MECHANISMS OF EXOCYTOSIS

Calcium-regulated Exocytosis in
Neuroendocrine Cells: Intersectin-1L
Stimulates Actin Polymerization and
Exocytosis by Activating Cdc42
Fanny Momboisse, Stephane Ory, Valerie Calco,
Magali Malacombe, Marie-France Bader,
and Stephane Gasman
Departement Neurotransmission et Secretion Neuroendocrine, Institut des Neurosciences
Cellulaires et Integratives, Centre National de la Recherche Scientifique et Universite
Louis Pasteur, Strasbourg, France

Actin cytoskeleton remodeling is a critical step of regulated exocytosis in many secretory cell types, including neuroendocrine cells. While the classical model considers the
cortical actin network as a physical barrier preventing the uncontrolled recruitment of
secretory granules to the plasma membrane docking sites, recent evidence supports
the idea that actin polymerization also plays a more active role in the late stages of
exocytosis. However, the molecular machinery underlying this positive function of actin
in the course of exocytosis remains largely unknown. Here, we propose that the neuronal guanine nucleotide exchange factor, intersectin-1L, activates the GTPase Cdc42,
which in turn provides de novo actin filaments that are important for calcium-regulated
exocytosis in PC12 cells.
Key words: actin; Cdc42; exocytosis; intersectin; PC12 cells

Introduction
Calcium-regulated exocytosis requires a fine
and rapid remodeling of peripheral actin filaments. For a long time, the classical view of
actin rearrangements in most secretory cells
were based on the actin-physical-barrier model
whereby local disassembly of the cortical actin
network permitted secretory granules to gain
access to exocytotic sites at the plasma membrane.1 However, we and others have recently
questioned this model by describing that actin
polymerization may also play an important active role in the final stages of exocytosis.1
Address for correspondence: Stephane Gasman, Departement Neurotransmission et Secretion Neuroendocrine, Institut des Neurosciences
Cellulaires et Integratives (UMR 7168), Centre National de la Recherche
Scientifique et Universite Louis Pasteur, 5 rue Blaise Pascal, 67084 Strasbourg, France. Voice: +33 388 45 67 12; fax: +33 388 60 16 64.
gasman@neurochem.u-strasbg.fr

Over the past years, we have focused our efforts on elucidating the functional importance
of Rho GTPases, which are well-known regulators of actin cytoskeleton dynamics in a
wide range of membrane trafficking processes.2
Using chromaffin and PC12 cells, we found
that activation of Cdc42 stimulates secretion
by inducing the formation of actin structures at
the interface between granules and the plasma
membrane.3 In addition, we demonstrated that
intersectin-1L, a Rho guanine nucleotide exchange factor (GEF) from the Dbl family, modulates hormone release in PC12 cells through
the activation of Cdc42.4 However, the participation of intersectin-1L in the actin remodeling
events accompanying the exocytotic process remained to be investigated.
Intersectin-1L is mainly expressed in neuronal cells and contains, in addition to the Dbl
homology domain (DH)-plekstrin homology

Mechanisms of Exocytosis: Ann. N.Y. Acad. Sci. 1152: 209214 (2009).

C 2009 New York Academy of Sciences.
doi: 10.1111/j.1749-6632.2008.03998.x 

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Annals of the New York Academy of Sciences

domain (PH) region that catalyzes nucleotide

exchange on Rho GTPases, two N-terminal
Eps15 homology domains (EH1 and EH2), a
central coiled-coil region, five Src homology
3 (SH3) domains, and a carboxy-terminal C2
domain.5 Because of its capacity to promote
actin polymerization6,7 and to interact with
various endocytic proteins, such as dynamin,
Eps15, synaptojanin, and stonin,810 intersectin
has been essentially described as a scaffolding
protein that couples the assembly of endocyticprotein complexes to the actin network reorganization at the sites of clathrin-mediated
endocytosis. In the present study, we demonstrate the importance of intersectin-1L in the
actin cytoskeletal rearrangements required for
exocytosis in neuroendocrine cells. We also
show that the C-terminal part of intersectin1L, containing DH, PH, and the C2 domain,
stimulates exocytosis by promoting actin polymerization in the cell periphery through the
activation of Cdc42.
Results and Discussion
In view of their well-established function on
actin organization, Rho GTPases are ideal candidates to promote actin structures required for
calcium-induced exocytosis in neuroendocrine
cells. Using an interference RNA strategy, we
previously demonstrated that intersectin-1L is
the GEF responsible for the activation of Cdc42
that occurs during hormone release in PC12
cells.4 To directly probe the importance of
the GEF domain of intersectin-1L for exocytosis, we compared the secretory response of
PC12 cells expressing intersectin-1S (a shorter
splice variant lacking DH-PH and C2 domains),5 intersectin-1L, or the isolated tail region of intersectin1-L containing the DH, PH,
and C2 domains (Fig. 1A). Using the growth
hormone (GH) release assay,4 we observed
that expression of the tail DH-PH-C2 construct enhanced the K+ -evoked secretion by
approximately 40%, whereas the full-length
intersectin-1S had no effect (Fig. 1B). These

