Am J Physiol Endocrinol Metab 309: E611–E620, 2015.
First published August 18, 2015; doi:10.1152/ajpendo.00268.2015.
Review
The skeleton in the closet: actin cytoskeletal remodeling in ␤-cell function
Caroline Arous and Philippe A. Halban
Department of Genetic Medicine and Development, University of Geneva Medical Center, Geneva, Switzerland
Submitted 11 June 2015; accepted in final form 11 August 2015
pancreatic islets; ␤-cells; insulin secretion; actin cytoskeleton; myosin; focal
adhesions
Putting the Skeleton in the Closet: Early Studies on the
␤-Cell Cytoskeleton and Its Role in Insulin Secretion
of the microtubular-microfilamentous cytoskeleton in promoting and guiding ␤-cell granule movement
and exocytosis was first proposed in 1968 (67), and supported
experimentally one year later by morphological studies (91).
Subsequent functional/pharmacological studies throughout the
1970s consolidated this central hypothesis (68, 77, 86, 130 –
132). Interestingly, pioneering morphological studies identified
actin-containing protrusions on the surface of ␤-cells that were
modified by glucose (76), resembling the glucose-mediated
focal adhesion remodeling described in the past few years (see
below). In that same visionary paper, the authors concluded
presciently that the microfilamentous web (underlying the
plasma membrane) is “not necessarily limited to a restrictive
role” and that this web “might represent, like a sphincter, both
a barrier to and an effector of emiocytosis”. This same group
showed that microtubules were important for the transfer of
newly synthesized proinsulin/insulin from the site of synthesis
to that of release (75), with the equally prescient proposal as
early as 1979 that “ѧthe microtubular apparatus serves as a
guiding cytoskeleton for the oriented translocation of secretory
granules, whereas the microfilamentous cell web may control
THE INVOLVEMENT
Address for reprint requests and other correspondence: C. Arous, Dept. of
Genetic Medicine and Development, Univ. of Geneva Medical Center, 1 rue
Michel-Servet, 1211 Geneva 4, Switzerland (e-mail: [email protected]).
http://www.ajpendo.org
the eventual access of the granules to exocytotic sites” (113);
the same year saw the first direct demonstration in vitro of
an association between ␤-cell granules and F-actin (48).
Remarkably, these early observations and postulates have
withstood the test of time and are fully integrated into
today’s working models of the cellular and molecular biology of insulin secretion. It was next shown that secretagogues induced contractile movements at the surface of the
cells, reflecting actin-like microfilament activity (reviewed
in Ref. 49). During the same period, involvement of the
cytoskeleton in exocytosis was being studied in other secretory cell types (for review see Ref. 7), and several key
proteins known to interact with actin were localized by
immunofluorescence to the cell periphery (7, 8, 21, 28, 99,
153). The classical “barrier” hypothesis of the actin cytoskeleton in exocytosis was refined, with the earliest suggestion of two granule pools, one available for rapid release, the
other reserve pool being deeper in the cell (7, 114). During
the 1990s, other postulated roles of actin were suggested,
including vesicle transport when actin is coupled with myosin (13) or generation of contractile forces to facilitate the
expulsion of secretory material (111). A more complete
understanding of the molecular events underlying actin
remodeling (both disassembly and assembly) in exocytosis
has emerged progressively with time (32, 74, 93, 122, 123).
Meanwhile, the skeleton remained in the closet as researchers focused on other aspects of ␤-cell function.
