct them to generate a stratified epidermis, make hair follicles, or make sweat and sebaceous glands. And if we can understand how this happens, then how are stem cells born, and how do they replace dying cells or regenerate tissue after injury? And, finally, how does this process change during malignant progression or in other aberrant skin conditions? In tackling tissue morphogenesis, I have had to forgo knowing the details of each tree and instead focus on the forest. There are many times when I stand back and can only admire those who are able to dissect beautiful cellular mechanisms with remarkable precision. But I crossed that bridge some years ago in tackling a problem that mandates an appreciation of nearly all the topics covered in Bruce Alberts’ textbook Molecular Biology of the Cell. I am now settled comfortably with the uncomfortable, and the problem of tissue morphogenesis in normal biology and disease continues to keep me more excited about each year’s research than I was the previous year. Perhaps the difference between my days as a student, postdoc, and assistant professor and now is that my joy and excitement is as strong for those I mentor and have mentored as it is for myself. E. B. Wilson Medal | 21 MBoC | PERSPECTIVE Biosecurity in the age of Big Data: a conversation with the FBI Keith G. Kozminski Department of Biology, University of Virginia, Charlottesville, VA 22904; Department of Cell Biology, University of Virginia, Charlottesville, VA 22908 ABSTRACT New scientific frontiers and emerging technologies within the life sciences pose many global challenges to society. Big Data is a premier example, especially with respect to individual, national, and international security. Here a Special Agent of the Federal Bureau of Investigation discusses the security implications of Big Data and the need for security in the life sciences. INTRODUCTION “The FBI is reading our poster!” Granted, this is not a typical refrain heard at the annual meetings of the American Society for Cell Biology, but it is heard frequently at other research meetings, for example, in the field of synthetic biology. I admit that it was head turning when I first heard these words spoken a few years ago at the International Genetically Engineered Machines (iGEM) Jamboree, which is an annual, global, intercollegiate synthetic biology competition. In format, the iGEM Jamboree is much like the annual meeting of any major scientific society. But why, in the aisles, were there suited people with badges? Perhaps a new age has dawned upon the research community. The contents of this special issue of Molecular Biology of the Cell, with an emphasis on Big Data, certainly suggest that this is true. Nonetheless, overt governmental examination of research beyond the standard purview of granting agencies and its program officers can only raise questions. To answer some of these questions, I invited, on behalf of Molecular Biology of the Cell, Supervisory Special Agent (SSA) Edward You, who heads the Biological Countermeasures Unit (BCU) at Federal Bureau of Investigation (FBI) Headquarters in Washington, D.C., and frequently addresses the synthetic biology community, to have a conversation on DOI:10.1091/mbc.E14-01-0027. Mol Biol Cell 26, 3894–3897. Address correspondence to: Keith G. Kozminski (kkoz@virginia.edu). Abbreviations used: AAAS, American Association for the Advancement of Science; BCU, Biological Countermeasures Unit; BWC, Biological Weapons Convention; CDC, Centers for Disease Control and Prevention; DoD, Department of Defense; FBI, Federal Bureau of Investigation; iGEM, International Genetically Engineered Machines; NIH, National Institutes of Health; SSA, Supervisory Special Agent; WMD, weapons of mass destruction. © 2015 Kozminski. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0). “ASCB®,” “The American Society for Cell Biology®,” and “Molecular Biology of the Cell®” are registered trademarks of The American Society for Cell Biology. 22 | K. G. Kozminski Monitoring Editor David G. Drubin University of California, Berkeley Received: Jul 30, 2015 Accepted: Aug 5, 2015 biosecurity, especially with respect to Big Data (Figure 1). This conversation was recorded on July 17, 2015, and is presented here, abridged and edited for clarity and considerations of space. ONE FOOT IN NATIONAL SECURITY; ONE FOOT IN THE LIFE SCIENCES MBoC: Agent You, before you talk about Big Data, please tell our readers about your scientific background and path to the FBI. SSA You: I got my bachelor’s degree in the biological sciences from the University of California at Irvine, then a master’s degree in biochemistry and molecular biology at the University of Southern California. All that has served me well; it does show that there is life without a PhD. Before joining the Bureau, I came from the laboratory setting. I had six years of graduate research in human gene therapy, with a focus on retrovirology, and three years in the biotech sector at Amgen, where I did oncology research. Then I decided to go into public service and apply to the FBI. MBoC: What are your responsibilities at the FBI? What is your mission today? SSA You: I sit at headquarters at the Weapons of Mass Destruction (WMD) Directorate in the Biological Countermeasures Unit. The WMD Directorate is one of the newest divisions of the FBI. It was born out of the events of September 11, 2001. On the heels of that terrorist event, we had the anthrax mailings. It was a serious wake-up call for the U.S. government and the FBI in particular. Since then, as a law enforcement service, our priority has become one of prevention rather than being reactive, just going in and investigating a crime or incident. Now our number one priority is to prevent in particular a 9/11 from happening again. Safeguarding science is the theme of my mission. Part of that is reaching out proactively to different members of the scientific community, ranging from the private sector, biotech and the pharmaceutical industry; Molecular Biology of the Cell FIGURE 1: FBI SSA Edward You. In addition to heading the FBI’s BCU, he is a Working Group member of the National Security Council Interagency Policy Committee on Countering Biological Threats and an ex officio member of the NIH National Science Advisory Board for Biosecurity. He also serves on two National Academies committees: the Institute of Medicine’s Forum on Microbial Threats and the Committee on Science, Technology, and Law’s Forum on Synthetic Biology. SSA You also serves on the Strategic Advisory Board for the Synthetic Biology and Engineering Research Center and as an instructor for the United Nations Interregional Crime and Justice Research Institute. He can be reached at (202) 324-0236 or Edward .You@ic.fbi.gov. to universities; to the iGEM; and even to the amateur community, the sprawling Do-It-Yourself bio community, showing how members of law enforcement and the life science community have a shared responsibility of safeguarding the development and very beneficial applications of the life sciences. I find myself in a unique position, where I have one foot in national security and another in the life sciences. I seek very hard to ensure that we are able to support both at the same time. BIG DATA WORRIES AT THE FBI MBoC: You mentioned synthetic biology and have been involved in that community. However, it seems more recently that the FBI has been showing more overt concern toward the security of Big Data in the life sciences. Why does the FBI have concern? SSA You: If you take my consideration of how to protect the life sciences in a proactive manner, it is our responsibility to identify emerging areas. Six years ago the emerging area was synthetic biology. That is why you have seen all this activity and outreach occurring, especially at iGEM. The reason why Big Data has become very significant is that it is the next evolutionary step that synthetic biology will take, meaning that all applications and technologies coming out of this field will be completely dependent upon data—all the various omics. 