Figure 1. The DH-PH-C2 domain of intersectin1L stimulates exocytosis in PC12 cells. (A) Schematic
representation of the intersectin-1 constructs depicting the position of the various functional domains.
EH, Eps15 homology domain; DH, Dbl homology
domain; PH, pleckstrin homology domain. (B) PC12
cells were transfected with the indicated intersectin-1
constructs along with the plasmid encoding growth
hormone (GH). At 48 h posttransfection, cells were
maintained under resting conditions in Lockes solution for 10 min or stimulated for 10 min with
59 mM K+ (Lockes containing 59 mmol/L KCl and
85 mmol/L NaCl) and GH release assay was performed as described.4 GH release is expressed as
the percentage of total GH present in the cells before
the 10-min stimulation period. GH release detected
in resting cells (approximately10%) was unchanged
and was subtracted from the GH release evoked by
59 mmol/L K+ to obtain the net secretory response.
Data are given as the mean values SEM (n =
3), P < 0.001, NS (not significant) compared to
control cells (ANOVA using Minitab statistical software).

results are in line with a role of the guanine

nucleotide exchange activity of intersectin-1L
in its regulation of exocytosis. However, we
also observed that expression of intersectin1L full length did not affect exocytosis. One
possible explanation is that intersectin-1L remained inactive because it is maintained in an
auto-inhibited conformation by intramolecular bonds between the SH3 and the DH domain, which would block Cdc42 binding.11 In

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Figure 2. The tail DH-PH-C2 domain of intersectin-1L stimulates actin filament formation
at the cell periphery. PC12 cells expressing green fluorescent protein (GFP) (control) or the
GFP-tagged tail DH-PH-C2 were maintained in Lockes solution (resting) or stimulated for
10 min with 59 mmol/L K+ , fixed, and processed for actin-filament staining as previously
described.3 (A) Confocal images representing the distribution of GFP-DH-PH-C2 and actin
filaments visualized with rhodamine-conjugated phalloidin. Asterisks indicate untransfected
cells. (B) Semiquantitative analysis of the F-actin content in cells transfected with the indicated
plasmid. The amount of F-actin present in PC12 cells was measured and expressed as average
fluorescence intensity normalized to the corresponding surface area and divided by the total
surface of each cell.3 Data are given as the mean values SEM (n = 25 cells) and expressed
in arbitrary units (AU). P < 0.001, NS (not significant) compared to control cells (ANOVA
using Minitab statistical software).

agreement with this possibility, the isolated tail

region of intersectin-1L has been previously
shown to be more efficient than the full-length
protein at both activating Cdc42 and stimulating the formation of filopodia.6
Aside from Cdc42, intersectin directly interacts with various proteins that have been functionally linked to actin organization, such as
Eps15, N-WASP, and dynamin.6,810 N-WASP
and the yeast homologue of Eps15, Pan1, can
promote actin assembly through the activation of the Arp2/3 complex.12,13 In addition
to the established role of dynamin in fission
of clathrin-coated pits, it has been shown to
function in the formation of actin comets tails,
which trigger endosomal compartment transport.14,15 Moreover, dynamin interacts with
cortactin, a protein that has been also shown to
activate the nucleating activity of the Arp2/3
complex.16 Interestingly, we have previously
demonstrated the presence of both dynamin2 and the Arp2/3 complex at the surface of

secretory granules in chromaffin and PC12

cells.3,17 Thus, given its capacity to promote
actin polymerization in various cells and to
mediate secretagogue-induced Cdc42 activation in PC12 cells, it is tempting to propose
that intersectin-1L could be a component of
the exocytotic machinery, which may be linked
either directly or via Cdc42 to the actin rearrangements needed for exocytosis. To test
this hypothesis, we expressed the green fluorescent protein (GFP)-tagged tail DH-PH-C2
domain of intersectin-1L and examined its effect on the cortical actin network organization
in PC12 cells. In chromaffin and PC12 cells, the
majority of actin filaments are concentrated in
the subplasmalemmal region, forming a continuous cortical actin network that is partially
disassembled upon activation of exocytosis.18
Accordingly, phalloidin staining revealed intact
and disrupted actin networks in resting cells
and stimulated cells, respectively (Fig. 2A, B).
In contrast, expression of the DH-PH-C2