0193-1849/15 Copyright © 2015 the American Physiological Society
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Arous C, Halban PA. The skeleton in the closet: actin cytoskeletal remodeling
in ␤-cell function. Am J Physiol Endocrinol Metab 309: E611–E620, 2015. First
published August 18, 2015; doi:10.1152/ajpendo.00268.2015.—Over the last few
decades, biomedical research has considered not only the function of single cells
but also the importance of the physical environment within a whole tissue,
including cell-cell and cell-extracellular matrix interactions. Cytoskeleton organization and focal adhesions are crucial sensors for cells that enable them to rapidly
communicate with the physical extracellular environment in response to extracellular stimuli, ensuring proper function and adaptation. The involvement of the
microtubular-microfilamentous cytoskeleton in secretion mechanisms was proposed almost 50 years ago, since when the evolution of ever more sensitive and
sophisticated methods in microscopy and in cell and molecular biology have led us
to become aware of the importance of cytoskeleton remodeling for cell shape
regulation and its crucial link with signaling pathways leading to ␤-cell function.
Emerging evidence suggests that dysfunction of cytoskeletal components or extracellular matrix modification influences a number of disorders through potential
actin cytoskeleton disruption that could be involved in the initiation of multiple
cellular functions. Perturbation of ␤-cell actin cytoskeleton remodeling could arise
secondarily to islet inflammation and fibrosis, possibly accounting in part for
impaired ␤-cell function in type 2 diabetes. This review focuses on the role of actin
remodeling in insulin secretion mechanisms and its close relationship with focal
adhesions and myosin II.
Review
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ACTIN CYTOSKELETAL REMODELING IN ␤-CELL FUNCTION
Discovering More About the Skeleton in the Closet: New
Evidence for the Role of Cell Adhesion in Secretion
Letting the Skeleton Out of the Closet
Adhesions in ␤-cell function. Integrin-ECM adhesions affect
␤-cell function through modulation of Ca2⫹ fluxes (15, 54)
(Fig. 2, event 1). We have further demonstrated that when rat
␤-cells are placed on ECM there is mild and transient activation of NF-␬B downstream of integrin engagement and FAK
activation, leading to proliferation and improved glucose-stimulated insulin secretion (GSIS) (4, 94, 101). Another study
demonstrated that cadherin-mediated (cell-cell) adhesion induces an asymmetric distribution of cortical actin in ␤-cells
while improving insulin secretion from human ␤-cells (96),
and given the documented cross-talk between cadherin and
integrin signaling (23), this most likely arises through actin
remodeling.
We have observed glucose-stimulated morphological
changes at the ␤-cell surface that are reminiscent of actin and
FA remodeling in cell migration (103, 104), and adhesion to a
biologically compatible ECM enhances both GSIS and ␤-cell
survival (16, 42). Glucose-stimulated spreading of ␤-cells
coincides with reorganization of actin stress fibers into thick
networks and the phosphorylation of the two main FA proteins
FAK and paxillin (Fig. 1). Upon glucose stimulation, activated
FAK-paxillin-ERK1/2 complexes are incorporated into newly
formed FAs in intimate association with the extremities of
actin fibers (104), and these events are linked to actin depolymerization (18) (Fig. 2 event 2). Additionally, GSIS was
significantly decreased and glucose-induced actin remodeling
disrupted by either knockdown of paxillin or chemical inhibition of FAK activation, showing for the first time that FA
remodeling is a critical event for regulated insulin secretion.
These observations in primary rat ␤-cells were completed by
work on (transformed mouse) MIN6B1 cells, showing that
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In tissues, cells are in contact with extracellular matrix
(ECM) and continuously communicate with their extracellular
environment. Actin can assemble into branched or bundled
fibers that provide mechanical stability or elasticity to cells
(35) and extend to points of cellular contact with the ECM.
Actin cytoskeleton remodeling in secretion mechanisms is
tightly linked to integrin-dependent ECM adhesions, also
known as focal adhesions (FAs), which are sites of mechanical
linkage between the actin cytoskeleton (136) and their engagement with the ECM, and activation requires association with
the actin cytoskeleton (116). Simultaneously, maturation of
FAs depends upon the orderly recruitment of myriad proteins
and adaptors centered around focal adhesion kinase (FAK) and
dependent upon integrin activation (3). FAK is considered a
key player in cellular communication with the external physical environment and is involved upstream of multiple signaling
pathways leading to myriad biological end points, including, as
we shall see, exocytosis (110). While the extracellular domain
of integrins binds to ECM, a short cytoplasmic domain recruits
adapter proteins providing either a mechanical link to the
cytoskeleton (i.e., talin and vinculin) or intracellular signaling
(i.e., FAK and paxillin) (142, 143, 156). Kindlins bind to and
activate integrins while also binding to FA proteins including
FAK and ␣-actinin (for reviews see Refs. 63 and 81).
