Guía seminarios 2014 primera parte

Transcription

INTRODUCCIÓN A LA
FISIOLOGÍA MOLECULAR
GUIA DE SEMINARIOS 2014
PRIMERA PARTE
IFM 2014
Seminario 1: Homeostasis. Membrana y transporte
-Bibliografía obligatoria: “Glucose homeostasis with infinite gain: further lessons from the
Daisyworld parable?” J.H. Koelag et al. Journal of Endocrinology 154:187–192 (1997).
-Bibliografía sugerida: “Biología Celular y Molecular” de Lodish y colaboradores. 5ta
Edición. Capítulo 5: “Biomembranas y Arquitectura Celular”. Capítulo 7: “Transporte de
iones y moléculas pequeñas a través de las membranas celulares”.
-Bibliografía de consulta opcional: “Hormonal Rein Control” en:
http://sun025.sun.ac.za/portal/page/portal/Health_Sciences/English/Departments/Biomedical
_Sciences/MEDICAL_PHYSIOLOGY/Essays/Hormonal_rein_control
1) ¿Qué diferencia hay entre un sistema en equilibrio dinámico y un sistema en estado
estacionario?
2) El concepto de homeostasis implica poner en juego un sensor y un efector. Explique cuál
es la interacción entre estos dos elementos genéricos de todo sistema homeostático. Dé
ejemplos de la vida diaria y de la fisiología de este tipo de sistemas, e identifique en cada
caso el sensor y el efector.
3) ¿A qué se refieren los términos retroalimentación positiva y retroalimentación negativa?
Clasifique los ejemplos que dio en el punto anterior de acuerdo a este criterio.
4) ¿Qué caracteriza a un sistema homeostático con ganancia infinita? ¿Qué garantiza este
tipo de control?
5) ¿Es necesario que un sistema homeostático sea un sistema abierto? ¿Por qué? Dé una
explicación en términos energéticos.
6) En el artículo de de Koelag y colaboradores se repasa el concepto de homeostasis desde
un conocimiento básico. Los principios de control homeostático serán recurrentes en cada
uno de los tópicos de la Fisiología Animal que se verán a lo largo del curso. El artículo debe
leerse como un texto „auto contenido”, donde los temas de la fisiología que se abordan no
requieren de un conocimiento más amplio que el que se plantea en el manuscrito.
Solo a modo de introducción cabe
tener en cuenta que las islas de
Langerhans
del
pancreas
se
componen de células
y . Las
células producen glucagon y las
insulina.
7) Señale con una X las aseveraciones correctas, y exponga un enunciado correcto para el
caso de las aseveraciones incorrectas:
La porción polar de los fosfolípidos está formada por péptidos y fosfato.
Los esfingolípidos y los glicolípidos comparten una estructura molecular similar, pero se
diferencian en la cabeza polar.
Las membranas biológicas están formadas por una bicapa cuya composición de
fosfolípidos es asimétrica.
Las flipasas son enzimas que favorecen la pérdida de la asimetría entre las hemicapas de la
membrana, utilizando el gradiente de concentración de los fosfolípidos entre ambas
hemicapas como fuente de energía para transportarlos.
% de
células
% de células doblemente marcadas
8) La figura que se muestra a continuación expresa los resultados de un experimento
realizado por Frye y Edidin en 1970. En este trabajo se provocó la fusión de células de ratón
con células humanas. Posteriormente se trató a estas células con dos anticuerpos
fluorescentes, cada uno de los cuales reconocía antígenos sólo en uno de los tipos celulares
fusionados. Cada anticuerpo estaba asociado a un fluoróforo que emitía en una longitud de
onda diferente. Las barras indican el % de células que presentaron: una tinción homogénea
que resultó de la mezcla de ambos fluoróforos (mosaico), o una tinción en la cual ambos
fluoróforos se distribuyeron aisladamente uno de otro (no mosaico). Se muestra como varían
ambos porcentajes en función del tiempo para el experimento realizado a 37°C. El tiempo
cero corresponde al inicio de la incubación que da comienzo a la fusión.
no-mosaico
mosaico
100
50
5
10
25
40
120
tiempo de incubación a 37º (min)
a. Interprete el gráfico.
b. ¿Qué se concluye, cualitativa y cuantitativamente, de este experimento respecto de la
movilidad de las proteínas presentes en la bicapa lipídica?
c. Grafique los resultados esperados si este experimento se realizara en las siguientes
condiciones:
i) Las células se incuban a 20°C.
ii) Las células fueron preincubadas con -ciclodextrina hasta reducir en un 50% el
contenido de colesterol en la membrana plasmática. La -ciclodextrina tiene un interior
hidrofóbico que interactúa y secuestra colesterol.
En cada caso explique las modificaciones que espera observar respecto de lo mostrado en la
figura de Frye & Edidin (1970).
9) La viscosidad de la membrana plasmática es dependiente de la composición lipídica y
proteica de la misma. La movilidad de una proteína de membrana se encuentra íntimamente
ligada a su coeficiente de difusión en la bicapa lipídica. Con el fin de medir el efecto de la
viscosidad de la membrana sobre la difusión lateral de la proteína HRas (GTPasa involucrada
en la división celular) se utilizaron células COS7 (línea celular derivada del riñón de mono)
en las que se expresó la proteína HRas unida a GFP (green fluorescent protein). La imagen en
A muestra una imagen obtenida con un microscopio de fluorescencia donde se puede
observar la expresión de la proteína fluorescente en la superficie membranal. Mediante la
técnica de FRAP (del inglés Fluorescence Recovery After Photobleaching, es decir,
recuperación de la fluorescencia después del fotoblanqueado) se estudió la movilidad de la
proteína en la membrana celular. En los paneles B-D se muestran los resultados para la
condición control y en el panel E se muestra la recuperación de la fluorescencia en
condiciones control y en dos tratamientos.
a. De acuerdo a los datos presentados de recuperación de florescencia en función del
tiempo, explique las fases de la curva presentada en el panel E.
b. Teniendo en cuenta los factores y/o tratamientos que afectan la viscosidad de la
membrana plasmática proponga un candidato para cada tratamiento referido en los
gráficos E y F.
E
F
Control
Trat 1
Trat 2
Figura. (A-D) Imágenes de un experimento de FRAP en células expresando GFP-HRas. Se indican
los tiempos después del fotoblanqueado. Escala, 10 µm. (E) Curva de recuperación de la florescencia
en función del tiempo en condiciones control (círculos negros), tratamiento 1 (cuadrados blancos) y
tratamiento 2 (triángulos negros). (F) Cuantificación del coeficiente de difusión (D) de la proteína en
la membrana plasmática en condiciones control y en cada tratamiento.
c. ¿Qué parámetro biofísico permite calcular con precisión la técnica de FRAP? ¿Cree que
será útil para estudiar la movilidad de proteínas solubles en citoplasma? Explique su
respuesta.
10) Si dos proteínas se mueven libremente en la bicapa lipídica, ¿qué factores afectan la
probabilidad de que éstas se encuentren?
11) Si dos proteínas están asociadas a un LIPID RAFT, ¿cómo espera que varíe la distancia
entre ellas a lo largo del tiempo?
12) Se le pide determinar la localización en la membrana plasmática del receptor de tipo II
para la fracción Fc de IgG Fc RII) en células B del sistema inmune. Se sabe que la
asociación del receptor Fc RII con fosfolípidos presentes en los RAFTs resiste el tratamiento
con soluciones de detergentes (Triton X-100) en concentraciones de 0.2 a 1%. Utilizando
anticuerpos específicos se analizó la co-localización de Fc RII con proteínas asociadas
(CD55, Fig 1, A-C) o proteínas no asociadas (TfR, Fig 1, F-G) a los RAFTs y se obtuvieron
los siguientes resultados:
Figura 1. Distribución de
Fc RII, CD55 y TfR en las
membranas de las células B. A.
Distribución de Fc RII. B.
Distribución de CD55. C.
Superposición de A y B. F.
Distribución de Fc RII. G.
Distribución de TfR. H.
Superposición de F y G. E
Gráfico del perfil de intensidad
de fluorescencia en las series AB (gráfico superior) y F-G
(gráfico inferior). En gris se
grafica el perfil de Fc RII y en
negro el perfil de CD55
(superior) y TfR (inferior).
Barra de escala, 5 m. (ver
versión a color en la página
Web de la materia).
a. De acuerdo a los resultados, ¿cuál esperaría que fuera la localización de Fc RII en
la membrana plasmática? Explique su razonamiento
b. El tratamiento con ciclodextrina (fármaco que elimina el colesterol de las
membrana) hace que los LIPID RAFTs se desensamblen. Proponga qué resultados
se obtendrían en la figura 1 luego de este tratamiento.
c. ¿Qué papel podrían cumplir los LIPID RAFTs en la fisiología celular?
13) ¿Por qué son necesarias las proteínas canal para el flujo de iones a través de la membrana
plasmática? ¿Por qué resulta funcional que los canales sean proteínas transmembranales de
múltiples pasos transmembrana?
14) ¿Cuál/es de las siguientes moléculas difunden a través de la bicapa lipídica sin una
proteína de membrana que medie el proceso? ATP, CO2, serina, agua, glucosa, Na+.
¿Por qué mecanismo atraviesan la membrana las restantes moléculas mencionadas que no
difunden libremente? Explique el funcionamiento de cada mecanismo.
15) ¿Cuál de los siguientes tipos de proteínas carrier es la Na+/K+-ATPasa? Simporter,
uniporter, antiporter. Mencione ejemplos de los dos tipos restantes.
1. El transporte de glucosa acopla la captación de Na+ con la de glucosa en el dominio apical
de una célula epitelial del intestino.
a. ¿El movimiento de Na+, es a favor o en contra del gradiente electroquímico?
b. ¿Y en el caso de la glucosa?
c. Discutir en base a la respuesta dada si este proceso de transporte es activo o pasivo.
d. La glucosa una vez acumulada dentro de la célula, sale de ésta por la cara basolateral
por medio de un transportador de glucosa. ¿Qué tipo de transporte es éste?
e. ¿Cómo se explica que la entrada de glucosa no produzca una acumulación de Na +
dentro de la célula epitelial?
16) Haga un esquema de la membrana plasmática e indique las concentraciones de Na +, K+ y
Ca2+ en los compartimentos intra y extra celular. ¿Están estos iones en equilibrio? ¿Están en
estado estacionario? Explique
17) Dentro de un rango de concentración de soluto biológicamente relevante el transporte de
moléculas a través de una proteína transportadora (carrier) alcanza una velocidad máxima.
¿Sucede lo mismo con el transporte a través de una proteína canal? Explique.
18) ¿Cuál/es de las siguientes afirmaciones sobre el antiport de Na +/Ca2+ en células
musculares cardíacas es falsa? Justificar.
a. En estado de reposo, este sistema de transporte lleva al Ca 2+ fuera de la célula muscular
cardíaca, mientras que el Na+ es simultáneamente llevado dentro de la célula muscular.
b. Sin un gradiente electroquímico de Na+ mantenido por la actividad de la Na+/K+ ATPasa,
esta forma de transporte activo secundario del calcio no existiría.
c. El tratamiento de las células musculares cardíacas con ouabaína (bloqueante de la bomba
Na+/K+ ATPasa) resulta en un incremento de la concentración intracelular de Ca 2+ y en una
disminución en la concentración intracelular de Na+.
d. El tratamiento de las células musculares cardíacas con digoxina (bloqueante del
transportador Na+/Ca2+) resulta en un incremento en la concentración intracelular de Na + y
Ca2+.
e. Este intercambiador produce el transporte neto de una carga positiva desde el exterior al
interior celular por ciclo.
2. Se desea analizar la permeabilidad de una membrana al cloro y para ello reemplaza todas
las sales que contienen cloruro por sales sulfato (SO42-). La composición de la solución
fisiológica normal es (en mM): NaCl, 115; KCl, 4; MgCl2, 1; CaCl2, 2, Tris (pH, 7.4), 5 y
glucosa, 10. Calcule la concentración de sales sulfato que forme una solución isotónica
respecto de la solución normal, manteniendo similar relación entre los iones.
3. Un axón gigante de calamar se dializa con una solución salina que contiene Ca2+ radiactivo
(Dipolo & Beauge, 1991. Annals of the NY Acad 639:100). Para estimar el eflujo de Ca 2+ a
través de la membrana se mide a intervalos regulares la cantidad de Ca 2+ radiactivo
acumulado por unidad de tiempo en la solución externa en presencia y ausencia de Na+
(círculos blancos) en la solución externa (Ext) y en ausencia o presencia de ATP (círculos
negros) en la solución interna (Int), según se indica en la figura.
a. ¿Le parece que la salida de calcio radiactivo desde el interior al exterior del axón (eflujo)
se debe a un flujo pasivo, activo o a ambos? Justifique su respuesta.
b. De los resultados observados en la figura los autores deducen que el requerimiento de ATP
para el transporte de Ca++ no es absoluto. ¿Cómo se puede explicar este fenómeno?
c. A qué le parece que podría deberse el efecto del ATP sobre: (i) el eflujo de Ca 2+
independiente de la presencia de Na+ externo; (ii) el eflujo de Ca2+ dependiente de la
presencia de Na+ externo.
19) ¿Cuál de las siguientes afirmaciones es aplicable a la comparación entre una solución 0.5
M NaCl y una solución 1 M de glucosa?
a) Ambas tienen la misma osmolaridad.
b) Ambas ejercen la misma presión osmótica
c) Son isotónicas entre sí si están a ambos lados de una membrana que es impermeable
a ambos solutos pero permeable al agua.
d) Todas las aseveraciones anteriores son ciertas.
187
Commentary
Glucose homeostasis with infinite gain: further lessons from the
Daisyworld parable?
J H Koeslag, P T Saunders1 and J A Wessels
Department of Medical Physiology and Biochemistry, University of Stellenbosch, Tygerberg 7505, South Africa and 1Department of Mathematics,
King’s College, Strand, London WC2R 2LS, UK
(Requests for offprints should be addressed to J H Koeslag, Department of Medical Physiology and Biochemistry, PO Box 19063, Tygerberg, 7505,
South Africa)
Abstract
A major unresolved physiological problem is how the rate
of hepatic glucose production is increased to match the
increased rate of glucose utilization during exercise without a change in arterial blood glucose level. A homeostat
with such capabilities is said to have infinite gain.
Daisyworld is an imaginary planet orbiting a variable
star. The only life is black and white daisies. Black daisies
retain heat, slightly warming the planet; white daisies cool
it. When the two types of daisies grow best at slightly
different temperatures, variations in solar luminosity (over
a wide range) cause the ratio of white:black daisies to vary
in a manner that keeps the planetary temperature constant.
This model therefore achieves infinite gain by having two
opposing but interdependent controllers.
Here we suggest that the pancreatic islet á- and â-cells
might act as black and white daisies. For the analogy to
Introduction
A major unresolved physiological problem is how the rate
of hepatic glucose production is increased to match the
increased rate of glucose utilization during exercise without a change in arterial blood glucose level (Ahlborg 1969,
Felig & Wahren 1975, Zinman et al. 1977, Winder et al.
1979, Kjaer et al. 1991, Roy et al. 1991, Coggan et al.
1995). There are two classic answers. The first involves
‘feedforward control’ (Riggs 1963, Guyton & Hall 1996).
This means that the anticipation and performance of
muscular exercise, working via the autonomic nervous
system, cause the pancreatic islets to secrete less insulin and
more glucagon. Anticipatory or feedforward responses
characterize many physiological systems. However, feedforward systems consist of open loops. The magnitude of
the response to a given stimulus is subject, therefore, to
‘guess-work’, and is, on its own, not self-adjusting. The
Journal of Endocrinology (1997) 154, 187–192
0022–0795/97/0154–0187 $08.00/0
apply, glucagon and insulin must not only have opposing
effects on the blood sugar concentration, but the secretion
of the one has, at some quantum level, to be at the expense
of the other. Electrical coupling between heterocellular
groups of á- and â-cells within the pancreatic islets
suggests that this might indeed be the case. á-Cell activity
must, furthermore, promote secretory activity in other
á-cells; similarly with â-cells. This is probably mediated
via pancreastatin and ã-amino butyric acid (GABA) which
are paracrinically co-secreted with glucagon and insulin,
respectively. á-Cell activity spreads (at the expense of
â-cell activity) when the blood glucose level is below set
point, while â-cell activity progressively replaces á-cell
activity above set point. At set point changes in the ratio of
á:â-cell activity are inhibited.
response is, at best, therefore only approximately correct or
appropriate.
The second possibility involves ‘integral control’
(Milsum 1966). Integral controllers are closed loop,
negative feedback systems. They differ, however, from the
negative feedback systems described in the standard physiology textbooks. A standard negative feedback system
responds only when a sensor detects an error in the
controlled quantity (blood sugar, arterial blood pressure,
body temperature etc.). The greater the error the greater
the response (Cannon 1960, Riggs 1963, 1970, Milsum
1966, Guyton & Hall 1996). A zero error always produces
a zero response. Thus, a standard controller is incapable of
totally correcting an error in the face of steady state
disturbances, such as the increased rate of glucose utilization during exercise. Instead, a standard controller merely
reduces the error that would have occurred if that controller had not been operational (Riggs 1963). If the error
? 1997 Journal of Endocrinology Ltd Printed in Great Britain
188
Commentary
is reduced to half of what it would have been in the
homeostat’s absence, then the homeostatic ‘gain’ is said to
be two (the reciprocal of half; Riggs 1963, 1970, Guyton
& Hall 1996, Milsum 1966). Homeostasis with infinite
gain implies perfect control.
An integral controller responds not simply to the error
in the controlled quantity, but to the error multiplied by
the time it persists. It is, at present, the only known type
of homeostat with infinite gain (Milsum 1966). Thus,
during exercise, an integral controller would respond with
progressively increasing degrees of hypoinsulinaemia and
hyperglucagonaemia until normoglycaemia is restored. On
reaching normoglycaemia, the hypoinsulinaemia and
hyperglucagonaemia persist until the high rate of glucose
utilization starts to wane. This then reverses the process.
However, the mechanism for such integral control is
unknown. It is striking, however, that most of the body’s
integral controllers operate via counterregulatory pairs of
effectors (hormones or nerves).
The Daisyworld parable
The sun is probably about 30% warmer today than it was
3000 million years ago when life first appeared on this
planet (Margulis & Sagan 1995). Yet, remarkably, the
Earth’s surface temperature has probably never varied by
very much more than about 5 )C from its present level.
This is almost certainly due to the metabolic activities of its
biota. Thus, if life were suddenly to disappear today,
the amount of atmospheric oxygen and nitrogen would
decline to trace levels in an atmosphere of water vapour
and carbon dioxide (Lovelock 1972). The Earth’s surface temperature would consequently rise to 80–90 )C,
rendering the planet uninhabitable.
To illustrate how even a simple biota might regulate the
surface temperature of a planet, Watson & Lovelock
(1983) devised an imaginary planet called Daisyworld. It
orbits a star very much like our own sun. Daisies are the
only form of life. There are only two species, one white
and one black. Otherwise they are identical. Both grow
best at 22·5 )C, and neither can grow at temperatures
below 5 )C or above 40 )C (Watson & Lovelock 1983,
Saunders 1994). The temperature on Daisyworld depends
on solar luminosity and the planet’s albedo (or reflectivity).
Black daisies have a lesser albedo and so absorb more heat
than bare earth. Hence for a given solar luminosity a planet
covered in black daisies will be warmer than a lifeless one.
Conversely, a planet covered in white daisies will be cooler
than its dead counterpart. If both daisies are present, it is
warmer where there are black daisies and cooler where
there are white ones.
It turns out that these very ordinary properties have
remarkable consequences. Consider a planet which is
gradually warming under the influence of an evolving sun.
When the temperature reaches 5 )C daisies begin to
Figure 1 The temperature response (heavy black lines) to changes
in solar luminosity on a Daisyworld with microclimates. The thin
line indicates the temperature changes on the bare planet.
Temperature changes on the planet with black and white daisies
are resisted over a wide range of solar luminosities by changes in
the proportion of black:white daisies on the planet (see text).
germinate. The white ones cool their immediate environment to below 5 )C and die. The black ones, however,
warm their surroundings, encouraging more daisies to
germinate. This makes the planet progressively warmer,
encouraging more black daisies and, now, even a few
white daisies to germinate. The process stops when the
planet is covered with flowers. The ratio of black:white
daisies, that have germinated, ensures, surprisingly, that
the temperature on Daisyworld is now just above 22·5 )C
(Saunders 1994).
If, at this point, solar luminosity were to decrease again,
life does not disappear. Instead, black daisies replace the
white daisies that have germinated, lowering the planet’s
albedo, and raising the overall temperature slightly. With
an increase in solar luminosity the opposite happens. This
is because a change in solar luminosity causes an initial
change in planetary temperature. With a rise in temperature, for instance, the black daises, which already live in
microclimates somewhat above 22·5 )C, are disadvantaged, compared with the white daisies, which live in
microclimates slightly below optimum, and, therefore,
benefit from the rise in temperature. Thus, white daisies
replace the black daisies, and will continue to do so until the
overall temperature returns to approximately 22·5 )C.
Over a comparatively wide range of solar luminosities,
which would otherwise cause a planetary temperature
change of 50 )C, there is, when daisies are present,
temperature homeostasis (Fig. 1).