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Figure 3. Intersectin 1L-mediated actin remodeling during exocytosis requires a functional

interaction with Cdc42. (A) The tail DH-PH-C2 domain of intersectin-1L coprecipitates with
Cdc42 in PC12 cells. Cell lysates from PC12 cells transfected with the pCB6-GFP-DH-PHC2 plasmid were incubated with anti-GFP antibodies and then with A/G Sepharose beads.
Immunoprecipited proteins (IP) or initial cell lysate extract (total) were separated by gel electrophoresis and transferred to nitrocellulose. Immunoblots were probed with anti-Cdc42 and
anti-GFP antibodies. Bands at 50 and 25 KDa correspond to the heavy and light chains of
immunoglobulin (IgG), respectively. (B) Cells were cotransfected with pCB6-GFP (control) or
pCB6-GFP-DH-PH-C2 (DH-PH-C2) along with the pSuper vector either empty (empty vector) or
encoding the small hairpin (sh)RNA for Cdc42 [Cdc42 small interfering (si)RNA]. Cells were
then maintained in resting condition or stimulated for 10 min with 59 mmol/L K+ (K+ stimulated), fixed, and stained with rhodamine-conjugated phalloidin. Confocal image analysis of
the F-actin content was then performed as previously described.3 Data are given as the mean
values SEM (n = 25 cells) and expressed as arbitrary units (AU). P < 0.001 (ANOVA
using Minitab statistical software).

domain enhanced the phalloidin fluorescence

in the cell periphery in stimulated cells, indicating that this domain triggers actin polymerization in response to secretagogue stimulation
(Fig. 2A, B). This effect is similar to what we
observed in cells transfected with the GTPasedeficient Cdc42Q61L mutant.3
To further probe the possibility that
intersectin-1L regulates actin organization
through the activation of Cdc42, we next examined whether the tail DH-PH-C2 region
of intersectin-1L interacts with Cdc42. Endogenous Cdc42 could be precipitated with
intersectin-1L DH-PH-C2 from PC12 cells expressing a GFP-tagged DH-PH-C2 construct
(Fig. 3A), in line with a possible interaction
between intersectin-1L and Cdc42. In an attempt to assess the role of Cdc42 in the stim-

ulatory effect of intersectin-1L on actin filament polymerization, Cdc42 expression was silenced by interference RNA. PC12 cells were
cotransfected with the plasmid containing the
DH-PH-C2 region and either an empty vector
or a plasmid coding for the small interfering
(si)RNA against Cdc42. Interestingly, we found
that expression of the tail region of intersectin1L, which has nucleotide exchange activity, did
not significantly modify the amount of peripheral F-actin in cells depleted of endogenous
Cdc42 (Fig. 3B). These data taken together with
our previous results3,4 indicate a causal relationship between the intersectin-1L-mediated
activation of Cdc42 and the de novo formation
of actin filaments during the exocytotic process
in neuroendocrine cells. The role of these newly
formed actin filaments at the site of exocytosis

Momboisse et al.: Intersectin and Actin Dynamics in Regulated Exocytosis

remains an open question. One particularly interesting feature of intersectin-1L is its bifunctional aspect, which allows both the regulation
of the actin cytoskeleton and recruitment of
the endocytic machinery. Hence, the actin cytoskeleton plays a key role in various types of
endocytic processes.19 To maintain a constant
cell surface area and to permit secretory granule recycling, regulated exocytosis is followed by
compensatory endocytosis.20 Whereas synaptic
vesicle recycling has been extensively explored,
little is known concerning the molecular basis for large dense-core granule endocytosis after exocytosis. The few studies performed on
neuroendocrine cells suggest that there is a fast
spatial and temporal coupling of compensatory
endocytosis with exocytosis.2022 Intersectin-1L
is associated with exocytotic sites in neuroendocrine cells where it activates Cdc424 and,
as shown here, promotes actin polymerization
upon cell stimulation. Therefore it is tempting
to speculate that intersectin-1L couples the activation of Cdc42 and the subsequent actin remodeling necessary for hormone release to the
recruitment of the endocytotic machinery for
granule membrane retrieval. Further research
is required to establish the validity of this rather
attractive hypothesis.
Acknowledgments

We wish to thank Dr. Nancy Grant for critical reading of the manuscript and Dr. Peter
S. McPherson (McGill University, Montreal,
Canada) for his fruitful collaboration and for
providing us the intersectin constructs. This
work was supported by a Human Frontier Science Program (HFSP) grant (RGY40-2003C)
and an Agence Nationale de la Recherche grant
(ANR-07-JCJC-088-01) to SG. We acknowledge the confocal microscopy facilities of Plateforme Imagerie In Vitro of Institut Federatif de
Recherche 37.
Conflicts of Interest

The authors declare no conflicts of interest.

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