2015 ASCB Award Essays, Selected Perspective, and MBoC Paper of the Year A very good example is precision personalized medicine, where you are seeing tremendous investments in drug development, particularly in cancer research and metabolic disease, where very large data sets are leveraged. If you are looking at an individual’s genome, it is just one snap shot. What are needed are data over time, during exposure to the environment, for example. From the human standpoint, maybe this is looking at your lifestyle—daily diet or exercise. It all goes into helping determine potential health vulnerabilities and appropriate therapies. If you set that as a stage and then look at potential policy aspects, there is a lot of activity looking at privacy, but not a whole lot looking specifically at security. So, back in April 2014, I partnered with the American Association for the Advancement of Science (AAAS) and the United Nations Interregional Crime and Justice Research Institute (UNICRI). We kicked off a meeting where the theme was national and transnational security implications of Big Data in the life sciences. We really wanted to tackle some of the security implications in the area of Big Data, where biology has almost a complete overlap with the digital world. At this meeting I had representatives from Microsoft, Intel, IBM, Google, and Amazon, the entities leveraging this Big Data bio-innovation future, and challenged them at the outset to identify potential security issues. We did find some significant issues and published some reports that are now publically available. MBoC: You had an incredible lineup of expertise contributing to the AAAS report National and Transnational Security Implications of Big Data in the Life Sciences (Berger and Roderick, 2014). Was there any specific event that motivated the FBI to launch this reflection on biosecurity or was this entirely a proactive endeavor? SSA You: The anthrax mailing in 2001 was a huge seminal event. Security discussions in the past tried to overlay security structures that were used in the nuclear or chemical realm. Completely locking down certain areas of expertise or materiel is completely antithetical to how the life sciences operate. If our mission is one of preventing the misuse, exploitation, or abuse of the life sciences, how do we approach security without becoming a hindrance to the life science enterprise? Over the last two years, we have had the issues with regard to the Centers for Disease Control and Prevention (CDC) and Department of Defense (DoD). A lot of discussion also came when the J. Craig Venter Institute synthesized that bacterial genome. There were a lot of calls and discussions about the scientific community needing more ethics training and the need to develop a greater culture of responsibility. From a law enforcement perspective those are necessary but not sufficient. What has been lacking is the scientific community being provided security awareness—something that augments how they approach the life sciences. Individuals, no matter where they are in the world or when they enter the life sciences, always start with the premise, “Do No Harm,” taking a page from the Hippocratic Oath. Unfortunately there are groups and individuals who do not subscribe to the same ethics and norms and agreements to integrity that we all take for granted and are almost innate for us. How do we graduate from “Do No Harm” to “Not On My Watch”? It means you take an active role in being sentinels for what you are doing and preventing the abuse, misuse, and exploitation of the life sciences. If you are not fully aware what the security vulnerabilities are, then that becomes a true vulnerability for all of us. We also have a Biological Weapons Convention (BWC). It is amazing to me that we have an international treaty to which we are all beholden, yet there are very few programs, if any, in which incoming biology students are exposed to it or the fact that the BWC exists because biology had been absolutely used and exploited for Biosecurity in the age of Big Data | 23 offensive purposes, even by the United States. If we do not teach that little bit of history and other security aspects, then it becomes really a challenge in the future on how to better protect biology. It is not about ethics; it is not about responsibility; it really is about having a healthy appreciation of some of the security concerns. MBoC: How real is the threat? The aforementioned AAAS report read, “very little, if any, information exists about the theft, manipulation, or exploitation of Big Data in the life sciences.” SSA You: That is the key question. One of the goals of generating this report was to galvanize people to start thinking about security because quite honestly I do not think we really appreciate how deep or how wide the security vulnerabilities are in leveraging these large data sets or Big Data in general. Referring to my prior comments about precision medicine, it all hinges on genetic information and longitudinal data over time. You are only as good as the size of the data set. You need a large data set because when you do an analysis you need statistical significance to know whether your results are right. As you think about that, let me walk you back to some of the most significant cyber-intrusions this past year. In August 2014, there was a Community Health Systems hack with 4.5 million patient records accessed; a few months after that was the large Anthem Blue Cross hack with 80 million individuals impacted; and then a month after that, the Premera Blue Cross hack in which 11 million patient records were hit. This is when it keyed off for me. In the Premera Blue Cross hack, clinical data were accessed too. Across the government, with these particular intrusions, the focus has been only on the potential loss of personal identifying information, the risk of fraud, and identity theft. I do not want to give that short shrift, because we are talking about tens, hundreds of millions of dollars in potential loss. However, if you think about the critical data—a beautiful longitudinal data set, containing an individual’s demographics, disease state, drugs administered, and treatment received—someone now has a treasure trove of clinical trial information. Unfortunately, all of those hacks were allegedly attributed to a hacking group based out of China. It has become not just fraud anymore. There is a much broader security vulnerability, the potential loss of our ability to stay globally competitive in the new drug market. Now somebody out there has the brass ring—this gigantic data set, where the only limitation is deriving the analytical tools to make all that data useful. There are a couple of issues now. One is to identify how much we have given up. We have to get beyond the paradigm of just looking at the financial loss. In the area of Big Data with specific applications to the life sciences, information taken could potentially be used for exploitation or extortion. A second is that, with the analytical tools that are coming online today, it will be almost impossible to deidentify information in the future. This was a key takeaway from the meeting with the AAAS-UNICRI last year. If you have any short genetic sequence of an individual, you can effectively deanonomize it in fewer than three steps with publically available tools. MBoC: Privacy does not exist anymore? SSA You: Correct. If you are part of an institutional review board, you are in big trouble in maintaining compliance and keeping up with protecting human subject information. That is just one regulatory hurdle that will be coming up. MBoC: Where is the greatest security threat to Big Data through hacking? Is it through the lone wolf, companies engaged in industrial espionage, or is it from state-sponsored activities? SSA You: My answer is “yes.” The vulnerabilities are across the spectrum. MBoC: Are the threats to Big Data greater for private Big Data, for example Pharma, or for academic Big Data? 24 | K. G. Kozminski SSA You: It is all of the above. Our AAAS meeting came to the crux of it: whoever has the largest and most diverse data set is going to win. That means we really need to start thinking in a more holistic manner what security means with a data set. MBoC: Does the FBI define Big Data in terms of volumes of data or analytical functions? Is the threat against the volumes of data or the ability to analyze data? SSA You: It is both. I do not want to go too far into definitions because one of the issues is how to define Big Data. From a life sciences standpoint, we need to be going into this with our eyes wide open. How do we do anything? A thorough assessment of potential security vulnerabilities is a first step. Second, identify how to mitigate them up front. Finally, ask whether we have to come up with novel ways to address security in this bio-future. The power of the life sciences is open source, open sharing, but in it there is the added dimension of an individual’s very intimate information. So there may be a call to redefine how we address security in the future. It may not be building up secure walls, whether they are physical or virtual, that protect data like our financial data. In this world of the life sciences, which is inherently open, we are going to have to rethink