Commentary
The Watson & Lovelock model (1983), which is
primarily an ecological one, can be simplified to make it
more generally applicable. The microclimates are unnecessary if winds equalize the temperature over the
whole planet, and if black daisies grow optimally at a
slightly lower temperature (e.g. 20 )C) than the white
ones do (e.g. 25 )C). Then, irrespective of the shapes of
the two growth curves, the temperature on Daisyworld
stabilizes at exactly the cross-over point of the two growth
curves, over a wide range of solar luminosities. The
homeostatic gain is infinite. The reason is easy to see. At
the cross-over point, say at 22·5 )C, both species grow
equally well. If the temperature rises even minutely to,
say, 22·6 )C the white daisies grow faster than the black
ones. White daisies therefore replace the black ones, and
continue to do so for as long as the temperature on
Daisyworld remains above 22·5 )C. But, in spreading, the
white daisies increase the planet’s albedo, causing it
eventually to cool to 22·5 )C. At this point the black and
white daisies grow equally well again, and the replacement
process stops. Thus, seen from space, the planet responds
to changes in solar luminosity by changing colour from
black (at low solar luminosities) to white (at high solar
luminosities), keeping its surface temperature robustly at
22·5 )C.
We believe that the physiology of the insulin:glucagon
counterregulatory pair of hormones is strongly reminiscent
of white and black daisies.
Pancreatic islets
The human islets of Langerhans contain glucagon secreting á-cells, insulin secreting â-cells, and somatostatin
secreting D-cells. The â-cells are situated in the core of a
typical islet. The glucagon-secreting á-cells form a peripheral rim one to three cells in thickness (Unger et al. 1978).
In humans, rows of á-cells (together with D-cells) project
into the central portions of the islets along capillary axes,
forming lobulations in which the á- and D-cells remain
peripheral to the centrally assembled â-cells. The D-cells
are situated between the outer mantle of á-cells and the
core of â-cells. In addition, there are heterocellular regions
in each islet, with á-, â- and D-cells in close proximity
(Orci & Unger 1975).
A characteristic feature of the islet cells are membrane
specializations that may involve intercellular relationships.
These specializations consist of tight junctions, desmosomes and gap junctions (Orci et al. 1973, 1975). Gap
junctions, through which molecules of less than 1000 Da
can move from the cytoplasm of one cell to that of an
adjacent cell without entering the intercellular space, have
been found between á- and â-cells (Orci & Unger 1975,
Orci et al. 1975, Orci 1976, Meda et al. 1982), as well as
between D-cells and á- or â-cells (Unger et al. 1978,
Michaels & Sheridan 1981, Meda et al. 1982). Thus, gap
junctions make functional syncytia of small groups of
homologous and heterologous islet cells (Meda et al. 1982).
Each islet probably consists of a collection of such syncytial
aggregates.
The â-cells are unequivocally glucose sensitive. The
mechanism may be summarized as follows (Ashcroft et al.
1994, Dunne et al. 1994). High plasma glucose concentrations lead to high rates of glycolysis. Some product of
this metabolism, now generally accepted to be ATP,
brings about closure of the ATP-sensitive K+ channels
in the plasma membrane, causing depolarization. This
leads to the opening of voltage-dependent Ca2+ channels,
and increases in the intracellular free Ca2+ concentration.
This initiates exocytosis of insulin-containing secretory
vesicles (Prentki & Matschinsky 1987, Ashcroft et al. 1994,
Berggren & Larsson 1994, Dunne et al. 1994). Insulin
secretion, for any given â-cell, appears to be all-or-none
(Pipeleers et al. 1994).
The mechanisms for the stimulus-secretion coupling in
the glucagon producing á-cells are unclear. The rate of
glucose metabolism is only about 15–20% of that observed
in the â-cell (Rorsman et al. 1991). Furthermore, although
in vitro studies have indicated that á-cells are to some
extent glucose sensitive (Sumida et al. 1994), it is unclear
whether variations in glucose concentration in the physiological range have a direct effect on á-cells in vivo (Asplin
et al. 1981, Rorsman & Helman 1988, Rorsman et al.
1991). Indeed, the á-cells of patients with insulindependent diabetes mellitus continue to secrete glucagon
at normal resting rates despite severe hyperglycaemia
(Unger et al. 1970, Raskin et al. 1976, Powell et al. 1993).
These patients also do not increase glucagon secretion
during hypoglycaemia (Asplin et al. 1981, Bolli et al. 1983,
Powell et al. 1993). The á-cells therefore probably receive
their glycaemic cues primarily from the â-cells, presumably via electrical coupling (via gap junctions) between
these cells (Orci et al. 1975, Michaels & Sheridan 1981,
Meda et al. 1982, 1986).
Insulin and glucagon responses to hyper- and
hypoglycaemic clamps
When the endocrine pancreas is exposed to a hyperglycaemic clamp (e.g. 11 or 16 mmol glucose/l), there is
an initial spike of insulin release lasting for less than
10 min. This is followed by a slow continuous rise in
insulin secretion, over the next 2 h, to very high levels
(Fig. 2) (Grodsky 1972, Gerich et al. 1974, Bolaffi et al.
1986, Tsuchiyama et al. 1992). A hypoglycaemic clamp
(1 mmol/l) produces a similar spike in glucagon secretion,
followed by a gradual rise in glucagon production (Weir
et al. 1974). The mathematical characteristics of the acute
response led Grodsky (1972) to postulate that â-cells
operate as on-off units with differing blood glucose
thresholds. Direct evidence for such a model has recently
189
190
Commentary
Figure 2 The results of an archetypical hyperglycaemic clamp
experiment (Grodsky 1972, Gerich et al. 1974, Bolaffi et al. 1986,
Tsuchiyama et al. 1992). If the isolated pancreas is perfused with
blood containing a constant 15 mmol glucose per litre, then there
is an immediate spike of insulin release, followed by a prolonged
*second phase’ of progressively increasing rates of insulin release
for more than 2 h. This *second phase’ is characteristic of an
integral response, indicating that the different â-cell thresholds
(Pipeleers 1992, Pipeleers et al. 1994) are functions of
time#hyperglycaemic stimulus. This result would also typify the
white daisy response to a heat clamp experiment on Daisyworld.
been demonstrated (Schuit et al. 1988, Pipeleers 1992,
Hellman et al. 1994, Pipeleers et al. 1994).
However, the slowly rising ‘second phase’ of insulin
release in response to a hyperglycaemic clamp cannot be
due to simple differences in â-cell glucose thresholds,
as suggested by Grodsky (1972), Pipeleers (1992) and
Pipeleers et al. (1994). Instead, the evidence suggests that
high steady state blood glucose concentrations recruit more
and more active â-cells with time. The â-cell ‘thresholds’
are therefore, somehow, for different integrals (stimulus
magnitude#time) of the stimulus with respect to time.
If â-cells operate as on-off units, and are functionally
linked to á-cells, then the two probably operate as a
flip-flop mechanism, which secretes either insulin (in the
aB mode) or glucagon (in the Ab mode). Both are secreted
maximally or not at all (Schuit et al. 1988, Pipeleers 1992,
Hellman et al. 1994, Pipeleers et al. 1994). Only under
special, near-normoglycaemic circumstances would the
system be modulated (via autonomic nerves, the gut and
adrenal hormones) to secrete both hormones, or neither.
Glucose homeostasis
During the recent past it has become evident that
chromogranin-A (CgA) and its proteolytic products are
costored and cosecreted with most peptide hormones,
including insulin and glucagon (Cohn et al. 1984, Efendic
et al. 1987, Eiden 1987, Greeley et al. 1989, Huttner et al.
Figure 3 The proposed model of pancreatic islet function. The
functional units of the endocrine pancreas are suggested to be
sub-islet heterologous syncytial aggregates of electrically coupled
á- and â-cells, which operate as flip-flop mechanisms. They are
either in the Ab (glucagon secreting) or aB (insulin secreting)
mode. Ab]aB and aB]Ab transitions occur spontaneously, but
are influenced by paracrine secretions from neighbouring units
(see text). The relative effectivenesses of the two types of
paracrine secretion are determined by the blood sugar
concentration.
1991, Helle & Angeletti 1994, O’Connor et al. 1994,
Huttner & Natori 1995). Pancreastatin, the proteolytic
fragment of CgA found in pancreatic á-cells (Schmidt et
al. 1988, Winkler & Fischer-Colbrie 1992), is a powerful
paracrine inhibitor of insulin secretion. ã-amino butyric
acid (GABA) has been shown to colocalize with insulin in
the â-cells (Gerber & Hare 1980, Garry et al. 1986) and
has been shown to inhibit á-cell activity (Rorsman et al.
1991). CgA and CgB have similarly been localized in
â-cells (Winkler & Fischer-Colbrie 1992). An as yet
uncharacterized CgA or CgB fragment might therefore
also paracrinically inhibit glucagon secretion.
The existence of these strongly inhibitory paracrine
secretions, and the arrangement of á- and â-cells into
syncytial aggregates, suggest a novel, Daisyworld-like form
of blood glucose homeostasis.
Thus, we propose that the individual syncytial aggregates flip-flop spontaneously between the insulin (aB) and
glucagon (Ab) secreting modes (Fig. 3). Aggregates which
are in the Ab (glucagon-secreting) mode co-release pancreastatin, which promotes aB]Ab transitions among
their immediate neighbours. Ab units (like black daisies)
therefore tend to proliferate locally. aB units, which
cosecrete GABA with insulin, promote Ab]aB transitions. They too proliferate locally (like white daisies). At
a blood sugar concentration of 5 mmol/l these proliferative
tendencies are equal (Fig. 3). Thus, although individual
aggregates are flipping randomly from one mode to the
other, there is no overall change in the Ab:aB ratio. Both
Commentary
insulin and glucagon are therefore present in the blood of
normoglycaemic persons. (In contrast, a standard homeostat would require neither hormone to be present.) A fall
in the blood glucose concentration, due to a sudden
increase in the rate of glucose utilization, would, according
to our hypothesis, simply potentiate pancreastatin’s action.
This would increase the probability of aB]Ab flips,
thereby progressively increasing the proportion of units in
the Ab mode. The glucagon:insulin ratio in the blood will
therefore increase with time, as is typical of exercise (Wahren
et al. 1973, Koeslag et al. 1980, 1982). Only when
normoglycaemia has been completely restored will the bias
towards aB]Ab transitions cease. Normoglycaemia in the
presence of the new, high Ab:aB ratio persists for as long
as there is an increased rate of glucose utilization. This is
glucose homeostasis with infinite gain.
An increase in the blood glucose concentration has the
opposite effect. There is now a bias towards Ab]aB
transitions which continues until normoglycaemia has
been restored. During hyperglycaemic clamp experiments
the blood glucose concentration cannot be corrected.
Ab]aB transitions then continue for more than 2 h,
producing progressively higher and higher rates of insulin
secretion (Fig. 2). The process stops when all the units are
presumably in the aB mode.
In summary, therefore, we propose that glucose
homeostasis, with infinite gain, is critically dependent on a
pair of counterregulatory hormones (which is superfluous
in a standard homeostat). Our model also accords, for the
first time, full physiological significance to the puzzling
syncytial connections between sub-islet sets of á- and
â-cells, as well as to the presence of pancreastatin and
other inhibitory paracrine secretions in the pancreatic
islets. Standard homeostats benefit from neither of these
features.
References
Ahlborg B 1969 Blood glucose during prolonged physical exercise in
man. Förvarsmedicin 3 (Suppl 1) 85–99.
Ashcroft FM, Proks P, Smith PA, A
} mmälä C, Bokvist K & Rorsman P
1994 Stimulus-secretion coupling in pancreatic B cells. Journal of
Cellular Biochemistry 55S 54–65.
Asplin CM, Paquette TL & Palmer JP 1981 In vivo inhibition of
glucagon secretion by paracrine beta cell activity in man. Journal of
Clinical Investigation 68 314–318.
Berggren P-O & Larsson O 1994 Ca2+ and pancreatic B-cell function.
Biochemical Society Transactions 22 12–18.
Bolaffi JL, Heldt A, Lewis LD & Grodsky GM 1986 The third phase
of in vitro insulin secretion: Evidence for glucose insensitivity.
Diabetes 35 370–373.
Bolli G, DeFeo P, Compagnucci R, Cartechini MG, Angeletti G,
Santeusanio F, Brunetti P & Gerich JE 1983 Abnormal glucose
counterregulation in insulin dependent diabetes mellitus. Interaction
of anti-insulin antibodies and impaired glucagon and epinephrine
secretion. Diabetes 32 134–141.
Cannon WB 1960 The Wisdom of the Body. New York: WW Norton.
Coggan AR, Raguso CA, Williams BD, Sidossis LS & Gastaldelli A
1995 Glucose kinetics during high intensity exercise in
endurance-trained and untrained humans. Journal of Applied
Physiology 78 1203–1207.
Cohn DV, Elting JJ, Frick M & Elde R 1984 Selective localization of
the parathyroid secretory protein I/adrenal medulla chromogranin A
protein family in a wide variety of endocrine cells of the rat.
Endocrinology 114 1963–1974.
Dunne MJ, Harding EA, Jaggar JH & Squires PE 1994 Ion channels
and the molecular control of insulin secretion. Biochemical Society
Transactions 22 6–12.
Efendic S, Tatemoto K, Mutt V, Quan C, Chang D & O
} stenson C-G
1987 Pancreastatin and islet hormone release. Proceedings of the
National Academy of Sciences of the USA 84 7257–7260.
Eiden E 1987 Is chromogranin a prohormone? Nature 325 301.
Felig P & Wahren J 1975 Fuel homeostasis in exercise. New England
Journal of Medicine 293 1078–1084.
Garry DJ, Sorenson RL, Elde RP, Maley BE & Madsen A 1986
Immunohistochemical colocalization of GABA and insulin in â-cells
of rat islets. Diabetes 35 1090–1096.
Gerber JC & Hare TA 1980 GABA in peripheral tissues: presence and
actions in pancreatic function. Brain Research Bulletin 5 (Suppl 2)
341–346.
Gerich JE, Charles MA & Grodsky GM 1974 Characterization of the
effects of arginine and glucose on glucagon and insulin release from
the perfused rat pancreas. Journal of Clinical Investigation 54 833–841.
Greeley GH, Thompson JC, Ishizuka J, Cooper CW, Levine MA,
Gorr S-U & Cohn DV 1989 Inhibition of glucose-stimulated
insulin release in the perfused rat pancreas by parathyroid secretory
protein-I (chromogranin-A). Endocrinology 124 1235–1238.
Grodsky GM 1972 A threshold distribution hypothesis for the package
storage of insulin and its mathematical modeling. Journal of Clinical
Investigation 51 2047–2059.
Guyton AC & Hall JE 1996. Textbook of Medical Physiology.
Philadelphia: WB Saunders.
Helle KB & Angeletti RH 1994 Chromogranin A: a multipurpose
prohormone? Acta Physiologica Scandinavica 152 1–10.
Hellman B, Gylfe E, Grapengiesser E, Lund PE, Berts A, Tengholm
A, Pipeleers DG & Ling Z 1994 Glucose induces oscillatory Ca2+
signalling and insulin release in human pancreatic beta cells.
Diabetologia 37 (Suppl 2) S11–S20.
Huttner WB & Natori S 1995 Helper proteins for neuroendocrine
secretion. Current Biology 5 242–245.
Huttner WB, Gerdes H-H & Rosa P 1991 The granin
(chromogranin/secretogranin) family. Trends in Biochemical Sciences
16 27–30.
Kjaer M, Kiens B, Hargreaves M & Richter EA 1991 Influence of
active muscle mass on glucose homeostasis during exercise in
humans. Journal of Applied Physiology 71 552–557.
Koeslag JH, Noakes TD & Sloan AW 1980 Postexercise ketosis.
Journal of Physiology 301 79–90.
Koeslag JH, Noakes TD & Sloan AW 1982 The effects of alanine,
glucose and starch ingestion on the ketosis produced by exercise
and starvation. Journal of Physiology 325 363–376.
Lovelock JE 1972 Gaia as seen through the atmosphere. Atmospheric
Environment 6 579–580.
Margulis L & Sagan D 1995. What is life? London, Weidenfeld &
Nicolson.
Meda P, Kohen E, Kohen C, Rabinovitch A & Orci L 1982 Direct
communication of homologous and heterologous endocrine islet cells
in culture. Journal of Cell Biology 92 221–226.
Meda P, Santos RM & Atwater I 1986 Direct identification of
electrophysiologically monitored cells with intact mouse islets of
Langerhans. Diabetes 35 232–236.
Michaels RL & Sheridan JD 1981 Islet of Langerhans dye coupling
among immunocytochemically distinct cell types. Science 214
801–803.
Milsum JH 1966 Biological Control Systems Analysis. New York:
McGraw-Hill.
191
192
Commentary
O’Connor DT, Wu H, Gill BM, Rozansky DJ, Tang K, Mahata SK,
Mahata M, Eskeland NL, Videen JS, Zhang X, Takiyyuddin MA
& Parmer RJ 1994 Hormone storage vesicle proteins. Transcription
basis of the widespread neuroendocrine expression of chromogranin
A, and evidence of its biological actions, intracellular and
extracellular. Annals of the New York Academy of Sciences 733
236–245.
Orci L 1976 The microanatomy of the islets of Langerhans. Metabolism
25 (Suppl) 1303–1313.
Orci L & Unger RH 1975 Hypothesis: Functional subdivisions of the
islets of Langerhans and the possible role of the insular D-cells.
Lancet 2 1243–1244.
Orci L, Unger RH & Renold AE 1973 Structural coupling between
pancreatic islet cells. Experientia 29 1015–1018.
Orci L, Malaisse-Lagae F, Ravazzola M, Rouiller C, Renold AE,
Perrelet A & Unger RH 1975 A morphological basis for
intercellular communication between A- and B-cells in the
endocrine pancreas. Journal of Clinical Investigation 56 1066–1070.
Pipeleers DG 1992 Heterogeneity in pancreatic B-cell population.
Diabetes 41 777–781.
Pipeleers D, Kiekens R, Ling Z, Wilikens A & Schuit F 1994
Physiologic relevance of heterogeneity in the pancreatic beta-cell
population. Diabetologia 37 (Suppl 2) S57–S64.
Powell AM, Sherwin RS & Shulman GI 1993 Impaired hormonal
responses to hypoglycaemia in spontaneously diabetic and
recurrently hypoglycemic rats. Journal of Clinical Investigation 92
2667–2674.
Prentki M & Matschinsky FM 1987 Ca2+, cAMP, and phospholipidderived messengers in coupling mechanisms in insulin secretion.
Physiological Reviews 67 1185–1248.
Raskin P, Aydin I & Unger RH 1976 Effect of insulin on the
exaggerated glucagon response to arginine stimulation in diabetes
mellitus. Diabetes 25 227–229.
Riggs DS 1963 The Mathematical Approach to Physiological Problems.
Baltimore: Williams & Wilkins.
Riggs DS 1970 Control Theory and Physiological Feedback Mechanisms.
Baltimore: Williams & Wilkins.
Rorsman P & Hellman B 1988 Voltage-activated currents in guinea
pig pancreatic á2 cells: Evidence for Ca2+-dependent action
potentials. Journal of General Physiology 91 223–242.
Rorsman P, Ashcroft FM & Berggren P-O 1991 Regulation of
glucagon release from pancreatic á-cells. Biochemical Pharmacology 41
1783–1790.
Roy JY, Bongbélé J, Cardin S, Brisson GR & Lavoie J-M 1991
Effects of supramaximal exercise on blood glucose levels during
subsequent exercise. European Journal of Applied Physiology 63 48–51.
Saunders PT 1994 Evolution without natural selection: Further
implications of the Daisyworld parable. Journal of Theoretical Biology
166 365–373.
Schmidt WE, Siegel EG, Lamberts R, Gallwitz B & Creutzfeldt W
1988 Pancreastatin: molecular and immunochemical characterization
of a novel peptide in porcine and human tissues. Endocrinology 123
1395–1404.
Schuit FC, In’t Veld PA & Pipeleers DG 1988 Glucose stimulates
proinsulin biosynthesis by a dose dependent recruitment of
pancreatic beta cells. Proceedings of the National Academy of Sciences,
USA 85 3865–3869.
Sumida Y, Shima T, Shirayama K, Misaki M & Miyaji K 1994 Effects
of hexoses and their derivatives on glucagon secretion from isolated
perfused rat pancreas. Hormone and Metabolism Research 26 222–225.
Tsuchiyama S, Tanigawa K & Kato Y 1992 Impaired glucose priming
of insulin from perfused pancreas in aged female rats. Proceedings of
the Society for Experimental Biology and Medicine 201 54–58.
Unger RH, Aguilar-Parada E, Müller WA & Eisentraut AM 1970
Studies of pancreatic alpha cell function in normal and diabetic
subjects. Journal of Clinical Investigation 49 837–848.
Unger RH, Dobbs RE & Orci L 1978 Insulin, glucagon, and
somatostatin secretion in the regulation of metabolism. Annual
Reviews of Physiology 40 307–343.
Wahren J, Felig P, Hendler R & Ahlborg G 1973 Glucose and amino
acid metabolism during recovery from exercise. Journal of Applied
Physiology 34 838–845.
Watson AJ & Lovelock JE 1983 Biological homeostasis of the global
environment: the parable of Daisyworld. Tellus 35B 284–289.
Weir GC, Knowlton SD & Martin DB 1974 Glucagon secretion from
the perfused rat pancreas. Studies with glucose and catecholamines.
Journal of Clinical Investigation 54 1403–1412.
Winder WW, Hickson RC, Hagberg JM, Ehsani AA & McLane JA
1979 Training-induced changes in hormonal and metabolic
responses to submaximal exercise. Journal of Applied Physiology 46
766–771.
Winkler H & Fischer-Colbrie R 1992 The chromogranins A and B:
the first 25 years and future perspectives. Neuroscience 49 497–528.