FAs are dynamic structures and their composition can
change in response to mechanical stimulation, such as actomyosin contraction, ECM stiffness or cytokine-mediated signaling
(97, 98, 142). For such dynamic structures, disassembly is
obviously just as important as assembly. Even if less well
understood (3, 84, 110), FA disassembly could arise through
dephosphorylation of FAK and paxillin (31, 44) as well as their
proteolytic cleavage by calpain (24, 26). It is also known that
FA adaptors and associated kinases including FAK, and more
downstream proteins including Src, p130cas, paxillin, ERK,
and MLCK (myosin light chain kinase), are themselves critical
not only for assembly but also disassembly (141). Over the
years, the extraordinary complexity of FAs has become increasingly apparent, with an ever-growing catalog of associated proteins, culminating in the first detailed description of the
FA proteome (65, 70, 115). Careful evaluation of the FA
proteome (65) reveals dynamic association of several proteins
potentially implicated in exocytosis (note that this study was
performed on human foreskin fibroblasts that do not secrete via
the regulated but only the constitutive secretory pathway),
including the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins syntaxin 2 and 4, and
VAMP3 (key players in the exocytotic process in all neuroendocrine cells), as well as proteins involved in endocytosis, an
event known to be tightly coupled to exocytosis of insulin
granules (83, 125). Similarly, the presence of a voltage-dependent calcium channel subunit suggests that ␤-cell L-channels
may be associated with FAs during glucose stimulation, allowing for localized increases in Ca2⫹ to stimulate exocytosis;
indeed, in ␤-cells, glucose-induced increases in intracellular
Ca2⫹ are more pronounced close to actin-rich filopodia (36).
Intriguingly, the Ca2⫹ transients seen at the leading edge of
moving cells are mediated by TRPM7 (a member of the
melastatin subfamily of transient receptor potential of cation
channels that regulate multiple cell functions through Ca2⫹/
Mg2⫹ homeostasis) and generated through actomyosin contractility (144), while in islet cells both TRPM2 (127) and
TRPM5 (69) have been shown to contribute toward insulin
secretion, probably similarly driven by actin-myosin II tension
at sites of exocytosis.
Additional compelling evidence for direct involvement of
actin remodeling in regulated (neuroendocrine cell) secretion
comes from chromaffin cells, with colocalization through cortical F-actin of L-type Ca2⫹ channels and key components of
the secretory machinery including granules themselves (120).
Another link is provided by a study showing that neuritogenesis depends on the exocytotic machinery, and here again one
mechanism was shown to involve the FAK/Src-Arp2/3-VAMP
axis (41). Finally, the association of Src with FAK, an important early binding event in FA remodeling, has been suggested
to depend upon translocation of Src to the plasma membrane
through SNAP-23 (112), a ubiquitous t-SNARE protein that is
expressed in the ␤-cell and shown to deputize for SNAP-25 in
insulin secretion (108). In addition to the complexity of the FA
proteome described above, one has to consider changes over
time and space, with molecular organization differing even
within the same cell at any given time. This reflects the
functional heterogeneity of FAs, which, in addition to providing adhesion, also promote cell spreading (100), survival (109),
and many biological other processes, including insulin secretion (104, 105), as we shall now review.
