Cortical Filamentous Actin Disassembly and Scinderin
Redistribution during Chromatfin Cell Stimulation Precede
Exocytosis, A Phenomenon Not Exhibited by Gelsolin
M. L. Vitale, A. Rodriguez Del Castillo, L. Tchakarov, and J.-M. Trifar6
Secretory Process Research Program, Department of Pharmacology, Faculty of Medicine, University of Ottawa,
Ottawa, Ontario, Canada K1H 8M5
Abstract. Immunofluorescence and cytochemical
studies have demonstrated that filamentous actin is
mainly localized in the cortical surface of the chromaffin
cell. It has been suggested that these actin filament
networks act as a barrier to the secretory granules,
impeding their contact with the plasma membrane.
Stimulation of chromaffin cells produces a disassembly
of actin filament networks, implying the removal of the
barrier. The presence of gelsolin and scinderin, two
Ca2+-dependent actin filament severing proteins, in the
cortical surface of the chromatfin cells, suggests the
possibility that cell stimulation brings about activation
of one or more actin filament severing proteins with
the consequent disruption of actin networks. There-
fore, biochemical studies and fluorescence microscopy
experiments with scinderin and gelsolin antibodies and
rhodamine-phalloidin, a probe for filamentous actin,
were performed in cultured chromaffin cells to study
the distribution of scinderin, gelsolin, and filamentous
actin during cell stimulation and to correlate the pos-
sible changes with catecholamine secretion. Here we
report that during nicotinic stimulation or K+-evoked
depolarization, subcortical scinderin but not gelsolin is
redistributed and that this redistribution precedes cate-
cholamine secretion. The rearrangement of scinderin
in patches is mediated by nicotinic receptors. Cell
stimulation produces similar patterns of distribution of
scinderin and filamentous actin. However, after the
removal of the stimulus, the recovery of scinderin cor-
tical pattern of distribution is faster than F-actin reas-
sembly, suggesting that scinderin is bound in the corti-
cal region of the cell to a component other than F-actin.
We also demonstrate that peripheral actin filament dis-
assembly and subplasmalemmal scinderin redistribution
are calcium-dependent events. Moreover, experiments
with an antibody against dopamine-~-hydroxylase sug-
gest that exocytosis sites are preferentially localized to
areas of F-actin disassembly.
sponse to cholinergic stimulation and upon Ca 2+ entry, the
granules fuse with the plasma membrane and release their
soluble contents to the cell exterior by exocytosis (Trifar6,
1977; Viveros, 1974). Immunofluorescence and cytochemi-
cal studies have described the presence of a mesh of filamen-
tous actin (F-actin) underneath the chromattin cell plasma
membrane (Lee and Trifar6, 1981; Trifar6 et al., 1984;
Cheek and Burgoyne, 1986). It has been proposed that actin
networks act as a barrier to the secretory granules by block-
ing their movement towards the plasma membrane (Trifar6
et al., 1982, 1984, 1989; Cheek and Burgoyne, 1986, 1987;
Burgoyne and Cheek, 1987; Burgoyne et al., 1989). Evi-
HROMAFFIN cells of the adrenal medulla store their
secretory products in specialized organelles, the chro-
marlin granules (Smith, 1968; Trifar6, 1977). In re-
M. L. Vitale is on leave from the CONICET-Buenos Aires, Argentina.
A. Rodrfguez Del Castillo is on leave from the University of La Laguna,
La Laguna, Spain. L. Tchakarov is a postdoctoral fellow from the Medical
Research Council of Canada.
dence obtained from different experimental approaches has
demonstrated that stimulation of chromaflin cells brings
about a disassembly of cortical F-actin networks, suggesting
the removal of the physical barrier to granule movement
(Cheek and Burgoyne, 1986, 1987; Burgoyne and Cheek,
1987; Burgoyne et al., 1989; Trifar6 et al., 1989). The exis-
tence of actin-binding proteins that regulate the dynamics
of actin networks (Yin and Stossel, 1979; Craig and Pol-
lard, 1982; Stossel et al., 1985; Maekawa et al., 1989;
Rodriguez Del Castillo et al., 1990) strongly suggests a role
for these proteins in the disassembly of actin filaments trig-
gered by cell stimulation. Therefore, it was of interest to in-
vestigate the participation in this process of gelsolin (Yin and
Stossel, 1979) and scinderin (Rodriguez Del Castillo et al.,
1990) two Ca~+-dependent actin-binding proteins that con-
trol actin filament length.