Zinman B, Murray FT, Vranic M, Albisser AM, Leibel BS, McClean
PA & Marliss EB 1977 Glucoregulation during moderate exercise
in insulin treated diabetics. Journal of Clinical Endocrinology and
Metabolism 45 641–652.
Received 29 July 1996
Revised manuscript received 5 November 1997
Accepted 22 January 1997
IFM 2014
Seminario 2: Interacciones intercelulares
-Bibliografía de lectura obligatoria: “Mechanisms of Epithelial Cell–Cell Adhesion and Cell
Compaction Revealed by High-resolution Tracking of E-Cadherin–Green Fluorescent Protein”
Adams et al. The Journal of Cell Biology 142:1105–1119 (1998).
-Biblografía Sugerida: “Biología Celular y Molecular” de Lodish y colaboradores. 5ta Edición.
Capítulo 6: “Integración de células en tejidos”. Capítulo 13: “Señalización en la superficie
celular”.
1. ¿En qué se diferencian las uniones adherentes directas e indirectas? Dé un ejemplo de cada
una de éstas.
2. ¿Cuál es la relación entre las uniones adherentes directas e indirectas con el citoesqueleto?
Sugiera cuál puede ser la función fisiológica de dicha relación.
3. Dibuje el esquema de un epitelio y señale dónde esperaría encontrar las uniones oclusivas o
tight junctions.
a. ¿Cuál es la función principal de estas uniones en los epitelios?
b. Indique en dicho esquema el recorrido del flujo paracelular y transcelular.
c. ¿Qué condiciones deben darse para que una sustancia X atraviese un epitelio por la vía
paracelular o por la vía transcelular?
4. ¿Qué es una unión comunicante o gap junction? Indique cuál es su composición molecular.
¿Qué función cumple? Proponga un experimento que permita contrastar la hipótesis de que
existen gap junctions entre las células de un determinado tejido.
5. En el organismo las células pueden comunicarse de forma parácrina, endócrina o a través de
sinapsis (considerar solamente las químicas).
a. ¿Qué tienen en común estas tres vías de comunicación; y en qué se diferencian?
b. ¿En cuáles de estas vías de comunicación es más crítico que la célula blanco exprese
receptores altamente específicos para la sustancia transmisora u hormona?
c. Los receptores para las moléculas transmisoras pueden ser una proteína de membrana o una
proteína intracelular. ¿Qué determina que se trate de uno u otro tipo de proteína?
d. ¿En qué se distinguen los receptores ionotrópicos de los metabotrópicos?
e. El fosfatidilinositol es un lípido de membrana que participa en un mecanismo de señalización
intracelular. Explique este mecanismo.
f. ¿Qué otras vías de señalización por segundos mensajeros conoce?
6. ¿Qué tipo de transmisores son almacenados en vesículas? ¿Qué tipo de mecanismo de
tranporte tiene la vesícula para poder acumular a los transmisores en su interior? ¿Qué tipo de
mecanismo de transporte tienen las células para liberarlos?
7. La liberación de vesículas conteniendo moléculas transmisoras u hormonas depende de la
concentración intracelular de calcio. Indique en un esquema cuáles son los mecanismos
celulares por los cuales se puede modificar abruptamente la concentración citosólica de este
catión divalente. ¿Y cuales son los mecanismos que tienden a mantener las concentraciones
basales de Ca2+ constantes?
8. ¿En qué se diferencia un antagonista competitivo de uno no-competitivo? Describa un
experimento por el cual puede distinguir si una sustancia dada actúa como antagonista de uno u
otro tipo.
Análisis del artículo
Para tener en cuenta: Este artículo fue elegido porque presenta evidencias experimentales que
ilustran la difusión en el plano de la membrana de la molécula de adhesión E-cadherina en el
contexto de la formación de contactos intercelulares. La metodología descripta solo será
discutida en términos de los resultados fisiológicos. Comprender el artículo no requerirá
profundizar en la construccion del cDNA inyectado. Las técnicas de imágenes utilizadas ilustran
métodos modernos para el estudio de la fisiología de membranas y se les pide que se concentren
en enteder cómo se las emplea para estudiar la formación de uniones adherentes.
En la pagina de la materia hay una versión pdf del artículo que se sugiere mirar para ver las
figuras en mejor detalle, incluyendo imágenes en color.
1) En el artículo se identifican las siguientes proteínas: E-cadherina, -catenina, -catenina,
actina. Indique dónde se ubican estas proteínas (membrana, citosol, núcleo) y cuál es el
vínculo entre ellas.
2) Los autores extraen EcadGFP utilizando el detergente Triton X-100. ¿Por qué se selecciona
este detergente en particular?
3) ¿Por qué fue necesario realizar los experimentos de la Figura 1 si ya en la introducción se
recuerda la interacción entre las proteínas E-cadherina, -catenina, -catenina y actina? ¿Por
qué se analiza la interacción en presencia y ausencia de calcio?
4) En la discusión los autores explican que la presencia de -catenina, -catenina en relación
estequiométrica 1:1:1 con EcadGFP es un indicio del rol funcional de la proteína
experimentalmente expresada. ¿Cuál puede ser la explicación de este argumento?
5) Los autores definen tres formas de agregación de las moléculas de E-cadherina: los „puncta‟,
las placas y los „vertex‟. Identifíquelas en la Figura 2.
6) En la Figura 3 se estudian dos ejemplos de la interacciones intercelulares en estudio.
a. ¿En que se diferencian estos dos ejemplos?
b. ¿Cuál es su relación con los perfiles de fluorescencia mostrados en la Figura 3D.
7) La figura 4B muestra un gráfico en tres dimensiones que permite medir la velocidad de
difusión de los agregados de EcadGFP.
a. Identifique el inicio en la formación de cada puncta.
b. A medida que la zona de contacto se agranda, el número de puncta aumenta.
Identifique como se evidencia esto en este gráfico.
8) ¿En qué se diferencia el experimento descripto en la figura 5 del de la figura 4?
9) Mientras que en la figura 4 los autores argumentan que los puncta se forman de novo, en la
figura 6 argumentan que las placas se producen por agregación de puncta. Identifique la
evidencia que avala este argumento.
10) La citocalasina es un inhibidor de la elongación de los microfilamentos de actina. Actúa
inhibiendo la polimerización de la actina en su extremo de crecimiento (“barbed ends” en el
artículo)
a. ¿Cómo se relaciona el efecto de citocalacina con el estadío de formación del contacto
celular?
b. ¿Qué aporta la tinción que marca la actina y la -catenina?
11) Los autores argumentan que el fotoblanqueo no afectó el contacto celular ni interfirió con la
organización de la Ecadherina en la membrana.
a. ¿Qué evidencias avalan este conclusión?
b. ¿Por qué es importante esta constatación?
12) ¿Qué demuestra la figura 8B?
13) Los autores concluyen que la difusión de las Ecad depende de la edad del contacto. ¿Cómo
avala la figura 8E esta conclusión?
Published August 24, 1998
Mechanisms of Epithelial Cell–Cell Adhesion and Cell
Compaction Revealed by High-resolution Tracking
of E-Cadherin–Green Fluorescent Protein
Cynthia L. Adams, Yih-Tai Chen, Stephen J Smith, and W. James Nelson
Department of Molecular and Cellular Physiology, Stanford University School of Medicine, Stanford, California 94305-5345
Abstract. Cadherin-mediated adhesion initiates cell re-
C
ell–cell adhesion is crucial for the development
and survival of multicellular organisms (Townes
and Holtfreter, 1955; Takeichi, 1991; Steinberg,
1996). Members of the cadherin superfamily of Ca21dependent cell–cell adhesion proteins are expressed in
most organs and tissues of vertebrates and invertebrates.
Different cadherins are expressed in specific tissues, cell
layers, and neuronal cell types consistent with their roles in
distinct cellular recognition and sorting processes (Takeichi, 1987; Nose et al., 1988; Takeichi, 1991; Steinberg and
a circumferential actin cable that circumscribes both
cells, and is embedded into each E-cadherin plaque at
the contact margin. At this stage, the two cells achieve
maximum contact, a process referred to as compaction.
These changes in E-cadherin and actin distributions are
repeated when additional single cells adhere to large
groups of cells. The third stage of adhesion occurs as
additional cells are added to groups of .3 cells; circumferential actin cables linked to E-cadherin plaques on
adjacent cells appear to constrict in a purse-string action, resulting in the further coalescence of individual
plaques into the vertices of multicell contacts. The reorganization of E-cadherin and actin results in the condensation of cells into colonies. We propose a model to
explain how, through strengthening and compaction,
E-cadherin and actin cables coordinate to remodel initial cell–cell contacts to the final condensation of cells
into colonies.
Key words: cadherin • actin • cell–cell adhesion • epithelia • microscopy
The current address of Cynthia L. Adams is Cytokinetics, Inc., 280 East
Grand Ave., South San Francisco, CA 94040.
Address all correspondence to Stephen J Smith, Department of Molecular and Cellular Physiology, Beckman Center, Stanford University
School of Medicine, Stanford, CA 94305-5345. Tel.: 650-723-6799; Fax:
650-498-5286; E-mail: sjsmith@leland.stanford.edu
Takeichi, 1994; Fannon and Colman, 1996; Uchida et al.,
1996; Martinek and Gaul, 1997). While recent studies have
attempted to elucidate the molecular and mechanical properties of cadherin-mediated adhesion (Brieher et al. 1996;
Yap et al., 1997), little is known about the physical or
molecular dynamics of cell–cell adhesion (cell stickiness),
compaction (maximization of adhesive contacts), or condensation (aggregation of large cell colonies) during tissue
formation.
Cadherins are single transmembrane–spanning proteins.
The extracellular domain (amino terminus) is composed
of five repeats that have similar structures and contain
Ca21-binding motifs (Shapiro et al., 1995; Aberle et al.,
1996). While homotypic binding between the extracellular
domains of cadherins on adjacent cells is clearly important
for cell–cell recognition (Nose et al., 1988), the affinity of
 The Rockefeller University Press, 0021-9525/98/08/1105/15 $2.00
The Journal of Cell Biology, Volume 142, Number 4, August 24, 1998 1105–1119
http://www.jcb.org
1105
Downloaded from jcb.rupress.org on August 12, 2014
organization into tissues, but the mechanisms and dynamics of such adhesion are poorly understood. Using
time-lapse imaging and photobleach recovery analyses
of a fully functional E-cadherin/GFP fusion protein, we
define three sequential stages in cell–cell adhesion and
provide evidence for mechanisms involving E-cadherin
and the actin cytoskeleton in transitions between these
stages. In the first stage, membrane contacts between
two cells initiate coalescence of a highly mobile, diffuse
pool of cell surface E-cadherin into immobile punctate
aggregates along contacting membranes. These E-cadherin aggregates are spatially coincident with membrane attachment sites for actin filaments branching off
from circumferential actin cables that circumscribe
each cell. In the second stage, circumferential actin cables near cell–cell contact sites separate, and the resulting two ends of the cable swing outwards to the perimeter of the contact. Concomitantly, subsets of
E-cadherin puncta are also swept to the margins of the
contact where they coalesce into large E-cadherin
plaques. This reorganization results in the formation of
1. Abbreviations used in this paper: CD, cytochalasin D; DIC, differential
interference contrast; EcadGFP, E-cadherin fused to green fluorescent
protein; synGFP, synthetic jellyfish green fluorescence protein; TIP, time,
intensity, and position.
The Journal of Cell Biology, Volume 142, 1998
1106
Even less is known about the molecular mechanisms of
compaction and condensation of single cells into multicellular colonies. On the other hand, much work has been
done to elucidate the mechanisms involved in the healing
of wounded monolayers and tissues (Martin and Lewis,
1992; Bement et al., 1993). Small wounds in cell monolayers rapidly form circumferential actin cables at the wound
perimeter, which then slowly cinch together with a pursestring action (Bement et al., 1993; Brock et al., 1996). Similarly, tissue explants with ragged edges will round up in
vitro over extended periods of time, presumably by a similar purse-string action. The mechanisms that mediate
these events could play a role in the genesis of multicellular monolayers, but previous studies have not established
similarities among these processes.
The experimental approaches used in the studies described above have not provided much insight into the dynamic processes by which cadherins, catenins, and the actin cytoskeleton cooperate to initiate, strengthen, and
compact cell–cell contacts between cells initiating adhesion or reorganizing within colonies. In previous studies,
we used differential interference contrast (DIC) timelapse imaging coupled with retrospective immunocytochemistry to examine the distributions of E-cadherin,
catenins, and actin in adhering MDCK cells (McNeill et al.,
1993; Adams et al., 1996). We showed that during the first
hour of cell–cell adhesion, E-cadherin, b-catenin, and
a-catenin coaccumulated into Triton X-100–insoluble aggregates (puncta) that are associated with thin actin bundles
(Adams et al., 1996). However, from these previous studies many problems related to initiation of cell–cell adhesion were unresolved. For example, we were unable to determine the relationship of Triton X-100 insolubility and
clustering of E-cadherin, the source of E-cadherin in
puncta, the role of the actin cytoskeleton in the spatial organization of puncta, the dynamics and fate of puncta
within the contact, or the dynamics of actin and cadherin
reorganization in older contacts. Furthermore, we were unable to investigate the role(s) of these E-cadherin puncta in
strengthening and compacting initial adhesive contacts, or
the mechanisms of reorganization of cell–cell contacts as
cells condensed into colonies to form a multicell monolayer. Answers to these problems are at the core of understanding mechanisms involved in cell–cell adhesion.
In the present study, we examined the dynamics of cell–
cell adhesion using a fully functional protein comprising
E-cadherin fused to green fluorescent protein (EcadGFP).1 EcadGFP was stably expressed in MDCK epithelial cells, and was examined with time-lapse imaging and
photobleach-recovery analysis. We define three sequential
stages in cell–cell adhesion, and show how the distributions of E-cadherin and the actin cytoskeleton are remodeled to coordinate transitions in cell–cell adhesion from
initial contacts, to strengthening and compaction, to the final condensation of cells into colonies. Our results provide
new detailed insights into the dynamics and mechanisms
involved in regulating epithelial cell–cell adhesion in vivo.
binding (z1 mM; Shapiro et al., 1995) may not be sufficient to promote the strong cell–cell adhesion necessary to
maintain tissue integrity. X-ray diffraction studies of crystals of the amino-terminal repeat domains of E-cadherin
and N-cadherin revealed the presence of dimers and
higher ordered complexes (Nagar et al., 1996; Shapiro et al.,
1995). Formation of higher-order complexes between extracellular domains of parallel-oriented cadherins on single cells and clustering of extracellular E-cadherin between cells might cooperatively increase the strength of
adhesion (Brieher et al., 1996; Yap et al., 1997), but little is
known about how, when, or where clustering of cadherins
occurs during cell–cell adhesion in vivo.
The cytoplasmic domain of cadherin is also required for
cell–cell adhesion. The amino acid sequences of the cytoplasmic domain of different cadherins are very similar, and
contain a highly conserved binding site for a family of sequence-related cytosolic proteins: b-catenin, plakoglobin,
and p120CAS (Aberle et al., 1994; Jou et al., 1995; Ozawa
et al., 1989; Reynolds et al., 1994). The cadherin/b-catenin
complex binds via b-catenin to another cytosolic protein,
a-catenin (Herrenknecht et al., 1991; Jou et al., 1995;
Ozawa and Kemler, 1992), which interacts with actin filaments either directly (Rimm et al., 1995) or through other
actin-associated proteins such as a-actinin (Knudsen et al.,
1995). Binding of this protein complex to the actin cytoskeleton is consistent with the appearance of a pool of cadherins and catenins at cell–cell contacts that is resistant to
extraction by the nonionic detergent Triton X-100 (Nagafuchi and Takeichi, 1988; Ozawa et al., 1989).
In individual motile cells, actin filaments are continually polymerizing at the free cell edge of lamellae, and depolymerizing in a transition zone between the cell body
and lamellae. This transition zone is often characterized
by a conspicuous ring of actin that is called a circumferential actin cable (see review, Small et al., 1996). In single
motile epithelial cells, such actin cables often circumscribe the cell in the form of a continuous ring. In polarized epithelial cells, actin filaments are also organized
into a much thinner, much more peripherally disposed circumferential ring at the apical surface in association with
the adherens junction (Hirano et al., 1987); this actin organization is also referred to as a circumferential actin cable. While circumferential actin structures are thus characteristic of both motile and tissue forms of epithelial
cells, a dramatic structural transformation in actin organization occurs during the formation and stabilization of
cell–cell contacts. Recent studies have suggested that circumferential actin cables in single cells either passively
become parallel to cell–cell contacts (Yonemura et al.,
1995) or they actively reorganize to the cell periphery
(Gloushankova et al., 1997). These studies have not provided significant insight into how actin filaments in lamellae and circumferential actin cables reorganize when two
lamellae interact in forming a cell–cell contact. Thus,
while the actin cytoskeleton is known to be involved in
cadherin-mediated cell–cell adhesion, little is known
about how actin participates in the initiation and strengthening of cell–cell adhesion, or how the organization of the
actin cytoskeleton in single motile cells becomes incorporated into the actin organization observed in monolayers
of polarized epithelial cells.
Materials and Methods
Construction of E-cadherin–GFP Recombinant cDNA
and Stable Transfection of MDCK Cells
Expression of EcadGFP in Mouse L-Cells
To express EcadGFP in Ltk-cells stably, the E-cad-GFP cDNA was released from pUGFP2 by Hind3 and Not1 digestion, and was subcloned
into the retroviral vector LZRS-pBMN-Z (Kinsella and Nolan, 1996) after
restriction by Hind3 and Not1. Production of virus and infection of Ltk2
cells was performed as previously reported (Kinsella and Nolan, 1996).
Delivery of EcadGFP to the Basal-lateral Membrane of
Polarized MDCK Cells
Confluent monolayers of MDCK cells stably transfected with U-GFP2
were grown on Transwell™ filters (Costar Corp., Cambridge, MA) for 7 d,
and were treated with 5 mM sodium butyrate for 24 h before an experiment. Cells were first incubated with Met/Cys-free medium for 45 min,
and then cells on one 24-mm diameter petri dish were labeled with 250
mCi of [35S]Met/Cys for 1 h. After labeling, plasma membrane domain–
specific biotinylation was performed as previously described (Wollner et al.,
1992). Cells were lysed in Triton X-100 extraction buffer, and proteins in
the lysate were immunoprecipitated with mAb 3G8. Biotinylated protein
from the immunoprecipitates was collected with immobilized avidin
(Pierce, Rockford, IL). Precipitated proteins were resolved by 7.5% SDSPAGE. Radioactive signals were detected by fluorography.
Cell Culture
Stably transfected EcadGFP MDCK cells were maintained at low density
in DMEM (Gibco Laboratories, Grand Island, NY) supplemented with
10% FBS (Summit, Ft. Collins, CO). 12–24 h before experimentation, 5 mM
sodium butyrate in DMEM/FBS was added to 90% confluent cultures of
cells to further induce expression of EcadGFP. Cells were removed with
trypsin and plated at low density in DMEM/FBS on collagen-coated coverslips for 2–5 h. For time-lapse imaging, cells were removed from the incubator, and the media was replaced with imaging buffer (DME without
phenol red, 10% FBS, 50 mM Hepes). The coverslip was placed in a custom-built imaging chamber with 500 ml of imaging buffer. Cytochalasin D
(Sigma Chemical Co.) solution was diluted 1:2,000 from a stock solution in
DMSO to 2 mM in the imaging buffer.
Cell-Cell Adhesion Mediated by EcadGFP
Aggregation. Ltk-cells and Ltk-cells stably transfected with U-GFP1 were
tested for their ability to aggregate in suspension culture. Approximately
0.5 3 106 single cells were plated in DMEM containing 5 mM Ca21 and
10% FCS that had been dialyzed extensively against PBS, or normal
DMEM/FBS (1.8 mM Ca21) on 35-mm tissue culture dishes coated with
2% agarose (Sigma Chemical Co., St. Louis, MO) and left in a tissue culture incubator overnight. The organization of cells in clumps were recorded 18 h after plating using a Zeiss Axiovert microscope equipped with
a 103 phase-contrast objective.
Kinetics of aggregation. Wild-type MDCK type G cells, MDCK cells expressing EcadGFP, and MDCK cells expressing EcadGFP treated for 24 h
with 5 mM sodium butyrate were suspended with trypsin/EGTA treatment, counted, and adjusted to a density of 7 3 106 cells/ml in imaging
buffer (DMEM without phenol red, 10% FBS, 50 mM Hepes). Cells were
allowed to recover for 2 h before aggregation measurements. Cells were
vortexed for 30 s immediately before loading into the cuvette of the aggregometer. Cells were immediately placed in an optical aggregometer
(Model 440; Chrono-log, Corp., Havertown, PA) at 378C. 270 ml of cells
were transferred to a 0.6-mm diameter cuvette, and were kept in suspension by constant stirring at 300 rpm. Spontaneous aggregation was measured by monitoring the increase in OD of the cell suspension on a stripchart recorder for up to 6 h with cell-free buffer as a reference. Maximal
aggregation was determined by the value when changes in the transmitted
light were zero. The half-time was manually determined as the time at
half-maximal aggregation.