Review
ACTIN CYTOSKELETAL REMODELING IN ␤-CELL FUNCTION
Crop
Fig. 1. Glucose induces ␤-cell focal adhesion (FA) and actin remodeling. Rat
primary ␤-cells were cultured on extracellular matrix (ECM; 804G) for 3 h in
low glucose (2.8 mM) and then for a further 15 min with high glucose (16.7
mM). Cells were subsequently fixed and stained for paxillin (red) and actin
(phalloidin in green). Note the presence of numerous paxillin-containing
protrusions (spikes) on the cell surface that resemble FAs and develop at the
tips of actin fibers in response to a glucose stimulus. FA and actin remodeling
have been shown to be necessary for full development of glucose-stimulated
insulin secretion. Scale bar, 10 ␮m.
glucose-induced phosphorylation and activation of FAK, paxillin, and ERK1/2 in ␤-cells is mediated by ␤1-integrins (105).
These data are consistent with the role of ␤1-integrin observed
in the expansion and spreading of pancreatic ␤-cells on the
matrix protein laminin-5 in vitro (16, 30, 62, 95), and was
confirmed in a study using conditional ␤1-integrin knockout
mice (102), pointing to FAK-MAPK-ERK as the major pathway. FAK activation would furthermore seem to liberate
SNAP-25 from its association with the actin cytoskeleton,
allowing this t-SNARE protein to participate in granule exocytosis (117). TIRF-mediated visualization of insulin-secretory
vesicles revealed a reduction in the number of insulin-containing granules adjacent to the plasma membrane due to FAK
inhibition, which is accompanied by a reduction of glucoseinduced phosphorylation and activation of Akt and its substrate
AS160 in ␤-cells (105) (Fig. 2 event 3). More recently, robust
GSIS has been shown to require myosin IIA-dependent remodeling of F-actin, necessary for the recruitment of phosphopaxillin, FAK, and ERK to newly formed FAs (5). These in
vitro observations have been supported by a study in transgenic
mice with selective knockout of FAK in ␤-cells confirming that
FAK is critical for ␤-cell viability and function (22).
Glucose is not the only stimulus of insulin secretion that
acts, at least in part, through actin and FA remodeling. Similar
paxillin-dependent FA remodeling has been observed in primary rat ␤-cells in response to GLP-1 (with high glucose),
phorbol esters, and KCl (104). Interestingly, the islet endocan-
nabinoid system, too, appears to enhance insulin secretion
through activation of FAK and cytoskeletal remodeling (78).
Actin regulates insulin granule trafficking and exocytosis.
As suggested by historical studies and now confirmed in
multiple secretory cell types including ␤-cells, granules use
actin filaments as “rails” to move toward the basal membrane
(5, 135), whereas cortical actin acts as a “barrier” regulating
secretory granule access to the membrane for exocytosis (6, 7,
137) (Fig. 2, middle). Actin has further been shown to regulate
exocytosis in other secretory cell types in partnership with
myosin (2, 12, 79, 82). The pharmacological agents latrunculin
and jasplakinolide, that depolymerize and stabilize F-actin,
respectively, differentially influence insulin secretion. Initial
confusion of the effect of these two agents has now been
resolved by an elegant study demonstrating that their impact on
stimulated insulin secretion depends on the stimulus, and
specifically the origin of elevated cytosolic Ca2⫹, from outside
the cell via voltage-gated channels or through mobilization of
intracellular stores (46). There are, furthermore, two insulin
granule pools that are differentially regulated through actin
remodeling, depending again upon the source of Ca2⫹ (45),
and the rate of intracellular movement and of secretion of
young and old insulin granules is modulated by actin coating
(47). The role of the actin cytoskeleton and its dynamic
remodeling following a secretory stimulus is thus even more
complex than originally postulated, seeming to impact every
aspect of the insulin exocytotic apparatus.
