New Insights into the Role of the Cortical Cytoskeleton in Exocytosis from Neuroendocrine Cells

Abstract

The cortical cytoskeleton is a dense network of filamentous actin (F-actin) that participates in the events associated with secretion from neuroendocrine cells. This filamentous web traps secretory vesicles, acting as a retention system that blocks the access of vesicles to secretory sites during the resting state, and it mediates their active directional transport during stimulation. The changes in the cortical cytoskeleton that drive this functional transformation have been well documented, particularly in cultured chromaffin cells. At the biochemical level, alterations in F-actin are governed by the activity of molecular motors like myosins II and V and by other calcium-dependent proteins that influence the polymerization and cross-linking of F-actin structures. In addition to modulating vesicle transport, the F-actin cortical network and its associated motor proteins also influence the late phases of the secretory process, including membrane fusion and the release of active substances through the exocytotic fusion pore. Here, we discuss the potential interactions between the F-actin cortical web and proteins such as SNAREs during secretion. We also discuss the role of the cytoskeleton in organizing the molecular elements required to sustain regulated exocytosis, forming a molecular structure that foments the efficient release of neurotransmitters and hormones.

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From: Luis M. Gutiérrez, New Insights into the Role of the Cortical Cytoskeleton in
Exocytosis from Neuroendocrine Cells. In Kwang W. Jeon, editor:
International Review of Cell and Molecular Biology, Vol. 295,
Burlington: Academic Press, 2012, pp. 109-137.
ISBN: 978-0-12-394306-4
© Copyright 2012 Elsevier Inc.
Academic Press

CHAPTER THREE
New Insights into the Role of the
Cortical Cytoskeleton in Exocytosis
from Neuroendocrine Cells
Luis M. Gutie
´rrez
Contents
1. Introduction 110
2. Organization of Cortical Cytoskeleton 110
3. Changes Associated with Exocytosis 111
4. Forces Driving Cytoskeletal Changes Related to Exocytosis 113
4.1. Molecular motors influencing F-actin dynamics 113
4.2. Proteins modifying F-actin polymerization, fragmentation,
and cross-linking 114
5. Cytoskeletal Control of Vesicle Transport 116
5.1. Contribution of F-actin and microtubules to the
transport systems 116
5.2. Myosins II and V as molecular motors of vesicle motion 118
6. Role of Cytoskeleton in Membrane Fusion 119
6.1. Role of myosin II in exocytotic fusion 120
6.2. Role of filamentous actin in membrane fusion 121
6.3. Interactions between cytoskeleton and secretory machinery 122
6.4. Association of F-actin cortex with ion channels involved in
exocytosis 124
7. Cortical Cytoskeleton as an Organizer of Molecular
Cytoarchitecture of Exocytosis 125
8. Conclusion 128
Acknowledgments 128
References 128
Abstract
The cortical cytoskeleton is a dense network of filamentous actin (F-actin) that
participates in the events associated with secretion from neuroendocrine cells.
This filamentous web traps secretory vesicles, acting as a retention system that
International Review of Cell and Molecular Biology, Volume 295 #2012 Elsevier Inc.
ISSN 1937-6448, DOI: 10.1016/B978-0-12-394306-4.00009-5 All rights reserved.
Instituto de Neurociencias, Centro Mixto Universidad Miguel Herna
´ndez-CSIC, Sant Joan d’Alacant,
Alicante, Spain
109
Author's personal copy

blocks the access of vesicles to secretory sites during the resting state, and it
mediates their active directional transport during stimulation. The changes in
the cortical cytoskeleton that drive this functional transformation have been
well documented, particularly in cultured chromaffin cells. At the biochemical
level, alterations in F-actin are governed by the activity of molecular motors like
myosins II and V and by other calcium-dependent proteins that influence the
polymerization and cross-linking of F-actin structures. In addition to modulating
vesicle transport, the F-actin cortical network and its associated motor proteins
also influence the late phases of the secretory process, including membrane
fusion and the release of active substances through the exocytotic fusion pore.
Here, we discuss the potential interactions between the F-actin cortical web and
proteins such as SNAREs during secretion. We also discuss the role of the
cytoskeleton in organizing the molecular elements required to sustain regu-
lated exocytosis, forming a molecular structure that foments the efficient
release of neurotransmitters and hormones.
Key Words: Cytoarchitecture, Neuroendocrine cells, Chromaffin vesicles,
Exocytosis, F-actin, Myosins, Phosphorylation, Vesicle transport,
SNAREs. ß2012 Elsevier Inc.
1. Introduction
Cytoskeletal structures play fundamental roles in many aspects of cell
biology, influencing both basic structure and functional specialization. At
the cell periphery in particular, the F-actin cortical cytoskeleton influences
the cell’s shape, topology, motility, and signaling. In endocrine and neuro-
endocrine cells, this dense network of actin filaments not only provides
architectural support but it also entraps secretory vesicles that contain
neurotransmitters and hormones, playing an important regulatory role in
the exocytotic process. In this review, we summarize our current under-
standing of the cortical cytoskeleton’s role in the physiology of neuroendo-
crine cells. Specifically, we focus on chromaffin cells, an important model
that has significantly influenced our current understanding of the role of the
cortical F-actin network in regulated exocytosis.
2. Organization of Cortical Cytoskeleton
In the early 1980s, a number of studies using immunofluorescence
microscopy techniques demonstrated that cytoskeletal proteins such as
actin, caldesmon, and myosin localize to the cortex of cultured chromaffin
cells, forming a peripheral ring (Aunis and Bader, 1988;Aunis et al., 1980;
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Burgoyne et al., 1986;Lee and Trifaro, 1981;Trifaro et al., 1985). This
distribution was consistent with earlier descriptions of a dense web of
F-actin beneath the plasma membrane in other endocrine systems, such as
pancreatic b-cells (Orci et al., 1972). Accordingly, it was inferred that this
dense cortical cytoskeleton was continuous throughout the cell periphery,
preventing secretory vesicles having free access to the plasma membrane
(Aunis and Bader, 1988;Burgoyne and Cheek, 1987;Trifaro et al., 1985).
The three-dimensional ultrastructural architecture of this cortical net-
work was elegantly demonstrated using quick-freeze, deep etch electronic
microscopy (Nakata and Hirokawa, 1992). This technique revealed that
F-actin filament bundles were heterogeneously distributed, coinciding with
the plasma membrane in some areas but being sparse in others. The contin-
uous nature of the cortex was a source of some controversy, even in
unstimulated cells, particularly given the potential of chemical fixation
and low temperature procedures that accompanied fluorescence and
electronic microscopy to affect the integrity of the “native” cortex.
