http://www.elsevier.com/locate/bba

Biochimica et Biophysica A

Review

Coupling actin and membrane dynamics during calcium-regulated

exocytosis: a role for Rho and ARF GTPases

Marie-France Bader*, Frederic Doussau, Sylvette Chasserot-Golaz,

Nicolas Vitale, Stephane Gasman

CNRS UPR-2356 Neurotransmission and Secretion Neuroendocrine INSERM, 5 rue Blaise Pascal, 67084 Strasbourg, France

Received 1 July 2004; received in revised form 22 September 2004; accepted 24 September 2004

Available online 12 October 2004

Abstract

Release of neurotransmitters and hormones occurs by calcium-regulated exocytosis, a process that shares many similarities in neurons and

neuroendocrine cells. Exocytosis is confined to specific regions in the plasma membrane, where actin remodelling, lipid modifications and

protein–protein interactions take place to mediate vesicle/granule docking, priming and fusion. The spatial and temporal coordination of the

various players to form a bfast and furiousQ machinery for secretion remain poorly understood. ARF and Rho GTPases play a central role in

coupling actin dynamics to membrane trafficking events in eukaryotic cells. Here, we review the role of Rho and ARF GTPases in supplying

actin and lipid structures required for synaptic vesicle and secretory granule exocytosis. Their possible functional interplay may provide the

molecular cues for efficient and localized exocytotic fusion.

D 2004 Elsevier B.V. All rights reserved.

Keywords: ARF; Rho; Actin; Lipid; Exocytosis; Chromaffin cell; Neuron

1. Introduction

Exocytosis, i.e., the fusion of membrane-bound secre-

tory vesicles/granules with the cell plasma membrane and

consequent expulsion of vesicular contents, is a funda-

mental cellular process involved in many physiological

functions including cell migration, wound repair, neuro-

transmission, enzyme secretion and hormone release.

Exocytosis occurs constitutively in all eukaryotic cells

and is up-regulated in some cell types like neurons and

endocrine cells in response to extrinsic stimuli leading to

elevation in cytosolic calcium. Calcium-regulated exocy-

tosis has been the topic of intense investigation for

decades [1–6]. In most cells specialized for calcium-

activated secretion, exocytotic vesicles/granules are present

in at least two compartments, the release-ready vesicle

* Corresponding author. Tel.: +33 388 45 67 13; fax: +33 388 60 16 64.

E-mail address: bader@neurochem.u-strasbg.fr (M.-F. Bader).

0167-4889/$ - see front matter D 2004 Elsevier B.V. All rights reserved.

doi:10.1016/j.bbamcr.2004.09.028

pool and the reserve pool comprising the vast majority of

vesicles. The traffic of vesicles between these two

compartments is subject to a fine regulation by the actin

cytoskeleton [7–12]. Vesicles at the plasma membrane are

docked in apparently two stages: non-primed (fusion

incompetent) and primed (fusion competent). ATP is

required for priming whereas Ca2+ triggers the late ATP-

independent fusion reaction [13,14]. Details of the

molecular machinery underlying some of these steps have

been described. For instance, interaction of vesicles with

the plasma membrane is mediated by soluble N-ethyl-

maleimide-sensitive factor (NSF) attachment protein recep-

tors (SNAREs) found on secretory vesicles (v-SNAREs)

and on the plasma membrane (t-SNAREs). These proteins

form a stable complex with coiled-coil interactions [15],

providing thereby sufficient energy to pull membranes into

close proximity and dock vesicles to the plasma membrane

[16]. ATP-dependent priming reactions are then required to

render docked vesicles competent for calcium-triggered

fusion [14]. These involve NSF-mediated priming of

cta 1742 (2004) 37–49

M.-F. Bader et al. / Biochimica et Biophysica Acta 1742 (2004) 37–4938

SNARE protein complexes, ATP-dependent synthesis of

phosphoinositides and protein kinase-mediated protein

phosphorylations. Proteins that function specifically in

vesicle priming have been discovered, including Munc13,

a major priming factor in neurons and neuroendocrine

cells [14,17], and CAPS, a phosphoinositide-binding

protein required at a pre-fusion step in dense-core granule

exocytosis [18]. The mechanism of membrane fusion per

se is an aspect that continues to be debated [19,20].

SNAREs are able to drive liposome fusion in vitro [16],

but with rather slow kinetics suggesting the requirement of

additional factors to achieve physiological membrane

fusion. Hence, many observations are in agreement with

a lipid nature of the fusion reaction and the addition of

exogenous lipids affects various biological fusion reactions

at a stage that lies downstream of SNARE complex

formation [21,22]. It is therefore likely that lipids are

essential partners for proteins in the basic fusion machi-

nery [21–24].