Because an interesting and comprehensive review was published recently concerning signaling pathways involved in
actin cytoskeleton regulation of insulin secretion (56), we shall
provide here only a brief overview and update. The small Rho
family GTPases Cdc42, Rho, and Rac1 are central players in
the interaction between the plasma membrane and cortical
actin (29). They are recruited to filopodia upon integrin activation, are involved in FA remodeling (66, 90), and have been
shown to positively regulate both phases of insulin secretion
through actin cytoskeletal network reorganization (6, 43, 88).
Cdc42 is implicated in two distinct pathways involved in actin
(de)polymerization and recruitment of granules to the membrane, Cdc42:N-WASP:Arp2/3:cofilin (129) and Cdc42:
PAK1:Raf1:ERK1/2 (58), with upstream regulation by the Src
family kinase YES (154) and differential involvement in the
first and second phases of insulin secretion (129) (Fig. 2 event
4). Rac1 may mediate cAMP potentiation of insulin secretion,
again perhaps through actin cytoskeleton remodeling (117).
There is direct interaction between actin and t-SNARE
proteins implicated in insulin secretion that is disrupted by
glucose stimulation (55, 117). More specifically, SNAP-25 and
syntaxin 1 are localized to FAs through binding to F-actin;
upon glucose stimulation BAG3 (BCL2-associated athanogene
3 known to act as a co-chaperone for the heat-shock protein
Hsp70 and to be involved in various cell functions including
cytoskeleton organization) is phosphorylated by FAK and
dissociates from SNAP-25, allowing it now to interact with
syntaxin 1a in the SNARE complex (Fig. 2, event 5). These
events destabilize F-actin to facilitate insulin release (52, 104,
105). The Ca2⫹-activated actin-severing gelsolin is also involved in insulin secretion (118), forming complexes with
syntaxin 4, which are disrupted by glucose stimulation, freeing
the t-SNARE to promote exocytosis and gelsolin to remodel
F-actin (57) (Fig. 2, event 6). Meanwhile, syntaxin 1a interacts
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16.7mM glucose
2.8mM glucose
Actin-green
Paxillin-red
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ACTIN CYTOSKELETAL REMODELING IN ␤-CELL FUNCTION
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Ca2+
1
ER
Nucleus
+
F-actin
Myosin-IIA
MLCK
ROCK
FA
FA
FA
FA
FA
FA
FA Focal adhesion complex
Integrins
ECM
LOW GLUCOSE
ERK1/2
3
AS160 GDP
Rab
Insulin
F-actin
Readily
releasable pool
AKT
Reserve pool
MLCK
VAMP/
synaptobrevin
Myosin IIA
α-actinin
Insulin
FA
BAG3
Gelsolin
SNAP
Syntaxin
FA
K
Talin
ECM
6
BAG3
Gelsolin
SNAP
Syntaxin
PAX
α β
Vinculin
α β
Engaged integrin
t-SNARE
5
Talin
t-SNARE
α β
Disengaged integrin
HIGH GLUCOSE
3
MyoVa
7
GTP
P Rab27a
I
R
MY
P
AKT
P
AS160
P
Insulin
MLCK
P
P
8
VAMP/
synaptobrevin
10
P
4
ECM
P
Δ
P
Talin
ERK1/2
p130cas
Vinculin
α β
Engaged (activated) integrin
6
P
SNARE
core complex
Gelsolin
P
FA
K
Kindlin
Cdc42
2 P
P
Sr
PA c
X
FA
N-WASP
Arp2/3
α-actinin
P BAG3
9
5
SNAP
Syntaxin
FA
FA
α β
α β
t-SNARE
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Fig. 2. ␤-Cell remodeling events downstream from the increase in cytosolic Ca2⫹
evoked by a shift from low (middle) to high
glucose (bottom); other key elements of
␤-cell stimulus secretion coupling that may
also impact on these remodeling events have
been omitted for the sake of clarity. The FA
complex comprises numerous proteins; only
those shown to be directly implicated in
␤-cell secretion and mentioned in the text
are depicted here. Note the dissociation and
recruitment of diverse proteins from FAs
following stimulation by high glucose that is
tightly linked to actin remodeling. FA and
actin remodeling are dynamic processes
with changes in space and time that cannot
be captured in such single snapshots at low
and high glucose. Top: localization of key
players in FA and actin cytoskeleton remodeling in ␤-cells. Middle: inset of a ␤-cell at
low glucose (basal). FAs are connected to
the actin cytoskeleton through integrins. Actin serves as a barrier to limit basal insulin
secretion and to prevent recruitment of insulin granules to the readily releasable pool.