Gelsolin is an actin filament capping and severing protein
found in many cells, including chromaffin cells, and in ex-
tracellular fluids (Yin and Stossel, 1989; Yin et al., 1981;
Stossel et al., 1985; Trifar6 et al., 1985; Bader et al., 1986).
© The Rockefeller University Press, 0021-9525/91/06/1057/II $2.00
The Journal of Cell Biology, Volume 113, Number 5, June 1991 1057-1067 I057
Previous work from our laboratory has described the pres-
ence in chromaflin cells of another actin binding protein that
can be eluted by an EGTA containing buffer from actin-
DNase I affinity columns along with gelsolin (Bader et al.,
1986). Recently we have isolated, characterized, and given
the name of "scinderin" to this new protein (Rodriguez Del
CastiUo et al., 1990). Scinderin is an 80-kD cytosolic pro-
tein that shortens actin filament length provided Ca 2+ is pres-
ent in the medium (Rodriguez Del Castillo et al., 1990).
The fact that, when chromaffin ceils are stimulated there
is an entry of Ca 2+ (Douglas, 1968) with a consequent in-
crease in its intracellular level (Cheek et al., 1989) prompted
us to investigate whether this condition would influence gel-
solin and/or scinderin distributions in a way that could be
correlated to actin filament disassembly. This paper describes
biochemical and immunocytochemical experiments performed
in cultured chromaflin ceils. We have studied the cellular lo-
calization of scinderin and gelsolin under different experi-
mental conditions and compared their su.bcellular redistribu-
tion with F-actin disassembly and catecholamine secretion.
The present experiments demonstrate that during cell stimu-
lation, subplasmalemmal scinderin, but not gelsolin, is re-
distributed in chromaflin cells, that this redistribution pre-
cedes exocytosis, and that exocytosis sites are preferentially
localized to areas of F-actin disassembly. The redistribution
of scinderin is mediated by nicotinic receptors. The results
also show that the redistribution of scinderin and F-actin dis-
assembly are Ca2+-dependent events and that similar patterns
of distribution for scinderin and F-actin are observed during
stimulation. A preliminary account of this work has been
presented elsewhere (Vitale, M. L., A. Rodrfguez del Cas-
tillo, L. Tchakarov, M. L. Novas, and J.-M. Trifar6. 1990.
J. Cell Biol. 111:424a).
Materials and Methods
(a) Chromaffin Cell Culture
Bovine adrenal glands were obtained from a local slaughterhouse and
chromaffin cells were isolated by collagenase digestion and further purified
using a Percoll gradient (Trifar6 and Lee, 1980). Cells were plated on
collagen-coated glass coverslips contained within plastic Petri dishes at a
density of 0.25 x 106 cells/35-mm dish for fluorescence microscopy
studies or in collagen-coated plastic Petri dishes at a density of 0.5 × 10 ~
cells/35-mm dish for catecholamine release studies. Cells were grown at
37°C in a humidified incubator under a CO2 + air atmosphere for 48 h as
previously described (Trifar6 and Lee, 1980).
(b) Source of Antibodies
Polyclonal antibodies were raised in rabbits against purified bovine scinde-
tin, gelsolin, and dopamine-~-hydroxylase (D/~H) 1 as previously described
(Bader et al., 1986; Rodriguez Del Castillo et al., 1990; Trifar6 et al.,
1976). Scinderin antiserum 6 thus obtained does not recognize gelsolin and
gelsolin antiserum does not cross-react with scinderin (Bader et al., 1986;
P-,odriguez Del Castillo et al., 1990; Tchakarov et al., 1990). Moreover,
scinderin was the only protein immunoprecipitated from an adrenal medul-
lary cytosolic preparation by antiserum 6 (Tchakarov et al., 1990). Anti-
D/3H IgG has been previously characterized (Trifiu-6 et al., 1976). A mouse
mAb against gelsolin (clone GS-2C4) was purchased from Sigma Chemical
Co. (St. Louis, MO). This antibody is specific for an epitope localized on
a 47-kD peptide derived from a chymotryptic cleavage of human gelsolin
(Chaponnier et al., 1986).
1. Abbreviations used in this paper: D/~H, dopamine-~-hydroxylase.
(c) Extraction of ChromaOin Cell
Chromaflin cell cytoskeleton was prepared essentially as described by
Bader et al. (1984). Briefly, chromaflin cells were cultured in a 100-mm-
diam plastic Petri dish at a density of 60 x 106 cells/dish. After 24 h in
culture, the cells were resuspended by washing several times with buffer A
(PBS: 100 mM sodium phosphate, 130 mM NaC1, pH 7.2 containing 5 mM
EGTA, and 2 mM PMSF). Cells were collected by centrifugation at 5,000 g
for 10 rain. The sediment thus obtained was resuspended in 1 ml buffer A
containing 600 mM KCI, 10 mM MgC12, 1% Triton X-100, and 0.5 nag
DNAse l/ml. The mixture was centrifuged at 27,000 g for 30 rain and the
sediment containing Triton-insoluble proteins (cytoskeleton) was washed
three times with buffer A, resuspended in 1 ml electrophoresis buffer and
boiled for 5 rain.