A convergence of biochemical and cellular studies

supports the notion that the actin cytoskeleton is coupled

to calcium-regulated exocytosis in neuronal cells although

the precise roles played by actin filaments are not yet clear

[7,12,25]. Early ultrastructural studies of actively secreting

cells, such as pancreatic or chromaffin cells, reveal a dense

subplasmalemmal actin network [26,27]. Though primarily

needed to give cells their shape, one could also predict that

this zone poses an obstacle that impedes exocytosis and

subsequent endocytosis. Hence, active cytoskeletal rear-

rangements have been shown to accompany vesicle trans-

port and fusion events in a variety of cell systems

[7,28,29]. The data from many of these experiments have

been interpreted in support of the actin-physical-barrier

model and that transient depolymerization of F-actin is

needed so vesicles can gain access to their appropriate

docking and fusion sites [7]. Most recent findings confirm

that actin filaments play a major role in the function of the

exocytotic machinery although they do not support the

simplified actin-barrier model but reveal that actin plays

probably multiple, both inhibitory and activatory, roles in

exocytosis [25,30,31].

Within the past 10 years, two families of small

GTPases, namely ADP-ribosylation factor (ARF) and

Rho proteins, have emerged as master players in coupling

actin dynamics to membrane trafficking events in eukary-

otic cells. There are six mammalian ARFs and many more

ARF-like proteins. ARF1 and its activities at the Golgi

complex have been extensively studied [32]. ARF6 is

often located at the cell periphery where it influences

membrane trafficking at the plasma membrane [33]. ARFs

are thought to act through the recruitment of cytosolic coat

proteins onto membranes to facilitate sorting and vesicle

formation, activation of lipid-modifying enzymes and

modulation of actin structures. Rho proteins are also

established regulators of actin cytoskeletal dynamics

[34]. Around 20 Rho family proteins have so far been

identified and of these, RhoA, Rac1 and Cdc42 have been

the most widely studied for their effects on actin

organization. Like most small GTPases, ARF and Rho

proteins cycle between GDP-bound, inactive and GTP-

bound, active states. Thus, they represent acutely regulated

signal transducers that respond to upstream signals

originating from membrane receptors to further propagate

them to downstream effector molecules to carry out their

functions.

Here, we will review current evidence concerning the

role of ARF and Rho proteins in providing actin structures

and lipids required for calcium-regulated exocytosis. Addi-

tionally, we shall discuss the possible role of specialized

scaffolding proteins in spatially and temporally coordinating

ARF and Rho-signalling pathways, thereby imparting to the

exocytotic machinery efficiency and site specificity [35–37].

2. ARF-dependent lipids at the exocytotic sites in

neuroendocrine cells

ARF GTPases have been linked to regulated secretion in

various cell types including melanotrophs [38], adipocytes

[39], neutrophils [40], mast cells [41], gastric parietal cells

[42] and insulin-secreting pancreatic h cells [43]. In

chromaffin and PC12 cells, ARF6 is associated with the

dense core secretory granules [44,45]. Evidence that ARF6

plays a role in chromaffin granule exocytosis came first

from studies showing that myristoylated peptides corre-

sponding in sequence to the amino-terminal end of ARF6

inhibit noradrenaline secretion when introduced in the

cytosol of permeabilized chromaffin cells [44]. These

results were then substantiated by the fact that, among

various inactive or constitutively active ARF mutants,

ARF6 is the sole protein able to modify the secretory

response in PC12 cells [45]. It is interesting to note that the

activation/inactivation cycle of ARF6 is intimately linked to

the exocytotic reaction. Subcellular fractionation experi-

ments revealed that ARF6 bound to secretory granules is in

its inactive GDP-bound state whereas active GTP-bound

ARF6 was detected only in plasma membrane-containing

fractions prepared from secretagogue-stimulated cells [45].

Accordingly, several results indicate the presence of

ARNO, a guanine nucleotide exchange factor promoting

the activation of ARF6, on the plasma membrane in

chromaffin and PC12 cells [45,46]. Moreover, expression

of a catalytically inactive ARNO mutant in PC12 cells or

introduction of anti-ARNO antibodies in the cytoplasm of

permeabilized chromaffin cells inhibits secretion to an

extent similar to the inactive GDP-bound ARF6 mutant

[46], implicating ARNO in the exocytotic pathway through

the regulation of ARF6. Since ARNO is specifically

associated with the plasma membrane [45,46], ARF6 can

be activated only following the recruitment and docking of

granules to the plasma membrane in secretagogue-stimu-

lated cells.

M.-F. Bader et al. / Biochimica et Biophysica Acta 1742 (2004) 37–49 39

The specific activation of the granule-associated ARF6

at the plasma membrane is an appealing feature as it

implies that effector activation occurs near the exocytotic

sites. Two lipid-modifying enzymes have been proposed as

the effectors mediating the function(s) of ARF6 in

regulated exocytosis, namely phosphatidylinositol 4-phos-

phate 5-kinase (PIP5-kinase) leading to the production of

phosphatidylinositol 4,5-bisphosphate (PIP2) and phospho-

lipase D (PLD) producing phosphatidic acid (PA). In PC12

cells, expression of the ARF6(N48I) mutant that cannot

activate PLD inhibits secretion, demonstrating that activa-

tion of PLD is a major downstream effect of ARF6 that

leads to exocytosis [45]. The role of PLD in secretion has

been investigated by using primary alcohols, which

interfere with the PLD-induced production of PA, and by

expressing various wild-type and mutated PLD proteins.