The use of the transmitted light channel of confocal scanning microscopes
in conjunction with high numerical aperture objectives provided an alterna-
tive means of analyzing the cortical network, based on the differential refrac-
tion index presented by F-actin in the gel and sol states (Giner et al., 2005). In
living intact chromaffin cells, this technique revealed the cortex as an intricate
meshwork of cytoskeletal cages, which contained infrequent openings that
permit vesicles to access the membrane. Subsequentthree-dimensional recon-
structions of transmitted light images of the cortical area of unstimulated living
cells revealed numerous areas devoid of the cytoskeleton that resemble “cra-
ters” covering the cell’s surface (Fig. 3.1A; Giner et al., 2007).
3. Changes Associated with Exocytosis
Immunocytochemical analyses provided the first evidence that the
cortical cytoskeleton was a dynamic structure, demonstrating the redistri-
bution of fodrin, a spectrin-like protein, after cell stimulation (Perrin and
Aunis, 1985). Indeed, when F-actin was visualized with phalloidin-coupled
rhodamine, rapid fragmentation after cell stimulation with nicotine was
evident (Cheek and Burgoyne, 1986). The same authors later reported
that different secretagogues decrease the F-actin/G-actin ratio and they
suggested that this change is necessary prior to exocytosis (Burgoyne and
Cheek, 1987). The initial reports of cortical fragmentation were followed
by other studies using conventional epifluorescence (Marxen and Bigalke,
1991;Vitale et al., 1991) and confocal microscopy (Gil et al., 2000;Nakata
and Hirokawa, 1992). The hypothesis that F-actin disassembly was linked to
secretion was further investigated using a variety of natural chemicals that
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stabilize (e.g., jasplakinolide, phallotoxins) or disrupt polymerization (e.g.,
cytochalasins, latrunculins, clostridium C2 and iota toxins, snake venom
toxins) in a variety of cellular systems, including pancreatic b-cells (Orci
et al., 1972), melanotrophs (Chowdhury et al., 1999), mast cells (Pendleton
and Koffer, 2001), lactotropes (Carbajal and Vitale, 1997), acinar cells
(Jerdeva et al., 2005), chromaffin cells (Gasman et al., 2004;Gil et al.,
2000), and the related PC12 line (Matter et al., 1989).
Many of these studies demonstrated that the role of F-actin in exocytosis was
far more complex than that of a simple retention system that prevents granule
access and fusion. For example, by promoting F-actin depolymerization with
0 s
10 s
50 s
1–5 s
10–30 s
40–80 s
C
ABD
Figure 3.1Structure of the cortical cytoskeleton and its response to stimulation in
chromaffin cells. (A) Three-dimensional reconstruction of the F-actin cortex of a
cultured chromaffin cell based on transmitted light images from different planes. The
image shows the characteristic network and cages formed by the F-actin cortex in the
resting state. (B) Magnitude of the stimulation-induced changes. In the same cell
depicted in panel A, open spaces devoid of F-actin are generated by cell depolarization
for 30s. (C) Phases of the cytoskeletal changes. The scheme shows the different phases
of F-actin reorganization observed in a typical cell when stimulated. From the resting
state, cortical disruption occurs soon (1–5s) after stimulation, leading to the formation
of wider cytoskeletal depleted areas (10–30s) and the subsequent regeneration of the
cortical barrier. (D) Example of the cortical structures seen during stimulation. This
panel shows transmitted light images of the F-actin cortex in resting and stimulated
states. During stimulation, channel-like structures conduct vesicles to the secretory
sites. Scale bars¼10mm (panel A) and 1mm (panel D).
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the botulinum toxin C2, low levels of F-actin ADP-ribosylation were seen to
promote secretion, while very high levels significantly reduced exocytosis,
indicating an active role for F-actin during secretion (Matter et al., 1989).
Moreover, analyzing vesicle motion in the cortical area by evanescent light
microscopy revealed that, during stimulation, F-actin filaments mediate the
active transport of vesicles within 300nm of the cell’s limits (Lang et al., 2000;
Oheim and Stuhmer, 2000).
How does the F-actin cortex accomplish this dual role? This was
recently revealed in simultaneous transmitted light scanning and confocal
fluorescent microscopy studies of F-actin structural dynamics and granule
motion (Giner et al., 2005). This technique revealed major reversible
changes in the cortical cytoskeleton during stimulation, as illustrated in
the three-dimensional reconstructions in Fig. 3.1. Hence, the resting state
is characterized by a dense cytoskeletal web (Fig. 3.1A) that reorganizes to
reveal extensive areas devoid of F-actin (Fig. 3.1B). Dynamic changes occur
a few seconds after stimulation, with the formation of cortical disruptions
and channel-like structures (Fig. 3.1D), which allow vesicles access to
docking sites. If the stimulus persists, these alterations can continue for up
to 40s, with the formation of extended subplasmalemmal areas devoid of
cytoskeleton. The cortex finally reorganizes to impede vesicle from acces-
sing the plasma membrane (see Fig. 3.1C and D). In this cortical remodeling
scenario, F-actin transiently redistributes in a calcium-dependent manner,
acting as a structural barrier and a functional vesicle carrier during the
secretory cycle. The formation of cytoplasmic areas devoid of F-actin
during prolonged stimulation was previously reported in electron micros-
copy studies (Nakata and Hirokawa, 1992;Tchakarov et al., 1998).
4. Forces Driving Cytoskeletal Changes
Related to Exocytosis
The dynamic nature of the cortical network suggests that F-actin
plasticity must be exquisitely controlled to adapt to the dual effects of
stimulation. This control may be dependent on intracellular calcium levels
and accomplished by regulating actin activity via two different elements:
molecular motors such as specific myosin subtypes that control actin
dynamics and a variety of auxiliary proteins that regulate polymerization,
fragmentation, and F-actin cross-linking.