security. MBoC: How should life scientists, faculty members at universities, respond to the worries of the FBI in terms of biosecurity? What do you see people doing to improve the situation? SSA You: To me, the strategy is that once we build trusted partnerships with the scientific community, first with the FBI reaching out and providing the security awareness and education, something really profound happens. We have seen it happen in synthetic biology. You see the scientific community doing their own assessments of their technologies, self-identifying potential security vulnerabilities and then providing notification to the FBI—to my unit or other partners at the FBI. So the tables have turned. The scientific community educates the FBI on emerging vulnerabilities. They do us a favor, helping us to be better informed to better protect the life sciences, universities, and communities. Even better, the community will then develop security solutions based on their expertise, which is the best of both worlds. How powerful would that be when the experts, who are developing these powerful tools and applications of the future, immediately, on the front end, start developing and implementing security measures within these applications? That is where we want to be; that is where the future has got to be. So there is absolutely a very necessary and important partnership between law enforcement and the scientific community. It is just not a one-way street. Take, for example, the scientific papers regarding CRISPR/Cas9 and gene drives and most recently the genetically modified yeast producing opioids. Scientists drafted the scientific manuscript and a companion editorial piece calling out the potential security vulnerabilities. That is powerful; that is a home run. We have successfully empowered the scientific community to understand security and then to take some proactive actions of their own. MBoC: It seems one of the concerns of your unit, the BCU, is dual use of data. Does the BCU have formal relationships or work with the National Institutes of Health (NIH) National Science Advisory Board for Biosecurity or the CDC? SSA You: Thank you for that question. It goes to the background of the WMD Directorate. One of the cornerstone aspects of our program is the really important position called the WMD Coordinator. These are men and women, Special Agents, trained in chemical, biological, radiological, and nuclear matters. We have at least one stationed in each of our 56 field offices across the United States. The WMD Coordinator’s role, as the name implies, is to coordinate and Molecular Biology of the Cell lead the notification protocols with state and local law enforcement, public health, partner with other federal entities, and then build relationships with universities, companies, and institutions within their jurisdiction. So if anything did occur, a local university, for example, would then know they have a local federal representative that can respond. If there is ever a biological incident or actual bio-crime, then those WMD Coordinators become critical in the response. They actually have been a big part of the action over the years with the DoD and CDC events, the discovery of smallpox at NIH at a Food and Drug Administration laboratory, the two high-profile ricin mailings almost two years ago, and the incident at Georgetown University where a student was manufacturing ricin in his dorm room. In all of these different incidents, those WMD Coordinators were called in and were part of the response. No matter where in the government, the Coordinator is there to help and assist with either preventive training or, if anything did occur, the response. MBoC: How do people find the WMD Coordinators should they ever need one? SSA You: They can just call the FBI field office in their jurisdiction and ask to be referred to the WMD Coordinator. Should any suspicious or criminal activity be observed that puts personnel, institutions, or materials at risk, contact your local FBI WMD Coordinator to help with any assessments. Think of them as being a resource to the scientific community. If you call them, it is not immediately the opening of an investigation. They are someone specifically within the FBI who is familiar with the life sciences community and with whom you can just touch base to see if something passes the sniff test. MBoC: Although many readers of Molecular Biology of the Cell are gaining a greater awareness of Big Data, their own research does not take them into the realm of Big Data. For those readers, does the FBI have biosecurity concerns that lie with small data or is the focus really on Big Data? SSA You: The focus on Big Data is because it is an emerging area. If you see all of our activities, the overall theme is safeguarding science, whether you are working in large data analytics, with select agents, yeast, or Escherichia coli. We are not honing in on a specific subgroup or subtopic of the life sciences. It is really preventing the misuse of the life sciences in general. MBoC: The FBI’s primary role is safeguarding the homeland, the United States. Many of our readers are not Americans. Is there a separate, special, or additional message for people doing life science research, especially Big Data research, outside the boundaries of the United States? SSA You: Safeguarding science is universally applicable. I hope for a future when biologists are working as WMD Coordinators in other law enforcement agencies around the world. We need that. The 21st century will see the same leaps and bounds with the life sciences that we saw in the 20th century with the Internet and personal computing. If there is going be a global impact from the life sciences, there is absolutely a call to action for biologists wherever they are in the world to be guardians of science. However, we need to come to a realization first that there will be issues. We have to start discussing these things now before it is too late, before any attempts at security will be too little, too late. We are much better 2015 ASCB Award Essays, Selected Perspective, and MBoC Paper of the Year served tackling issues sooner. I hate to say it, but, if we are not careful and there is a complete overlap of the life sciences and the digital world, we might see ourselves with our security as we are facing cyber-security right now, and we do not want to be in that position. FROM BENCH TO BADGE—ARE YOU HIRING? MBoC: It is clear there is a lot of work ahead, not just for the scientific community, but for the FBI as well. What are the career opportunities for cell biologists in the FBI, whether they have Big Data experience or not? SSA You: We are most definitely hiring. You can be a Special Agent like me, or there are support positions such as the scientists who work in our laboratory division. These are individuals who develop the tools for forensic analysis. A key piece of our mission is looking at intelligence; that is an analyst position. There will absolutely be a need for folks with a biology background. You do not need to have law enforcement experience. I did not. I will be completely candid, upfront—our hiring is a very competitive process. Prior to 9/11, the FBI’s focus was on hiring individuals with law enforcement or military experience, lawyers, or accountants because the primary mission was tackling organized crime. In this day, when our number one priority is prevention, there is an absolute critical need for hiring individuals with background in computer science, foreign languages, and especially the natural sciences. If you have a chemistry or biology background, you are in the running. The minimal criteria are a bachelor degree and at least three years of real-world experience. More than anything else, if you can articulate and show how you excelled in your specific field, then you are a good candidate. Your field does not necessarily have to be Big Data. You need to be passionate about what you do because in doing so, you inherently excel. The key is to set yourself in a position where you can really excel so when we begin talking to your coworkers and managers about who you really are, you have put them in a position where they can say you are an integral part of the team and made significant contributions. That will be a good selling point for a future career in the FBI. THE TAKE-AWAY SSA You: Partnerships between the FBI and the scientific community to build security awareness are essential. Big Data in the life sciences is taking the biosecurity discussion beyond pathogens and toxins. Historically, the conversation almost always fell on pathogens and almost exclusively on select agents. We have to widen the