In the basal condition, t-SNARE proteins
(SNAPs and syntaxins) are linked to inhibitor proteins BAG3 and gelsolin (events 5
and 6, respectively). In parallel, AS160 is
associated with Rab-GDP proteins, which
prevents AS160 interaction with and activation by Akt, so inhibiting insulin granule
translocation to the basal membrane (event
3). These molecular pathways seem to be
regulated by FA kinase (FAK). Bottom: inset of a ␤-cell following a brief period of
stimulation by high glucose. Glucose stimulation induces an increase in cytosolic
Ca2⫹ leading to activation and recruitment
at remodeled FAs of (among others) cytoskeleton adaptors (talin, vinculin), integrin
activator proteins (␣-actinin, kindlins), and
intracellular signaling proteins (Src, cdc42
FAK, paxillin, ERK) (events 2 and 4). Such
remodeling of FA and actin leads to insulin
granule movement along the actin cytoskeleton with the assistance of myosin Va/Rab
27a/MYRIP (event 7) and allows granules to
approach toward t-SNARE complexes
newly dissociated from their inhibitors,
BAG3 (event 5) and gelsolin (event 6).
MLCK (myosin light chain kinase) phosphorylates myosin IIA (event 8), which induces its relocalization and association with
the actin ring surrounding granules [important for maintenance of the fusion pore in an
open conformation and for providing energy
for discharge of granule contents (event 9)
as well as cortical actin remodeling (event
10)] to allow insulin granules to approach
the basal membrane.
Ca2+
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ACTIN CYTOSKELETAL REMODELING IN ␤-CELL FUNCTION
basal membrane. By contrast, ROCK regulates actin stress
fibers at the center of the cell to restrain insulin granule access
to the plasma membrane without impacting FA remodeling (5).
In accord with this, Rho-ROCK signaling has been shown to
contribute to actin cytoskeleton stabilization and inhibits glucose and GLP-1-induced insulin secretion (43, 64). To conclude, NMII is a key player in GSIS. Its activity is modulated
principally via light chain phosphorylation by MLCK and by
ROCK that appears to manage actomyosin organization in
separate cellular compartments, leading to positive or negative
effects on trafficking of different pools of insulin granules.
Working model explaining the role of actin and FA remodeling in insulin secretion. Taking all this information into
consideration, we have developed a working model for the
central role of actin remodeling in insulin secretion (Fig. 2).
This model takes into consideration actin’s dual role as “barrier” or “rail” depending on where F-actin is localized in the
cell, FA remodeling, motor protein isoforms, and which signals
trigger and reverse the remodeling event over space and time.
Cytoskeleton and FA Remodeling in Type 2 Diabetes:
Parallels Between Insulin Secretion and Insulin Signaling
Type 2 diabetes is characterized by relative insulin insufficiency in the face of insulin resistance, the result of decreased
␤-cell function and most likely mass. Physiological, integrinmediated cell-ECM communication is critical not only normal
␤-cell function and indeed survival, as discussed here, but also
for insulin action on its target tissues. Pathological modification of the ECM in type 2 diabetes, for example by high-fat
diet, hyperglycemia-induced advanced glycation end-products
(AGEs), or inflammatory cytokine-induced fibrosis, has thus
been shown to participate in the insulin resistance state of
insulin-targeted tissues (muscle, adipose tissue, and liver) and
could impair ␤-cell function in a similar fashion (4, 50, 59,
107, 138, 146, 147). Deleterious cytokines that are elevated in
the circulation of individuals with type 2 diabetes directly
affect the expression of proteins of different functional classes
including the actin cytoskeleton in ␤-cells (103); perhaps this
may also occur in target cells to modulate their insulin sensitivity.