PLD activation was found to be an important event for

exocytosis in many secretory cell types [41,47–49]. We

previously microinjected a catalytically inactive PLD1

mutant into chromaffin cells and monitored secretion by

amperometry [48]. Analysis of individual exocytotic events

in injected cells revealed an apparent reduction in the

initial rate of release, possibly reflecting a defect in a late

step of exocytosis close to fusion pore formation and/or

expansion [48]. The precise role of PLD-produced PA at

the site of fusion remains to be elucidated. PA is a

Fig. 1. Hypothetical model for the role of PLD1-produced phosphatidic acid

hydrolyzes the membrane phospholipid phosphatidylcholine to generate the co

membranes are brought in close proximity through the formation of SNARE comp

fusion intermediates required for the formation of exocytotic fusion pores.

multifunctional lipid that has been proposed to serve as

protein attachment site, to activate selected enzymes or to

represent the starting material for the production of

additional signalling lipids. PA is also a cone-shaped lipid

and a predicted effect of its generation at the granule

docking site would be to promote a negative curvature of

the cytoplasmic plasma membrane leaflet (Fig. 1), thereby

facilitating the formation of the hemi-fusion intermediates

required for the fusion of two membranes [21–23].

Additional experiments are now required to prove or refute

any of these possibilities.

ARF6 is also a direct activator of PIP5-kinase

responsible for generating PIP2 [50], a major plasma

membrane phosphoinositide involved in many membrane

trafficking and actin rearrangement events [51–53]. In

chromaffin and PC12 cells, PIP5-kinase has been impli-

cated in the synthesis of the plasma membrane PIP2

required for the ATP-dependent priming reactions preced-

ing fusion [54,55]. Although the general role of PIP2 in

exocytosis remains to be defined, it is likely that PIP2

recruits the specific PIP2-binding proteins acting in the

exocytotic pathway, such as CAPS involved in the priming

of docked secretory granules for fusion [18]. Indeed,

ARF6 has been described as playing its role in exocytosis

through the regulation of PIP5-kinase for the synthesis of

the plasma membrane pool of PIP2 [43,56]. Surprisingly

(PA) in membrane fusion. ARF6 and/or Rac-dependent PLD1 activation

ne-shaped PA and choline. In stimulated cells, when vesicle and plasma

lexes, the local elevation of PA promotes membrane bending and the hemi-

M.-F. Bader et al. / Biochimica et Biophysica Acta 1742 (2004) 37–4940

for an ARF-effector complex [50,57], the interactions

between ARF6 and PIP5-kinase in PC12 cells were

reported to be governed by a calcium-dependent mecha-

nism involving the phosphorylation of the PIP5-kinase

rather than the guanine nucleotide-bound state of ARF6

[56]. This is an intriguing observation but it may shed

some light on the mechanism by which ARF6 could

control two distinct lipid-modifying pathways at the

exocytotic sites. For instance, ARF6-dependent activation

of the plasma membrane-bound PLD might occur through

a classical GTP-dependent switch of conformation whereas

the activation of the ARF6/PIP5-kinase complex might be

regulated by calcium signals. In other words, in the

sequence of events leading to exocytosis in stimulated

cells, recruitment and docking of secretory granules to the

plasma membrane in response to cytosolic calcium

elevation may first result in the activation of PIP5-kinase

by the granule-bound ARF6. Consequently, PIP2 formed

at the plasma membrane may contribute to the recruitment

of PIP2-dependent priming factors (CAPS for instance)

and ARNO to the granule docking sites. ARNO then

stimulates nucleotide exchange on ARF6, which in turn

activates PLD1 and the formation of PA required for the

final fusion event. Whether this is a universal mechanism

applicable to all secretory cell types remains to be

established. Nevertheless, these observations place ARF6

Fig. 2. Effect of GTP-loaded RhoA and Cdc42 on peripheral actin and secreta

Cdc42L61 and RhoAV14 stimulate the formation of actin filaments in the periphery

HA-RhoAV14 were stimulated for 10 min with 59 mM K+, fixed and stained with

indicate non-transfected cells displaying a classical disruption of cortical actin in re

and RhoAV14 on GH release from PC12 cells. Cells co-transfected with GFP-Cdc4

10 min in calcium-free Locke’s solution (basal release) or stimulated for 10 min w

basal release from the K+-evoked release. Basal release ranged from 5% to 8%. As

are given as mean valuesFS.E. (n=3).

at a strategic position to provide adequate lipids for the

exocytotic machinery.