(d) Preparation of Total Proteins and Actin-binding
Proteins from Bovine Adrenal Medulla
Bovine adrenal medullae (60 g) were washed in ice-cold Locke's solution to
remove the blood and then homogenized in 300 mM sucrose, 20 raM Tris-
HC1, pH 7.5, 100 mM KC1, 5 mM DTT, 1 m.M PMSF, 5 ram N-ethyl-
maleimide, I mM EGTA and 1 mM Na-ATP (1 g of medulla in 2 mi of solu-
tion), using a Sorvail omnimixer (Sorvall Instruments Div., Newton, CT).
The homogenate was centrifuged at 1,000 g for 10 rain. The supernatant
thus obtained (total protein sample) was centrifuged at 100,000 g for 60 rain
and CaC12 was added to obtain a final concentration of 2 raM. The prepa-
ration was then applied to a DNase I-Sepharose 413 column prepared as de-
scribed by Bader et al. (1986). The column was pre-cquilibrated with buffer
A (20 mM Tris-HCl pH 7.5, 2 mM CaC12, 0.5 mM Na-ATP, 1 mM DTT,
and 1 mM PMSF) containing 100 mM KCI. The column was then washed
extensively (300 ml) with the same buffer A but this time containing 300
mM KCI. Finally the Ca2+-dependent actin binding-proteins were eluted
with buffer B (20 mM Tris-HC1 pH 7.5, 100 mM KC1, 1 mM DTT, 2 mM
EGTA, and 1 mM PMSF). Samples from either total proteins or EGTA elu-
ares were used in the experiments.
(e) Preparation of Scinderin
Scinderin was purified from bovine adrenal medullae following a chromato-
graphic procedure previously described (Rodriguez Del Castillo et al.,
1990) except that the last chromatographic step (HPLC) was omitted.
Therefore, the preparation obtained in this case also contains small amounts
of gelsolin and other cae+-dependent actin-binding proteins.
09 Electrophoresis and Immunoblotting
Monodimensional SDS-PAGE was performed according to Doucet and
Trifar6 (1988); gels were usually run at 60 V overnight in a Bio-Rad Protean
I apparatus (Bio-Rad Laboratories, Inc., Richmond, CA). The protocol for
immunoblotting was as described by Towbin et al., (1979). After electro-
phoresis, proteins were electrotransferred onto nitrocellulose membranes
(Hoefer Scientific Instruments, San Francisco, CA). Membranes were first
blocked with 3 % BSA in PBS and then incubated with scinderin antiserum
6 (1:500 dilution) or gelsolin antiserum (1:500 dilution) for 60 rain. Mem-
branes were next incubated with goat antirabbit immunoglobulin G-alkaline
phosphatase conjugate (1:3,000 dilution) for another 60 min. Color was de-
veloped by treatment with a mixture ofp-nitroblue tetrazolium chloride a,d
5-bromo-4-chloride-3-indolyl phosphate-toluidine salt.
(g) Fluorescence Microscopy
Single Staining. Experiments were started by rinsing the cultured cells
three times with regular Locke's solution (in millimolar: NaC1, 154; KCI,
2.6; K2HPO4, 2.15; KI-I2PO4, 0.85; MgCI2, 1.2; CaCI2, 2.2; and glucose,
10.0; pH 7.2). Cells were then incubated for different periods of time with
Locke's solution in the absence (control) or presence (stimulated) of differ-
ent compounds. Chromaffin cells were fixed in 3.7% formaldehyde in
Locke's solution for 20 rain at different times after initiated the stimulation
and processed for fluorescence microscopy as described previously (Lee
and Trifart, 1981). Briefly, cells were permeabilized by three successive ex-
posures of 5 rain each to 50, 100, and 50% acetone. Preparations were
washed several times with PBS and then incubated at 370C with either
scinderin antiserum 6 (1:20 dilution), gelsolin antiserum (1:20 dilution) or
mouse monoclonal antibody against gelsolin (1:40 dilution) for 60 rain.
The Journal of Cell Biology, Volume 113, 1991 1058
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Vitale et al. Actin Disassembly and Scinderin Redistribution in Exocytosis