There is some evidence that FA remodeling may be altered
in islets from people with type 2 diabetes, based on a decrease
in FAK phosphorylation (27) (Fig. 2, symbolized by ⌬), while
the activity of the ERM family scaffolding proteins ezrin,
radixin, and moesin, which bind F-actin and promote granule
movement/insulin secretion, is decreased in diabetic mouse
islets (73). Additional indirect evidence for possible malfunction of FA remodeling in diabetes comes from a study on
glucotoxicity in mouse embryonic stem cell-derived ␤-like
cells, showing a lack of actin-stress fibers linked to FAs
through vinculin, and decreased GSIS (152). A further possible
link with type 2 diabetes, and an interesting overlap with
signaling in ␤-cells, stems from work indicating involvement
of FAK signaling in insulin action and resistance in classical
target tissues. FAK is a substrate of both the insulin and IGF-I
receptors, and its phosphorylation is dependent upon cellular
architecture (11, 33), whereas actin cytoskeleton remodeling
impacts insulin action by subcellular redistribution of signaling
molecules such as the p85 subunit of PI3K and IRS-1, which
are recruited to sites of insulin-induced actin reorganization in
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reversibly with KATP channels (61), and both syntaxin and
SNAP-25 also bind to L-type voltage-gated Ca2⫹ channels (60,
145). This is reminiscent of vascular smooth muscle cells, in
which integrin engagement can stimulate L-type Ca2⫹ channels via FAK (151). Phosphatidylinositol 4,5-bisphosphate
[PtdIns(4,5)P2] plays a pivotal role in actin remodeling and
␤-cell secretion (73, 118, 119) and has further been shown to
cluster FAK at FAs, leading to its activation (38). Such
evidence for compartmentalization reinforces the notion of
localized increases in cytosolic Ca2⫹ close to sites of exocytosis and FAs and indeed in the possibly analogous setting
of antigen activation of mast cells, coordinated oscillations
of Ca2⫹, PtdIns(4,5)P2, and cortical F-actin increase secretion (150).
Central role of myosin. The movement of vesicles clearly
depends on motor proteins (39) that may also influence the
polymerization dynamics of microtubules (51). In the ␤-cell,
kinesin is the principle motor protein driving microtubular
movement of granules from the deeper reserve pool to the
periphery (10, 80, 133, 134). However, myosins have also been
implicated in granule trafficking (reviewed in Ref. 14). Specifically, myosin II appears important for approach and fusion
of granules, but myosin V for docking and priming (14).
Myosin Va has been extensively studied in ␤-cells (see Ref.
106 for review). Studies in transformed ␤-cells indicate a
general role in movement of granules through the cortical actin
web following a stimulus (135). This requires Huntingtinassociated protein-1 (140) and appears more important for the
second phase of secretion, with passive movement underlying
the first phase (53). Granules appear physically linked to
myosin Va via Rab27a and Slac-2c/MYRIP (53, 85, 135), and
it is used by all regulated secretory cell types for granule
locomotion (20) (Fig. 2, event 7).
Non-muscle myosin II (NMII) is well recognized for its
universal role in cell spreading and migration (reviewed in
Refs. 25 and 136), but this myosin (there are three isoforms in
humans, each with a different heavy chain associated with a
common regulatory light chain, MRLC) is now known to be
critical for regulated secretion, too. NMII activity is regulated
directly by phosphorylation of both the heavy and light chains
(136), with suggested spatial resolution of the latter: MRLC
phosphorylation by ROCK toward the center of the cell, and by
MLCK toward the periphery, with distinct effects on plasma
membrane ruffling and FA dynamics (121) (Fig. 2, event 8).