3. Rho GTPases and the remodelling of actin in the

course of exocytosis

In view of their well-established effects on actin

organization, Rho proteins appear as ideal candidates to

provide actin structures required for exocytosis. In

chromaffin and PC12 cells, RhoA is associated to

secretory granules whereas Rac1 and Cdc42 are found

in the subplasmalemmal region [58]. Using Clostridium

botulinum exo-enzyme C3 (an ADP-ribosyltransferase that

specifically inactivates Rho), we found that RhoA is a

downstream partner of the granule-associated Go protein,

an heterotrimeric GTPase that regulates the ATP-depend-

ent priming of secretory granules in chromaffin cells

[59,60]. Activation of RhoA through the stimulation of

Go stabilized the cortical actin cytoskeleton visualized

with rhodamine-conjugated phalloidin in secretagogue-

stimulated cells [60]. Similarly, expression of an active

GTP-bound RhoA mutant in PC12 cells enhanced the

rhodamine-phalloidin fluorescence in the periphery of

stimulated-cells, suggesting the formation of actin fila-

ments (Fig. 2A). GTP-bound Cdc42 had a comparable

gogue-evoked growth hormone (GH) secretion in PC12 cells. (A) Active

of secretagogue-stimulated cells. PC12 cells expressing GFP-Cdc42L61 or

rhodamine-conjugated phalloidin to visualize actin filaments. The asterisks

sponse to K+-stimulation. Bar, 5 Am. (B) Effect of dominant active Cdc42L61

2L61 or HA-RhoAV14 along with a plasmid encoding GH were incubated for

ith 59 mM K+. The net secretory response was obtained by subtracting the

control, GH release was measured in cells expressing an empty vector. Data

M.-F. Bader et al. / Biochimica et Biophysica Acta 1742 (2004) 37–49 41

effect on peripheral actin in PC12 cells: it enhanced the

rhodamine-phalloidin staining in stimulated cells, suggest-

ing the de novo formation of actin filaments in the

subplasmalemmal region (Fig. 2A). Yet, these two

GTPases produced opposite effects on PC12 cell exocy-

tosis: expression of the dominant active RhoA mutant

inhibited secretion whereas the active Cdc42 mutant

significantly stimulated it (Fig. 2B). This observation

suggests that these two members of the Rho family form

distinct actin structures unresolved at the optical level,

and can play negative and positive roles in the exocytotic

machinery. In other words, the actin cortex, classically

viewed as a barrier that hinders the movements of

granules to the plasma membrane, may as well play a

secondary active role to drive the late steps of the

exocytotic process. In line with this idea, agents that

completely depolymerize actin filaments (like latrunculin

B or C. botulinum C2 toxin) increase secretion at low

concentrations and progressively inhibit it at higher doses

as reported in chromaffin and PC12 cells [61,62], or

completely inhibit the exocytotic response in pancreatic

cells [63,64] and in mast cells [65]. In the latter, the

appearance of newly synthesized actin filaments simulta-

neously with the disassembly of the cortical network has

been observed during degranulation [66]. This dual role

of actin was also illustrated in PC12 cells using

evanescent wave microscopy, in which actin was found

to both hinder and mediate movements of GFP-labeled

secretory granules in the subplasmalemmal region [30].

Thus, it is tempting to imagine that the actin remodelling

needed for exocytosis may include a rearrangement to

form structures that provide either tracks permitting

docking of secretory granules to appropriate sites or a

force which spatially constrains components of the

exocytotic machinery.

Although the precise role of actin and the mechanisms

underlying its dynamic regulation remain to be dissected,

the findings that Rho proteins play a role in secretion

provide a potential link to the pathway coupling actin

reorganization to exocytosis. In chromaffin cells, experi-

ments designed to identify the effectors by which the

secretory granule-associated RhoA protein inhibits exocy-

tosis and affects the cortical actin cytoskeleton led us to

propose that RhoA exerts its control on actin dynamics

through a phosphatidylinositol 4-kinase (PI 4-kinase) also

associated with secretory granules [67]. PI 4-kinase

produces phosphatidylinositol 4-phosphate, a phosphoino-

sitide that can be subsequently phosphorylated by the

PIP5-kinase to generate granule-bound PIP2. PIP2 inter-

acts with and regulates numerous cytoskeletal proteins

[68]. Local production of PIP2 on membranes has also

been shown to initiate actin nucleation and regulate

membrane–cytoskeleton interactions [69,70]. Thus, activa-

tion of the RhoA/PI 4-kinase pathway and formation of

PIP2 residing on the granule membrane may promote the

formation of granule-associated actin filaments and/or

contribute to the stabilization of the peripheral actin

barrier by decreasing the severing activity of scinderin

known to be involved in the actin reorganization under-

lying exocytosis [71]. In other words, PIP2 produced at

the surface of secretory granules may be involved in

maintaining a standby position of the exocytotic machi-

nery whereas the PIP2 generated at the plasma membrane

may define the active sites of exocytosis. This rather

attractive hypothesis remains, however, to be explored

experimentally.