4.1. Molecular motors influencing F-actin dynamics
Many cellular processes depend upon the mechanical properties of the
cytoskeleton, such as cell adhesion and motility, in which cytoplasmic
reorganization requires the contractile force provided by actomyosin
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complexes (Bershadsky et al., 2006;Borisy and Svitkina, 2000). Cortical
reorganization appears to be dependent on contractile systems, and indeed,
the motion of cytosolic cytoskeletal structures under scanning transmitted
light microscopy is reduced by wortmannin and 2,3-butanodione mono-
xime (BDM), inhibitors of myosin II (Giner et al., 2005). Moreover,
chromaffin cells contain cortical myosin II (Trifaro et al., 1984), a non-
processive motor that binds to F-actin and undergoes a conformational
change after ATP hydrolysis. This protein self-associates to form minifila-
ments (Verkhovsky et al., 1999), which act as “disordered micromuscles”
after binding to F-actin bundles, producing local contractions in the cortical
meshwork. The expression of an inactive nonphosphorylatable form of the
regulatory light chain of myosin II inhibits cytoskeletal motion in the
chromaffin cell cytosol (Neco et al., 2004), suggesting that the
phosphorylation–dephosphorylation cycle sustains F-actin cytoskeletal
dynamics. Accordingly, calcium-dependent phosphorylation of the myosin
II light chain accompanies the secretory cycle (Cote et al., 1986;Gutierrez
et al., 1988, 1989), while inhibition of the myosin light chain kinase
(MLCK) partially blocks secretion from intact and permeabilized chromaf-
fin cells (Kumakura et al., 1994;Reig et al., 1993).
Among other factors regulating contractile force that have been detected
in chromaffin granule preparations (Burgoyne et al., 1986), caldesmon is a
protein that stabilizes actin filaments against actin-severing proteins and
inhibits actomyosin ATPase activity. The inhibitory activity of caldesmon
is regulated by calcium and ATP through phosphorylation (Wang, 2008),
and when the calcium concentration is elevated to the levels reached during
the secretory process in chromaffin cells, caldesmon and tropomyosin
dissociate from F-actin gels (Cheek et al., 1986).
Molecular motors, such as myosin II, are abundant in the cell cortex
of endocrine and neuroendocrine cells, cross-linking F-actin in an ATP-
dependent manner. These proteins change the physical nature of the cyto-
solic sol–gel to that of an “active gels,” dynamic structures that induce
the instability of cortical actin ( Joanny and Prost, 2009), and they affect
organelle motion in a manner that will be discussed later.
4.2. Proteins modifying F-actin polymerization,
fragmentation, and cross-linking
Fodrin, a neural form of spectrin, has long been known to be a key factor in
cortical fragmentation (Perrin and Aunis, 1985). This protein cross-links
F-actin at low concentrations of intracellular calcium, but when cells are
stimulated and the intracellular calcium levels rise, fodrin dissociates from
actin and there is a local dissolution of the cortex. The role of this protein in
exocytosis was further demonstrated when calcium-induced secretion from
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digitonin-permeabilized cells was partially inhibited by purified antibodies
against this protein (Perrin et al., 1987).
The search for factors that regulate the disassembly of chromaffin cortex
upon stimulation led to the discovery of the calcium-dependent F-actin
severing protein, scinderin (Rodriguez del Castillo et al., 1990). Scinderin is
a protein localized in the subplasmalemmal region of chromaffin cells that is
found in a variety of secretory tissues including the brain, pituitary,and salivary
glands, as well as in platelets, and it alters the viscosity of F-actin gels. Interest-
ingly, scinderin is redistributed during chromaffin cell stimulation, following
the pattern of F-actin fragmentation, which is a feature not attributed to gelsolin
(Vitale et al., 1991). Scinderin contains 2 actin and 2 phosphatidylinositol 4,5
bisphosphate-binding domains (PIP2) and a relatively high degree of homol-
ogy to the gelsolin and villin genes (Marcu et al., 1994). The role of scinderin in
exocytosis was witnessed by the increased calcium-induced secretory response
of permeabilized chromaffin cells induced by recombinant scinderin, an effect
blocked by peptides competing with the actin PIP2-binding domains (Zhang
et al., 1996). These findings suggest that in resting conditions, PIP2 binding to
scinderin sequesters the protein in the plasma membrane, the correct cortical
location for this protein. Cell stimulation and the subsequent increase
in intracellular calcium activate actin-severing activity and induce F-actin
fragmentation in the region just below the plasma membrane.
The control of F-actin polymerization (Yin and Janmey, 2003) and the
regulation of the cytoskeletal–plasma membrane interactions (Meiri, 2004)
are associated with the production of phosphatidylinositol and its phos-
phorylated derivatives. In permeabilized chromaffin cells, manipulating
phosphoinositide levels with phospholipase C implicated phosphatidylino-
sitol polyphosphates in exocytosis (Eberhard et al., 1990). Two enzymes
that maintain the PIP2 levels, the phosphatidylinositol transfer protein and
phosphatidylinositol 4-kinase, are also required to sustain secretion in PC12
cells (Hay and Martin, 1993;Hay et al., 1995).
In terms of the signaling pathway that mediates PIP2 generation in
chromaffin and other secretory cells, a number of findings point to the
central role of the Rho GTPase family in this process. Specifically, RhoA is
associated with secretory granules and it is a downstream partner of vesicu-
lar-associated Go, a trimeric GTPase that regulates the priming steps of
exocytosis (Gasman et al., 1997;Vitale et al., 1996). Activation of RhoA via
the Go pathway (Gasman et al., 1997), or expression of a constitutively
activated GTP-RhoA mutant, impairs secretion and stabilizes the F-actin
cortical barrier in PC12 (Frantz et al., 2002) and chromaffin cells (Bader
et al., 2004). The phosphatidylinositol 4-kinase enzyme that associates with
chromaffin granules is thought to be the effector mediating RhoA inhibi-
tion of secretion (Gasman et al., 1998). Thus, the increase in PIP2 in the
granular membrane after RhoA activation may be involved in the nuclea-
tion of actin (Yin and Janmey, 2003), the regulation of cytoskeleton–plasma
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membrane interactions (Meiri, 2004), or even in the regulation of vesicle
motion in the vicinity of secretory sites (Holz and Axelrod, 2002).
Other GTPases may regulate actin dynamics by promoting F-actin
polymerization. For example, the constitutively active GTP-loaded mutants
of Rac1 (Rac1L61) and Cdc42 (Cdc42L61) facilitate secretion in chromaf-
fin and PC12 cells, respectively, suggesting that these GTPases play an active
role in exocytosis (Gasman et al., 2004;Li et al., 2003). The active Cdc42
L61 mutant also enhances F-actin polymerization in the subplasmalemmal
region (Gasman et al., 2004), indicating that Cdc42 triggers F-actin forma-
tion in the vicinity of active sites, thereby enhancing secretion efficiency.
