Molecular Biology of the Cell
Vol. 16, 4519–4530, October 2005
Myosin-Va Regulates Exocytosis through the
Submicromolar Ca2?-dependent Binding of Syntaxin-1A
Michitoshi Watanabe,*†‡Kazushige Nomura,*‡§?, Akihiro Ohyama,‡§¶#
Ryoki Ishikawa,@Yoshiaki Komiya,§Kohei Hosaka,** Emiko Yamauchi,††
Hisaaki Taniguchi,††Nobuyuki Sasakawa,‡‡Konosuke Kumakura,‡‡
Tatsuo Ushiki,§§Osamu Sato,??¶¶Mitsuo Ikebe,??and Michihiro Igarashi*†
Divisions of *Molecular and Cellular Biology and§§Microscopic Anatomy and Bio-Imaging, Graduate School
of Medical and Dental Sciences and†Center for Trans-disciplinary Research, Niigata University, Niigata,
Niigata 951-8510, Japan; Departments of§Molecular and Cellular Neurobiology,?Orthopedic Surgery,
¶Anesthesiology and Reanimatology, and@Molecular and Cellular Pharmacology, Gunma University School
of Medicine, Maebashi, Gunma 371-8511, Japan; **Basic Sciences for Medicine, Gunma University School of
Health Sciences, Maebashi, Gunma 371-8514, Japan;††Division of Enzyme Physiology, Institute for Enzyme
Research, University of Tokushima, Tokushima, Tokushima 770-8503, Japan;‡‡Life Science Institute, Sophia
University, Chiyoda-ku, Tokyo 102-8554, Japan; and??Department of Physiology, University of Massachusetts
Medical School, Worcester, MA 01655-0127
Submitted March 25, 2005; Revised June 30, 2005; Accepted July 11, 2005
Monitoring Editor: Anthony Bretscher
Myosin-Va is an actin-based processive motor that conveys intracellular cargoes. Synaptic vesicles are one of the most
important cargoes for myosin-Va, but the role of mammalian myosin-Va in secretion is less clear than for its yeast
homologue, Myo2p. In the current studies, we show that myosin-Va on synaptic vesicles interacts with syntaxin-1A, a
t-SNARE involved in exocytosis, at or above 0.3 ?M Ca2?. Interference with formation of the syntaxin-1A–myosin–Va
complex reduces the exocytotic frequency in chromaffin cells. Surprisingly, the syntaxin-1A-binding site was not in the
tail of myosin-Va but rather in the neck, a region that contains calmodulin-binding IQ-motifs. Furthermore, we found that
syntaxin-1A binding by myosin-Va in the presence of Ca2?depends on the release of calmodulin from the myosin-Va
neck, allowing syntaxin-1A to occupy the vacant IQ-motif. Using an anti-myosin-Va neck antibody, which blocks this
binding, we demonstrated that the step most important for the antibody’s inhibitory activity is the late sustained phase,
which is involved in supplying readily releasable vesicles. Our results demonstrate that the interaction between
myosin-Va and syntaxin-1A is involved in exocytosis and suggest that the myosin-Va neck contributes not only to the
large step size but also to the regulation of exocytosis by Ca2?.
Myosin-V, a processive molecular motor, conveys vesicles
and other organelles along F-actin (Mercer et al., 1991; Espre-
afico et al., 1992; Cheney et al., 1993; Reck-Peterson et al.,
2000; Vale, 2003). This unconventional myosin is a member
of the class-V myosins, which are expressed in all eukaryotic
species from yeast to mammals (Reck-Peterson et al., 2000;
Matsui, 2003; Vale, 2003). Myosin-V is a dimeric protein
(Cheney et al., 1993). Each monomer is composed of a head
region, a long neck domain containing six tandem IQ-motifs,
and a tail region (Reck-Peterson et al., 2000). The head acts as
a plus-end ATPase-dependent molecular motor to move
myosin-V along F-actin (Reck-Peterson et al., 2000). The long
neck region is thought to act as a lever arm to regulate the
motor activity of the head and to maintain the large step size
of myosin-V via bound light chains. In higher eukaryotes,
the light chains consist mainly of calmodulin (CaM) bound
to the IQ-motifs in the neck domain (Cheney et al., 1993;
Vale, 2003). In addition, the globular tail of myosin-V inter-
acts with membrane-bound vesicles. In these ways, myo-
sin-V plays a central role in intracellular polarized transport
(Reck-Peterson et al., 2000; Matsui, 2003; Vale, 2003).
Among the three isoforms of myosin-V in higher verte-
brates, myosin-Va is the most abundant, and it is highly
enriched in the brain (Espreafico et al., 1992), particularly in
the neurons (Tilelli et al., 2003). Several lines of evidence
indicate that synaptic vesicles, which undergo the Ca2?-
regulated exocytosis, are one of the most important cargoes
for myosin-Va (Prekeris and Terrian, 1997; Bridgman, 1999;
Tilelli et al., 2003). In addition, Myo2p, a yeast homologue of
myosin-Va, directs intracellular transport during secretion
This article was published online ahead of print in MBC in Press
on July 19, 2005.