We recently investigated the molecular cascade by which

Cdc42 is able to enhance actin polymerization and secretion

in PC12 cells [31]. Several functionally distinct Cdc42

effectors have been identified [72]. Among them, the neural

Wiskott–Aldrich syndrome protein (N-WASP) links Cdc42

to actin polymerization though the actin-related protein-2/3

(Arp2/3) complex, which promotes actin nucleation and

polymerization [73,74]. We found that Cdc42 is activated at

the plasma membrane in secretagogue-stimulated PC12

cells [31]. Expression of a constitutively active Cdc42

mutant increased the secretory response, recruited cytosolic

N-WASP to the plasma membrane, and enhanced actin

polymerization in the subplasmalemmal region [31] (Fig. 2).

Moreover, expression of N-WASP produced a stimulatory

effect on secretion that was comparable to the effect

obtained with active Cdc42, by a mechanism dependent

on its ability to induce actin polymerization at the cell

periphery. Co-expression of a N-WASP mutant unable to

polymerize actin completely abolished the stimulatory effect

of GTP-bound Cdc42 on secretion, demonstrating that N-

WASP lies downstream of Cdc42 in the exocytotic pathway

[31]. Finally, we observed that Arp2/3 is associated with

secretory granules and that it accompanies granules to the

docking sites at the plasma membrane upon cell activation

[31]. All together, our results provide for the first time a

molecular support for the de novo formation of actin

filaments in the course of exocytosis. Secretagogue-evoked

stimulation induces the sequential ordering of Cdc42, N-

WASP and Arp2/3 at the interface between granules and the

plasma membrane, thereby providing a way to specifically

target local actin filament polymerization at the granule

docking sites. What might be the role of these newly formed

actin filaments at the sites of exocytosis? In yeast, Cdc42p/

WASP triggers the formation of actin filaments that

accumulate on docked vacuoles and seem to be involved

in the terminal steps leading to homotypic membrane fusion

[75]. In Xenopus eggs, Cdc42/N-WASP-induced actin

filaments formed at the surface of exocytosing cortical

granules are able to drive closure of the exocytotic fusion

pore [76]. Recent work has established that for dense core

granules in endocrine cells, the fusion pore opening–closing

time can be modulated by various signalling pathways,

allowing a control of the amount of hormones released per

granule [77,78]. Thus, the possibility that actin filaments

formed at the granule docking site regulate expansion and/or

closure of the fusion pore, thereby providing a molecular

M.-F. Bader et al. / Biochimica et Biophysica Acta 1742 (2004) 37–4942

basis for control of quantal release, will be an interesting

future issue.

In summary, actin plays a composite function in the

exocytotic process in neuroendocrine cells. Both RhoA and

Cdc42 emerge as important signalling molecules for

regulating granule exocytosis and the accompanying actin

cytoskeletal rearrangements. Through their ability to interact

with a number of downstream targets, RhoA and Cdc42 can

coordinate the actin-based processes to the successive

molecular tasks required to drive a secretory granule to

membrane fusion.

4. ARF, Rho and their downstream partners in

neurotransmitter release

Evoked release of neurotransmitter in neurons has

many homologies with hormone secretion performed by

neuroendocrine cells; in both cell types, secretion arises

from an entry of Ca2+ that in turn triggers the fusion of a

sub-population of primed vesicles docked at the plasma

membrane [79]. In general, the localization and functions

of the molecular components participating to the release

machinery are conserved between neurons and neuro-

endocrine cell. However, neurons stand out from other

secretory cells by an extremely fast kinetic of fusion and

by their ability to sustain secretion over a wide range of

frequency. To be able to maintain neurotransmitter release

during prolonged synaptic activity, neurons must rapidly

refill the releasable pool of vesicles. As a consequence,

the recycling machinery and the movement of vesicles

inside the nerve terminals need to be tightly coupled to

the exocytotic process [80]. Because ARF and Rho-

related proteins are well-known regulators of various

organelle trafficking processes, they have been involved

in neurotransmitter release and pre-synaptic plasticity,

both at the stage of exocytosis and in the endocytotic

counterpart that underlie the cycling of synaptic vesicles

in nerve endings.

Among the ARF family, ARF6 is the sole member that

has been found in nerve terminals and implicated in

synaptic vesicle trafficking and neurotransmission. In

cortical neurons, GTP-bound ARF6 was found concen-

trated at synapses [81]. Subcellular fractionation of brain

synaptosomes indicated that ARF6 is present on the

plasma membrane and associated to clathrin-coated

vesicles [81], suggesting a role for ARF6 in synaptic

vesicle endocytosis. This hypothesis was supported by

experiments showing that the constitutively active GTP-

bound ARF6 mutant increases the binding of proteins of

the endocytotic machinery, like AP-2 and clathrin, on

purified synaptosomal membranes [81]. Furthermore, pull-

down experiments from rat brain extracts revealed that

GTP-bound ARF6 interacts with the PIP5-kinase Ig, an

isoform highly enriched in brain [82]. This interaction

leads to the stimulation of PIP5-kinase Ig, and in turn, to

the formation of PIP2 at the presynaptic plasma mem-

brane. Since a large body of data implicates PIP2 in the

regulation of clathrin mediated endocytosis [83], it has

been suggested that ARF6 regulates synaptic vesicle

trafficking and neurotransmission by promoting the

budding of nascent endocytotic vesicles via the formation

of PIP2 micro-domains at the pre-synaptic endocytotic

sites. Several arguments support also the participation of

ARF6 in synaptic vesicle exocytosis. For instance, in

Xenopus spinal neurons, microinjection of a guanine

nucleotide exchange factor for ARF proteins (mSec7-1)

increases the frequency of spontaneous release [84].