The annexin family of proteins may be particularly important in exocy-
tosis. These proteins are characterized by their calcium-sensing and phos-
pholipid-binding domains (Geisow et al., 1987), and some subtypes interact
with actin, such as types II and VI (Hayes et al., 2004). Translocation of
these proteins from the cytosol to the plasmalemma may dynamically link
these two structures (Mangeat, 1988). Annexin II and annexin VI (synexin)
activity in chromaffin cells is essential to sustain regulated exocytosis
(Burgoyne et al., 1991;Creutz et al., 1982;Pollard et al., 1982;Roth
et al., 1993), suggesting that the translocation of tetramers of these proteins
to the plasma membrane is an essential step in the formation of the lipid
microdomains required for exocytosis (Chasserot-Golaz et al., 2005).
5. Cytoskeletal Control of Vesicle Transport
Early studies investigating the influence of antimitotic drugs on adre-
nal gland secretion suggested a key role for microtubules in the exocytotic
transport of vesicles (Douglas and Sorimachi, 1972;Poisner and Bernstein,
1971). Thus, the relative importance of the two major cytoskeletal-
associated systems for organelle transport is central to understanding vesicle
transport in neuroendocrine cells.
5.1. Contribution of F-actin and microtubules to the
transport systems
The classical view proposes that cytoskeletal elements play a central role in the
transport of organelles from the inner cytoplasm to the cell periphery. In
neuroendocrine cells, immature vesicles follow the exocytotic pathway
through the endoplasmic reticulum to mature in different compartments of
the Golgi apparatus (Winkler, 1977). In these early phases, F-actin structures
support the architecture of the cisternae and they cooperate with microtubules
in transporting vesicles (Caviston and Holzbaur, 2006;DePina and Langford,
1999). In chromaffin cells, vesicle movement visualized by dynamic confocal
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microscopy appears to be sensitive to chemicals that affect microtubules in
regions adjacent to the chromaffin cell nucleus (Neco et al., 2003). By contrast,
F-actin inhibitors affect granule motionboth in the interior (Giner et al., 2005;
Neco et al., 2003) and in the cortical region (Allersma et al., 2006;Giner et al.,
2007;Lang et al., 2000;Oheim and Stuhmer, 2000). This observation may
reflect the distribution and density of these cytoskeletal elements since micro-
tubules have a radial distribution in chromaffin cells, concentrating in internal
regions and contacting the cell periphery tangentially (Neco et al., 2003).
Conversely, F-actin is localized in the cortical zone, and consequently, it
influences granule transport in this area. Accordingly, chemicals that disrupt
F-actin affect both the fast and slow secretory phases, whereas microtubule
inhibitors have a minor impact on secretion and they only affect the slow
secretory components recruited by repetitive stimulation (Neco et al., 2003).
Indeed, the microtubule-depolymerizing and stabilizing agents, colchicine
and nocodazole, inhibit the late and sustained phase of granule secretion in
b-pancreatic cells (Farshori and Goode, 1994). It is noteworthy that a variety
of antimitotic agents have a clear inhibitory effect on secretion in cultured
chromaffin cells (Cooke and Poisner, 1979;McKay et al., 1991), probably
reflecting their effects on the cytoskeleton.
The fundamental role of F-actin in directing or limiting the movement
of vesicles in the cortical region was clearly established in the first total
internal reflection fluorescence microscopy studies. Vesicle motion within
the cortical region can be described in detail by TIRFM, as the evanescent
field spans a region of 100–300nm from the cell–coverslip interface. This
technique reveals that the movement of chromaffin granules becomes more
restricted when approaching the plasma membrane ( Johns et al., 2001;Lang
et al., 1997;Oheim and Stuhmer, 2000;Steyer and Almers, 1999), princi-
pally due to two events: the interaction of vesicles with docking or tethering
elements (Lopez et al., 2009;Toonen et al., 2006) and vesicle entrapment in
the cytoskeletal network (Oheim and Stuhmer, 2000;Steyer and Almers,
1999). Recently, the use of transmission light scanning microscopy has
provided compelling evidence that vesicle motion is influenced by the
organization of the F-actin cortex into small polygonal cages (Giner et al.,
2007). Vesicles move in association with F-actin structures, and thus their
movement is restricted to the space available within the cages.
The consensus from multiple studies performed in neuronal, neuroendo-
crine, and endocrine cellular systems is that microtubules and kinesins are
important for the transport of secretory vesicles and other organelles from the
cell interior to the cortical region, where they are transferred to F-actin trails
(Fig. 3.2), the dominant system that controls the movement of vesicles within
the cell cortex. The possible interaction between the molecular motors of the
microtubule (kinesin) and F-actin-based (myosin V) transport systems may
create a “transportome” complex, facilitating the distribution of their cargo
(DePina and Langford, 1999).
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5.2. Myosins II and V as molecular motors of vesicle motion
Members of two classes of the myosin family are especially abundant in
chromaffin cells, myosins II and V (Neco et al., 2002;Rose et al., 2002).
Myosin II associates with F-actin and concentrates in the cell cortex, whereas
myosin V colocalizes with vesicles and it is therefore more widely distributed
in the cell cytoplasm. Class V myosins are strong candidates to participate in
vesicle transport. They possess a tail region designed for interaction with
cargo (Mooseker and Cheney, 1995), they are regulated by calcium (Sellers
1. Actin-mediated granule tensional pressure
2. Actin-mediated pore dilation
3. Direct influence on fusion machinery
Fusion
F-actin
Myosin II
Myosin Va
Microtubules
Kinesin
Maturation
Vesicle transport
2
1
3
Figure 3.2Cytoskeletal proteins involved in vesicle transport and the late phases of
the secretory process. Microtubules and kinesin are key players in the initial stages of
vesicle transport from the cell interior to the cortical zone. F-actin mediated transport
dominates in the cortex, characterized by two processes: F-actin dynamic remodeling
by myosin II and processive motion along F-actin trails by myosin V motors. These
molecular motors may play active roles during the maturation associated with exocyto-
sis. Membrane fusion is also influenced by F-actin-myosin II, and three possibilities
mechanisms are depicted: (1) exertion of a tensional pressure over the entire vesicle,
driving the release of the vesicular content; (2) F-actin-myosin II control of the
expansion of the exocytotic pore, acting as a cytoskeletal scaffold; (3) F-actin or myosin
motors directly affect the secretory machinery (SNARE and associated proteins).