‡These authors contributed equally to this work.
Present addresses: Departments of#Ophthalmology and¶¶Phar-
macology, Juntendo University School of Medicine, Hongo 2-1-1,
Bunkyo-ku, Tokyo 113-8421, Japan.
Address correspondence to: Michihiro Igarashi (tarokaja@med.
Abbreviations used: AFM, atomic force microscopy; CaM, calmod-
ulin; CaMKII, Ca2?/CaM-dependent protein kinase II; GST, gluta-
thione S-transferase; RU, resonance units.
© 2005 by The American Society for Cell Biology4519
and budding through interactions with other proteins (Mat-
sui, 2003). However, the roles of myosin-Va in secretion and
Ca2?-regulated exocytosis are not as clear, probably because
the myosin-Va-interacting molecules have not been identi-
fied in neurons (Reck-Peterson et al., 2000; Matsui, 2003).
In the current studies, we found a novel interaction be-
tween myosin-Va, which is present on cortical synaptic ves-
icles (Prekeris and Terrian, 1997; Bridgman, 1999) and syn-
taxin-1A, a t-SNARE that participates in exocytosis (Duman
and Forte, 2003; Li and Chin, 2003), in presence of micromo-
lar levels of Ca2?. We also found that this unique interac-
tion, linked to Ca2?-dependent release of CaM from the neck
region of myosin-Va, is involved in Ca2?-regulated exocy-
MATERIALS AND METHODS
Immunoprecipitation using an antibody against the globular tail of myo-
sin-Va (gift of P. C. Bridgman, Washington University School of Medicine, St.
Louis, MO; Evans et al., 1998) was performed as described previously
(Ohyama et al., 2002) except that the buffer contained 1 ?M CaCl2, 2 mM
MgCl2, and 0.5 mM ATP. In some experiments, immunoprecipitation was
performed with a pool of myosin-Va and syntaxin-enriched fractions. This
material was obtained as fractions 20–26 from a 5–40% sucrose density
gradient centrifugation of hypotonically-treated brain P2fraction, carried out
as described by Ohyama et al. (2002) for glycerol density gradient centrifu-
gation. The antibody against myosin-Va tail used for immunoblotting was
kindly provided by V. I. Gelfand (University of Illinois, Urbana, IL; Karcher
et al., 2001).
Identification of Myosin-Va
The brain homogenate (S2fraction) was prepared as described by Fujita et al.
(1998). Ca2?-dependent syntaxin-1A binding proteins from rat brain were
detected using PreScission protease (GE Healthcare, Uppsala, Sweden) as
described previously (Ohyama et al., 2002). The 190-kDa syntaxin-1A binding
protein was digested with trypsin and analyzed by mass spectrometry. This
protein contained the sequence YFATVSGSASEANVEEK, which corresponds
to amino acids 179–195 of myosin-Va.
Ternary Complex Formation between Myosin-Va, F-actin,
Cosedimentation experiments were performed as described by Nascimento et
al. (1996). Purified brain myosin-Va (50 nM) was mixed with 500 nM of F-actin
in the presence of 10?6M Ca2?. In some experiments, the mixture was added
to glutathione S-transferase (GST)-syntaxin-1A (50 nM). Cosedimentation was
confirmed by centrifugation of the protein mixture at 100,000 ? g for 1 h
(Nascimento et al., 1996), and the pellet and supernatant were analyzed by
SDS-PAGE followed by staining with Coomassie Brilliant Blue.
We also examined whether actin can access the complex between myo-
sin-Va and syntaxin-1A to form a ternary complex. Myosin-Va (5 nM) and
either actin (50 nM) or syntaxin-1A (5 nM) were first mixed together and
incubated for 1 h at 4°C in the presence of 10?6M Ca2?. The missing third
component (syntaxin-1A or actin, respectively) was then added, and the
mixture was incubated for another 1 h. Next, immunoprecipitation was
carried out as described above using an anti-myosin-Va antibody (1:200) or an
anti-syntaxin-1A antibody (1:200). Myosin-Va, syntaxin-1A, and actin were
detected by immunoblotting.