Time-lapse Imaging
The Smith Mark IV multisite laser scanning confocal microscope, designed by Stephen Smith and built by Stanford’s Physiology Instrument
Shop, has been described elsewhere (Adams et al., 1996). In brief, it is an
optical bench design using argon (488 nm) and helium-neon (633 nm) lasers, a mirror galvanometer–based scanning unit, a GaAs photomultiplier
and silicon photodiode photodetectors, and a PC-based control and data
acquisition unit. It allows collection of multispectral fluorescence and
transmitted light (e.g., Nomarski DIC) images in perfect spatial register as
determined by a single laser scanning geometry. The multisite feature (see
Cooper and Smith, 1992; McNeill et al., 1993; Adams et al., 1996; software
written by Dr. Noam Ziv) provides for the acquisition of time-lapse sequences at multiple specimen areas by frequent automated motions of a
motorized specimen stage combined with use of an autofocusing algorithm. For observing the maturation of cell–cell contacts, time-lapse sequences were collected at 6–12 stage-position sites with consecutive DIC
and fluorescence images every 2–10 min for 2–23 h at 378C. For tracking
the dynamics of new puncta, images were collected at single sites every 0.9 s
to 1 min for 3–100 min.
Image Analysis
HEK 293-EBNA cells (Invitrogen Corp., Carlsbad, CA) were transfected
with the E-cadherin-GFP plasmid, U-GFP2, using lipofectamine (GIBCO
BRL, Gaithersburg, MD). 24 h after transfection, cells in one 35-mm dish
To generate time, intensity, and position graphs (TIP scans), images were
rotated in Adobe Photoshop (Adobe Systems, Inc., Mountain View, CA)
so that the long axis of the contact in the last image of the time series was
parallel to the horizon. The rotated images were imported into MetaMorph software (Universal Imaging Corp. West Chester, PA), and the developing contact area was boxed off by 50–200 adjacent horizontal regions, 2 pixels high by 60–200 pixels wide, depending on the relative
position and length of the contact through the time sequence. MetaMorph
software automatically collected maximum and integrated intensity data
from contact, noncontact, and noncell (background) regions for all time
Adams et al. Dynamics of EcadGFP
1107
Binding of Catenins to EcadGFP in HEK 293 Cells and
MDCK Cells
Recombinant cDNA of a synthetic jellyfish green fluorescence protein
(synGFP; Haas et al., 1996; a gift from Dr. Brian Seed) was used as the
PCR template to amplify the complete synGFP coding region, with Xho1
and Not1 sites added to the 59 and 39 ends, respectively. The PCR product
was subcloned into CDM8FluTag (Chen et al., 1993) through Xho1/Not1
sites that generated the plasmid HA-synGFP. A second PCR reaction using canine E-cadherin cDNA as the template amplified the complete coding region of E-cadherin (without a stop codon) with a Hind3 site added at
the 59 end and a Xho1 site added at the 39 end. The PCR product was subcloned into HA-synGFP through Hind3 and Xho1 sites after restriction of
HA-synGFP with Hind3 and Xho1 to release the influenza virus hemagglutin tag (Chen et al., 1993), and resulted in an in-frame fusion of E-cadherin and synGFP with the Xho1 site as the linker (U-GFP1). Most of the
extracellular domain of E-cadherin–GFP fusion protein cDNA was then
replaced with a Hind3/Bgl2 fragment from the cDNA of E-cadherin
pCEcad1 after restricting U-GFP1 with Hind3/Bgl2 enzymes, to generate
U-GFP2. Accordingly, there is no sequence difference between the coding
regions for E-cadherin–GFP fusion protein in U-GFP1 and U-GFP2, but
most of the extracellular domain encoded by U-GFP2 is from the E-cadherin cDNA instead of the PCR product. The E-cadherin-GFP fusion
cDNA in U-GFP2 was confirmed by restriction mapping and DNA sequencing, and was used for transfection. MDCK IIG cells were transfected with U-GFP2 using Ca21 phosphate (Graeve et al., 1990) with
pSV2neo (Southern et al., 1982) as the selection marker. Eight positive
clones expressing the E-cadherin–GFP fusion cDNA in U-GFP2 (EcadGFP) were isolated using cloning rings, and all clones gave identical patterns of EcadGFP fluorescence and Western blot profiles.
were labeled with 250 mCi of [35S]Met/Cys (Amersham Life Science, Inc.,
Arlington Heights, IL) for 24 h. At the end of the labeling period (48 h after transfection), cells were lysed in Triton X-100 lysis buffer (10 mM TrisHCl, pH 7.5, 120 mM NaCl, 25 mM KCl, 2 mM EDTA, 2 mM EGTA,
0.5% Triton X-100), and proteins were immunoprecipitated with mAb
3G8, which binds to the extracellular domain of E-cadherin. The immunoprecipitate was resolved by 7.5% SDS-PAGE, and the radioactive signals
were detected by fluorography. Identical labeling and immunoprecipitation experiments were performed with MDCK IIG cells stably transfected
with U-GFP2.
points. These data were then imported into Excel (Microsoft, Redmond,
WA) and were corrected for photobleaching during the imaging period
(,10%). Three-dimensional graphs were generated in Excel, and the
pseudocolor intensity scale was created for ease of interpretation by colorblind persons and after photocopying (Livingstone, 1988). Intensity values
from regions not in the contact were set to zero (black).
To determine the extent of clustering of existing vs. accumulation of
new EcadGFP to the developing plaque area, measurements of the maximum fluorescence intensity and the sum of fluorescence intensities of all
pixels in an area of fixed size were monitored. For analysis, time-lapse images were passed through a 3 3 3 convolution filter. A region of z20 mm2
was placed over an area where multiple small puncta were coalescing into
a plaque at the edge of a developing cell–cell contact. The maximum and
integrated intensities were measured using MetaMorph during the timelapse series.
Photobleach Recovery
Figure 1. EcadGFP has properties similar to those of endogenous E-cadherin. (A and B) Catenins were coimmunoprecipitated with EcadGFP from HEK 293 cells transiently transfected
with EcadGFP (A) or stably transfected MDCK cells (B). Cells
were labeled with [35S]Met/Cys for 24 h before immunoprecipitation. (A) Proteins in cadherin immunoprecipitates are compared
among mock-transfected HEK 293 cells (No DNA), HEK 293
cells transfected with canine E-cadherin (Ecad), and HEK 293
cells transfected with EcadGFP, and (B) between untransfected
MDCK cells (No DNA) and MDCK cells stably transfected with
EcadGFP. (C) EcadGFP fluorescence and b-catenin immunofluorescence from HEK 293 cells transiently transfected with EcadGFP. Bar, 30 mm. (D) Preferential delivery of newly synthesized
EcadGFP to the basal-lateral plasma membrane of fully polarized MDCK cells stably transfected with EcadGFP, or of endogenous E-cadherin in untransfected cells (No DNA); A, apical
membrane; B, basal-lateral membrane. (E) Phase contrast images of L-cells and L-cells expressing EcadGFP after 18 h of aggregation in suspension culture in the presence or absence of extracellular Ca21. Bar, 60 mm.
To examine directly the dynamics of E-cadherin in living
cells, we constructed a fusion protein composed of full-
length canine E-cadherin fused at the carboxyl terminus to
GFP (EcadGFP). EcadGFP was expressed in MDCK cells
(Fig. 1, B and D), HEK 293 EBNA cells (Fig. 1, A and C),
and L cells (Fig. 1 E). In all cell types, EcadGFP had an apparent molecular mass of z150 kD (Fig. 1, A and B) consistent with the combined molecular masses of the fused
proteins; a minor protein band of z160 kD was the
likely precursor. Expression of endogenous E-cadherin in
MDCK cells was suppressed to some extent in the presence of EcadGFP (Fig. 1 B); thus, EcadGFP contributes
significantly to cell–cell adhesion in these MDCK cells.
Analysis of EcadGFP-immunoprecipitated protein complexes showed the presence of three additional bands at
z102, 98, and 86 kD (Fig. 1 B), corresponding to the molecular weights of a-, b-, and g-catenin (plakoglobin), respectively. The stoichiometry of the EcadGFP/catenin
complex was similar to that of the endogenous E-cadherin/
catenin complex (see Fig. 1, A and B).
Expression of EcadGFP in HEK 293 EBNA cells that
do not normally express cadherin resulted in the formation of condensed cell colonies in the presence of extracellular Ca21 (Fig. 1 C), but cell–cell attachments were not
1108
2
D = 0.224* r ⁄ t d
where r is the radius of the bleached region. Mobile fraction constants
were calculated according to equation in Axelrod (1976; equation 9) at indicated times after recovery. Measurements of the slower photobleach recovery timecourses were subject to potential errors due to gradual redistribution motions of puncta and plaques during recovery phases. TIP
scans of photobleach recoveries were generated (see Fig. 9) and used to
reject data from any experiment where such errors might have been significant.
Results
EcadGFP Binds Catenins, is Targeted to
Cell–Cell Contact Sites, and has Adhesive Properties
Identical to Those of E-cadherin
Method. Photobleaching experiments were performed on the scanning laser confocal microscope described above, and with EcadGFP cells prepared as for time-lapse experiments. The Smith Mark IV microscope incorporates a digitally controlled acousto-optic shutter (NEOS, Melbourne,
FL). In conjunction with appropriate interface electronics and control
software, this high-speed shutter allows the scanning laser beam to be
turned on or off anywhere within the imaging area on a pixel-by-pixel basis. For the present experiments, the shutter was used for controlled photobleaching of circular areas within the scanning raster pattern. Typically,
circular spots of a 120 pixel radius were generated with a 380 3 240 pixel
scan raster. The actual spot radius at the specimen was varied by adjusting
pixel sizes within the range of 0.01–0.05 mm. Scan geometries and beam
intensities were varied independently as necessary during successive baseline acquisition, photobleaching, and recovery acquisition episodes. Scanning frame rates were varied to suit the needs of individual experiments
between 0.33 and 3 Hz. Photobleaching energies were adjusted by varying
exposure intensities and durations to achieve depths of photobleaching
ranging from 30 to 70%. Calibrations of the exact position and dimensions
of the photobleached area were collected using fixed specimens and aminopropysilane coverslips (Sigma Chemical Co.) coated with fluoresceinisothiocyanate (data not shown). The region of the cell to be photobleached was controlled by adjusting the x, y stage on the microscope
during low-power imaging until the desired target area on the cell was in
the middle of the video monitor.
Analysis. Digital images from the photobleaching experiments were imported into MetaMorph and passed through a 3 3 3 low-pass convolution
filter. Average fluorescence intensity data from the bleached regions, two
non-cell regions, and two non-bleached cell regions were collected for all
frames. Data were imported into Excel and corrected for the minor photobleaching caused by the fluorescence recording process by normalization to a region outside the main photobleach pulse spot. The photobleaching recovery part of the data was then imported into Igor Pro,
and the characteristic diffusion time (td) was calculated using equations
for diffusion into a circular disk (Axelrod et al., 1976) and modified for total recovery (Soumpasis, 1983; equation 16). The diffusion coefficient (D)
was calculated according to the equation
formed in the absence of extracellular Ca21 (data not
shown), demonstrating that the adhesion was mediated by
EcadGFP. In HEK 293 EBNA cells, EcadGFP and b-catenin accumulated at the lateral membrane of cell–cell contacts (Fig. 1 C), similar to the distributions of endogenous
E-cadherin and b-catenin in MDCK cells (Näthke et al.,
1994). In addition, newly synthesized EcadGFP was directly targeted to the basal-lateral membrane of polarized
MDCK cells, similar to endogenous E-cadherin (Fig. 1 D).
Also, expression of EcadGFP in mouse L-cells, which do
not normally express cadherin, resulted in the formation
of large cell aggregates in suspension culture in the presence of extracellular Ca21, but not in the absence of extracellular Ca21 (Fig. 1 E). Finally, the kinetics of aggregation
in suspension were identical for wild-type MDCK cells (t1/2 5
15.362.1 min; n 5 2) and MDCK cells expressing EcadGFP (t1/2 5 15.963.0 min; n 5 2), and faster for cells overexpressing EcadGFP. The properties of EcadGFP are
indistinguishable from those of endogenous E-cadherin.
Thus, EcadGFP can substitute for endogenous E-cadherin
in cell–cell adhesion.
EcadGFP Distribution Changes During
Cell–Cell Adhesion
1109
Figure 2. Distribution of EcadGFP during monolayer formation.
A single confocal image was collected from EcadGFP expressing
cells every 10 min for 12 h at 0.12 mm/pixel at 12 sites. Five representative images from each time-lapse are shown. Elapsed time is
indicated on top of each column in h (0, 2, 4, 6, and 8, respectively). The circles in A highlight the edges of a cell–cell contact
that have developed large aggregates of EcadGFP plaques. The
arrows in B–F, columns 0 or 2 h point to the well-separated
plaques at the edges of developing cell–cell contacts that reorganize into a multicellular vertex by 8 h. Note that the 0-h designation is arbitrarily set as the first time point shown. Bar, 15 mm.
EcadGFP expressing MDCK cells were imaged for 10 h to
observe the dynamics of the localization of EcadGFP during
formation of contacts between single cells, and during formation of small multicell colonies (Fig. 2). Expression of
EcadGFP in single cells was relatively uniform over the
plasma membrane with some increased intensity in a circumferential ring at the cell periphery (Fig. 2, A and B, 0 h). During the formation of cell–cell contacts between two (Fig. 2 A)
or three (Fig. 2 B) cells, or single cells and larger cell clusters (Fig. 2 C), EcadGFP fluorescence became significantly
more intense at the cell–cell contact during the first 2 h. After at least 2 h, the largest and brightest regions of EcadGFP
fluorescence were at the edges of cell–cell contacts; we call
these structures plaques (Fig. 2, circles). The fluorescence
intensity of EcadGFP plaques was 6–10 times greater than
that of EcadGFP in noncontacting membranes, and 2–4
times greater than that of EcadGFP in areas of the membrane in the middle of the contact (Fig. 2, A–C; 8 h).
When three or more cells developed cell–cell contacts
(Fig. 2, B–D), EcadGFP plaques from two noncontacting
cells often moved towards each other and eventually coalesced to form a vertex of E-cadherin between multiple
cells (Fig. 2, B–D; compare 2 and 8 h). After forming such
a multicellular vertex, EcadGFP reorganized into the center of the colony (Fig. 2 E). As more cells formed contacts,
this sequential formation of contacts (puncta and plaques)
and coalescence of nonadjacent EcadGFP plaques into
vertices caused cells to become engulfed into the developing cell monolayer. These steps resulted in the formation
of a circumferential ring of both EcadGFP (Fig. 2 F) and
actin (data not shown) around each cell, similar to the organization of E-cadherin and actin between cells in monolayers of polarized MDCK cells (Näthke et al., 1994).
These results demonstrate that we are able to observe with
EcadGFP the complete transition of initial contacts between cells through compaction to the establishment of
E-cadherin/actin organizations characteristic of a complete epithelium. In general, transitions between initial
cell–cell contact (formation of puncta) to E-cadherin
plaque formation, to condensation of plaques into multicell vertices were on the time scale of 2–3 h.
To better understand the evolution of these distinct patterns of E-cadherin, the distribution of EcadGFP during
development of cell–cell contacts was examined in multisite time-lapse confocal images taken over the course of 3 h
(we initially focused on the formation of puncta and
plaques during the first two stages of adhesion see below).
The cells were then fixed and stained with phalloidin,
(which labeled F-actin) and mAb 3G8 (which recognized
the extracellular domain of endogenous E-cadherin and
EcadGFP), and were imaged. Fig. 3 shows representative
contacts from one time-lapse recording. Column 1 of Fig. 3
A shows the formation of a contact between two cells over
71 min. During cell–cell adhesion, EcadGFP fluorescence
appeared at cell–cell contacts, and then increased in intensity with time and as the contact lengthened. The distribution of EcadGFP and endogenous E-cadherin were re-
EcadGFP Clusters into Puncta Upon Cell–Cell Contact
Our observation that EcadGFP initially aggregated into
puncta during formation of cell–cell contacts is similar to
our previous observations in which E-cadherin distributions were determined retrospectively by immunolabeling
cells after time-lapse DIC imaging (Adams et al., 1996).
However, in that previous study we could not determine
the relationship of Triton X-100 insolubility and clustering
of E-cadherin, the source of E-cadherin in puncta, the role
of the actin cytoskeleton in the spatial organization of
puncta, or the dynamics and fate of puncta within the contact. Using EcadGFP, we were able to address these critical problems directly.
The reorganization of EcadGFP during cell–cell adhesion was examined quantitatively by measuring EcadGFP
fluorescence intensity over time after initial contact between cells. The graphs in Fig. 3 D show quantitative representations of the maximum E-cadherin fluorescence intensity vs. position in the contacts shown in Fig. 3 C. The
younger contact shows multiple small peaks of fluorescence along the contact, each corresponding to a punctum
(Fig. 3, column 1D). In contrast, the older contact shows
two pronounced peaks of fluorescence intensity at the
edge of the contact, each corresponding to an EcadGFP
plaque (Fig. 3, column 2D). In addition, there were multi-
Figure 3. Redistribution of EcadGFP during cell–cell contact occurs in two stages and correlates with reorganization of the actin
cytoskeleton. Three 0.3-mm z-sections were collected from EcadGFP-expressing cells every 4.2 min at 0.5 micron/pixel at 6 sites.
(A) Combined stacks from two sites are shown (Contact 1 and
Contact 2). The age of each contact is displayed in min; the zero
time point defines when a stable cell–cell contact had formed.
Bar, 12 mm. The arrow points to a circumferential pattern of EcadGFP observed in single cells. (B and C) Immunofluorescence of
the same cells stained, after formaldehyde fixation, with
rhodamine phalloidin (actin; B) and mAb 3G8/CY5 (E-cadherin;
C). (D) EcadGFP organization shown in C is plotted as a function of fluorescence intensity (y axis, arbitrary fluorescence units)
vs. position in the contact (x axis, mm). The dashed gray lines run-
ning between C and D approximately register the edges of the
contact in the image and graph. (E and F) Triton X-100 extracted
wild-type MDCK cells (i.e., without EcadGFP) that had formed a
contact for ,1 h (Contact 1) or .2 h (Contact 2) stained with
FITC-phalloidin and mAb 3G8/CY5, respectively. Bar, 10 mm.
1110
corded retrospectively with mAb 3G8 (Fig. 3, 1C),
showing that both proteins had coincident distributions as
expected (see Fig. 1). Retrospective actin staining (Fig. 3,
1B) shows that circumferential actin cables were organized parallel to the cell–cell contact interface at this time.
Fig. 3 A (2) shows similar images of another cell–cell
contact that formed over 2 h. At 38 min (Fig. 3, 2A),
EcadGFP was distributed in puncta evenly spaced along
the length of the forming contact. However, after 122 min
(Fig. 3, 2A) EcadGFP was prominently localized to plaques
at either end of the contact. Retrospective actin staining
shows that the most prominent actin cables were also greatly
rearranged so that they terminated at each bright plaque
of EcadGFP located at the margins of the contact, and
were perpendicular to the contact (Fig. 3, 2B). A much
thinner and more discrete line of F-actin staining (Fig. 3, 1B
and 2B) remained in the orientation parallel to the contact
in association with some remaining EcadGFP puncta.
Fig. 3, E and F shows staining of actin and E-cadherin,
respectively, in wild-type MDCK cells (i.e., not expressing
EcadGFP) at a contact that is ,1 h old (Fig. 3, 1E and 1F),
and at a contact that is .2 h old (Fig. 3, 2E and 2F). The
distributions of actin and endogenous E-cadherin in cell–
cell contacts are very similar to those of actin and EcadGFP shown in the contacts in Fig. 3, B and C, respectively,
supporting the general conclusion that EcadGFP is fully
functional. Furthermore, during cell–cell adhesion, changes
in the organization/distribution of EcadGFP are very similar, if not identical, to those of endogenous E-cadherin.
ple smaller peaks between the plaques that represented
residual puncta within the cell–cell contact.
To gain information about the genesis, lifetime, and position of EcadGFP during initiation of contact formation, a
single field of EcadGFP-expressing cells was imaged rapidly at high resolution. Fig. 4 A shows representative images from one of these time-lapse recordings. An arrow
follows the position of a bright EcadGFP fluorescent
punctum at the cell–cell interface. To provide an objective
nonbiased format for the quantitative representation of
dynamic data like that illustrated in Fig. 4 A, we developed
the type of representation shown in Fig. 4 B. The fluorescence intensity profiles along the length of the contact
(e.g., Fig. 3 D) were color-coded and combined for each
time-lapse frame to provide a color map of EcadGFP intensity distribution along the length of the contact as
the contact lengthened. We term such graphs TIP scans.
By providing a clear representation of time-dependent
changes in EcadGFP fluorescence along the cell–cell contact interface, TIP scans make it relatively easy to discern
the organization of EcadGFP during contact formation.
Background fluorescence in the TIP scan is contributed by
overlapping regions of plasma membrane. Areas of the
contact that are brighter than the background cell fluorescence correspond to brighter clusters of EcadGFP. The
TIP scan in Fig. 4 B shows that the contact in Fig. 4 A grew
to a length of z12 mm in z20 min. The contact then grew
more slowly to reach a length of z35 mm after 90 min.
EcadGFP puncta could be identified in the TIP scan as
areas that displayed twice the fluorescence intensity of
background. EcadGFP puncta at first appeared close to
the initial site of the cell–cell contact, while later they appeared at the margins of the contact (Fig. 4 B). We never
observed any hint of the insertion of preassembled E-cadherin puncta from the cytoplasm into the membrane at
cell–cell contacts, suggesting that E-cadherin puncta originate by de novo aggregation at sites of cell–cell contact,
and not from translocation of preassembled aggregates
from some other cellular site(s). An arrow in the TIP scan
in Fig. 4 B also marks the punctum tracked by an arrow in
Fig. 4 A. The punctum appeared de novo at the contact
site and gradually gained intensity over 10 min. By 10–15
min after formation, the intensity level of the punctum remained constant. Such gradual punctum formation was
observed in essentially all cases analyzed (see other examples in Fig. 4 B), which supports the idea that puncta form
in situ from aggregation of molecular subunits.