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and Knight, 2007), and they associate with chromaffin (Rose et al., 2003)
and PC12 vesicles (Rudolf et al., 2003). Moreover, expression of a domi-
nant-negative tail of myosin V alters the distribution of vesicles, which
appear to dissociate from the cell cortex (Rudolf et al., 2003). The small
GTPase Rab27 and the MyRIP proteins appear to mediate the interaction
between myosin V, vesicles, and actin filaments (Desnos et al., 2003).
Consequently, altering the function of these proteins affects the movement
of the vesicles in the subplasmalemmal area. Nevertheless, myosin V does not
appear to be simply associated with vesicle transport, as it appears to partici-
pate in the maturation process (Kogel et al., 2010), and in the docking of
dense vesicles (Desnos et al., 2007).
In addition to myosin V, there is clear evidence of a role for myosin II in
the transport of vesicles in neuroendocrine systems. This conventional
nonmuscle myosin colocalizes with cortical F-actin (Neco et al., 2002;
Rose et al., 2003), and its inhibitors alter the motion of vesicles in the
cortical region, such as BDM and blebbistatin (Berberian et al., 2009;Neco
et al., 2002). This effect can be observed by confocal and evanescent wave
fluorescent microscopy, and moreover, an inactive nonphosphorylatable
mutant of the myosin II regulatory light chain drastically reduces vesicle
movement within the cytoplasm of chromaffin cells (Neco et al., 2004).
How does conventional myosin II influence vesicle motion? As men-
tioned earlier, myosin II binds to actin filaments and forms cross-linked
structures, the so-called active gels ( Joanny and Prost, 2009). These struc-
tures are static in the absence of ATP, but in its presence, they display
dynamic fluctuations and induce the movement of small colloidal spheres
(Mizuno et al., 2007). Indeed, expression of a ATP-insensitive light chain of
myosin II causes F-actin structures to become static and prevents vesicular
motion in the chromaffin cell cytoplasm (Neco et al., 2004).
Taken together, these findings indicate that the movement of secretory
vesicles in the cortex of neuroendocrine systems depends on the activity of
two molecular systems: nonprocessive motion mediated by the F-actin-
myosin II network and processive movement generated by the activity of
myosin V along F-actin trails (concepts summarized in Fig. 3.2, which also
illustrates the cooperation of F-actin structures and microtubules in the
interior of the cytosol to transport chromaffin granules from the inner
region, adjacent to the nucleus, to the peripheral cortex).
6. Role of Cytoskeleton in Membrane Fusion
While F-actin and myosin motors clearly participate in vesicle trans-
port, the role of cytoskeletal elements in exocytosis is far more complex than
solely ensuring the access of vesicles to docking sites. This first became
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evident through the effects of botulinum toxin C2, an F-actin ribosylating
agent, on secretion in PC12 cells (Matter et al., 1989), and subsequently, it
was shown that inhibitors of the MLCK affect the fast phases of exocytosis
associated with fusion of primed vesicles (Kumakura et al., 1994). These
findings were supported by studies on endocrine cells such as insulin-
secreting lines (Iida et al., 1997).
6.1. Role of myosin II in exocytotic fusion
The direct involvement of a member of the myosin family in vesicle fusion
during the final stages of secretion was demonstrated by analyzing single
fusion kinetics in bovine chromaffin cells, expressing a nonphosphorylatable
form of the regulatory light chain of myosin II (Neco et al., 2004). In these
cells, the kinetics of amperometric events was slowed by the expression of
this mutated myosin, which also reduced the rate of fusion of the ready
releasable pool of vesicles. Patch amperometry, probably the most efficient
technique to analyze membrane fusion kinetics (Albillos et al., 1997), was
later used to demonstrate a direct link between myosin II phosphorylation
and the rate of expansion of the fusion pore, the structure that initially
connects the intravesicular space with the cell exterior (Neco et al., 2008).
The role of the F-actin-myosin II contractile system in controlling single
vesicle fusion kinetics was further studied using specific inhibitors. This
study suggested that both the rate of expansion of the fusion pore and the
increase in its lifespan enhanced the degree of catecholamine dissociation
from the vesicular matrix (Berberian et al., 2009).
The complex role of the cortical cytoskeleton in controlling the final
phases of membrane fusion was elegantly demonstrated when kiss and run
and full collapse events were discriminated by altering the frequency of
electrical stimulation in mouse chromaffin cells (Doreian et al., 2008).
Accordingly, disrupting the F-actin cortical cytoskeleton through myosin
II phosphorylation favors the full collapse of chromaffin granules, whereas
kiss-and-run events are stabilized by F-actin structures. These conclusions
are supported by data from PC12 cells expressing vesicular proteins of
variable size, and hence with different diffusion rates through the fusion
pore (Aoki et al., 2010). Using TIRFM, myosin II was clearly seen to
control the duration of fusion pore opening. Taken together, these findings
indicate that F-actin-myosin II interactions govern vesicle transport and that
they are essential to ensure “normal” vesicular fusion kinetics, ending in full
collapse. The regulation of this contractile system appears to determine the
type of release: from incomplete kiss-and-run events to full fusion (the
possible molecular mechanisms underlying this regulatory activity are
described in Fig. 3.2). F-actin-myosin II may influence fusion by exerting
mechanical tension over the entire vesicle or alternatively (mechanism 1);
this tension may only affect a hypothetical cytoskeletal scaffold required for
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pore dilation (mechanism 2). By contrast, F-actin and myosin II (or other
myosin classes) molecules may directly affect the proteins responsible for the
formation and expansion of the fusion pore (probably SNARE proteins,
mechanism 3).
In addition to myosin II, several observations suggest that other classes of
myosin, such as myosins V, VI, and 1c/1e, fulfill similar roles in the final
stages of secretion (Bond et al., 2011). Thus, it should be stressed that a
variety of motor enzymes may play essential roles in fusion pore opening or
in driving the release of active compounds in diverse secretory systems.
6.2. Role of filamentous actin in membrane fusion
Exocytosis is inhibited when more than 60% of F-actin is sequestered by
ADP-ribosylation suggesting that a threshold of F-actin is required for
exocytosis (Matter et al., 1989). However, the source of this F-actin remains
unclear, yet it seems likely that after F-actin reorganization, sufficient
F-actin remains in the proximity of secretory sites to fulfill this role.