mSec7-1 was found to enhance the amplitude of evoked

responses without affecting the size of the quantum, and

it increased the depression rate of synaptic depression

induced by repetitive stimulation [84]. From these data, it

has been proposed that mSec7-1, most likely by activat-

ing ARF6, facilitates the probability of a mature vesicle

to fuse in response to Ca2+ entry and/or increases the size

of the readily releasable pool of synaptic vesicles. In

neurons, Munc 13-1, implicated in the priming of

synaptic vesicles, and synaptotagmin, supposed to be

the Ca2+-sensor of exocytosis, are both potentially

regulated by phosphoinositides [85–87]. Thus, ARF6

may modulate neurotransmitter release by inducing PIP2

micro-domains near the release sites and thereby regulat-

ing synaptotagmin, Munc 13-1 or any other pre-synaptic

phosphoinositide-binding protein required for synaptic

vesicle exocytosis.

Several members of the Rho family have been found in

pre-synaptic nerve terminals. These include RhoA, RhoB

and Rac1 present in cytosolic and membrane-bound

fractions isolated from rat brain synaptosomes [88]. Using

highly purified synaptic vesicles, we demonstrated that

only Rac1 is directly associated with these organelles [88].

The pre-synaptic functions of Rho-related proteins have

been probed by using various clostridial toxins that

specifically and differentially inactivate members of the

Rho family by covalently fixing a sugar or a nucleotide

near the effector loop [89]. Exoenzyme C3 from C.

botulinum, toxin B from C. perfringens and lethal toxin

from C. sordellii are all inhibitors of neurotransmitter

release when injected in Aplysia cholinergic neurons [88].

The most potent effects were obtained with lethal toxin,

which inactivates various Ras-related GTPases and Rac

[90]. In Aplysia neurons, lethal toxin acts with an

efficiency that is comparable to tetanus toxin which is

known to block neurotransmitter release through the

proteolytic cleavage of the vesicle-associated SNARE

protein VAMP [91], indicating that lethal toxin blocks a

crucial step of the synaptic vesicle cycle. The use of

several variants of lethal toxin that affect a different

spectrum of small GTPases has led to the idea that Rac

inactivation accounts for the lethal toxin-induced inhibition

of neurotransmitter release [92]. Interestingly, the blockade

of acetylcholine release induced by lethal toxin can be

M.-F. Bader et al. / Biochimica et Biophysica Acta 1742 (2004) 37–49 43

fully reversed in about 1 s by applying sustained

stimulations [88]. This time scale is smaller than the

replenishment kinetics of the release sites [93] and the

residency time of synaptic vesicles on the plasma

membrane before fusion estimated around 2–5 s [94,95].

Therefore, recovery from lethal toxin-induced blockade

involves most likely synaptic vesicles that are recruited

from a population that is already docked at the plasma

membrane. By inference, this observation suggests that

Rac is involved in a post-docking step in neuronal

exocytosis. In line with this idea, analysis of the variance

of the post-synaptic response amplitudes, which allows to

determine several parameters of quantal release [96–99],

indicated that the inhibitory action exerted by lethal toxin

on neurotransmitter release was neither due to an alteration

of the release probability nor to a reduction in the quantal

size [92]. The only consequence of lethal toxin action was

the reduction of the apparent number of active release sites

[92], in agreement with a role of Rac in a stage close to the

final fusion event.

The implication of PLD1 in neuroendocrine cell exocy-

tosis led us to probe the idea that PLD1 fulfills a role in

neurotransmitter release. PLD1 is present in membrane

fractions prepared from rat brain synaptosomes and it co-

localizes with synaptophysin, a marker of synaptic vesicles,

in cultured cerebellar granule cells [99]. Thus, PLD1 is

present in nerve terminals in areas specialized in neuro-

transmitter release. To determine the possible involvement

of PLD1 in neurotransmitter release, a catalytically inactive

PLD1 mutated protein was injected into Aplysia cholinergic

neurons. This mutant was found to be a potent inhibitor of

acetylcholine release [99] and, as revealed by analyzing the

fluctuations in amplitude of postsynaptic responses, it

affected the number of release sites. Thus, by analogy with

the function proposed in hormonal secretion, PLD1 may

induce lipid modifications in the pre-synaptic plasma

membrane required for fusion of synaptic vesicles and

neurotransmitter release (Fig. 1). The mechanisms regulat-

ing PLD1 activity in neuronal exocytosis remain to be

explored. PLD1 is known being regulated by ARF and Rho

proteins [100,101] but the possible functional links between

ARF6, Rac and PLD1 in neurons have not yet been

investigated. It is, however, interesting to note that lethal

toxin, which inactivates Rac, and the inactive PLD1 mutant

similarly inhibit acetylcholine release from Aplysia neurons

by reducing the number of functional release sites without

affecting the probability of release or the size of the

quantum [92,99]. Thus, the synaptic vesicle-associated

Rac might well be an activator of the plasma membrane-

bound PLD1 in the cascade leading to neurotransmitter

release.