aperture of what we mean by biosecurity in the future. ACKNOWLEDGMENTS I thank John Fleischman, American Society for Cell Biology Senior Science Writer, for tips in the preparation of this interview. REFERENCE Berger KM, Roderick J (2014). National and transnational security implications of Big Data in the life sciences. Available at www.aaas.org/report/ national-and-transnational-security-implications-big-data-life -sciences (accessed 19 March 2015). Biosecurity in the age of Big Data | 25 MBoC | ARTICLE 2015 MBoC PAPER OF THE YEAR Subcellular optogenetic inhibition of G proteins generates signaling gradients and cell migration Patrick R. O’Neilla and N. Gautama,b a Department of Anesthesiology and bDepartment of Genetics, Washington University School of Medicine, St. Louis, MO 63110 ABSTRACT Cells sense gradients of extracellular cues and generate polarized responses such as cell migration and neurite initiation. There is static information on the intracellular signaling molecules involved in these responses, but how they dynamically orchestrate polarized cell behaviors is not well understood. A limitation has been the lack of methods to exert spatial and temporal control over specific signaling molecules inside a living cell. Here we introduce optogenetic tools that act downstream of native G protein–coupled receptor (GPCRs) and provide direct control over the activity of endogenous heterotrimeric G protein subunits. Light-triggered recruitment of a truncated regulator of G protein signaling (RGS) protein or a Gβγ-sequestering domain to a selected region on the plasma membrane results in localized inhibition of G protein signaling. In immune cells exposed to spatially uniform chemoattractants, these optogenetic tools allow us to create reversible gradients of signaling activity. Migratory responses generated by this approach show that a gradient of active G protein αi and βγ subunits is sufficient to generate directed cell migration. They also provide the most direct evidence so for a global inhibition pathway triggered by Gi signaling in directional sensing and adaptation. These optogenetic tools can be applied to interrogate the mechanistic basis of other GPCR-modulated cellular functions. INTRODUCTION A cell’s function often depends on its ability to sense gradients of external cues and generate a polarized response such as directed migration or neurite initiation. There is a limited understanding of how dynamic networks of intracellular signaling molecules generate polarized cell behaviors. Network motifs have been proposed that can give rise to some of the features of cell migration, such as directional sensing, adaptation, and amplification of an external gradient (Xiong et al., 2011; Wang et al., 2012). However, existing experimental methods have provided mostly static information on the relevant signaling molecules, making it difficult to examine whether This article was published online ahead of print in MBoC in Press (http://www .molbiolcell.org/cgi/doi/10.1091/mbc.E14-04-0870) on June 11, 2014. Mol Biol Cell 25, 2305–2314. Address correspondence to: N. Gautam (gautam@wustl.edu). Abbreviations used: CRY2, cryptochrome 2; GAP, GTPase-accelerating protein; GPCR, G protein–coupled receptor; GRK, G protein–coupled receptor kinase; OA, optical activation; RGS, regulator of G protein signaling. © 2014 O’Neill and Gautam. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0). “ASCB®,” “The American Society for Cell Biology®,” and “Molecular Biology of the Cell®” are registered trademarks of The American Society of Cell Biology. 2015 ASCB Award Essays, Selected Perspective, and MBoC Paper of the Year Monitoring Editor Peter Van Haastert University of Groningen Received: Apr 9, 2014 Revised: May 16, 2014 Accepted: Jun 5, 2014 and how specific molecular interactions map onto these dynamic network motifs. In particular, there has been a lack of methods to exert spatial and temporal control over the activity of select signaling molecules inside a cell. Optical manipulation of signaling presents an attractive approach for achieving such control (Toettcher et al., 2011). We recently used color opsins to spatially confine G protein–coupled receptor (GPCR) activity to a selected region of a single cell and gain optical control over immune cell migration (Karunarathne et al., 2013b) and neurite initiation and extension (Karunarathne et al., 2013a). The opsin approach optically activates an entire signaling pathway to orchestrate cell behavior, but new tools that provide optical control of downstream signaling molecules are required to dissect the network of dynamic interactions triggered inside a cell. Here we create new optogenetic tools that enable light-triggered inhibition of endogenous G protein subunits in a selected region of a cell. We use them to generate reversible intracellular signaling gradients in cells exposed to a uniform extracellular stimulus. We apply this approach to study cell migration in a macrophage cell line, RAW 264.7. GPCRs control migration of a wide variety of cell types, but the dynamic roles of the G protein α and βγ subunits in directing cell 27 one side of a cell. We applied two approaches: light-triggered acceleration of GTP hydrolysis on the α subunit, and optical recruitment of a βγ-sequestering domain. Design of an optically controlled GTPase-accelerating protein GTPase-accelerating proteins (GAPs) act allosterically on G protein α subunits to accelerate the transition from active αGTP to inactive αGDP (Ross and Wilkie, 2000). Spatially localized acceleration of GTP hydrolysis at the α subunit can potentially reduce signaling by both the α and βγ subunits because deactivated αGDP rapidly rebinds the βγ complex and prevents its interaction with effectors (Lin and Smrcka, 2011). We sought to gain optical control over regulator of G protein signaling 4 (RGS4), which has GAP activity on both the αi and αq subunit types (Berman et al., 1996; Hepler et al., 1997). In yeast, exogenously expressed RGS4 has been shown to localize to the plasma membrane and inhibit the GPCRregulated mating pathway (Srinivasa et al., 1998). A truncated mutant, RGS4(Δ1-33), FIGURE 1: Generating intracellular signaling gradients by localized optical inhibition. (A) Optical did not localize to the plasma membrane recruitment of an RGS protein to a spatially confined region of the plasma membrane generates and did not exhibit GAP activity. Its function, however, was rescued by addition of localized GAP activity, resulting in deactivation of the α subunit and the βγ complex. (B) Local inhibition of βγ signaling by optical recruitment of a βγ-sequestering peptide. Both approaches an alternative, C-terminal membrane–tarprovide spatial control over G protein subunit activity downstream of uniform GPCR activation. geting domain (Srinivasa et al., 1998). These results suggested that it might be possible to gain optical control over the GAP activity of RGS4 by replacing its migration remain unclear. Signaling by βγ subunits is generally recnative membrane-targeting domain with a light-induced memognized as a requirement for GPCR-stimulated chemotaxis (Bagorda brane-targeting domain. and Parent, 2008), and multiple βγ effectors have been implicated in The CRY2PHR and CIBN domains from Arabidopsis thaliana cell migration (Li et al., 2003; Yan et al., 2012; Runne and Chen, proteins cryptochrome 2 (CRY2) and CIB1 exhibit blue light–depen2013). However, it is unknown whether a gradient of active βγ is sufdent binding and can be used for light-triggered recruitment of a ficient to trigger a directional response. Recent work in neutrophils CRY2-fused protein to the plasma membrane (Kennedy et al., suggests that βγ signaling may be primarily involved in controlling 2010). We fused CRY2PHR-mCherry to RGS4(Δ1-33) to make CRY2the motility rather than the directionality of a migrating cell mCh-RGS4Δ. We then coexpressed this construct in HeLa or RAW (Kamakura et al., 2013). Meanwhile, there have been conflicting re264.7 cells with a construct containing CIBN fused to the plasma ports on the requirement of G protein αi subunit signaling in membrane–targeting C-terminal domain from KRas (CIBN-CaaX; chemotaxis (Neptune et al., 1999; Kamakura et al., 2013), and there Idevall-Hagren et al., 2012). We found that CRY2-mCh-RGS4Δ remains the possibility that GPCR activation of non–G protein pathtranslocated from the cytosol to the plasma membrane on photoways also contributes to chemotaxis (Neptune et al., 1999; Van activation with 445-nm light (Figures 2 and 3). Haastert and Devreotes, 2004). Here we use our new optogenetic tools to address fundamental Optical control over the GAP activity of an