F-actin remodeling results in the redistribution but not the net destruction
of F-actin structures, and vesicles are associated with F-actin structures for
the duration of the secretory cycle (Giner et al., 2005). Further, although
exocytosis requires contact with the walls of cytoskeletal structures
(Torregrosa-Hetland et al., 2011), this does not preclude the possibility
that de novo F-actin polymerization occurs during secretion. In fact, in
pancreatic acinar cells, vesicles appear to be coated with new F-actin during
exocytosis (Valentijn et al., 2000), stabilizing the structures required sus-
taining exocytosis (Nemoto et al., 2004). However, a subsequent study
proposed that F-actin coating occurs following exocytosis, as active coating
only occurred in granules undergoing exocytotic pore formation (Turvey
and Thorn, 2004).
The possible existence of a molecular cascade leading to de novo F-actin
synthesis has been investigated in different secretory cells. The Rho GTPase
family may be involved in this cascade as Cdc42 is a positive regulator of
mast cell degranulation and it controls exocytosis in pancreatic b-cells
(Abdel-Latif et al., 2004;Hong-Geller and Cerione, 2000). In chromaffin
cells, constitutive activation of Cdc42 triggers the formation of F-actin
filaments in the subplasmalemmal area (Gasman et al., 2004), a process
accompanied by an increased secretory activity. This recruitment of
F-actin appears to be mediated by N-WASP and the Arp2/3 complex,
two factors that govern actin nucleation during the propulsion of secretory
and endocytic vesicles (Malacombe et al., 2006;Taunton et al., 2000).
Overexpression of N-WASP stimulates secretion in a manner that is depen-
dent on its F-actin polymerizing activity (Gasman et al., 2004).
There is as yet no consensus regarding the requirement of de novo F-actin
recruitment for exocytosis in secretory systems. Active coating has only
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been demonstrated in slow secretory systems characterized by large granules
or insoluble vesicles, a form of exocytosis termed “kiss and coat” (Sokac and
Bement, 2006). Further, it remains unclear whether these structures are
involved in generating extrusion forces or in the subsequent recovery of the
membrane by endocytosis. It has been suggested that F-actin coating may
simultaneously drive closure of the fusion pore and compression of the
secretory compartment (Sokac et al., 2003). Indeed, F-actin local assembly
was proposed to stabilize the secretory compartment during docking and
drive the posterior compensative endocytosis, triggered by a molecular
mechanism that detects the mixing of vesicular and plasma membranes
(Yu and Bement, 2007). Following the incorporation of diacylglycerol
from the plasmalemma, the bisoform of protein kinase C would be
recruited into the granular membrane, which would otherwise phosphory-
late Cdc 42. Finally, the Arp2/3 complex controls the exocyst in the final
phases of F-actin-mediated exocytosis (Zuo et al., 2006). The exocyst is a
multiprotein evolutionary conserved complex that, along with other possi-
ble protein complexes, mediates the tethering of vesicles to the plasma
membrane (Lipschutz and Mostov, 2002).
6.3. Interactions between cytoskeleton and
secretory machinery
Protein–protein interactions probably provide the specificity required for
granule docking, as well as contributing the essential machinery necessary
for vesicular fusion. These proteins include the plasma membrane proteins
(t-SNAREs) and their counterparts in the vesicular membrane (v-SNAREs:
Sollner et al., 1993a,b; Weber et al., 1998). Indeed, a ternary complex
consisting of syntaxin 1 (a t-SNARE), SNAP-25, and the vesicular synap-
tobrevin II constitutes the neuronal secretory machinery (Sollner et al.,
1993a,b) and has being described in chromaffin cells (Hodel et al., 1994;
Roth and Burgoyne, 1994) as well as in other endocrine and neuroendo-
crine secretory systems.
There is evidence that SNARE proteins interact with the F-actin
cytoskeleton, and, for example, the binding of syntaxin 4 to F-actin is
disrupted by stimulation in insulin-secreting cells (Thurmond et al.,
2003). F-actin readily binds to the first two coiled-coil domains of this
t-SNARE, apparently inducing granule accumulation prior to fusion
(Jewell et al., 2008). This mechanism is implicated in the targeting of
insulin-containing granules to secretory sites, and it involves the association
of cytoskeletal and SNARE proteins, and the small GTPase participating in
F-actin remodeling, Cdc42 (Daniel et al., 2002). Similarly, Cdc42 associates
with synaptobrevin II during vesicle transport, and a trimeric complex of
Cdc42, synaptobrevin II, and syntaxin 1 may arise (Nevins and Thurmond,
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2005). Associations between cytoskeletal elements and SNARE proteins
during transport are further demonstrated by the calcium-dependent bind-
ing of myosin V to syntaxin 1A, a process implicated in vesicle tethering
(Watanabe et al., 2005). In addition, other proteins that interact with either
individual SNAREs or the SNARE complex may be involved in the nexus
between the secretory machinery and the cortical cytoskeleton. The associ-
ation of Munc-18A with syntaxin-1 (Han et al., 2010;Hata et al., 1993;
Haynes et al., 1999;Jahn, 2000;Katagiri et al., 1995) has been implicated in
the morphological docking of dense vesicles controlled by the F-actin
cortical cytoskeleton (de Wit, 2010a,b). Interestingly, overexpression of
this protein strongly influences the density of the submembrane F-actin
layer (Toonen et al., 2006), indicating that Munc-18 is a protein that
influences the interaction between the F-actin cortical cytoskeleton and
SNARE proteins.
These findings suggest that the secretory machinery interacts with
F-actin and cytoskeleton-related proteins, probably via SNAREs and
other factors. But are these interactions essential for fusion? To answer
this question, we must consider studies of the porosome, a structural
element that aggregates the molecular factors required for the formation
of the fusion pore during secretion. During exocytosis, the intravesicular
and extracellular spaces communicate through a pore structure that permits
the entrance of water molecules, generating an electrical conductance that
can be measured by electrophysiology (Alvarez de and Fernandez, 1988;
Breckenridge and Almers, 1987;Chow et al., 1992;Spruce et al., 1990).
Studies in mast and chromaffin cells, and in other cellular models, proposed
the existence of a scaffold formed by a group of proteins that bring together
membranes implicated in vesicle fusion (Monck and Fernandez, 1992;
Monck et al., 1995). This concept assumed the transient formation of
temporal pores with a conductance similar to that of an ion channel,
which induced either kiss and run (Ales et al., 1999;Artalejo et al., 1998;
Schneider, 2001;Stevens and Williams, 2000) or full collapse of vesicles.