As the contact lengthened, new EcadGFP puncta appeared sequentially such that the number of puncta remained constant with respect to the length of the contact
1111
Figure 4. EcadGFP puncta are formed and
stabilized along newly formed cell–cell contacts. (A) 16 time-lapse images of EcadGFP
cells taken from a sequence recorded every 1
min for 100 min at 0.11 mm/pixel; time in min
after formation of the contact is shown. Arrows follow a single punctum. (B) TIP scan of
all of the time-lapse images from the experiment represented in A. The contact was divided into 129 0.22-mm sections, and the maximum fluorescence intensity at 100 time
points was collected for a total of 12,900 data
points. The contact originates at 0 min and 0
mm. Small fluctuations in the apparent intensity of stable puncta are near the limits of instrumental noise sources such as laser output
fluctuations and noise processes in the photomultiplier tube detector. (C) Double immunofluorescence of the same contact extracted
with Triton X-100, fixed with formaldehyde,
and stained with rhodamine phalloidin and
E-cadherin mAb 3G8/CY5. The arrows in all
panels point to the same punctum. Bars: (A)
10 mm; (C) 5 mm. (B) 0–210 gray scale fluorescence intensity units divided into 15 colors.
puncta organize and become stabilized around actin filaments located close to the contacting membranes.
Formation of EcadGFP Plaques at the Contact Margins
and Reorganization of the Actin Cytoskeleton
Next, we asked how early E-cadherin puncta are reorganized with actin to further strengthen cell–cell adhesion,
and then to cause condensation of cells into multicell colonies. Over longer times (.2 h), EcadGFP and endogenous
E-cadherin became organized into large plaques at the
margins of the contact (Fig. 2 and Fig. 3, column 2). Fig. 5
A shows representative images from a longer time-lapse
experiment. After z1.5 h, two regions of increasing EcadGFP fluorescence appeared and migrated out with the
edges of the contact at velocities of up to 0.5 mm/min (Fig.
5). These regions gradually gained up to 103 the average
punctum fluorescence intensity over the course of 1 h, in
contrast to a punctum that reached maximum fluorescence
intensity within 30 min of formation. Approximately 2.5 h
after contact nucleation, EcadGFP was heavily concentrated in discrete fluorescent plaques at the margins of the
Figure 5. Two large plaques of EcadGFP
form and move to the edges of the cell–cell
contact. (A) 16 time-lapse images of EcadGFP cells taken from a sequence recorded every 2 min for 2.7 h at 0.23 mm/pixel. Arrows
follow a single plaque. (B) TIP scan of all of
the time-lapse images from the experiment
represented in A. The contact was divided
into 101, 0.46-mm sections, and the fluorescence intensity at 85 time points was collected
for a total of 8,585 data points. The contact
originates at 0 min and 0 mm. Note that the
TIP scan at this reduced resolution shows a
relatively homogeneous distribution of EcadGFP within the contact during the first hour,
whereas the TIP scan at a higher resolution
revealed individual punctum (see Fig. 4). (C)
Double immunofluorescence of the same
contact stained with rhodamine phalloidin
and E-cadherin mAb 3G8/CY5. The arrows
in all panels point to the same plaque. (B) 0–
151 gray scale fluorescence intensity units divided into 15 colors. Bars: (A) 10 mm and (C)
5 mm.
1112
(z1 punctum per 1.5 mm contact length; see also Adams
et al., 1996). These new puncta also appeared de novo and
gradually increased in intensity with time (Fig. 4 B). Many
of these puncta were spatially stable in the contact interface over time, while some gradually changed position towards the edges of the contact. In general, the fluorescence intensity of puncta was brightest in the oldest part of
the contact near the site of initial cell–cell contact (marked
as 0 mm), and dimmest at the perimeter of the lengthening.
The EcadGFP puncta evident in these time-lapse sequences were Triton X-100–insoluble. Each EcadGFP
punctum observed in the final frames of the time-lapse sequences colocalized with the brightest Triton X-100–insoluble E-cadherin puncta (compare similarly oriented images in Fig. 4 A, 909; and Fig. 4 C). EcadGFP puncta were
associated with thin cables of actin filaments that emerged
from circumferential actin cables oriented parallel to the
contact (Fig. 4 C). In summary, while we have confirmed
our early observation that cell adhesion initiates the formation of E-cadherin puncta, the data presented here
demonstrate that a diffuse pool of E-cadherin clusters into
puncta in response to cell–cell contact, and that those
(i.e., the density of EcadGFP) and average fluorescence
intensity values (i.e., the total amount of EcadGFP) in
large (.10 mm diameter ) regions surrounding the edge of
the developing contact (Fig. 6 B). It is clear that the peak
density (Fig. 6 B, black diamonds), but not the total
amount (Fig. 6 B, gray circles) of EcadGFP increased in
the region of the membrane containing the forming
plaque. Thus, plaques most likely to arise by lateral clustering of a subset of EcadGFP puncta already formed
along the cell–cell contact, and are perhaps supplemented
by recruitment of additional EcadGFP molecules in the
area of plaque formation.
Circumferential Actin Cables Reorganize During
Maturation of Cell–Cell Contacts
To obtain information about the role of actin during the
formation and stabilization of cell–cell contacts, cells were
treated with the actin-capping agent cytochalasin D (CD).
After multisite time-lapse recording for 1 h, EcadGFP
cells were treated with 2 mM CD. New cell–cell contacts
did not form in the presence of CD (data not shown). Furthermore, young cell–cell contacts (Fig. 7, A and B) disassembled upon addition of CD (Fig. 7, C and D). Analogous to an intact monolayer of cells (Hirano et al., 1987),
cell–cell contacts within small colonies that were .1 h old
did not disassemble during CD treatment. Cells that were
in contact for ,1 h (Fig. 7 A, upper right and lower left)
rounded after CD treatment (compare Fig. 7, A and D).
Greater than 70% of 19 cell–cell contacts that were ,1 h
old disassembled after treatment with CD (Fig. 7 G). In
contrast, .15% of 48 contacts that were .1 h old disassembled (Fig. 7 G). During CD treatment, EcadGFP
formed aggregates that coincided with irregularities in the
DIC images (Fig. 7 D, arrow). These aggregates colocalized with actin and b-catenin (Fig. 7, E and F) and areas
that have been shown to be enriched in the barbed ends of
actin filaments (Verkhousky et al., 1997), suggesting that
the cadherin/catenin complex may associate with the
barbed ends of actin filaments. Furthermore, these data
indicate that capping the barbed ends of actin filaments
with CD disrupts the ability of E-cadherin puncta, but not
plaques to maintain the integrity of cell–cell contacts.
Direct Measurement of EcadGFP Mobility
by Photobleach Recovery at Different Stages of
Cell–Cell Adhesion
Figure 6. EcadGFP puncta cluster into plaques during transition
between early and late stages of adhesion. (A) Representative
images of a time-lapse sequence taken at 0.8 Hz for 300 s at 0.12
mm/pixel in a region of the cell–cell contact in which a plaque is
developing. Time is in min; arrows point to individual puncta;
bar, 2 mm. (B) Quantitative fluorescence intensities of EcadGFP.
The average (gray circles) and maximum (black diamonds) intensities in a 20-mm2 region surrounding a developing plaque area
are plotted over a 40-min period. The average fluorescence intensity of the same number of EcadGFP in a fixed area is constant
regardless of its distribution. However, the maximum fluorescence intensity increases as EcadGFP clusters into a smaller
region within that fixed area. For details, see Materials and
Methods.
To gain further insight into the assembly dynamics of E-cadherin puncta and plaques, we developed a photobleachingrecovery method to measure the diffusion coefficient,
mobile fraction, and redistribution of EcadGFP during different stages of contact development (see Materials and
Methods). Photobleaching of cell–cell contacts neither disrupts the organization of adhering plasma membranes, induces retraction of membranes, nor changes membrane
movements or dynamics (Fig. 8 A). Fig. 8 A shows the DIC
and EcadGFP images for a live cell before and after photobleaching. After photobleaching, the cells were immediately fixed and stained for E-cadherin and actin. The fluorescence images show that the actin cytoskeleton remained
intact, and that the fine spatial organization of E-cadherin
at the cell–cell contact was the same as that before pho-
1113
contact (Fig. 5, A and B, arrows). The region of the contact between the two plaques retained a thin line of
EcadGFP intensity (compare to Fig. 3, column 2B). Comparison of the last live EcadGFP images with the retrospective immunofluorescence of E-cadherin and actin
(Fig. 5 C) shows that EcadGFP plaques were resistant to
extraction with Triton X-100, and were sites at which circumferential actin cables terminated.
The gradual increase in the amount of EcadGFP in
these plaques might be the result of de novo clustering of
EcadGFP around new actin filaments exposed at the margins of the cell–cell contact, or from the aggregation and
migration of puncta that had preformed along the length
of the contact. To distinguish between these two possibilities, time-lapse images of EcadGFP plaques were recorded rapidly for 300 s at high resolution. Fig. 6 A shows
a representative montage of images in which a plaque was
observed forming from an area of membrane that contained many small puncta (Fig. 6 A, arrowheads). The
small puncta clearly merged together over time to form
the larger plaque. The fluorescence intensity of the plaque
increased concomitantly with the disappearance of individual puncta. We quantified changes in fluorescence intensity and plotted maximum fluorescence intensity values
ments (black bars). The number of those contacts that disassembled within 1 h after CD treatment was determined (striped bars).
The percentage of cell–cell contacts disassembled by CD treatment is 14% for old contacts and 73% for new contacts.
1114
Figure 7. Cytochalasin D selectively disassembles new cell–cell
contacts. Representative images of a time-lapse sequence taken
at 1 frame/2 min for 2 h at 0.4 mm/pixel before and after adding
2 mM CD. (A) 14 min before CD; (B) 1 min before CD; (C) 30
min after CD; (D) 60 min after CD. Immunofluorescence of the
same area is shown using rhodamine phalloidin (E) or b-catenin/
CY5 (F). The arrows in D–F point to CD-induced EcadGFP clusters; bar, 10 mm. (G) The number of cells that were in contact before the time-lapse experiment began (.60 min old), and those
that made contact during the imaging experiment (,60 min old)
were counted and the totals shown for three independent experi-
tobleaching. We also examined whether photobleaching of
EcadGFP was reversible. Fig. 8 B shows the effects on
EcadGFP after 1/2 of a cell was photobleached. The EcadGFP fluorescence was monitored in the photobleached
half of the cell (Fig. 8 B, blue), the nonphotobleached half
of the cell (Fig. 8 B, red), and the entire cell (Fig. 8 B,
green). These data show that the photobleached part of
the cell recovered EcadGFP fluorescence while, at the
same rate, the nonphotobleached part of the cell lost
EcadGFP fluorescence. The average intensity of EcadGFP fluorescence remained constant throughout the entire cell, reflecting the fact that the EcadGFP fluorescence
was irreversibly photobleached. These results also show
that the entire pool of EcadGFP in the cell was mobile and
exchanged within 45 min.
The EcadGFP diffusion coefficient in our system was
measured in the thin (1 mm) membrane lamellae between
the edge of the cell and circumferential actin cable. To validate our photobleaching methodology, we performed experiments to assess the dependence of fluorescence recovery on the diameter of the photobleached area (Fig. 8 C).
Table I summarizes data from photobleaching EcadGFP
in membrane lamella not involved in cell–cell adhesion using different-sized photobleach areas. These experiments
show that EcadGFP diffusion time was related to the
square of the photobleach radius as expected from simple
diffusion theory; the average value of the diffusion coefficient D was calculated to be 3.6 6 1.5 3 10210 cm2/s. This
value is similar to that measured for another transmembrane protein, Na1, K1-ATPase, in low-density MDCK
cells (Jesaitis and Yguerabide, 1986). We note that previous photobleach-recovery measurements of E-cadherin in
the apical membrane of polarized F7p cells yielded a
somewhat lower D value of 3.4 3 10211 cm2/s (Kusumi et al.,
1993), and single particle-tracking measurements of E-cadherin in L-cells yielded a D value of 5.2 3 10211 cm2/s
(Sako et al., 1998). The difference between these observed
diffusion coefficient values of E-cadherin is probably due
to differences in methodologies, cell types, and cytoskeletal states.
Our interest here is to compare E-cadherin mobility in
different membrane regions during cell–cell adhesion using identical methodologies on the same cell type under
carefully controlled conditions. Fig. 8 D shows representative EcadGFP images immediately before photobleaching,
immediately after photobleaching, and 10 min after recovery in four regions: (a) membranes not involved in cell–
cell contact; (b) new cell–cell contacts; (c) puncta; and (d)
plaques. Note that to measure EcadGFP mobility at cell–
cell contacts, we examined the kinetics of recovery of
EcadGFP fluorescence after photobleaching in thin lamellae between contacting cells; at those sites, the height of
the contact was minimal, EcadGFP fluorescence could be
photobleached through the contact, and the subsequent
recovery of EcadGFP fluorescence could be tracked with
high resolution. The recovery curves in Fig. 8 (C and E)
show that EcadGFP in either a contact-free area of the
membrane (Fig. 8 C) or a newly formed contact (Fig. 8 E,
blue) recovered .90% of fluorescence 15 min after photobleaching. In contrast, EcadGFP in either a punctum or
a plaque recovered 50% and ,10% fluorescence, respectively. These data indicate that an initially highly mobile
pool of EcadGFP becomes increasingly immobilized
within developing puncta and plaques.
Fig. 9 shows a TIP scan from an experiment where a 2.8mm–diameter area was bleached in a 1-h-old contact containing two very bright EcadGFP puncta (Fig. 9, arrow). It
is clear that bleached puncta partially recovered their fluorescence, concomitant with a partial loss of fluorescence in
nonbleached puncta. However, it is also obvious that individual puncta undergo gradual redistributions during
these slow recoveries. As the EcadGFP fluorescence recovered, the bleached puncta migrated out of the bleached
area, and puncta adjacent to the original bleached area
slowly migrated into the bleached area (Fig. 9).
Detailed TIP scan analyses of photobleach recovery
data allowed us to make the following generalizations
about the dynamics of EcadGFP: (a) puncta move as individual units within the cell–cell contact interface during
contact expansion; (b) puncta on the edge of a fluorescence bleach area recovered their fluorescence first; and
(c) adjacent nonbleached puncta sometimes exhibited a
decrease in fluorescence intensity. These data indicate that
the immobilized fraction of EcadGFP is associated with
the cytoskeleton, and that cytoskeletal associated EcadGFP moves and exchanges within the cell–cell contact interface.
1115
Figure 8. Photobleach-recovery analysis shows a highly
mobile pool of EcadGFP coalesces into immobile puncta.
A shows a live cell before
and after photobleaching.
The box indicates where the
cell was photobleached. The
arrow points to an area that
formed a contact during the
photobleach. The cells were
fixed in formaldehyde and
stained with phalloidin and
mAb 3G8. B shows the fluorescence recovery curves of a
single noncontacting cell in
which half of the cell was
photobleached (blue). EcadGFP fluorescence of the nonbleached region (red) and
the entire cell (green) was
monitored during recovery.
Notice that the EcadGFP fluorescence values equalize in
the photobleached and nonphotobleached areas. C shows
the first 3 min of photobleach-recovery data of noncontacting membrane regions photobleached with a
5.8-mm (pink) and 3-mm
(black) circle. The relative
fluorescence is scaled between the fluorescence intensity just after bleaching and
equilibrium. The lines show
the theoretical recovery
curves for each region with a
diffusion coefficient of 3 3
10210 cm2/s. Note that the
smaller photobleach circle (black line) recovers more quickly. D shows images taken before, 0.1 min after, and 10 min after photobleaching a 5.8-mm-diameter circle in a region of membrane not involved in cell–cell contact (Membrane), a region of membrane in a
,15-min-old contact (New contact), a region of membrane in the middle of a ,60-min-old contact (Puncta), and a membrane at the
edge of a .2-h-old contact (Plaque). For each experiment, 300 images were collected every 3.2 s at 0.11 mm/pixel. The circles mark the
photobleach region, and the colors correspond to the recovery curves shown in C (pink) and E. E shows photobleach-recovery data for
the bleached contact regions identified in D. The relative fluorescence is scaled to the pre-bleach intensity value. The mobile fraction
of EcadGFP in the new contact (blue), puncta (green), and plaque (orange) is 100, 50, and ,10%, respectively.
Table I. E-cadherin/GFP Diffusion Coefficient Measurements
Radius
D 3 10210
mm
cm2/s
4.1
2.4
1.7
all data
4.14
3.6
3.3
3.6
SD
n
1.4
1.4
1.7
1.5
7
10
11
26
Data are summarized from photobleaching EcadGFP in membrane lamella not involved in cell–cell adhesion using different-sized photobleach areas. Typically, circular spots of a 120-pixel radius were generated with a 380 3 240 pixel scan raster, and
the spot radius (1.7–4.1 mm) at the specimen was varied by adjusting pixel sizes
within the range of 0.01–0.05 mm. The photobleaching recovery part of the data was
inported into Igor Pro, and the diffusion coefficient (D) was calculated according to
the equation D 5 0.224* r2/to, where r is the radius of the bleached region. These experiments show that EcadGFP diffusion time was related to the square of the photobleach radius as expected from simple diffusion theory.
Discussion
In the first stage of adhesion, E-cadherin spontaneously
clusters into puncta at initial sites of developing cell–cell
contacts. The formation of E-cadherin puncta results in
decreased E-cadherin mobility. In new areas of cell–cell
contact (,15 min old), EcadGFP has a high mobile fraction (.90%) and a high diffusion coefficient (3.6 6 1.5 3
10210 cm2/s). However, where EcadGFP clusters into
1116
Figure 9. Mobility of E-cadherin puncta within the cell–cell contact interface. Fig. 9 shows a TIP scan of an entire contact during
a photobleach-recovery experiment. A newly developing plaque
in a 1.5-h-old contact was photobleached with a 2.8-mm-diameter
bleach circle (0 mins, 0 mm) on the TIP scan (arrow). Images
were collected every 16 s for 24 min at 0.11 mm/pixel. The fluorescence intensity scale bar ranges from 0–255 units divided into 15
colors.
process of cell–cell adhesion is normal even when levels of
endogenous E-cadherin are reduced in the presence of
EcadGFP (Fig. 1).
We present evidence for three sequential stages of cell–
cell adhesion that involve specific changes in E-cadherin
and actin cytoskeleton organization. These stages are: (I)
clustering of mobile E-cadherin into immobile puncta
along the length of the forming contact; (II) reorganization of E-cadherin puncta into plaques at the edges of the
contact; and (III) coalescence of E-cadherin plaques to the
vertices of contacts among three or more cells.
Stage I
E-cadherin plays important roles in cell–cell recognition
and adhesion. However, the dynamics of E-cadherin redistribution during the processes of initial cell–cell contact
through development of a polarized monolayer are unknown. It is thought that clustering of E-cadherin (Yap et al.,
1998) via extracellular homotypic binding (Nose et al.,
1988) is sufficient for initial cell–cell interactions: the role
of cadherin clusters during the development of older cell
colonies is less understood. Furthermore, the dynamic interactions of E-cadherin with intracellular proteins, especially the cytoskeleton, has not been described. By quantitatively analyzing EcadGFP redistribution and mobility in
epithelial cells during adhesion development, we provide a
new dynamic view of how E-cadherin and the actin cytoskeleton establish strong cell–cell adhesion.
GFP was attached to the COOH terminus of the E-cadherin cytoplasmic domain. The cytoplasmic domain of
cadherin binds to catenins, which are required for binding
the cadherin/catenin complex to the actin cytoskeleton.
Therefore, it was necessary to show that EcadGFP protein
has functions identical to those of endogenous E-cadherin.
We showed that EcadGFP is fully functional by the following criteria: EcadGFP formed a 1:1:1 stoichiometric
complex with a- and b-catenin; EcadGFP precisely colocalized with catenins at cell–cell contacts; EcadGFP was
targeted directly from the Golgi to the basal-lateral membrane in polarized MDCK cells; EcadGFP localized to
cell–cell contacts and entered a Triton X-100–insoluble
pool of proteins only at cell–cell contacts; and EcadGFP
induced Ca21-dependent cell–cell adhesion and condensation in transfected MDCK cells and nonadherent cells (fibroblasts) with kinetics that were qualitatively and quantitatively similar to those of endogenous E-cadherin (Fig.
1). In addition, the kinetics of assembly of EcadGFP
puncta in live cells was similar to that measured for the assembly of Triton X-100–insoluble E-cadherin puncta by
retrospective immunocytochemistry (Adams et al., 1996),
and the formation of cell–cell contacts between MDCK
cells expressing EcadGFP and wild-type MDCK cells appeared very similar (compare to Adams et al., 1996).
Therefore, the fact that EcadGFP has functions identical
to those of endogenous E-cadherin implies that EcadGFP
can substitute for endogenous E-cadherin, and that the
gins of the contacts at velocities of up to 0.5 mm/min (Fig.
5), which is similar to the velocity of translocation of
ConA beads along cell–cell contacts (Gloushankova et al.,
1997).
During the second stage of contact formation, circumferential actin cables rearrange from a parallel to a perpendicular orientation with respect to the cell–cell contact
(Fig. 5). The reorganization of actin appears to be different in MDCK cells and fibroblasts (Yonemura et al.,
1995), which raises the possibility that the strength of the
interactions between E-cadherin and actin might be responsible for specific differences in actin dynamics between these two cell types. A consequence of the reorganization of E-cadherin and the actin cytoskeleton is the
compaction of contacting cells, which is a clear sign of the
establishment of strong cell–cell adhesion and cells maximizing the contacting surfaces between them (Fig. 2). We
showed that these compacted cell–cell contacts are resistant to disassembly by CD, indicating either that these
contacts have become mechanically resistant to depolymerization of actin, or the barbed ends of the circumferential actin cables are firmly embedded within E-cadherin
plaques and are no longer accessible to CD.