RGS protein questions about chemotaxis: can a gradient of heterotrimeric G procan be demonstrated using a G protein βγ subunit tein subunit activity stimulate all of the processes required for GPCR translocation assay mediated chemotaxis, or is there an additional requirement for a We used a βγ subunit translocation assay to test whether light-actigradient of G protein–independent signaling stimulated by the revated recruitment of CRY2-mCh-RGS4Δ to the plasma membrane ceptor? Is a gradient of βγ activity sufficient for directional sensing? could regulate its GAP activity in a living cell. This assay leverages Does G protein subunit activity at one end of a cell lead to inhibition the αGDP-dependent plasma membrane targeting of βγ subunits to of responses such as increased phosphatidylinositol (3,4,5)-trisphosdetect changes in the relative amounts of αGTP and αGDP in a phate (PIP3) and lamellipodia formation at the opposite end? living cell. We previously showed that βγ subunits translocate reversibly RESULTS from the plasma membrane to intracellular membranes upon GPCR Creating intracellular signaling gradients using uniform activation (Akgoz et al., 2004; Azpiazu et al., 2006; Saini et al., 2007; ligand stimulation and confined optical inhibition Karunarathne et al., 2012). In unstimulated cells, G protein α and βγ The general scheme used in our experiments is shown in Figure 1. subunits are primarily found as heterotrimers anchored to the plasma We combined spatially uniform stimulation of GPCRs by a chemoatmembrane by the lipid modifications on the α and γ subunits tractant with confined optical inhibition of G protein signaling on 28 | P. R. O’Neill and N. Gautam Molecular Biology of the Cell detected as a loss of YFP fluorescence from the plasma membrane (Figure 2, B and C). Localized optical activation (OA) of CRY2 resulted in localized accumulation of CRY2mCh-RGS4Δ at the plasma membrane. This was accompanied by an increase of YFP-γ9 at the region proximal but not distal to the optically activated area. The light-triggered reverse βγ translocation occurred in the presence of continued receptor activity. No reverse βγ translocation was observed when CRY2-mCh lacking the RGS4 domain was optically recruited to the plasma membrane (Supplemental Figure S1). Thus the spatially confined reversal of βγ translocation detected here is consistent with optical recruitment of CRY2-mCh-RGS4Δ to the plasma membrane being able to locally trigger its GAP activity on α-GTP and thereby increase the concentration of α-GDP in that region. Optically generated Gi protein signaling gradients direct migration of RAW 264.7 macrophage cells The foregoing results showed that local optical activation of CRY2-mCh-RGS4Δ can trigger deactivation of αi-GTP and Gβγ in a selected area of a cell. This capability provides a way to create an intracellular G proFIGURE 2: Optical control of GTP hydrolysis with CRY2-mCh-RGS4Δ. (A) CXCR4 activation by tein subunit activity gradient and examine SDF-1α triggers G protein activation, dissociation, and βγ translocation to intracellular polarized cell behaviors. We used it to exmembranes. OA of CRY2-mCh-RGS4Δ recruits it to the plasma membrane, where it can amine the migratory response in RAW 264.7 accelerate GTP hydrolysis on the α subunit. The increased concentration of αGDP at the plasma macrophage cells. RAW cells are known to membrane results in reverse βγ translocation due to reformation of heterotrimers. (B) Live-cell exhibit GPCR stimulated chemotaxis (Wiege imaging of a HeLa cell transiently transfected with CRY2-mCh-RGS4Δ, CIBN-CaaX, and YFP-γ9. et al., 2012), and we found that their low Activation of endogenous CXCR4 receptors with 50 ng/ml SDF-1α triggered γ9 translocation basal motility compared with commonly from the plasma membrane to intracellular membranes. Photoactivation-stimulated studied HL-60 neutrophils and Dictyostetranslocation of CRY2-mCh-RGS4Δ to the plasma membrane. There it catalyzed hydrolysis of lium cells simplifies the use of localized OA αGTP to αGDP, leading to reverse translocation of γ9 back to the plasma membrane due to the to control membrane recruitment of CRY2 reformation of heterotrimers. Scale bar, 10 μm. (C) Time course of plasma membrane intensity for CRY2-mCh-RGS4Δ and YFP-γ9 in the photoactivated region. constructs. Directionally responsive spatial gradients of PIP3 are believed to be one of the mediators of chemotaxis (Cai (Wedegaertner et al., 1995). GPCR activation triggers nucleotide and Devreotes, 2011; Weiger and Parent, 2012). We examined exchange on the α subunit, resulting in dissociation of the αGTP whether local inhibition of G protein subunit activity could be used and βγ subunits (Bondar and Lazar, 2014). The prenylated C-terminal to direct the formation of PIP3 gradients in RAW cells exposed to a domain of the γ subunits provides βγ subunits some membrane afuniform extracellular stimulus. We examined the PIP3 response in finity, but it is insufficient for permanent anchoring in a membrane RAW cells transfected with CRY2-mCh-RGS4Δ, CIBN-CaaX, PH(Akt)(O’Neill et al., 2012). As a result, free βγ subunits diffusively transloVenus, and CXCR4. PIP3 dynamics in a live cell can be measured by cate to intracellular membranes (Saini et al., 2007; O’Neill et al., imaging the translocation of a PH(Akt)-Venus sensor from the cyto2012). When receptors are deactivated, rebinding of βγ to αGDP sol to the plasma membrane (James et al., 1996; Meili et al., 1999). results in their return to the plasma membrane. We used the chemokine receptor CXCR4 to activate G proteins Because reverse translocation of βγ subunits to the plasma memglobally, since activation of this receptor by a gradient of the chebrane occurs through rebinding to αGDP, accelerating GTP hydrolysis mokine SDF-1α stimulates migration in many cell types (Bleul et al., on the α subunit should be capable of triggering reverse βγ transloca1996; Klein et al., 2001; Molyneaux et al., 2003). tion even if the receptors remain activated. We leveraged this feature First, we used localized OA to recruit CRY2-mCh-RGS4Δ to the of βγ translocation to test whether optical recruitment of CRY2-mChplasma membrane at one side of a cell and followed this with global RGS4Δ to the plasma membrane can control its GAP activity. CXCR4 activation using 50 ng/ml SDF-1α (Figure 3). Before receptor We measured βγ translocation in HeLa cells by imaging a yellow activation, localized plasma membrane recruitment of CRY2-mChfluorescent protein (YFP)–tagged version of γ9, a fast-translocating RGS4Δ did not produce any detectable PIP3 generation or cell subunit (Figure 2). Consistent with previous observations (Karunarashape changes. On receptor activation, cells responded by generatthne et al., 2012; O’Neill et al., 2012), activation of endogenous ing PIP3 gradients and initiating migration in the direction opposite CXCR4 receptors with 50 ng/ml SDF-1α triggered βγ9 translocation to the CRY2-mCh-RGS4Δ gradient (Figure 3A and Supplemental from the plasma membrane to intracellular membranes, which was 2015 ASCB Award Essays, Selected Perspective, and MBoC Paper of the Year Optical inhibition of G protein subunits | 29 respond in opposite directions (Supplemental Figure S2). The same directional control was observed when OA was applied after the uniform extracellular stimulus, with migration being initiated at the time of OA (Supplemental Movie S2). Furthermore, the direction of PIP3 accumulation and lamellipodia formation could be reversed by switching the location of OA to the opposite side of the cell (eight of eight cells) (Supplemental Figure S3). Cells did not exhibit any of these directional responses to localized OA when a CRY2 construct (CRY2-mCh) without RGS4Δ was expressed in the cells or when a CRY2mCh construct containing the cDNA for a glycolytic enzyme, PGK1, was expressed (CRY2-mCh-PGK1; Figure 3, B and D, and Supplemental Figure S4). These cells exhibited uniform PIP3 responses (29 of 36 cells) or polarized spontaneously in directions that did not depend on the side of OA with reference to the cell (7 of 36 cells). Compared to neutrophils, spontaneous polarization in response to a uniform stimulus appears to be much less common in RAW macrophage cells. This is consistent with their general lack of basal polarization and their greatly reduced basal motility compared with neutrophils. These controls show that the directional responses observed with CRY-mChRGS4Δ are due to localized inhibition of αi and βγ subunit activity by RGS4Δ rather than a nonspecific effect due to localized OA or accumulation of the CRY protein at the membrane. We performed identical experiments using