However, permanent fusion pore structures, where secretory vesicles dock
and fuse to release their contents, have also been described using atomic
force and electron microscopy in a variety of secretory lines, including
exocrine and endocrine pancreatic cells, chromaffin and mast cells, and
neurons (Cho et al., 2002, 2009;Jena, 2005;Jena and Cho, 2002;Jeremic
et al., 2003;Schneider et al., 1997). These porosomes have a cup-shaped
morphology and they are of variable size, ranging from 10–15nm in
neurons to 150–200nm in pancreatic exocrine cells. The porosome has
been isolated to determine its composition (Cho et al., 2004;Jena et al.,
2003) and reconstituted in artificial membranes (Cho et al., 2004;Jeremic
et al., 2003). These supramolecular structures appear to be composed of
SNAREs (such as SNAP-25 and syntaxins, synaptotagmin), the ATPase
Role of Cortical Cytoskeleton in Exocytosis 123
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NSF, calcium channel subunits, and cytoskeletal proteins, such as actin,
a-fodrin, and vimentin. Actin is thought to associate with the neck of the
porosome (Schneider et al., 1997), indicating that fusion pore opening and
dilation may be directly regulated by cytoskeletal elements, as described for
myosin II (Berberian et al., 2009;Neco et al., 2004, 2008).
The association of F-actin with the secretory machinery was further
demonstrated in chromaffin cells overexpressing SNAREs (Villanueva
et al., 2010) or the native protein (Torregrosa-Hetland et al., 2011). In
these conditions, dynamic clusters form in the plasma membrane (Lopez
et al., 2009) that associate with the borders of cytoskeletal F-actin cages in
chromaffin cells. These clusters of SNARE proteins also appear to be
associated with L and P/Q-type calcium channels, though this association
varies between intact tissue and isolated cell cultures (Lopez et al., 2007).
6.4. Association of F-actin cortex with ion channels involved
in exocytosis
A number of studies suggest that actin filaments may modulate the activity
of a range of ion channels. For example, cytochalasin D alters the ion flux
associated with calcium channels ( Johnson and Byerly, 1993) and voltage-
dependent sodium channels (Cantiello et al., 1991). Moreover, calcium
channels are endocytosed when the F-actin cytoskeleton is disrupted
(Cristofanilli et al., 2007). This probably reflects the alterations of the
microdomains in the plasma membrane, in the form of cholesterol-rich
lipid rafts that are anchored to cytoskeletal structures, which help organize
the secretory machinery (Churchward and Coorssen, 2009). Indeed,
alpha-7 nicotinic acetylcholine receptors were recently shown to tether to
the postsynaptic membrane in cholesterol-rich domains via PDZ scaffolds
and actin filaments (Fernandes et al., 2010). Interestingly, the microdomains
of L-type voltage-dependent calcium channels exhibit a dynamic behavior
during the fusion of individual vesicles at the ribbon synapse (Mercer et al.,
2011). These domains move within a confined region that is modulated by
actin and cholesterol. After vesicular fusion, the domains spread to occupy a
larger region, although they quickly return to their original position within
a molecular scaffold in order to sustain efficient neurotransmission.
In endocrine cells, cytoplasmic loops II and III of L-type calcium
channels are essential for channels to localize to lipid rafts ( Jacobo et al.,
2009). Moreover, the potentiation of exocytosis by repetitive stimulation of
neuroendocrine chromaffin cells is dependent upon the clustering of L-type
calcium channels in lipid rafts (Park and Kim, 2009). The capacity of F-actin
to agglutinate other relevant molecules, forming clusters in a lipid raft
environment, suggests that the cytoskeleton organizes the cytoarchitecture
associated with secretion in different cellular systems.
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7. Cortical Cytoskeleton as an Organizer of
Molecular Cytoarchitecture of Exocytosis
Fast exocytosis in neurons is very precisely controlled in spatial and
temporal terms, occurring within a specialized region known as the active
zone (Couteaux and Pecot-Dechavassine, 1970;Landis et al., 1988). This is
an electron-dense structure composed of a cytoskeletal matrix and a variety
of associated molecular complexes, and it is involved in the tethering,
docking, and release of neuronal light secretory vesicles (for active zone
function and organization, see Rosenmund et al., 2003 and Schoch and
Gundelfinger, 2006). From the structural viewpoint, the active zone cyto-
matrix is formed by regular arrays of cone-shaped electron-dense particles
that extend 50nm into the neuronal cytosol (Bloom and Aghajanian, 1968;
Gray, 1963). These arrays are interconnected by a meshwork of cytoskeletal
proteins and long filamentous strands of other proteins, forming a scaffold
structure that holds together the constituents of the exo- and endocytotic
neuronal machinery. SNARE proteins are among the proteins that form
these organized structures (Munc-18), as well as scaffolding proteins (e.g.,
CASK, Mint, Bassoon and Piccolo), cell adhesion molecules (including
neurexins, cadherins, integrins), voltage-dependent calcium channels,
and cytoskeletal proteins (such as actin, tubulin, myosins, spectrin, and
b-catenin: Schoch and Gundelfinger, 2006).
Given this level of organization and the presence of specialized structures
that prevent the free diffusion of essential components of the secretory
machinery, there is no doubt that one of the major roles of submembranous
F-actin in neurons is its scaffold function (Sankaranarayanan et al., 2003).
Thus, it is pertinent question to ask whether the cytoskeleton plays a similar
role in endocrine and neuroendocrine cells, thereby organizing the essential
elements of the secretory machinery.
The description of “hot spots” in neuroendocrine cells provided the first
indication of organization of the secretory machinery (Robinson et al.,
1995). These spatially restricted calcium microdomains coincide with indi-
vidual exocytotic events that can be measured with thin amperometric
carbon electrodes in chromaffin cells. However, the sensitivity of secretion
to slow calcium buffers and the latency of secretion suggest that calcium
signals originate at distances within 300–600nm in most chromaffin gran-
ules (Klingauf and Neher, 1997). Interestingly, a fraction of granules (10%)
experienced much higher calcium signals, suggesting they colocalize with
calcium channels. Recently, simultaneous determination of calcium signals
and exocytosis using dual-color TIRFM revealed that most released vesicles
are located within 300nm of calcium microdomains (Becherer et al., 2003).