Studies in other secretory cell types suggest a role for NMII in
late events leading to exocytosis, involving maintenance of the
fusion pore in the open state (2, 12, 87) (Fig. 2, event 9). NMII
and F-actin are recruited to coat the surface of dense-core
granules during their fusion with the plasma membrane (79),
and contraction of this coat drives discharge of granule contents (82, 89). In transformed ␤-cells, MLCK colocalizes with
granules with activation of PKC leading to a common shift
toward the periphery (155), and F-actin and NMII heavy chain
A (MHCIIA) redistribute toward contact points in response to
stimulation by KCl (149) (Fig. 2, event 10). Nutrient stimulation elicits rapid threonine phosphorylation of MHCIIA (148),
that distributes within the cell in a similar way to F-actin (149).
In primary ␤-cells (5), acute glucose stimulation induces MHCIIA remodeling at the cell periphery and colocalization with
␥-actin. This is regulated by MLCK and mediates the FA
remodeling necessary for insulin granule movement to the
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myotubes (124). As expected from the canonical FAK signaling cascades, ERK and Akt are also implicated in insulin
signaling (and blunted in insulin resistance) (37, 40), just as
shown in ␤-cells for insulin secretion (104, 105, 118). There is
also a positive feedback regulatory loop, with FAK activating
and stabilizing IGF-IR (1), suggesting that similar pathways
may act in ␤-cells downstream of the insulin or IGF-I receptor,
both of which are known to be important for ␤-cell function,
survival, and proliferation (92, 128), acting in this cell type
through IRS-2 (19, 71, 72) and Akt/AS160 (17). Finally, there
are interesting parallels between the role of PAK1, which acts
downstream of Cdc42 in insulin secretion (see above) and in
skeletal muscle insulin stimulation of glucose uptake (126),
with a 80% decrease in PAK1 in human islets from subjects
with type 2 diabetes (139).
with FAs and involves different myosin isoforms in charge of
the various steps of insulin secretion, most specifically granule
trafficking and exocytosis. While no clear defect in these
processes has been demonstrated directly to contribute toward
␤-cell dysfunction in type 2 diabetes, evidence in other tissues
and preliminary observations in ␤-cells suggest that this may
well be the case.
Challenges in the Study of Focal Adhesion and Actin
Remodeling Mechanisms
GRANTS
Conclusion
Integrin-mediated cell-ECM interaction is a critical regulatory component of ␤-cell secretory function, acting through the
actin cytoskeleton to impact granule trafficking, docking, and
fusion events, under the upstream control of various signaling
relays triggered by glucose. Spatial organization of granules
within the cell is mediated by the actin cytoskeleton coupled
We thank the many collaborators who helped in this work over the past
years, especially the late Dr. Dominique Rouiller and Drs. Vincenzo Cirulli,
Domenico Bosco, Carmen Gonnelle-Gispert, Eva Hammar, Salomé Katengwa,
Géraldine Parnaud, Pascale Ribaux, Alejandra Tomas, Dieter Rondas, and
Barbara Yermen, as well as Melanie Cornut, Stephane Dupuis, and Katharina
Rickenbach for expert technical assistance. We apologize to those investigators
whose work was not cited due to space limitations or our oversight.
This work was supported, in part, by Swiss National Science Foundation
Grant 31003A-144092.
DISCLOSURES
No conflicts of interest, financial or otherwise, are declared by the author(s).
AUTHOR CONTRIBUTIONS
Author contributions: C.A. and P.A.H. conception and design of research;
C.A. and P.A.H. performed experiments; C.A. and P.A.H. analyzed data; C.A.
and P.A.H. interpreted results of experiments; C.A. and P.A.H. prepared
figures; C.A. and P.A.H. drafted manuscript; C.A. and P.A.H. edited and
revised manuscript; C.A. and P.A.H. approved final version of manuscript.
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