activation of endogenous C5 receptors to ensure that the migratory response induced by localized GAP activity was not peculiar to the CXCR4 receptor or due to overexpression of a GPCR. The anaphylatoxin C5a is known to stimulate chemotaxis of all myeloid cell lineages (Gerard and Gerard, FIGURE 3: Cell migration driven by localized Gi protein inhibition. (A) Image sequence of a live 1994), and it has been shown to induce cheRAW 264.7 cell transiently transfected with CRY2-mCh-RGS4Δ, CIBN-CaaX, PH(Akt)-Venus, and motaxis of RAW 264.7 cells (Wiege et al., CXCR4. Local OA was applied to generate a CRY2-mCh-RGS4Δ gradient before uniform 2012). We activated endogenous C5a readdition of SDF-1α. Scale bar, 10 μm. (B) Negative control expressing CRY2-mCh-PGK1 instead of CRY2-mCh-RGS4Δ. (C, D) The t-stacks corresponding to the data in A and B. Localization of ceptors with 10 μM FKP-(D-Cha)-Cha-r, a the RGS construct, but not the PGK construct, results in a PIP3 gradient, directional cell peptide derived from the C-terminus of the protrusions, and migration. White boxes correspond to OA regions. Yellow boxes show regions full-length, 74–amino acid C5a. It has been selected for generating the corresponding t-stacks. reported to be a full agonist of the C5a receptor, eliciting responses comparable to Movie S1). Of 43 cells that provided a PIP3 response, all exhibited those of full-length C5a in several assays, including chemotaxis PIP3 gradients and directed lamellipodia. Of these, seven migrated (Konteatis et al., 1994). Localized OA of CRY2-mCh-RGS4Δ with uniat least 1 cell diameter in 15 min, 10 migrated between 1/2 and form activation of endogenous C5a receptors generated directional 1 cell diameter, and 26 migrated <1/2 cell diameter. Of those that responses similar to those seen with uniform activation of transmigrated <1/2 cell diameter, five extended the front by at least 1/2 fected CXCR4 (Supplemental Figure S5). cell diameter but did not retract the back, and five initiated migraThe ability to locally inhibit G protein signaling and generate a tion before snapping back to their initial positions, perhaps due to migratory response in immune cells showed that an internal gradistrong adhesion to the uncoated glass surface. ent of αi and βγ activity is sufficient to direct cell migration in the The directional responses were not due to unintended SDF-1α absence of an external gradient. The results with endogenous C5a gradients, because two cells in close proximity could be made to receptors showed that these internal gradients are sufficient to drive 30 | P. R. O’Neill and N. Gautam Molecular Biology of the Cell FIGURE 4: Localized Gβγ inhibition directs PIP3 gradients and lamellipodia. (A) Light-triggered recruitment of CRY2mCh-GRK2ct to the plasma membrane allows for spatially confined inhibition of βγ signaling. (B) Live-cell imaging of a RAW cell expressing CRY2-mCh-GRK2ct, CIBN-CaaX, PH(Akt)-Venus, and CXCR4. (C) The t-stack corresponding to the data in B. Localization of the GRKct construct generates reversible lamellipodia and PIP3 responses. cell migration at levels of signaling activity normally achieved within a cell. Gβγ signaling gradients generated by CRY2-mCh-GRK2ct direct PIP3 gradients and lamellipodia formation To further dissect the roles of G protein subunits in cell migration, we sought to develop an optogenetic tool to specifically inhibit βγ signaling. We created a CRY2-mCh-GRK2ct construct that could be optically recruited to one side of a cell to produce a gradient of βγ activity (Figure 4A). The C-terminal domain of G protein–coupled receptor kinase 2 (GRK2ct) is capable of inhibiting responses downstream of βγ without inhibiting those generated by α subunit effectors (Koch et al., 1994). It has been widely used to sequester Gβγ and inhibit its activity, but this is the first time it was used asymmetrically within a single cell to study a polarized cell behavior. In RAW cells transiently transfected with CRY2-mCh-GRK2ct, CIBN-CaaX, PH(Akt)-Venus, and CXCR4, spatially confined OA resulted in localized recruitment of CRY2-mCh-GRK2ct from the cytosol to the plasma membrane. Subsequent activation of CXCR4 receptors with 50 ng/ml SDF-1α resulted in generation of a PIP3 gradient and lamellipodia toward the side of the cell that was opposite to the location of the OA (40 of 50 cells; Figure 4B). The direction of the lamellipodia and the PIP3 gradient could be reversed by switching the location of OA with reference to the cell (6 of 12 cells; Figure 4, B and C, and Supplemental Movie S3). 2015 ASCB Award Essays, Selected Perspective, and MBoC Paper of the Year To ensure that these responses occurred due to localized sequestration of βγ and not some peculiar effect of GRK2ct, we performed identical experiments with a homologue, GRK3ct. GRK3ct binds to βγ subunits in biochemical (Daaka et al., 1997) and live-cell imaging assays (Hollins et al., 2009). GRK2 and GRK3 sequences are 85% identical, but their βγ-binding regions are only 52% identical (Daaka et al., 1997). The CRY2-mCh-GRK3ct construct was capable of producing similar directional (15 of 19 cells) and reversible (9 of 13 cells) responses (Supplemental Figure S6). The ability of both CRY2-GRKct constructs to elicit these directional responses, but not CRY2-mCh or CRY2-mCh-PGK1, confirmed that the directional responses occurred due to sequestration of Gβγ. Similar CRY2-mChGRKct directed responses were also observed when endogenous C5a receptors were activated with 10 μM FKP-(D-Cha)-Cha-r (Supplemental Figure S7). Whereas the CRY2-mCh-GRKct constructs were capable of generating PIP3 gradients and directional lamellipodia similar to those generated by the CRY2-mCh-RGS4Δ construct, none of these cells exhibited appreciable cell migration. This difference could potentially be due to different magnitudes of βγ inhibition achieved by the GRKct versus RGS constructs, or it could it be that a gradient of αi activity is additionally required for migration. We suspect that the latter explanation is more likely, given that recent studies using a variety of cell types reported roles in chemotaxis for αi subunit interactions with proteins such as GIV (Ghosh et al., 2008), ELMO1/ Dock180 (Li et al., 2013), and AGS3/mInsc (Kamakura et al., 2013). Optical inhibition of G protein subunits | 31 FIGURE 5: Local optical inhibition of Gi activity after adaptation to uniform stimulus. (A) Image sequence showing a RAW cell transfected with CRY2-mCh-RGS4Δ, CIBN-CaaX, PH(Akt)-Venus, and CXCR4. Addition of uniform SDF-1α (2:05) resulted in PIP3 accumulation and generation of cell protrusions (3:20). After several minutes, the PIP3 level and cell shape resembled those seen before the uniform stimulus (11:40). Localized OA of CRY2-mCh-RGS4Δ (12:55) was applied in this adapted state to inhibit Gi activity at one end of the cell. This resulted in the generation of a PIP3 gradient and cell migration directed toward the far side of the cell. Scale bar, 10 μm. (B) An illustration of the expected time dependence of an activator (A), inhibitor (I), and downstream response (R) in a LEGI model (Xiong et al., 2010) in which Gi signaling generates both A and I. Uniform activation of Gi signaling produces a transient downstream response that returns to the basal level due to the delayed increase in I. Subsequent optical inhibition of Gi signaling at the back causes a reduction in the level of Ifront but not Afront due to the differential movement of I and A throughout the cell. This leads to an increase in R at the front of the cell, resulting in directional migration. Overall, these results suggest that a gradient of activated Gβγ subunits stimulated by endogenous receptors is sufficient to elicit directional PIP3 responses and cell protrusions in the absence of an external gradient. Generating light-triggered gradients in cells that have adapted to a uniform stimulus: evidence of global inhibition mediated by G protein subunits Directional sensing in migratory cells is believed to be intimately related to their ability to adapt to a spatially uniform stimulus (Parent and Devreotes, 1999; Van Haastert and Devreotes, 2004; Levchenko and Iglesias, 2002). In this context, adaptation refers to a cell’s ability to generate transient responses that return to near-basal levels after a uniform increase in chemoattractant concentration. This occurs through a mechanism other than desensitization, and it allows a cell to sense gradients over a wide range of background chemoattractant concentrations. The mechanisms that control adaptation in migratory cells are not fully understood. An incoherent feedforward loop (IFFL) has been identified as a signaling