The second stage of contact formation is distinguished by
the gradual emergence of much larger E-cadherin clusters
that we designate as plaques (Figs. 2, 5, and 6). Generally,
one plaque is observed to form at either end of the developing contact where the reorganized circumferential actin
cables terminate. Identical plaques were formed by endogenous E-cadherin in compacted MDCK cell–cell contacts
(Fig. 3). Using EcadGFP, we showed that these plaques
arose by lateral clustering of a subset of puncta that were
formed during the first stage of adhesion and the continual
immobilization of a mobile pool of EcadGFP (Fig. 9).
Plaque formation resulted in a further decrease in the mobile fraction of E-cadherin to ,10%. Using TIP scans, we
found that during plaque formation, EcadGFP puncta
traveled within the cell–cell contact interface to the mar-
Figure 10. A three-stage model for cell–cell adhesion and colony
formation. Stage I: multiple E-cadherin puncta form along the
developing contact and loosly hold contacting cells together. A
circumferential actin cable (thick red line) surrounds isolated
cells. As cells adhere, E-cadherin clusters into puncta within the
cell–cell contact interface (blue circle) and rapidly associates with
thin actin bundles and filaments (thin red lines). As the contact
lengthens, puncta continue to develop along the length of the
contact at a constant average density during the first 2 h. Stage II:
E-cadherin plaques develop at the edges of the contact which
compact and strengthen cell–cell interactions. Stabilization of actin filaments by E-cadherin puncta within the cell–cell contact results in gradual dissolution of the circumferential actin cable behind the developing contact and insertion of the circumferential
actin cables into the cell–cell contact accompanied by additional
clustering of E-cadherin puncta into E-cadherin plaques (green
ovals). Between cell plaques, E-cadherin is more diffusely distributed (green line) and associates with actin filaments oriented
along the axis of the cell–cell contact (red line). Stage III: E-cadherin plaques cinch together to form multicellular vertices, further condensing cell colonies. In multicellular colonies, contractility within the circumferential actin cable brings E-cadherin
plaques from adjacent cells together. Dynamics within the cytoskeleton result in continual rearrangement.
1117
Stage II
puncta and associates with the cytoskeleton, a smaller
fraction is mobile (,50% within a 26-mm area). The
puncta formed by EcadGFP are very similar in organization and distribution to structures formed by endogenous
E-cadherin and catenins that were previously characterized by retrospective immunocytochemistry (Adams et al.,
1996; see also Fig. 3).
Weak interactions between extracellular and juxtamembrane domains of cadherins may be sufficient to initiate
clustering of the protein in the membrane (Yap et al.,
1998). However, interactions between E-cadherin and the
actin cytoskeleton are initiated quickly upon cell–cell contact, and these interactions affect the organization of the
adhesion complex. We showed that as E-cadherin puncta
begin to form during this first stage, they always appear to
be associated with the ends of thin actin cables that are
oriented toward the contact (Fig. 4). These actin filaments
branch from circumferential actin cables that are organized parallel to the forming contact and circumscribe the
perimeter of single cells. We speculate that binding actin
filaments to E-cadherin/catenin complexes may cause further clustering and stabilization of puncta. This type of
cadherin/actin organization has been shown to provide a
mechanical linkage between fibroblasts (Ragsdale et al.,
1997). Quantitative measurements showed that this initial
stage of adhesion coincides with an exponential increase in
the strength of adhesion (Angres et al., 1996). Significantly, this strengthening stage was completely inhibited
by treatment of cells with CD (Angres et al., 1996; Fig. 7).
In the present study we showed that during this initial
stage, CD selectively disassembled contacts and caused
formation of aggregates that include cell-surface EcadGFP (this study) and probably the barbed ends of actin filaments (Verkhovsky et al., 1997). It is also interesting to
note that myosin is involved in the CD-induced aggregation of the barbed ends of actin filaments (Verkhovsky et
al., 1997), and that actin treadmilling ceases in areas of developing cell–cell contacts (Gloushankova et al., 1997).
We speculate that E-cadherin puncta gradually sequester
the barbed ends of actin filaments, and directly or indirectly anchor them to the membrane at cell–cell contacts,
resulting in the gradual strengthening of cell–cell adhesion. These changes in actin organization may also set up
cytoarchitectural cues for stage II of adhesion.
Stage III
1118
E-cadherin plaque formation coordinates circumferential
actin cables to cell–cell contact and maximizes the area of
membrane involved in the contact between two cells.
When more than two cells form contacts, E-cadherin
plaques and actin cables continue to reorganize to form a
more compact cell colony (Fig. 2). This process of condensation involves movement of E-cadherin plaques towards
each other until they have coalesced to form a vertex
among three or more cells (Fig. 2, C and D). We suggest
that E-cadherin plaques are cinched together by contraction of the actin cables that coordinate the plaques within
the multicell colony. This reorganization of E-cadherin
changed the shape of cells from rather cubiodal after
plaque formation to cone-shaped after cell condensation
in colonies; the vertices of E-cadherin were located at the
tip of the cone (Fig. 2, B–D). The formation of coneshaped cells is redolent of the effects of purse-string contraction of circumferential actin filaments during wound
healing in vitro (Bement et al., 1993) and in vivo (Martin
and Lewis, 1992; Brock et al., 1996). Previous studies have
shown that contraction of cell monolayers around a wound
coincides with reorganization of actin cables, myosin II,
tropomyosin, and other actin-associated proteins on the
membrane adjacent to the wound. It is assumed that actin–
myosin contraction pulls on membranes at the edge of the
wound to close the opening between cells. We suggest that
circumferential actin contraction initiated during cell–cell
compaction continues until an equilibrium, perhaps equal
tension, is reached between the circumferential actin bundles throughout the forming multicell colony. Studies are
underway to test the roles of actin and actin contractility in
this stage of adhesion and cell reorganization.
A Model
In summary, we suggest a model for how contacts between
cells are initiated, strengthened, compacted, and condensed as cells transform from the migratory phenotype of
a single cell to a sedentary phenotype of one cell in a multicell monolayer. Cell–cell adhesion is initiated by weak
binding between extracellular domains of E-cadherin that
are present in a highly mobile pool at the plasma membrane. At or near the same time, E-cadherin/catenin complexes attach to actin filaments that branch from actin cables that circumscribe the perimeter of migratory cells.
These two processes act synergistically to assemble puncta,
which, as a group, are sufficiently adhesive to hold the nascent cell–cell contact together (Fig. 10, stage I). Subsequently, there is a change in actin dynamics as actin treadmilling ceases in areas of cell–cell contact, perhaps due to
sequestration of the barbed ends of actin filaments into
E-cadherin puncta. We hypothesize that reduced actin
treadmilling causes the dissolution of the circumferential
actin cables immediately adjacent to the developing contact. It is also possible that a signaling event at the cell surface induced by cell–cell adhesion causes a change in the
organization or polymerized state of the circumferential
actin cables adjacent to the contact site. We suggest that
stabilization of actin via the clustered cadherin/catenin
complex engages the myosin II clutch (Suter et al., 1998),
thereby inducing translocation of circumferential actin cables and the rest of the cell body to the cell–cell contact interface and the rapid movement of associated E-cadherin
puncta into large plaques. This coordinated reorganization
of E-cadherin and the actin cytoskeleton results in the establishment of strong compacted cell–cell contacts and the
generation of an actin cable that circumscribes the free
edges of the newly contacting cells and is embedded into
either side of a E-cadherin plaque at the margins of the
contact (Fig. 10, stage II).
The third stage of adhesion (Fig. 10, stage III) is initiated once another cell joins a two-cell colony. Additional
cells join larger cell colonies using the same mechanisms
outlined in stages I and II. When three cells contact, two
cell–cell contacts and two free edges flank a center cell.
Two perimeter actin cables are localized to the free edges
of the center cell, and are further linked at E-cadherin
plaques to the circumferential actin cables from the two
flanking cells. This organization is unstable, and results in
further reorganization of E-cadherin puncta, and the circumferential actin cytoskeleton. This reorganization is initiated by the lateral translocation of the E-cadherin
plaques on one side of the colony towards each other until
they coalesce. This triangular organization of E-cadherin
undergoes a final rearrangement as the cells condense and
maximize contacts between each other (Fig. 10, stage III).
In stage III, we suggest that one of the perimeter actin cables of the center cell dominates, exerts tension on the
E-cadherin plaques, and slowly pulls the plaques from the
outside cells together. The colony continues to reorganize
into a stable configuration of a circle, with each cell connected together on one side sharing a common vertex in
the middle of the colony.
Additional cells systematically join a multicellular colony as described above. E-cadherin and actin in newly
forming cell–cell contacts reorganize, and then cinch together adjacent plaques attached to actin cables at the free
cell edges (Fig. 2, D and E). Eventually some cells will become engulfed by a multicellular colony such that there is
no longer a circumferential actin cable, and the cell is surrounded by contacts on all sides. There still exist larger
clusters of E-cadherin at the vertices of cells within the
multicellular colony, and the cells are probably still able to
exert tension on each other through these regions to maximize the contact area. The organization of actin in the
multicellular colony is now distinctly different from that in
a cell–cell contact that is ,1 h old, and is characterized by
thin actin bundles running parallel to the cell–cell contact
in association with E-cadherin (see Fig. 3). These E-cadherin and actin organizations are also characteristic of the
mature monolayer.
Our findings reveal a very tight coordination among
cadherin binding, aggregation, adhesion events, and dramatic reorganizations of the actin cytoskeleton. These reorganizations occur in parallel with transitions from weak
initial adhesion to strong adhesions associated with cell–
cell compactions, condensations of cells into colonies, and
formation of a belt of cadherin and actin as observed in
mature monolayers. These results provide a new framework for future studies aimed at identifying the effects of
other regulatory molecules (e.g., GTPases, kinases, and
cytoskeletal motors) and cadherin-associated proteins (e.g.,
p120CAS) on these distinct adhesive stages of cell–cell adhesion.
We thank members of the Nelson and Smith labs and Chris Hazuka for
their comments during the course of this work, Dr. Richard Lewis for help
programming diffusion solutions in Igor Pro, Dr. Lee Rubin (University
College London) for the canine E-cadherin cDNA clone, and Dr. Brian
Seed (Harvard Medical School) for the generous gift of humanized codonpreference-adjusted red-shifted GFP before its publication. We also thank
Dr. David Loftus for his help with the aggregation assay.
W.J. Nelson and S.J Smith were supported by grants from the National
Institutes of Health and The Mathers Charitable Foundation. Y.-T. Chen
is a recipient of a postdoctoral fellowship award from National Kidney
Foundation.
Received for publication 13 February 1998 and in revised form 1 June 1998.
References
1119
Aberle, H., S. Butz, J. Stappert, H. Weissig, R. Kemler, and H. Hoschuetzky.
1994. Assembly of the cadherin-catenin complex in vitro with recombinant
proteins. J. Cell Sci. 107:3655–3663.
Aberle, H., H. Schwartz, and R. Kemler. 1996. Cadherin-catenin complex: protein interactions and their implications for cadherin function. J. Cell. Biochem. 61:514–523.
Adams, C.L., W.J. Nelson, and S.J. Smith. 1996. Quantitative analysis of cadherin-catenin-actin reorganization during development of cell-cell adhesion.
J. Cell Biol. 135:1899–1911.
Angres, B., A. Barth, and W.J. Nelson. 1996. Mechanisms for transition from
initial to stable cell-cell adhesion: kinetic analysis of E-cadherin-mediated
adhesion using a quantitative adhesion assay. J. Cell Biol. 134:549–557.
Axelrod, D., D.E. Koppel, J. Schlessinger, E. Elson, and W.W. Webb. 1976.
Mobility measurement by analysis of fluorescence photobleaching recovery
kinetics. Biophys. J. 16:1055–1069.
Bement, W.M., P. Forscher, and M.S. Mooseker. 1993. A novel cytoskeletal
structure involved in purse string wound closure and cell polarity maintenance. J. Cell Biol. 121:565–578.
Brieher, W.M., A.S. Yap, and B.M. Gumbiner. 1996. Lateral dimerization is required for the homophilic binding activity of C-cadherin. J. Cell Biol. 135:
487–496.
Brock, J., K. Midwinter, J. Lewis, and P. Martin. 1996. Healing of incisional
wounds in the embryonic chick wing bud: characterization of the actin pursestring and demonstration of a requirement for Rho activation. J Cell Biol.
135:1097–1107.
Chen, Y.-T., C. Holcomb, and H.-P. Moore. 1993. Expression and localization
of two low molecular weight GTP-binding proteins, Rab8 and Rab10, by
epitope tag. Proc. Natl. Acad. Sci. USA. 90:6508–6512.
Cooper, M.W., and S.J. Smith. 1992. A real-time analysis of growth cone-target
cell interactions during the formation of stable contacts between hippocampal neurons in culture. J. Neurobiol. 23:814–828.
Fannon, A.M., and D.R. Colman. 1996. A model for central synaptic junctional
complex formation based on the differential adhesive specificities of the cadherins. Neuron. 17:423–434.
Gloushankova, N.A., N.A. Alieva, M.F. Krendel, E.M. Bonder, H.H. Feder,
J.M. Vasiliev, and I.M. Gelfand. 1997. Cell-cell contact changes the dynamics of lamellar activity in nontransformed epitheliocytes but not in their rastransformed descendants. Proc. Natl. Acad. Sci. USA. 94:879–883.
Graeve, L., A. Patzak, K. Drickamer, and E. Rodriguez-Boulan. 1990. Polarized expression of functional rat liver asialoglycoprotein receptor in transfected Madin-Darby canine kidney cells. J. Biol. Chem. 265:1216–1224.
Haas, J., E.C. Park, and B. Seed. 1996. Codon usage limitation in the expression
of HIV-1 envelope glycoprotein. Curr. Biol. 6:315–324.
Herrenknecht, K., M. Ozawa, C. Eckerskorn, F. Lottspeich, M. Lenter, and R.
Kemler. 1991. The uvomorulin-anchorage protein a-catenin is a vinculin homologue. Proc. Natl. Acad. Sci. USA. 88:9156–9160.
Hirano, S., A. Nose, K. Hatta, A. Kawakami, and M. Takeichi. 1987. Calciumdependent cell-cell adhesion molecules (cadherins): Subclass specificities
and possible involvement of actin bundles. J. Cell Biol. 105:2501–2510.
Jesaitis, A.J., and J. Yguerabide. 1986. The lateral mobility of the (Na1,K1)dependent ATPase in Madin-Darby canine kidney cells. J. Cell Biol. 102:
1256–1263.
Jou, T.-S., D. Stewart, J. Stappert, W. Nelson, and J. Marrs. 1995. Genetic and
biochemical dissection of protein linkages in the cadherin-catenin complex.
Proc. Natl. Acad. Sci. USA. 92:5067–5071.
Kinsella, T.M., and G.P. Nolan. 1996. Episomal vectors rapidly and stably produce high-titer recombinant retrovirus. Hum. Gene Ther. 7:1405–1413.
Knudsen, K.A., A.P. Soler, K.R. Johnson, and M.J. Wheelock. 1995. Interaction of a-actinin with the cadherin/catenin cell-cell adhesion complex via
a-catenin. J. Cell Biol. 130:67–77.
Kusumi, A., Y. Sako, and M. Yamamoto. 1993. Confined lateral diffusion of
membrane receptors as studied by single particle tracking (nanovid microscopy). Effects of calcium-induced differentiation in cultured epithelial cells.
Biophys. J. 65:2021–2040.
Livingstone, M.S. 1988. Art, illusion and the visual system. Sci. Am. 258:78–85.
Martin, P., and J. Lewis. 1992. Actin cables and epidermal movement in embryonic wound healing. Nature. 360:179–183.
McNeill, H., T.A. Ryan, S.J. Smith, and W.J. Nelson. 1993. Spatial and temporal dissection of immediate and early events following cadherin-mediated
epithelial cell adhesion. J. Cell Biol. 120:1217–1226.
Martinek, S., and U. Gaul. 1997. Neural development: how cadherins zipper up
neural circuits. Curr. Biol. 7:R712–R715.
Nagafuchi, A., and M. Takeichi. 1988. Cell binding function of E-cadherin
is regulated by the cytoplasmic domain. EMBO (Eur. Mol. Biol. Organ.) J.
7:3679–3684.
Nagar, B., M. Overduin, M. Ikura, and J.M. Rini. 1996. Structural basis of calcium-induced E-cadherin rigidification and dimerization. Nature. 380:360–364.
Näthke, I.S., L. Hinck, J.R. Swedlow, J. Papkoff, and W.J. Nelson. 1994. Defining interactions and distributions of cadherin and catenin complexes in polarized epithelial cells. J. Cell Biol. 125:1341–1352.
Nose, A., A. Nagafuchi, and M. Takeichi. 1988. Expressed recombinant cadherins mediate cell sorting in model systems. Cell. 54:993–1001.
Ozawa, M., H. Baribault, and R. Kemler. 1989. The cytoplasmic domain of the
cell adhesion molecule uvomorulin associates with three independent proteins structurally related in different species. EMBO (Eur. Mol. Biol. Organ.) J. 8:1711–1717.
Ozawa, M., and R. Kemler. 1992. Molecular organization of the uvomorulincatenin complex. J. Cell Biol. 116:989–996.
Ragsdale, K.G., J. Phelps, and K. Luby-Phelps. 1997. Viscoelastic response of
fibroblasts to tension transmitted through adherens junctions. Biophys. J. 73:
2798–2808.
Reynolds, A.B., J. Daniel, P.D. McCrea, M.J. Wheelock, J. Wu, and Z. Zhang.
1994. Identification of a new catenin: The tyrosine kinase substrate p120cas
associates with E-cadherin complexes. Mol. Cell. Biol. 14:8333–8342.
Rimm, D.L., E.R. Koslov, P. Kebriaei, C.D. Cianci, and J.S. Morrow. 1995.
a1(E)-catenin is an actin-binding and -bundling protein mediating the attachment of F-actin to the membrane adhesion complex. Proc. Natl. Acad.
Sci. USA. 92:8813–8817.
Sako, Y., A. Nagafuchi, S. Tsukita, M. Takeichi, and A. Kusumi. 1998. Cytoplasmic regulation of the movement of E-cadherin on the free cell surface as
studied by optical tweezers and single particle tracking: corralling and tethering by the membrane skeleton. J. Cell Biol. 140:1227–1240.
Shapiro, L., A.M. Fannon, P.D. Kwong, A. Thompson, M.S. Lehmann, G.
Grubel, J.-F. Legrand, J. Als-Nielsen, D.R. Colman, and W.A. Hendrickson.
1995. Structural basis of cell-cell adhesion by cadherins. Nature. 372:327–337.
Small, V.J., K. Anderson, and K. Rottner. 1996. Actin and the coordination of
protrusion, attachment and retraction in cell crawling. Biosci. Rep. 16:351–368.
Soumpasis, D.M. 1983. Theoretical analysis of fluorescence photobleaching recovery experiments. Biophys. J. 41:95–97.
Southern, P.J., and P. Berg. 1982. Transformation of mammalian cells to antibiotic resistance with a bacterial gene under control of the SV40 early region
promoter. J. Mol. Appl. Genet. 1:327–341.
Suter, D.M., L.D. Errante, V. Belotserkovsky, and P. Forscher. 1998. The Ig superfamily cell adhesion molecule, apCAM, mediates growth cone steering
by substrate-cytoskeletal coupling. J. Cell Biol. 141:227–240.
Steinberg, M.S., and M. Takeichi. 1994. Experimental specification of cell sorting, tissue spreading, and specific spatial patterning by quantitative differences in cadherin expression. Proc. Natl. Acad. Sci. USA. 91:206–209.
Steinberg, M.S. 1996. Adhesion in development: an historical overview. Dev.
Biol. 180:377–388.
Takeichi, M. 1991. Cadherin cell adhesion receptors as a morphogenetic regulator. Science. 251:1451–1455.
Takeichi, M. 1987. Cadherins: a molecular family essential for selective cell-cell
adhesion and animal morphogenesis. Trends Genet. 3:213–217.
Townes, P.L., and J. Holtfreter. 1955. Directed movement and selective adhesion of embryonic amphibian cells. J. Exp. Zool. 128:53–120.
Uchida, N., Y. Honjo, K.R. Johnson, M.J. Wheelock, and M. Takeichi. 1996.
The catenin/cadherin adhesion system is localized in synaptic junctions bordering transmitter release zones. J. Cell Biol. 135:767–779.
Verkhovsky, A.B., T.M. Svitkina, and G.G. Borisy. 1997. Polarity sorting of actin filaments in cytochalasin-treated fibroblasts. J. Cell Sci. 110:1693–1704.
Wollner, D.A., K.A. Krzeminski, and W.J. Nelson. 1992. Remodeling the cell
surface distribution of membrane proteins during the development of epithelial cell polarity. J. Cell Biol. 116:889–899.
Yap, A.S., W.M. Brieher, M. Pruschy, and B.M. Gumbiner. 1997. Lateral clustering of the adhesive ectodomain: a fundamental determinant of cadherin
function. Curr. Biol. 7:308–315.
Yap, A.S., C.M. Niessen, and B.M. Gumbiner. 1998. The juxtamembrane region of the cadherin cytoplasmic tail supports lateral clustering, adhesive
strengthening, and interaction with p120ctn. J. Cell Biol. 141:779–789.