In chromaffin cells, this distance was reduced in an activity-dependent
manner, indicating that vesicles approach calcium microdomains in
Role of Cortical Cytoskeleton in Exocytosis 125
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response to continuous stimulation, increasing the efficiency of exocytosis.
Direct analysis of the colocalization of L and P/Q calcium channels with
SNARE microdomains in cultured chromaffin cells (Lopez et al., 2007)
revealed that 30% of SNARE patches containing SNAP-25 and syntaxin-1
colocalize with calcium channel patches. However, the majority of these
SNARE microdomains are within 300nm of calcium channel clusters.
Further, the colocalization of calcium channel and SNARE microdomains
is elevated in intact tissue (adrenomedullary slices), suggesting that the
secretory machinery is more efficiently coupled in neuroendocrine cells of
the adrenal gland, and that cell dissociation and culturing may affect the
organization of the secretory machinery. This would explain the accelerated
secretory kinetics observed in adrenomedullary slices when compared with
dissociated cells (Moser and Neher, 1997).
Does the cortical F-actin cytoskeleton mediate the interaction between
calcium channel clusters and SNARE microdomains? The Cav 2.2 and Cav
2.2 subunits that form the P/Q and N-subtypes of voltage-dependent
calcium channels can directly interact with SNAREs via the synaptic
protein interaction motif (synprint: Sheng et al., 1994, 1997, 1998). These
interactions involve SNARE proteins, such as syntaxin-1A and SNAP-25,
as well as synaptotagmin-1, thought to sense calcium in exocytosis. Thus,
calcium channels appear to contain structural elements that integrate the
molecular machinery required for vesicle docking, priming, and fusion.
Interestingly, recent evidence indicates that the clusters formed by syn-
taxin-1/SNAP-25 dimers are associated with the borders of cytoskeletal
cages in chromaffin cells, as demonstrated by the immunocytochemical
detection of native clusters (Torregrosa-Hetland et al., 2011) and by study-
ing the expression of exogenous SNAP-25 (Villanueva et al., 2010). As
these t-SNARE microdomains colocalize with patches formed by voltage-
dependent calcium channels (Lopez et al., 2007), it is not surprising that
calcium channel microdomains are also found at the border of cytoskeletal
cortical structures (Torregrosa-Hetland et al., 2011). Therefore, despite the
absence of a proper active zone in neuroendocrine cells, it appears that the
cytoskeleton serves as a structural element that recruits calcium channels and
the secretory machinery microdomains to specific membrane regions
(Torregrosa-Hetland et al., 2010).
An important question arising from these observations relates to the
benefits of associating the components of the secretory machinery with a
network of cytoskeletal cavities? To answer this question, we proposed
mathematical models of secretory behavior based on Monte Carlo algo-
rithms (Segura et al., 2000), including the active zone as a domain of
cylindrical geometry where calcium channel clusters and in which secretory
sites are located either in the walls or the center. Of the two geometries, the
most robust initial rise [Ca
2þ
]
i
in the proximity of the active site occurs
when this site is close to the border of the simulated spherical cage. The
126 Luis M. Gutie
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predicted [Ca
2þ
]
i
distributions resulted in faster exocytotic responses when
the secretory machinery (SNAREs) and calcium channels form clusters
associated with the borders of cortical cytoskeletal cages. Therefore, as
seen in neurons, the association of components of the secretory machinery
with the F-actin cortical cytoskeleton, including calcium channels and
SNARE proteins, appears to define a special cytoarchitecture that shapes
secretory kinetics (see Fig. 3.3). This configuration would favor vesicle
docking at active sites in neuroendocrine cells where SNARE clusters
colocalize with VDCC microdomains, leading to fast release of a “ready
Vesicles
Snare clusters
F-actin structures
VDCC clusters
Figure 3.3Cytoarchitecture of the secretory machinery in neuroendocrine chromaf-
fin cells. As mentioned in Fig. 3.1, the cortical cytoskeleton of chromaffin cells forms an
intricate network characterized by the presence of polygonal cages. Recent findings
revealed the association of SNARE microdomains and voltage-dependent calcium
channels (VDCC) clustered along the borders of these cytoskeletal structures
(Torregrosa-Hetland et al., 2011;Villanueva et al., 2010). Other observations indicate
that vesicle fusion occurs in the precise sites occupied by SNARE clusters, and that
VDCC microdomains can colocalize with SNAREs (as observed for the vesicle on the
left). In other cases, VDCC microdomains localize within 300nm of the secretory
machinery (vesicle in the right: Lopez et al., 2007). The F-actin cytoskeleton organizes
the key elements of the secretory machinery to ensure rapid intracellular calcium rises in
the proximity of some active sites (ready releasable vesicle pool). In other populations
of docked vesicles, slower calcium elevations are induced by the diffusion of this second
messenger from nearby VDCC microdomains.
Role of Cortical Cytoskeleton in Exocytosis 127
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releasable” secretory component (Gillis et al., 1996;Heinemann et al.,
1994;Horrigan and Bookman, 1994). Alternatively, vesicles may dock at
secretory sites located at a distance from the closest calcium channel cluster,
which would involve a delay due to calcium diffusion, resulting in the
release of a slower “docked pool” of vesicles (Becherer et al., 2003;Chow
et al., 1992;Stevens et al., 2011).
8. Conclusion
The cortical F-actin cytoskeleton of neuroendocrine cells is a dynamic
structure that fulfills a variety of fundamental roles required for exocytosis.
This structure reorganizes during secretion in a calcium-dependent manner,
acting as a barrier to vesicles in the resting state and as a transport system to
facilitate vesicle access to exocytotic sites when cells are stimulated by
secretagogues. It is now evident that during the initial phases of vesicle
transport, the F-actin cortical network and molecular motors (such as
myosins II and V) play an essential role in both the dynamics of cytoskeletal
structures and in the processive motion of the vesicles along F-actin trails. In
addition to its expected role in vesicle transport, the cortical network and its
associated proteins also shape the kinetics of neurotransmitter release by
acting in the late membrane fusion steps of exocytosis. In addition, this
network organizes the molecular elements of the secretory machinery by
the forming of specific structures that promote the efficient coupling of
these elements.
ACKNOWLEDGMENTS
The author wishes to thank his mentors and especially his postgraduate students for their
dedication in the field of exocytosis. This work was supported by grants from the Spanish
Ministry of Science and Innovation (MICINN, BFU2008-00731) and the Generalitat
Valenciana (ACOMP2011/090).
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