motif capable of generating adaptive responses (Ma et al., 2009). In the IFFL, the input signal generates an activator with fast kinetics and an inhibitor with slower kinetics that converge on a downstream response such as PIP3. At short times after application of the stimulus, the activator generates an increase in PIP3 levels, but over time, the rising level of the inhibitor causes the PIP3 to decay back to its prestimulus level. Recent studies show that an IFFL 32 | P. R. O’Neill and N. Gautam can explain adaptation of PIP3 and Ras responses in Dictyostelium (Takeda et al., 2012; Wang et al., 2012) A local-excitation global-inhibition (LEGI) mechanism that incorporates the IFFL motif has been proposed that can account for both adaptation and directional sensing (Parent and Devreotes, 1999; Levine et al., 2006). In the LEGI model, the activator signals locally, while the inhibitor diffuses throughout the cell to signal globally. As a result, downstream responses adapt to a uniform stimulus but exhibit sustained intracellular gradients in response to a gradient stimulus. Several models of chemotaxis incorporate the LEGI motif to account for directional sensing and adaptation, combining it with motifs that account for additional features of chemotaxis, such as basal motility, cell shape changes, or amplification of the external gradient (Xiong et al., 2011; Wang et al., 2012; Shi et al., 2013). However, a specific global inhibitor has not yet been identified. It is not known whether an inhibitor is generated by Gi signaling or by an independent pathway triggered by the GPCR. We designed an experiment to determine whether Gi signaling by itself leads to global inhibition (Figure 5, Supplemental Figure S5, and Supplemental Movie S4). First we exposed RAW cells to a uniform chemoattractant, either 50 ng/ml SDF-1α to activate transfected CXCR4 or 10 μM FKP-(D-Cha)-Cha-r to activate endogenous C5a receptors. This resulted in translocation of PH(Akt) to the plasma membrane and generation of cell protrusions. After the cells had adapted, as indicated by PH(Akt) returning to the cytosol and the cell protrusions subsiding, CRY2-mCh-RGS4Δ was optically recruited Molecular Biology of the Cell to one side of the cell to induce localized inhibition of αi and βγ activities. This resulted in the formation of a PIP3 gradient and initiation of cell migration in a direction that was opposite to the location of the OA (seven of eight cells). The ability to generate responses at the front of a cell simply by inhibiting G protein activity at the back provides direct evidence that Gi signaling can act at a distance to inhibit “frontness” signaling pathways. This result is consistent with a LEGI model in which both the local activator and the global inhibitor are generated by Gi signaling. Figure 5B shows schematic plots that illustrate the time dependence of the activator, the inhibitor, and the downstream response. Application of a uniform input initially leads to rapid generation of the activator and the downstream response. The delayed accumulation of the inhibitor causes the response to return to its prestimulus level. The levels of both activator and inhibitor remain high throughout the cell in the adapted state. When the cell is in this state, applying localized OA to inhibit Gi signaling causes the levels of activator and inhibitor to decrease on one side of the cell. Because the inhibitor acts globally, the cell encounters the reduced level of inhibitor over its entire space. In contrast, the level of activator is only reduced on one side. As a result, the level of activator overwhelms that of the inhibitor on one side of the cell, leading to the generation of downstream signaling gradients that drive cell migration. et al., 1985; Hartmann et al., 1997). However, the dynamic roles for the αi and βγ subunits are not known, and it has not been possible to test whether a gradient in the activity of αi and βγ subunits is sufficient to generate cell migration. There have been suggestions that other G protein subunit types may also be required. For example, in N-formyl-methionyl-leucyl-phenylalanine (fMLP)–stimulated neutrophil chemotaxis, it has been reported that Gi signaling regulates “frontness,” whereas G12/13 regulates “backness” pathways (Xu et al., 2003). Cell migration could additionally require gradients of GPCR-triggered but G protein–independent signaling (Ge et al., 2003). It could also potentially require interactions between ligandbound GPCRs and accessory proteins that modulate G protein– mediated signaling, for example by bringing specific effector molecules closer to the activated G protein (Ritter and Hall, 2009). Here we activated receptors that couple to Gi heterotrimers. By breaking spatial symmetry downstream of the receptor, directly at the level of the Gi protein, we were able to identify molecular and cellular responses generated by a gradient of αi and βγ activity. The ability of optically localized CRY2-mCh-RGS4Δ to generate directional cell migration shows that a gradient of αi and βγ activity is sufficient to elicit the entire gamut of migratory responses, including generation of lamellipodia at the front of a cell, retraction of the back, directional changes, and ability to respond directionally after adapting to a uniform stimulus. DISCUSSION Optical control of cell signaling by inhibition of endogenous proteins Directional sensing by a Gβγ signaling gradient Most of the current information about signaling molecules involved in cell migration comes from genetic manipulations that establish whether a given protein is required for migration and biochemical studies that identify its relevant interactions. Imaging methods have provided additional information about the localization of several signaling molecules to the front or back of a migrating cell. This information is valuable, but new kinds of information are required in order to understand how a network of dynamic interactions shapes the cellular response. Obtaining this kind of information has been limited due to a lack of methods to exert spatial and temporal control over the activity of intracellular signaling molecules. Here we developed optogenetic tools that provide such control by locally inhibiting the activity of specific G protein subunits. We showed that light-triggered membrane recruitment of a truncated RGS4 can be used to spatially localize G protein subunit activity within a cell. We also showed that similar optical recruitment of GRK2ct to a spatially confined region of the plasma membrane can locally inhibit Gβγ-signaling activity. We combined the capabilities of these optogenetic tools with spatially uniform activation of GPCRs to generate intracellular gradients of G protein subunit activity. An advantage of the optical inhibition approach used here is that it enables spatial and temporal control over the activity of endogenous untagged proteins. Inhibition is achieved by expression of a CRY2-tagged protein, but the cellular response is elicited by a pathway that is entirely in its native state at the distal end of the cell with reference to the site of OA. This ensures that the targeted protein retains all of its native signaling properties. It also provides control over intracellular signaling at levels that reflect those driving native cell behavior because all of the signaling is done by endogenous proteins. A gradient of G protein αi and βγ activity is sufficient to drive cell migration Inhibition by pertussis toxin showed that Gi signaling is required for cell migration toward many different chemoattractants (Spangrude 2015 ASCB Award Essays, Selected Perspective, and MBoC Paper of the Year It has been shown that βγ inhibition by sequestering proteins (Arai et al., 1997; Neptune and Bourne, 1997) or small molecules (Lehmann et al., 2008; Kang et al., 2014) suppresses chemotaxis in many cell types. It was unknown, however, which features of cell migration are controlled by βγ signaling. Some reports implicated βγ signaling in directional sensing, whereas others proposed that it is primarily involved in controlling cell motility (Kamakura et al., 2013). Our results with CRY2-mCh-GRK2ct and CRY2-mCh-GRK3ct show that a gradient of activated βγ is sufficient to generate a PIP3 gradient and lamellipodia formation directed toward the side with a higher level of βγ activity. This directly demonstrates a role for βγ signaling in directional sensing. Overall these results with CRY2-RGS and CRY2-GRKct suggest that in immune cells sensing a chemoattractant gradient, the occurrence of a gradient of activated G protein subunits is sufficient to initiate directionally sensitive migration. Adaptation