The role of Rac in neurotransmitter release raises also

the question of the actin cytoskeleton as a downstream

effector of Rac in the neuronal exocytotic pathway. The

implication of actin in trafficking and fusion of synaptic

vesicles is still subject to debates [12]. Depending on the

type and/or the age of the synapse studied, actin has been

alternatively proposed to have no function at all [102], to

be positively or negatively involved in synaptic vesicle

mobilization before fusion [103–108] or to act after fusion

in the recycling of empty vesicles [109]. These contra-

dictory results may reflect the diversity in function, size

and morphology of the synapses studied. To date, there is

no direct evidence that actin filaments are required for the

fusion of synaptic vesicle to occur since drugs that

depolymerize (latrunculin, cytochalasin) or stabilize (phal-

loidin, jasplakinolide) actin have little or no effect on the

amount of neurotransmitter released per stimuli [103–

108]. Thus, because the inactivation of Rac leads to a

complete and fast inhibition of neurotransmitter release

[88,92], it is rather improbable that actin filaments serve

as downstream effectors of Rac in controlling the number

of pre-synaptic release sites. On the other hand, one

cannot completely exclude that a remodeling of actin

filaments controlled by Rac or any other member of the

Rho family participates in a fine regulation of synaptic

vesicle movement and trafficking inside the nerve

terminal. This sort of control cannot be observed in

neurons exposed to lethal toxin as the toxin completely

blocks the late fusion stages in neurotransmitter release. In

this context, it is interesting to note that N-WASP, the

downstream partner of Cdc42 in remodeling actin for

large dense-core granule exocytosis [31], has been

detected in pre-synaptic nerve terminals after subcellular

fractionation of brain synaptosomes [110]. Nerve terminals

are also enriched in LIM-kinase [111], a serine/threonine

kinase that blocks actin depolymerization upon Rac

activation [112,113]. At the postsynaptic site, a long-

lasting increase in F-actin content is observed in dendritic

spines after induction of Long-Term Potentiation (LTP)

[114]. Since LTP is impaired in hippocampal neurons of

the LIM-kinase1 knock-out mice [115] and in neurons

exposed to lethal toxin [116], LTP is likely to involve

actin filaments and the regulation of their dynamics via

the Rac/LIM-kinase pathway. The occurrence of a similar

molecular cascade at the pre-synaptic site is an interesting

line for future investigations.

5. Coupling ARF and Rho signalling pathways at the

sites of exocytosis

The functional characteristics of the sites of exocytosis

that ensure tethering of vesicles to the appropriate active

zones at the plasma membrane, as well as organization of

the exocytotic machinery for rapid and efficient release,

remain poorly understood. Spatial and temporal coordina-

tion of the components involved in exocytosis may be

achieved by dynamic macromolecular complexes, which are

based on protein–protein and/or protein–lipid interactions.

SNARE complexes in imparting site specificity seem

incompetent given their intracellular localization that is

M.-F. Bader et al. / Biochimica et Biophysica Acta 1742 (2004) 37–4944

not specifically restricted to zones of active exocytosis. In

neurons, several large scaffolding proteins specifically

localized in the presynaptic active zone have been identi-

fied. These proteins are typically composed of multiple

protein-binding domains that are able to link networks of

synaptic constituents and, in some cases, the underlying

cytoskeleton [117,118]. Given the role played by Rho and

ARF proteins in different stages of calcium-evoked exocy-

tosis, molecules that are able to couple Rho and ARF

signalling pathways at the granule docking and fusion sites

are also expected to assemble exocytotic sites into effective

functional units.

Scribble is a cytoplasmic protein, first characterized in

epithelial cells where it plays an important role in

maintaining apical polarity and in tumour suppression

[119]. Scribble contains several consensus binding motifs

including PDZ domains which have been described as

protein-interacting modules that play a central role in

organizing diverse signalling assemblies [120,121]. At

synaptic junctions, PDZ domain-containing proteins are

thought to be responsible for the organized assembly of

components involved neurotransmission and synaptic

plasticity [122]. Accordingly, a role for Scribble in

sustaining synaptic vesicle concentrations at their sites of

release during high-frequency stimulation has been

reported [123]. Interestingly, Scribble is able to directly

interact with h-PIX, a nucleotide exchange factor for Rac1

and Cdc42, and with GIT1, a GTPase activating protein

that inactivates ARF6 [124]. In PC12 cells, Scribble

present at the plasma membrane recruits cytosolic h-PIXand GIT1 to the cell periphery upon secretagogue-induced

stimulation [124]. Expression of mutated Scribble proteins

that remain in the cytosol and thereby sequester h-PIXaway from the plasma membrane significantly reduce

secretion. Moreover, h-PIX itself increases secretion

whereas a catalytically inactive mutant defective in

nucleotide exchange activity reduces it, implicating h-PIX in the exocytotic pathway in PC12 cells [124]. Taken

together, these results reveal that Scribble may act as a

membrane anchor for a h-PIX/GIT1 complex devoted to

regulated exocytosis. In other words, Scribble is a prime

candidate to define a spatial landmark in the plasma

membrane where Rho and ARF signalling pathways

converge to the sites of vesicle/granule docking and

fusion.