Yonemura, S., M. Itoh, A. Nagafuchi, and S. Tsukita. 1995. Cell-to-cell adherens junction formation and actin filament organization: similarities and differences between non-polarized fibroblasts and polarized epithelial cells. J.
Cell Sci. 108:127–142.
IFM 2014
Seminario 3: Estado de Reposo
-Bibliografía Sugerida: Capítulos “Ionic Basis of the Resting Potential” y “Neurons as
conductors of electricity” del libro “From Neuron to Brain” de Nicholls y col..
1. Conteste las siguientes preguntas
a. ¿Qué es el potencial de equilibrio de un ión?
b. ¿Qué ecuación puede utilizar para calcularlo?
c. ¿Sirve dicha ecuación para estimar el potencial de membrana? Discuta este punto.
d. ¿Por qué se dice que la bomba Na+/K+ ATPasa es electrogénica?
e. ¿Qué le pasaría al potencial de reposo si la bomba Na+/K+ ATPasa es bloqueada
farmacológicamente? Explique el origen de los cambios esperados.
2. Señalar cuál de las siguientes afirmaciones es correcta:
La ecuación de Goldman-Hodgkin-Katz es válida si se cumple, entre otras condiciones, que:
a. Todos los iones involucrados están en equilibrio termodinámico.
b. El sistema está en estado estacionario.
c. Todas las corrientes iónicas son nulas.
d. La corriente iónica neta es nula
3. Calcular el potencial de equilibrio del ion H+ a través de la membrana muscular, si el
citoplasma tiene un pH = 7.0 y el fluido extracelular tiene pH = 7.4. ¿Está en equilibrio el ion
H+ en el estado de reposo (ΔVm = -50mV)?
4. Considere un sistema modelo de dos compartimentos separados por una membrana permeable a
K+ y Cl-, pero no a los aniones orgánicos (A-). No hay bombas activas involucradas.
I
II
en mM
en mM
A100
0
+
K
150
150
Cl50
150
a. ¿Está el sistema en equilibrio electroquímico?
b. Si no lo está, ¿en qué dirección se moverá cada ión? ¿Cuáles son las concentraciones
finales que cada ión alcanzará en I y II?
c. ¿Cuál será la diferencia de potencial cuando se alcance el equilibrio? ¿Cómo se puede
calcular?
5. En la gran mayoría de las células el potencial de equilibrio del K+ es de aproximadamente
–100 mV y el del Na+ es de aproximadamente +40 mV. ¿Por qué se observan estas
diferencias si ambos son cationes?
6. Si en condiciones normales el potencial de reposo de una célula es Vm = -60 mV ¿Qué
valor tendría aproximadamente si invirtiéramos las concentraciones de K+ y Na+ entre el
exterior y el interior?
7. ¿Qué diferencia existe entre la permeabilidad y la conductancia de la membrana? ¿Qué
condición tiene que cumplir un soluto para que tenga sentido referirse a su conductancia?
8. Se tienen los siguientes datos para el axón gigante del calamar:
[K+]o = 20 mM
[Na+]o = 450 mM
[Cl-]o = 470 mM
----------------------PK : PNa : PCl = 1.0 : 0.04 : 0.45
[K+]i = 420 mM
[Na+]i = 50 mM
[Cl-]i = 40 mM
[A- org] = 430 mM (impermeables)
R*T/F = 25 mV
a. Calcular el potencial de reposo. Indicar la polaridad.
b. Calcule el potencial de equilibrio para el cloruro a las concentraciones dadas en el
cuadro. ¿Está en equilibrio electroquímico el cloro en este sistema?
c. Si la permeabilidad al Na+ aumenta en un factor de 500, ¿cuál será el nuevo potencial
de membrana? Indique su polaridad.
d. ¿Cuál será el efecto sobre el potencial de membrana de disminuir 10º C la
temperatura? Utilizar las concentraciones y las permeabilidades dadas inicialmente.
9. La membrana biológica puede ser homologable a un circuito RC.
a. ¿Qué elemento de la membrana puede ser analogable a un capacitor? Explique
b. ¿Qué elemento de la membrana puede ser analogable a una resistencia? Explique. ¿En qué
se parecen y en qué se diferencian?
c. ¿En qué configuración se ubican estos dos elementos (en serie, en paralelo)? Explique
funcionalmente por qué esta configuración describe adecuadamente el funcionamiento de la
célula.
d. Un sistema RC es considerado un sistema pasivo; la corriente que circula por el mismo
depende de que éste sea conectado a una batería. ¿Qué opera en la membrana como una
batería? Explique.
e. Grafique la variación de la carga almacenada por el capacitor frente a la aplicación de un
pulso de corriente cuadrado de una duración de 10 veces la constante temporal τ del
capacitor.
d. Realice el mismo gráfico del punto anterior pero para la diferencia de potencial entre
ambas placas del capacitor. Identifique τ en el gráfico. ¿Que representa matemáticamente esta
constante?
10. ¿Qué esperaría que ocurra con el potencial de membrana de una neurona determinada
frente a la aplicación de un pulso de corriente despolarizante si los valores de permeabilidad
de los canales de fuga normales para potasio y sodio se duplicaran? ¿Qué efecto tiene este
cambio sobre la constante de tiempo de la célula?
11. Considerando la figura
a. Explicar las diferencias observadas entre la relación teórica (T), obtenida por Nernst, y la
curva obtenida experimentalmente (E), para el comportamiento del potencial de membrana
(Vm) en el reposo para distintas concentraciones de K+ en el medio externo.
b. ¿ Qué ocurriría con esta curva luego de tratar a la célula con un bloqueante de los canales
de Na+?
c. ¿Qué sucede con esta relación luego de tratar a la célula con un bloqueante de los canales
de K+?
E
T
12. Durante el desarrollo aumenta la actividad del cotransportador de K+-Cl- causando un
cambio en la concentración intracelular del cloro. La siguiente figura muestra los valores del
potencial de equilibro del Cl- (ECl) en función de la edad postnatal para neuronas de
hipocampo.
a. Calcular la concentración intracelular de cloro para las neuronas a 2 y a 14 días de edad, si
la composición del medio extracelular en que se hicieron las mediciones fue (en mM): NaCl
130, KCl 4, MgCl2 1, CaCl2 2.
b. Si las neuronas, independientemente de la edad, tienen un potencial de reposo de -70 mV,
¿qué efecto espera que tenga un neurotransmisor como el GABA al actuar sobre receptores
ionotrópicos con permeabilidad selectiva para el cloro a 2 y a 14 días de edad?
IFM 2014
Seminario 4: Potencial de Acción
-Bibliografía Sugerida: Capítulo “Ionic Basis of the Action Potential” del libro “From
Neuron to Brain” de Nicholls y col..
1. ¿Qué significa que un canal iónico sea dependiente de voltaje? ¿Qué mecanismos
moleculares explican esa dependencia?
2. Durante el potencial de acción el potencial de membrana se acerca al potencial de
equilibrio del sodio ¿Por qué sucede esto? ¿Por qué luego de disparado un potencial de
acción el potencial de membrana no se mantiene en ese valor indefinidamente?
3. El desarrollo de un potencial de acción implica procesos de retroalimentación positiva y
procesos de retroalimentación negativa (feedback positivo y negativo, respectivamente).
¿Cuáles son?
4. En numerosas neuronas del sistema nervioso central de mamíferos la propagación de
potenciales de acción es saltatoria.
a. ¿A qué se refiere este término?
b. ¿Qué ventajas ofrece la conducción saltatoria, en comparación con la conducción continua
de potenciales de acción?
c. ¿Cómo afecta la cubierta de mielina que envuelve un axón a las propiedades eléctricas de
dicho axón?
d. ¿Qué desventaja representaría que una neurona tenga distancias internodales muy cortas?
¿Y si fueran muy largas, qué exigencias a las propiedades del axón implicaría?
5. Las propiedades eléctricas del axón de calamar se pueden reproducir satisfactoriamente
aún cuando el citoplasma es extraído y reemplazado por una solución cuya composición
iónica es similar a la del citoplasma. ¿Qué demuestra esta observación?
6. Un segmento de axón de calamar tiene un potencial de reposo de –70 mV. Cuando se
produce un potencial de acción, el pico llega a +40 mV. Las concentraciones de sodio y
potasio dentro del axón son de 50 mM y 100 mM, respectivamente.
a. Demuestre mediante cálculos que el movimiento de iones a través de la membrana
plasmática durante el potencial de acción no altera apreciablemente las concentraciones
iónicas.
b. Los valores que calculó son subestimaciones de los valores reales ¿Por qué?
c. ¿En qué casos el cambio de concentración iónica intracelular debido al flujo iónico podrá
ser apreciable? ¿Qué mecanismos tiene la célula para contrarrestar este cambio?
Datos: Dimensiones del segmento de axón: 2cm de longitud y 1mm de diámetro; Superficie del cilindro: S =
π.diámetro.longitud; Volumen del cilindro: Vol = π.r 2.longitud; Carga de un capacitor: Q = C.V; Capacitancia
de la membrana celular: C = 1µF/cm2; Constante de Faraday: F = 96487 C/mol; Número de Avogadro: N =
6.0225.1023
7. Las figuras muestran los cambios en las conductancias de sodio (g Na) y potasio (gK)
durante el cambio de potencial (V) que se produce cuando se desencadena un potencial de
acción.
a. En base a estos gráficos explique cada fase del potencial de acción incluyendo el fenómeno
de hiperpolarización tardía (after-hyperpolarization) que se puede apreciar en el registro.
b. Las gNa gK durante el potencial de acción tienen cursos temporales con características
similares: aumentan, llegan a un pico y luego disminuyen llegando a cero. ¿Estos
comportamientos generales se deben a las mismas causas?
8. En la figura se muestran registros intracelulares realizados en una neurona de hipocampo
en condiciones control (trazo más oscuro) y tratando al tejido con dos concentraciones de la
droga 4-AP.
Describa lo que se observa en los registros y sugiera cuál es el blanco de acción del 4-AP.
9. Se aplican tres electrodos en las posiciones A, B y C de un axón gigante de calamar, según
se indica en la figura.
A
B
C
a.
Si se aplica un estímulo supraumbral en A, indique con flecha/s en qué dirección
espera que se propague el potencial de acción iniciado por este estímulo
b.
Se aplican sendos estímulos supraumbrales en A y B en forma simultánea. Indique
cómo espera que se propaguen ambos potenciales de acción.
c.
Qué característica particular poseen los canales de sodio dependientes de voltaje que
explican el fenómeno que describió en la pregunta b?
d.
Se aplica un estímulo supraumbral en A y luego, con cierto retardo, un estímulo
supraumbral en B, y se mide la respuesta en C. Qué condición debe cumplir dicho retardo
para que en C, se observen: i) dos potenciales de acción; ii) un solo potencial de acción. ¿Qué
fenómeno ocurrió en este último caso? Explíquelo brevemente.
10. Esquematice un potencial de acción desencadenado por un estímulo supraumbral,
indicando la escala de voltaje y tiempo apropiadas; sobre este mismo gráfico dibuje la
respuesta que resultaría ante la misma estimulación si se estudia a la neurona en presencia de
un bloqueante de los canales de Na+ sensibles al voltaje (TTX) o de canales de K+ sensibles a
voltaje (TEA)?
11. En base al grafico
a. Esquematizar el potencial de membrana en función del tiempo, registrado en un axón
gigante de calamar en cada uno de los electrodos (1, 2, 3 y 4) de la figura luego de una
estimulación subumbral y de una supraumbral. Superponga los gráficos en un único par de
ejes.
b. Hacer un gráfico del potencial
Inyección de
de membrana máximo en función
corriente
de la distancia luego de ambas
estimulaciones. ¿Qué tipo de
propagación se observa en cada
caso? ¿De qué propiedades de la
membrana depende cada una?
Explique qué parámetro describe
el
cambio
del
potencial
subumbral con la distancia y
1
2
3
4 Electrodos de
registro
como se obtiene.
c. ¿Cómo se modificarían los
esquemas de la pregunta a) si se agregara TTX (inhibidor de canales de Na + dependientes de
voltaje) a la solución extra-celular?
d. Compare las características de las señales registradas por el electrodo 4 si se trata de un
axón mielínico o amielínico del mismo calibre.
12. Está en discusión si las dendritas son ramas de conducción pasiva mientras el axón es el
sitio de iniciación del potencial de acción. Sin
embargo estudios realizados en los últimos diez
años muestran que las membranas de las
dendritas de ciertas neuronas poseen canales de
Na+ y K+ Vm-dependientes. Aún no está claro en
qué medida contribuyen estas conductancias al
disparo de potenciales de acción medido en los
axones. En neuronas de la corteza de ratón se
lograron realizar registros simultáneos en las
dendritas y el soma (figuras de la izquierda). La
célula fue estimulada inyectando corriente tanto
en el soma (registros superiores) como en la
dendrita (registros inferiores). Los autores
concluyen que estos registros muestran que tanto
cuando se estimulan las dendritas como el soma,
el potencial de acción que se registra se origina
en el axón. Observe los registros y determine
cómo puede deducirse esto de los registros
mostrados, y grafique los resultados esperados si
las dendritas fueran sitios de iniciación de
potenciales de acción.
13. En neuronas de hipocampo, como en muchas otras, se observa la expresión de una
conductancia voltaje dependiente que se activa cuando el potencial de membrana es
desplazado a valores más negativos que -55 mV. Esta conductancia tiene un potencial de
equilibrio de alrededor de -20 mV y es conocida como Ih o „conductancia funny‟ If. La
figura muestra las respuestas de una neurona del hipocampo a la aplicación de una serie de
pulsos hiperpolarizantes (registros inferiores) en condiciones control (registros superiores) y
en presencia de un bloqueante de dicha conductancia (registros medios).
a. Calcule la resistencia de membrana en condiciones control y tratado con el bloqueante.
b. ¿Cómo se explica el cambio observado en la resistencia de membrana producido por el
bloqueo de la conductancia Ih?
control
Bloqueo de
Ih
corriente
c. ¿Cómo puede explicar que la neurona dispara potenciales de acción al ser repolarizada?
IFM 2014
Seminario 5: Sinapsis
Bibliografía Sugerida: Capítulos “Principles of synaptic transmission”, “Indirect Mechanisms
of Synaptic Transmision" y “Transmitter Release” del libro “From Neuron to Brain” de
Nicholls y col.. Ver también “Neurons as conductors of electricity” (Seminario 4).
1. Entrenamiento para desprevenidos:
Dibujar un esquema de los elementos presentes en una sinapsis química genérica y enumerar
los pasos más importantes involucrados en la liberación del neurotransmisor y la respuesta
post-sináptica.
a. ¿Qué diferencias anatómicas y funcionales existen entre sinapsis químicas y eléctricas?
b. ¿Qué determina la latencia entre el pico del potencial de acción en una neurona
presináptica y el pico de la respuesta postsináptica?
c. ¿A qué se refieren los términos receptor postsináptico y receptor presináptico? ¿Qué
funciones cumplen?
2. Un neurotransmisor activa un canal hipotético permeable tanto al Na + como al K+. El ENa =
+50 mV y el EK = -85 mV. Si tus experimentos determinan que el canal tuvo una
conductancia 3 veces mayor al Na+ que al K+, ¿cuál sería el potencial de reversión de la
respuesta sináptica?
3. La figura fue extraída del paper de Takeuchi & Takeuchi, 1960. En este experimento el
nervio fue estimulado a medida que se variaba el potencial de la fibra muscular. Los registros
fueron realizados sobre la fibra muscular en la configuración de fijación de voltaje (voltage
clamp). La figura muestra la relación entre la amplitud de las corrientes postsinápticas
evocadas por estimulación del nervio y el potencial de membrana, cuando la preparación fue
incubada en solución externa normal y en solución externa con bajo contenido de sodio.
Los símbolos blancos corresponden a la
solución Ringer normal (113.6 mM
Na+) y los símbolos negros a la
solución con una concentración de Na+
de 33.6 mM.
a. Se te pide que informes aproximadamente el E rev en la placa motora del músculo sartorius
de la rana en Ringer normal. ¿Por qué se modifica en Ringer con bajo Na+?
b. ¿Qué valor tomará el Erev cuando se baña la preparación con una solución Ringer que tiene
10 mM K+ y 113.6 mM Na+?
Datos: gNa = 1.29; gK = 1; Na+i = 15.5 mM; K+i = 126 mM
4. En un experimento realizado para estudiar las propiedades de la conexión sináptica entre
dos neuronas se registra, en la configuración de voltage clamp, la corriente postsináptica
medida a diferentes potenciales de membrana (A). En base a estos datos se graficó la
corriente postsináptica inhibitoria (IPSC) en función del potencial (B).
A
B
a. ¿Como identifica el potencial de reversión en cada una de estas figuras?
b. ¿Qué tipo de conductancia podría estar asociada a esta respuesta sináptica?
c. Proponga un experimento para evaluar la hipótesis que su respuesta a “b” plantea.
Grafique los resultados que esperaría obtener ante estos experimentos.
5. Los experimentos ilustrados en la siguiente figura (Katz & Miledi, 1967) se diseñaron con
el objetivo de determinar el papel del Ca2+ en la transmisión sináptica utilizando una
preparación de placa neuromuscular de rana (nervio frénico unido al músculo diafragma). La
preparación fue sometida a una perfusión constante con una solución que no contenía Ca 2+ y
a la cual se agregó TTX para bloquear el disparo de potenciales de acción. Se utilizó un
electrodo extracelular para estimular el nervio, otro electrodo extracelular conteniendo CaCl 2
a traves del cual se puede descargar calcio (de manera localizada y acotada en el tiempo) en
la zona de la placa neuromuscular y un electrodo intracelular en la fibra muscular. El
estímulo se aplica sin descargar calcio (izquierda), descargando calcio inmediatamente antes
de estimular (centro) o descargando el calcio después de estimular. La escala vertical indica 2
mV y la escala horizontal 5 ms.
calcio
stim
registro
A
estim
calcio
a. ¿Cómo explica las diferencias en la respuesta de la fibra muscular en las diferentes
configuraciones de estimulación?
b. ¿Que esperaría si la solución de perfusión hubiese sido suplementada con una alta
concentración de Mg2+?
6. Las dos figuras resumen los resultados obtenidos en un experimento que consistió en
evaluar la transmisión sináptica entre el nervio frénico y las fibras musculares que forman el
diafragma de un ratón. Para ello se estimuló el nervio 300 veces mientras se registró
mediante un electrodo intracelular el potencial de membrana de la fibra muscular. Para
realizar este experimento la preparación fue bañada en una solución con bajo Ca 2+ (0.6 mM)
y alto Mg2+ (6 mM).
La primera figura representa el histograma de amplitudes de las 300 mediciones realizadas en
una misma fibra, habiendo excluido los eventos donde no hay respuesta ante la estimulación
(fallas). En la figura de la derecha se realizó una interpolación polinómica para visualizar
mejor los diferentes picos de la figura de la izquierda.
a. Basados en los datos que se muestran en la figura, ¿cuál es la amplitud de los potenciales
de placa miniatura? Explique su razonamiento, incluyendo en su explicación el por qué del
tamaño relativo de los dos picos. ¿Cuál es el correlato anatómico de los potenciales
miniatura?
b. ¿Cuál es la razón por la cual el experimento se realizó en un medio conteniendo bajo Ca2+
y alto Mg2+?
c. ¿Qué esperarías obtener si repetís el experimento después del agregado de un bloqueante
de canales de K+ voltaje-dependientes?
d. ¿Cómo creés que se vería el histograma si se repite el experimento en un medio con 0.8
mM Ca2+?
7. La figura muestra tres registros de potenciales de placa obtenidos en una misma fibra
muscular esquelética.
a. Describir el curso temporal de la señal y explicar la dinámica observada.
b. ¿Qué relación tienen estos registros con los potenciales miniatura?
c. ¿Cómo se verían afectados los potenciales de placa en presencia de un inhibidor de la
enzima acetilcolinesterasa?
8. La figura ilustra los resultados de un experimento en el cual se midió la liberación de 3HGABA de sinaptosomas de cerebelo, utilizando un colector de fracciones y un contador de
centelleo.
Figura A: durante los intervalos indicados por las barras la preparación fue expuesta a una
solución con una alta concentración de potasio. El experimento se realiza en presencia de
inhibidores de la recaptación de GABA.
Figura B: similar al protocolo experimental descripto en A pero, en la segunda de las tres
aplicaciones de potasio, se co-aplicó una alta concentración de potasio con 100 µM GABA.
B
cpm
A
15
30
45
60
15
30
45
60
Tiempo (min)
a. Explicar los resultados obtenidos en A. ¿Cuál es el mecanismo por el cual actúa la solución
con alta concentración de potasio?
b. Explicar los resultados obtenidos en B y proponer un mecanismo que pueda explicar el
efecto producido por la co-aplicación de GABA.
c. ¿Por qué resultó útil elegir la segunda exposición a alto K+ para testear los posibles efectos
modulatorios en la liberación del neurotransmisor?