of cell migratory responses involves Gi-mediated global inhibition There is limited understanding of the molecular interactions that allow eukaryotic migratory cells to adapt to uniform stimulation. Dynamic control over receptor activation using microfluidics provides evidence that these cells use an IFFL network motif for adaptation (Takeda et al., 2012; Wang et al., 2012). Examining whether and how specific signaling molecules map onto the IFFL motif can be aided by methods that provide acute control over their activities within a living cell. Previously it had not been possible to test directly whether Gi activity generates the inhibitory signaling that is required for adaptation in a migratory cell. It was only known that an inhibitory pathway should be present downstream of the receptor. Our results show that Gi signaling is capable of triggering a delayed inhibitory pathway that acts throughout the entire space of a cell. The ability to generate a postadaptation PIP3 gradient by local suppression of αi and βγ activity shows that Gi stimulates a signaling pathway capable of inhibiting PIP3 globally. The response is reflected at the Optical inhibition of G protein subunits | 33 cellular level, because the cells demonstrate directional migration. Many downstream responses have been observed to adapt, and there is evidence that pathways acting in parallel to PIP3 signaling are involved in controlling cell migration. The ability of local Gi inhibition in an adapted cell to elicit a directional migratory response suggests that all of the relevant pathways are under control of G protein αi and βγ subunit activity. G proteins remain active when downstream responses adapt A fluorescence resonance energy transfer–based G protein sensor in Dictyostelium indicated that G protein heterotrimers remain dissociated after transient downstream responses such as PIP3 have adapted (Janetopoulos et al., 2001). This suggested that adaptation does not require deactivation of G protein subunits. There are examples, however, such as the response to mating pheromone in yeast, in which adaptation occurs at the level of the G protein (Cole and Reed, 1991). In the case of mammalian migratory cells, it has not been clear whether G protein subunit deactivation plays a role in adaptation. It has not been directly tested whether G protein subunit activity continues after an immune cell adapts to a uniform signal (Iglesias, 2012). Here, in immune cells that have adapted to a uniform stimulus, asymmetric G protein deactivation triggered a directional migratory response. This showed that in a fully adapted cell, G protein subunits continue to be in the activated state. Optical control over G protein subunits to dissect their dynamic signaling roles GPCRs have been implicated in other polarized cell behaviors, such as yeast budding (Bi and Park, 2012), neurite outgrowth (Fricker et al., 2005; Georganta et al., 2013), and orientation of asymmetric cell divisions (Yoshiura et al., 2012). The ability of our optogenetic tools to locally inhibit G protein subunits can be used to help determine their dynamic roles in these polarized responses. G protein subunits were classically believed to carry out all of their signaling functions at the plasma membrane, but mounting evidence suggests that they can have signaling activities at other locations within a cell (Hewavitharana and Wedegaertner, 2012). There is a lack of methods to determine the functions of G protein subunit signaling at intracellular locations. Existing methods to interfere with G protein signaling act over an entire cell. Optical recruitment of the CRY2-RGS to specific intracellular locations could be achieved through the use of appropriately targeted CIBN constructs. This could provide a means to acutely perturb G protein subunit activity at different locations within a cell. This could help dissect the functions of GPCR stimulated signaling at different locations of a cell. For example, signaling can be perturbed at a growth cone or a synapse. It can also be used to examine the temporal role of GPCR signaling in cell differentiation or development by inhibiting it at specific time points. MATERIALS AND METHODS Reagents SDF-1α/CXCL12 (S190; Sigma-Aldrich, St. Louis, MO) was dissolved to 10 μg/ml in 1× phosphate-buffered saline (PBS) containing 0.1% bovine serum albumin and stored as aliquots at −20°C. The C5a receptor agonist FKP-(D-Cha)-Cha-r (65121; Anaspec, Freemont, CA) was dissolved to 2.5 mM in 1× PBS containing 0.1% albumin and stored as aliquots at −20°C. DNA constructs CRY2PHR-mCh was obtained from AddGene (Cambridge, MA) (plasmid #26866). CIBN-CaaX was a kind gift from the lab of P. Di Camilli 34 | P. R. O’Neill and N. Gautam (Boyer Center for Molecular Medicine, Yale School of Medicine, New Haven, CT) (Idevall-Hagren et al., 2012). CXCR4 was a kind gift from the lab of I. Schraufstatter (Torrey Pines Institute for Molecular Studies, San Diego, CA) (Zhao et al., 2006). YFP-γ9 has been described before (Saini et al., 2007). A PCR product of PGK1 (38071; Addgene) was inserted into the KpnI and XbaI sites of CRY2PHR-mCh to create CRY2-mCh-PGK1. A PCR product of GRK2ct (Irannejad and Wedegaertner, 2010) was inserted into the KpnI and XbaI sites of CRY2PHR-mCh to make CRY2-mCh-GRK2ct. A PCR product of GRK3ct (Hollins et al., 2009) was inserted into the KpnI and XbaI sites of CRY2PHR-mCh to make CRY2-mCh-GRK3ct. A PCR product of RGS4 lacking residues 1–33 was inserted into the KpnI and XbaI sites of CRY2PHR-mCh to make CRY2-mCh-RGS4Δ. PH(Akt)–green fluorescent protein (GFP; 18836; Addgene) was cut with BamHI and XbaI to release GFP, and a PCR product of Venus was inserted in its place to make PH(Akt)-Venus. Tissue culture HeLa cells were obtained from ATCC and cultured in MEM (CellGro 10-010-CM) supplemented with 10% dialyzed fetal bovine serum (FBS; Atlanta Biologicals) and 1× antibiotic-antimycotic solution (CellGro) at 37°C and 5% CO2. RAW 264.7 cells were obtained from the Washington University Tissue Culture Support Center and cultured in DMEM supplemented with 10% dialyzed FBS and 1× antibiotic-antimycotic solution at 37°C and 5% CO2. RAW cells ranging from passage 3 to passage 12 were used for experiments. Transfections HeLa cells were transfected using Lipofectamine 2000. Cells were plated at 2 × 105 cells/dish in 29-mm glass-bottom dishes (In Vitro Scientific) 1 d before transfection. RAW cells were transfected by electroporation using Cell Line Nucleofection Kit V (Lonza) with a Nucleofector II device (Amaxa). For each sample, 2 × 106 cells were pelleted by spinning at 90 × g for 10 min, resuspended in 100 μl of Nucleofection solution containing between 0.2 and 2.5 μg of each plasmid DNA, depending on the specific construct (0.2 μg of PH(Akt)-Venus, 2 μg of CXCR4, and 2.5 μg of others), and electroporated using program D-032. Immediately after electroporation, 500 μl of prewarmed medium was added to the cuvette, and this was split among 29-mm glass-bottom dishes (8–10 dishes) containing 500 μl of prewarmed medium in the center well. After transfection, dishes were kept in a 37°C, 5% CO2 incubator until imaging. Live-cell imaging and optical activation All imaging was performed using a spinning-disk confocal imaging system consisting of a Leica DMI6000B microscope with adaptive focus control, a Yokogawa CSU-X1 spinning-disk unit, an Andor iXon electron-multiplying charge-coupled device camera, an Andor fluorescence recovery after photobleaching–photoactivation unit, and a laser combiner with 445-, 448-, 515-, and 594-nm solid-state lasers, all controlled using Andor iQ2 software. This system allows live-cell imaging to be combined with localized OA within a selected region of the sample that can be redefined in between images in a sequence. For OA of CRY2, the 445-nm laser was used at 5 μW and scanned across the selected region at a rate of 0.9 ms/μm2. This was performed once every 5 s. Solid-state lasers with wavelengths of 515 and 594 nm were used for excitation of Venus and mCherry, respectively. Emission filters were Venus 528/20 and mCherry 628/20 (Semrock). All images were acquired using a 63× oil immersion objective. A single confocal plane was imaged at a rate of 1 frame/5 s. All imaging was performed inside a temperature-controlled chamber held at 37°C. Imaging of HeLa cells was performed 1 d after Molecular Biology of the Cell transfection with Lipofectamine 2000. Imaging of RAW 264.7 cells was performed 2–10 h after electroporation. Before imaging, the culture medium was replaced with 500 μl of Hank’s balanced salt solution supplemented with 1 g/l glucose (HBSSg). 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