Piccolo is another potential applicant to fulfil the

integration of Rho and ARF signals. Piccolo is a large

(~530 kDa) protein that is spatially restricted to active

zones within nerve terminals, together with RIM1,

Munc13-1 and structurally related proteins such as

Bassoon [125,126]. RIM1 was originally identified as a

target protein of the small G protein Rab3A implicated in

the docking of synaptic vesicles [127] whereas Munc13-1

that binds both RIM1 and the SNARE protein syntaxin

regulates the priming of synaptic vesicles [128]. These

proteins associate into large molecular complexes to form

with the actin cytoskeleton a meshwork thought to

functionally organize the site of neurotransmitter release

[129]. In neuroendocrine cells, the role of Piccolo and

associated proteins in determining the sites of exocytosis is

less well understood. However, Piccolo is expressed in

PC12 cells and pancreatic h cells and recent studies

suggest that it might serve as a calcium sensor in insulin

secretion [130]. Interestingly, Piccolo directly interacts

with GIT1 through a small region that is not conserved

in the closely related protein Bassoon [131]. In cultured

neurons, Piccolo and GIT1 co-localize at synaptic sites

[131]. In brain, Piccolo forms a complex with GIT1 and

several GIT-associated proteins including hPIX [131].

From these observations, it is tempting to speculate that

the convergence of ARF and Rho signalling pathways to

the sites of exocytosis is based on a Piccolo-induced

protein network. The fact that Piccolo is a C2 domain-

containing protein with putative calcium-binding sites

might provide to the scaffold the calcium-sensitivity

expected for the spatial and temporal regulation of

exocytosis in neurons and neuroendocrine cells.

6. Conclusion

Chromaffin granule exocytosis differs in many phys-

iological aspects from neuronal synaptic vesicle exocy-

tosis. While different mechanisms are certainly operating

to suit the physiologically different cellular functions, it is

now clear that neurons and neuroendocrine cells use

similar proteins to mediate and regulate the docking and

fusion of synaptic vesicles and granules with the plasma

membrane. ARF and Rho GTPase signalling occurs in

neurons and neuroendocrine cells and they are likely to

represent additional components of the conserved machi-

nery for hormone and transmitter release (Fig. 3). Intimate

links between Rho and ARF GTPases have emerged

along with the evidence that many regulators and

effectors of ARF and Rho families are often intertwined.

These functional relationships between ARF and Rho

proteins could provide neurosecretory cells with a way of

tightly coordinating vesicular traffic and fusion under the

plasma membrane with the accompanying actin dynamics.

Spatial control of exocytosis requires also molecular

mechanisms that identify localized sites of membrane

fusion, and link them to the intracellular machinery of

vesicle targeting and fusion. The scaffolding proteins

described to interact with both ARF- and Rho-regulating

proteins are attractive candidates to act as molecular

devices that integrate the many components of the

exocytotic pathway into a finely tuned machinery for

secretion. We have to keep in mind, however, that most

of our models and paradigms derive from studies in tissue

culture cells. The challenge in the future will be to

determine whether regulated secretion in living organisms

obeys similar rules and mechanisms.

Fig. 3. Hypothetical model for the role of ARF6 and Rho GTPases in chromaffin granule (A) and neuronal synaptic vesicle (B) exocytosis. (A) In resting

chromaffin cells, secretory granule-associated RhoA contributes to the stabilization of cortical actin. Stimulation with a secretagogue triggers the recruitment

and docking of secretory granules to the plasma membrane. Docking allows the formation of Cdc42-dependent actin structures required for the late stages of

exocytosis. Granule docking permits also the activation of ARF6 by ARNO, which in turn stimulates PLD1 to produce membrane-localized PA. The local

elevation of PA at the granule docking site facilitates the formation of the exocytotic fusion pore. (B) In synaptic terminals, vesicle-associated Rac1 activates

membrane-bound PLD1 to render pre-synaptic release sites competent for fusion. ARF6 regulates neurotransmission by stimulating the formation of PIP2

micro-domains at the pre-synaptic membrane, thereby priming vesicles to fuse or promoting the formation of nascent vesicles.

M.-F. Bader et al. / Biochimica et Biophysica Acta 1742 (2004) 37–49 45

M.-F. Bader et al. / Biochimica et Biophysica Acta 1742 (2004) 37–4946

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