9. Los siguientes resultados son parte de un estudio sobre el efecto de la toxina -CgTX
sobre la respuesta sináptica de fibras musculares del diafragma al estimular el nervio frénico
(contiene a las neuronas motoras que inervan al diafragma). La hipótesis de trabajo es que
dicha toxina bloquea en forma selectiva a los canales de calcio activables por despolarización
de la membrana.
a. La figura de la izquierda muestra un potencial de placa en ausencia (a) y en presencia (b)
de la toxina (escala vertical 1 mV; escala horizontal 0.5 mseg). Basándose en lo que conoce
sobre la sinapsis neuromuscular: explique por qué estos resultados apoyan la hipótesis
mencionada previamente. ¿Dónde espera que actúe la toxina?
b. Con el fin de descartar un efecto de la toxina ω-CgTX sobre los receptores nicotínicos, los
investigadores aplicaron pulsitos de acetilcolina directamente sobre la placa en presencia y
ausencia de la toxina, y registraron los cambios de potencial postsinático. Explique la lógica
de este experimento. ¿Qué resultado espera observar si la toxina no tiene ningún efecto sobre
el receptor colinérgico nicotínico?
c. Con el fin de confirmar su hipótesis los investigadores realizaron un estudio utilizando una
dosis de 40 nM de la toxina en presencia de diferentes concentraciones de Ca 2+ extracelular
(representadas a la derecha de cada curva en el gráfico de la derecha). En este experimento se
estimuló el nervio frénico a diferentes tiempos a partir del agregado de la toxina y se
obtuvieron las curvas que se muestran en la figura, donde se grafica la amplitud (relativa al
valor de la respuesta al primer estímulo) del potencial de placa en función del tiempo (0 min
es el momento anterior a la aplicación de la toxina). Información importante: a una
concentración de 40 nM la toxina produce solo un bloqueo parcial de los canales. Explique
los resultados representados en el gráfico de la derecha en términos de la hipótesis de trabajo.
d. En este gráfico la amplitud de los potenciales de placa fue relativizada a la máxima
amplitud, obtenida antes de aplicar la toxina. ¿Cómo espera que varíe el valor absoluto de
estas amplitudes máximas en las diferentes concentraciones de calcio utilizadas?
e. Grafique la inhibición en función de [Ca2+ ] medida a los 30 min.
f. ¿Cómo espera que afecte la toxina -CgTX al potencial de acción de la neurona motora
que se proyecta en el nervio frénico?
10. Los inhibidores de la acetilcolinesterasa (como el gas nervioso) son mortales en altas
concentraciones. ¿Por qué? Sin embargo, cuando se usan en bajas concentraciones en
pacientes con miastenia gravis, estos inhibidores tienen efectos terapéuticos. Considerando
que la miastenia es un desorden autoinmune que reduce la densidad de receptores de
acetilcolina en las placas motoras del músculo esquelético, ¿por qué tendrían efectos
beneficiosos los anticolinesterásicos?
11. En la figura se observan 9 registros simultáneos de
potenciales de placa, realizados en una misma fibra muscular
curarizada con 9 electrodos colocados a diferentes distancias
de la placa.
a. Dibuje la configuración del registro.
b. ¿A qué puede deberse el decaimiento en la amplitud de la
señal?
c. ¿En que unidades está la escala horizontal?
d. Estime la constante de espacio ( )
12. La siguiente figura ilustra por qué
el potencial de acción sólo se produce
si las constantes de tiempo o espacio
son suficientemente grandes. Explique
esta aseveración.
13. La neurona esquematizada posee un potencial de membrana en el reposo de –60mV. Su
umbral para el disparo de potenciales de acción es de –50mV (registrado en el soma, sitio B).
Las sinapsis 1 y 2 son excitatorias. Frente a la activación de cualquiera de ellas por separado
se registra en B una despolarización de 6mV.
Dibuje los registros que obtendría en los sitios A, B y C en las siguientes condiciones
(incluya en su dibujo la escala de voltaje usando como referencia el Vreposo y Vumbral):
a. Se activa únicamente la sinapsis 1.
b. Se activan simultáneamente las sinapsis 1 y 2 (asuma que están equidistantes del soma).
c. Se activa tres veces consecutivamente la sinapsis 2.
d. Se activan simultáneamente las sinapsis 1 y 2 pero ahora considere que la constante de
espacio de la célula se redujo a la mitad.
14. Los resultados mostrados en las figuras A-C fueron extraídos de un artículo de Wu y
colaboradores (2004, J. Neurosci. 24:4625) en el cual se estudia la integración de señales
sinápticas en neuronas del colículo inferior (neuronas ICC) de rata que reciben señales
excitatorias e inhibitorias convergentes. La excitación está mediada por glutamato que
impacta sobre receptores glutamatérgicos, bloqueables por los inhibidores CNQX y APV; la
inhibición está mediada por GABA que impacta sobre receptores que se bloquean con
bicuculina. El estudio se realiza mediante registros intracelulares de las neuronas ICC en la
configuración de fijación de voltaje mientras se estimulan las fibras aferentes a esta región
mediante electrodos extracelulares. En este estudio las conductancias activadas por voltaje en
la neurona ICC fueron inhibidas mediante la inyección intracelular de bloqueantes
específicos. Datos en mM: [Na+]o = 129, [K+]o = 1.2, [Cl-]o =130, [Na+]i = 4, [K+]i= 110, [Cl-]i
=8
estimulación
Registro
extracelular
Registro intracelular
neuronas ICC
glu
GABA
El diagrama esquematiza la configuración
experimental
a. ¿Por qué se evitó aplicar bloqueantes de las conductancias activadas por voltaje
directamente en el medio externo de la preparación?
La figura A muestra un registro representativo de las respuestas de una neurona ICC a un
estímulo en las fibras aferentes (indicado por la flecha) en presencia de los bloqueantes de los
receptores glutamatérgicos CNQX+APV. La neurona fue estudiada a los potenciales de
membrana indicados a la izquierda de cada registro. La línea punteada representa la línea de
base de la corriente antes del estímulo. El gráfico B resume los datos mostrando la relación
entre la amplitud de la corriente sináptica (eje y) y el potencial de membrana (eje x).
b. Explique a qué se debe la variación en la respuesta de la neurona al estímulo en función del
potencial de membrana (figura A).
c. ¿Qué conclusión puede extraer sobre la conductancia asociada a la respuesta GABAérgica
de los resultados mostrados en A y B? Haga explícito su razonamiento.
En el gráfico Ci se muestra la respuesta a una ráfaga de 10 estímulos en solución normal sin
agregado de ningún agente farmacológico; en el gráfico Cii se muestra la respuesta a un
estímulo similar en presencia de los bloqueantes de los receptores glutamatérgicos; y en el
gráfico Ciii se muestra la respuesta en presencia del bloqueante de los receptores
GABAérgicos. En todos los registros mostrados en la figura C el potencial de membrana de
la neurona ICC fue fijado a -40 mV. La línea punteada representa la línea de base de la
corriente antes del estímulo.
d. ¿Qué conclusión puede obtener respecto de la integración de la respuesta sináptica de las
neuronas ICC según lo que se observa en la figura Ci solamente?
e. ¿Qué información agrega las figuras Cii y Ciii?
IFM 2014
Seminario 6: Sistemas sensoriales
-Bibliografía Sugerida: Del Libro “From Neuron to Brain” de Nicholls y col.: Capítulo
17, Transduction of Mechanical and Chemica Stimuli; Capítulo 18, Processing of
Somatosensory and Auditory Signals y Capítulo 19, Transduction and Signaling in the
Retina
1. Los receptores de las distintas modalidades sensoriales comparten la capacidad de
traducir un determinado cambio del medio ambiente en un potencial receptor. Para los
casos del gusto, oído, vista, tacto y olfato, ¿cuál es la conversión de energía que tiene
lugar en los receptores periféricos?
2. Discuta el concepto de campo receptivo en el sistema visual, táctil y en el olfativo.
3. Dos cualidades se distinguen en relación al sonido: el volumen y el tono.
a. Explique a qué se refiere cada término.
b. Cuál es la unidad en que se mide el volumen del sonido.
4. El oído se divide en tres regiones, oído externo, medio e interno.
a. Indique la función principal del oído medio, y cual es el mecanismo principal que le
permite cumplir con ésta.
b. Describa cómo y dónde a lo largo del proceso auditivo que se da en el oído se percibe
el volumen y el tono.
c. A qué se refiere el término proyección tonotópica.
5. Diseñe un experimento para determinar el campo receptivo de una célula somatosensorial de una rata.
6. Los siguientes registros muestran las respuestas de un mecanoreceptor de adaptación
lenta a estímulos mecánicos aplicados con una varilla de 2 mm de diámetro sobre la piel
(Rowe et al 2003. CBP 136:883-893). La dinámica temporal del estímulo (cómo varía la
presión en el tiempo) se grafica con la línea inferior y a la izquierda se indica la máxima
indentación (desplazamiento en el eje normal a la superficie de la piel) aplicada.
a. Defina el concepto de adaptación sensorial.
b. Explique las variaciones en la frecuencia de disparo del mecanoreceptor a lo
largo del estímulo y en función de esto indique por qué se lo clasifica como de
adaptación lenta.
c. Explique las variaciones observadas en los sucesivos registros.
d. ¿Cómo esperaría que fuera la respuesta si se tratara de un receptor de adaptación
rápida?
En la siguiente figura se analiza la respuesta de un receptor de adaptación rápida. En
este caso se utiliza un estímulo levemente diferente. Se aumenta paulatinamente la
indentación, pero al llegar al valor estable se aplica una variación sinusoidal alrededor
de este valor máximo.
e. Explique los resultados observados en la figura.
f. Se propone que los receptores de adaptación rápida son los responsables de la
percepción somatosensorial de texturas. Ensaye una explicación para esta
hipótesis en base a los resultados mostrados en la figura.
7. Discuta la siguiente afirmación: “Los campos receptivos de las neuronas sensoriales
de segundo o mayor orden son más amplios y más complejos que los de las neuronas
sensoriales primarias”. Ejemplifíquelo en el sistema visual.
8. La inhibición lateral permite resaltar los bordes. Explique esta aseveración teniendo
en cuenta el esquema siguiente.
9. Explique qué se entiende por representación somatotópica. ¿A qué sentidos puede
aplicarse?
10. En la retina de mamífero el proceso de visión se inicia con el impacto de los fotones
sobre las células fotorreceptoras.
a. ¿Qué tipo de células fotorreceptoras existen en la retina y cuáles son sus propiedades?
b. ¿Cuáles son las moléculas responsables de captar la luz?
c. Dibuje un esquema que explique el proceso completo de transducción de la señal que
ocurre en los fotorreceptores, desde que impacta la luz hasta la liberación del
neurotransmisor.
11. Conteste verdadero o falso. Fundamente las respuestas falsas:
a. En la oscuridad no existen corrientes entrantes ni salientes en el fotorreceptor.
b. La presencia de luz suprime la corriente entrante.
c. La luz hiperpolariza a los fotorreceptores alejándolos del E K+.
d. Los bastones se caracterizan por tener una alta resolución temporal (respuesta
rápida con tiempo de integración corto)
e. Los bastones tienen mayor concentración de fotopigmentos que los conos.
f. Los conos están especializados en visión diurna.
g. El daltonismo está causado por una pérdida congénita de bastones.
h. Las respuestas de las células bipolares de centro “on” o de centro “off” están
mediadas por diferentes neurotransmisores.
12. Una de las evaluaciones que se hacen sobre la visión en un organismo es la agudeza
visual
a. ¿A qué se refiere el término?
b. ¿De qué parámetros depende?
c. ¿Es homogénea en toda la retina?
13. Describir las propiedades neurofisiológicas de las células bipolares y las de sus
campos receptivos. Discutir la siguiente figura:
14. En el artículo publicado por Hubel y Wiesel en 1961 (J. Physiol. 155:385-398) se
describió la forma de los campos receptivos visuales de las neuronas de los núcleos
geniculados. Para ello se posicionó a un gato a 1.5 m de distancia de una pantalla en la
que se proyectaron haces de luz de diámetro variable por un periodo de 1 segundo
mientras se registró la actividad de las neuronas mediante registros extracelulares (solo
se registran los potenciales de acción). En A, B y C se proyecta un haz de 1º, 2º y 14º, y
en D se proyecta un ánulo de luz de 14º de diámetro externo y 2º interno.
a.
b.
c.
d.
¿Se trata de una célula ON u OFF? Justifique su respuesta
¿Cómo se explican los cambios que se observan entre A y B?
¿Cómo se explican los cambios que se observan entre B y C?
¿Cómo se explican los cambios que se observan entre C y D?
15. Flores-Herr y colaboradores (2001, J. Neurosci 21:4852) realizaron un estudio
similar al de Hubel y Wiesel, pero en este caso utilizaron la técnica de fijación de
voltaje (voltage clamp). En este estudio se proyectaron haces de luz sobre la retina de
conejos y se midió la respuesta de una neurona ganglionar ON como la corriente
eléctrica que se activa durante la respuesta al estímulo lumínico. En A la célula fue
fijada a -75 mV mientras se aplicaron haces de luz de diámetro variable (en µm, según
se indica a la izquierda de cada registro). La línea superior indica el momento de
aplicación de la luz. En B se grafica el promedio de las amplitudes normalizadas en
función del diámetro del haz. Tenga en cuenta solo el valor de la amplitud al pico (Peak
Amplitude)
A
B
a. Tomando la respuesta al haz de 150 µm mostrada en A, explique cómo se
condice el resultado con la aseveración del enunciado que indica que se trata de
una neurona de tipo ON.
b. Explique las variaciones observadas en A en función del diámetro del haz de luz.
c. ¿Qué variable fisiológica puede deducirse de los resultados descriptos en la
figura B?
C
D
En los registros de la figura C se muestra la respuesta de una neurona ganglionar ON a
un haz de luz de 200 µm cuando la neurona fue fijada a los potenciales indicados a la
derecha. Se informa que en estas células, el potencial de reversión para corrientes
mixtas de Na+ y K+ es de aproximadamente 0 mV, y el equilibrio del Cl- es de
aproximadamente -55 mV. En D se grafica la respuesta para experimentos realizados
con haces de diferente diámetro. Para construir esta figura se consideró como “corriente
excitatoria” la medida a un potencial de membrana de -55 mV y la inhibitoria a un
potencial de 0 mV.
d. En base al enunciado, justifique la estrategia adoptada por los autores para
discernir entre corriente excitatoria e inhibitoria.
e. Explique los resultados mostrados en la figura D.
IFM 2014
Seminario 7: Músculo
Bibliografía obligatoria: Capítulo 17 del libro Hill, Wyse y Anderson, 2004. Ed.
Panamericana.
Observaciones histológicas
Luego de analizar cada preparado hacer un esquema en donde se puedan identificar las
características morfológicas distintivas que se pudieron reconocer en cada tipo muscular
(al final de la guía encontrarán un glosario sobre las técnicas de tinción utilizadas).
Músculo Liso. Se observará en un corte transversal de vejiga teñido con tinción
tricrómica de Masson. Encontrar una zona del preparado en el que se observen células
musculares cortadas en forma longitudinal. Notar la forma fusiforme de las células
musculares y de sus núcleos. En las zonas de corte transversal, observar la localización
central de los núcleos y el tamaño variable de su diámetro, según a qué nivel han sido
rebanados por el corte. Las células que han sido cortadas en un plano superior o inferior a
la localización del núcleo aparentan ser anucleadas, si bien no lo son.
Músculo Esquelético. Se observará en un corte transversal de lengua teñido con eosina
hematoxilina. Identificar la disposición de las fibras musculares paralelas con las
miofibrillas en un arreglo ordenado. Identificar las bandas transversales claras y oscuras.
Observar que las fibras son multinucleadas, ¿Cómo es la localización de los núcleos:
central o periférica?
Músculo Cardíaco. Tinción de eosina hematoxilina. Notar que las fibras cardíacas
presentan ramificaciones que son evidentes tanto cuando son cortadas en su plano
longitudinal como en el transversal. Identificar en ambas orientaciones de corte los
núcleos grandes. ¿Cómo es la ubicación de los núcleos? En el corte longitudinal intentar
observar los discos intercalares ¿qué son, anatómica y funcionalmente?
Guía de preguntas
1. En los vertebrados distinguimos tres tipos de músculos: esquelético, cardíaco y liso.
Describa las semejanzas y diferencias en los siguientes aspectos:
a. Morfología
b. Cinética de contracción
c. Rol que juega el ion calcio
d. Inervación
e. Acople excitación contracción
2) Explique cómo es la organización anatómica del músculo esquelético desde el nivel de
tejido hasta las principales estructuras subcelulares que los conforman.
3. ¿Cuáles son los elementos principales que conforman un sarcómero y cómo ocurre la
contracción de los mismos?
4. La contracción de los músculos esqueléticos es un fenómeno que depende de ATP y de
Ca++. Sin embargo, la contracción puede producirse en presencia de muy baja
concentración de calcio en el medio externo, a que se debe esto? La relajación muscular
de que factores depende?
5. Grafique y explique cómo evoluciona la fuerza en función del tiempo durante una
contracción isométrica y durante una isotónica. Si usted intentara levantar un peso con su
brazo, ¿en qué caso su bíceps realizaría una contracción principalmente isométrica y en
qué caso una fundamentalmente isotónica?
6. La fuerza de contracción depende de la longitud de la fibra muscular en reposo. Trace
un gráfico de la relación fuerza total vs. longitud en reposo y explíquelo brevemente.
7. ¿Cómo se induce un proceso de contracción tetánica en el músculo esquelético?
Explique el porqué de dicho fenómeno y grafíquelo como fuerza vs. tiempo.
8. En una preparación experimental se aísla un músculo esquelético de ratón junto con un
segmento del nervio que lo inerva. El músculo es sujetado en un extremo a un punto fijo
y en el otro a un transductor de fuerza. Por su parte, el nervio es conectado a un
estimulador eléctrico. El músculo es sostenido a una longitud inicial (5 cm) igual a su
longitud normal (la longitud en el animal en ausencia de actividad motora). Se aplican
pulsos eléctricos al nervio de amplitud creciente hasta que se activan el 50% de las
unidades motoras contenidas en la preparación y se adopta este estímulo.
a. ¿Las contracciones registradas son de tipo isométricas o isotónicas?
b. Defina unidad motora.
c. Explique por qué aumentando la intensidad del estímulo que se aplica a un nervio se
puede activar un número creciente de unidades motoras.
d. En el análisis de la relación entre la actividad del nervio motor y la contracción del
músculo esquelético ¿qué efecto tendría la omisión del ion calcio durante un corto
período? Explique.
e. Se aplican ráfagas de 5 estímulos (del tipo que se describe en el párrafo inicial) a
frecuencias de 1, 5, 10 y 100 estímulos/seg. La siguiente figura muestra la respuesta
contráctil de este músculo a un solo estímulo.
Dibuje un gráfico que describa la tensión máxima ejercida por el músculo en función de
la frecuencia de disparo de la neurona motora (tensión vs. frecuencia). Explique
brevemente el comportamiento que describe el gráfico.
9. A diferencia del músculo esquelético el músculo cardíaco no está integrado por
unidades motoras, sino que se contrae siempre como un todo con intervención de todos
los miocitos. ¿Cómo es, entonces, que se regula fisiológicamente la contracción del
músculo cardíaco?
10. Existen reflejos espinales que protegen al músculo esquelético frente a situaciones
que puedan provocar un sobre-estiramiento del mismo. Describa cuál es el sensor, el
efector, y el circuito neuronal involucrado.
11. Shiels y colaboradores midieron en dos condiciones experimentales distintas la
evolución de la concentración interna de Ca++ (AF/F es una medida indirecta de la
concentración de Ca++) durante la contracción de un miocito aislado (figura A). Al
mismo tiempo estudiaron el desarrollo de tensión del mismo miocito (figura B).
AF/F
Tiempo (ms)
Tiempo (ms)
En relación a las propiedades del músculo estudiadas aquí, ¿A qué puede deberse que
para idénticas concentraciones de Ca++ se desarrolle distinta cantidad de fuerza? ¿Cómo
se explica este fenómeno?
Descripción de las técnicas utilizadas
Tinción con Hematoxilina-Eosina
La Hematoxilina-Eosina (H&E) es la técnica de tinción histológica de aplicación
general más usada. Una de sus principales ventajas radica en que se puede usar después
de cualquier tipo de fijación tisular. Es usualmente usada en histología y patología para
observar la morfología de diversos tejidos y células.
La hematoxilina, una tinción nuclear común, se aísla de un extracto de madera
(Haematoxylon campechianum). El primer éxito en la aplicación biológica de la
hematoxilina fue descrito por Bohmer en 1865. Esta tinción no tiñe directamente los
tejidos, sino que necesita de un “mordiente” para unirse a los tejidos. Este efecto es
provisto por un catión metálico como el hierro, aluminio o tungsteno. La variedad de
hematoxilinas disponibles se basa parcialmente en la elección del ión metálico usado. El
complejo ión metálico-hematatoxilina con carga positiva se combina con grupos de
fosfatos con carga negativa de ADN nuclear, formando un color azul-púrpura
característico de las tinciones con hematoxilina.
Las soluciones de eosina se utilizan para contratinciones en general. La eosina es
la tinción citoplásmica de uso más común. Ya en 1885 fue preconizada por List como
contratinción para verde metilo. También se utiliza junto con tintes azules básicos y,
cuando se utiliza en combinación con la hematoxilina, recibe la denominación de tinción
“H&E”. La eosina es una tinción ácida que interactúa con proteínas celulares ricas en
aminoácidos básicos. Se forma un complejo proteíco de tintes que se caracteriza por una
tinción citoplásmica de color rosa intenso.
Tinción tricrómica de Masson
Tinción modificada por De Carlo, que lleva como colorantes hematoxilina de Carazzi,
fucsina ponceau de xilidina y azul de anilina. En general, con esta coloración los núcleos
se ven violetas rojizos, los citoplasmas de acuerdo al tipo celular pueden ser rojos
(epitelios y músculo por ej.), naranjas (glóbulos rojos), azul (fibras colágenas), celeste
claro (mucinógeno de células mucosas).

Guía seminarios 2014 primera parte

Transcription

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