Biochemical and Molecular Biological Techniques for
Assessing Protein Binding
Native myosin-Va was purified from chick brain (Cheney, 1998). Recombi-
nant myosin-V (DHM5; [1-1193]) was produced in Sf9 cells as described
previously (Homma et al., 2000) or by in vitro translation (Promega, Madison,
WI) using the mouse dilute cDNA (gift of N. A. Jenkins, University of Sao
Paolo, Ribeirao Preto, Brazil; Mercer et al., 1991). DHM5 was detected with
anti-myosin-Va head antibody (gift of R. E. Larson, National Cancer Institute,
Frederick, MD; Nascimento et al., 1996; Evans et al., 1998). The binding
experiments were carried out using GST-syntaxin-1A fusion proteins immo-
bilized on glutathione-Sepharose (Ohyama et al., 2002). In some experiments,
His6-DHM5 fusion protein (Homma et al., 2000) was immobilized on a Ni2?-
chelating column. Various concentrations of Ca2?were generated using an
EGTA-Ca2?buffer with the required amounts of CaCl2and 4 mM EGTA
calculated using Max Chelator or WebMaxC (http://www.stanford.edu/
?cpatton/maxc.html) software. In the reconstitution study, the purified my-
osin-Va and GST-syntaxin 1A [1-262] were incubated together for 1 h, fol-
lowed by an additional 1 h with recombinant SNAP-25, VAMP-2 [1-96], NSF,
and ?-SNAP (Hohl et al., 1998). The concentration ratio of the proteins (except
for DHM5) was determined as described by Hohl et al. (1998). Synaptic
vesicles were purified from adult rat brain as described previously (Huttner
et al., 1983). Bacterial two-hybrid experiments were carried out using Bacte-
rioMatch (Stratagene, La Jolla, CA) according to the manufacturer’s instruc-
tions. The rabbit anti-myosin-V neck antibody was generated against the
neck domain sequence of mouse myosin-Va and was affinity-purified
using protein G-Sepharose (Sigma-Aldrich, St. Louis, MO).
Determination of the Stoichiometry for Binding
The binding stoichiometry between syntaxin-1A and myosin-Va was mea-
sured using a BIAcore3000 (BIAcore, Uppsala, Sweden) by immobilizing
myosin-Va on CH5 carboxymethyl chips and adjusting the resonance units
(RU) to ?10,000 as described in the manufacturer’s instructions. Next, syn-
taxin-1A [1-262] (0.1–25 ?M) in HEPES-buffered saline (10 mM HEPES, pH
7.4, 150 mM NaCl) containing 0.005% Tween 20, 0.1 mM dithiothreitol, and
pCa ? 5.5 was injected into the flow cells of a BIAcore 3000. The sensorgrams
were analyzed using BIA evaluation software version 3.1 (BIAcore). The
stoichiometry was calculated from changes in RU at the point between
association and dissociation on the compensated sensorgram and using 1200
RU as equal to 1.2 ng of mass per flow cell.
Amperometric measurement of exocytotic catecholamine release was per-
formed as described previously (Ohyama et al., 2002; Quetglas et al., 2002)
except that the chromaffin cells were stimulated with 60 mM KCl. Microin-
jection was performed using a 6-d-old culture of chromaffin cells on collagen-
coated coverslips. In each experiment, the cytosol of 50–150 cells was micro-
injected using an Eppendorf injection system. The cytosolic concentration of
the injected fragments was estimated to be 60–120 ?g/ml. For cells injected
with the syntaxin-1A fragment [191-240] or its L222E mutant, the microin-
jected cells were stimulated by 60 mM KCl for 4 s. Cells injected with the
anti-myosin-Va neck and normal antibodies were stimulated with KCl for 5
min, and the exocytotic frequency during the initial (0- to 1-min) and sus-
tained (1- to 5-min) phases was compared with determine the step of exocy-
tosis regulated by the interaction between myosin-Va and syntaxin-1A.
Effects of Syntaxin-1A Binding on Myosin-Va Properties
Myosin-Va ATPase activity was determined in a reaction mixture containing
50 ?g/ml myosin-Va, 420 ?g/ml F-actin, 20 mM imidazole-HCl, pH 7.2, 75
mM KCl, 2.5 mM MgCl2, 2 mM ATP, 4 mM EGTA, enough CaCl2to generate
the desired pCa (between 8 and 5) at 25°C, and the presence or absence of 30
?g/ml syntaxin-1A [1-262]. The time course was measured by removing an
aliquot every 3 min. The ATPase activity was calculated from the concentra-
tion of the released Pi (Chifflet et al., 1988) per mole of myosin-Va per second.
The assay of myosin-Va motility was carried out using rhodamine-phalloidin-
labeled F-actin as described previously (Rock et al., 2000). After blocking the
flow cells with bovine serum albumin, myosin-Va (20–30 ?g/ml) was added
and adsorbed to the cells for 2 min at room temperature. The flow buffer
contained Ca2?(pCa ? 6) in the presence or absence of 1 ?M syntaxin-1A.
Similar results were obtained with 1 ?M and higher concentrations (e.g., 10
?M) of syntaxin-1A (our unpublished data).
The 80-kDa SNARE complex (i.e., the SDS-resistant complex) was isolated
as described previously (Igarashi et al., 1997). Briefly, the immobilized 1 ?M
GST-syntaxin-1A was incubated for 1 h with an equal amount of recombinant
SNAP-25 and VAMP-2 and eluted by cleavage with PreScission protease. The
eluted proteins were then incubated with or without 1 ?M syntaxin-1A
[191-240] for 0.5 h and then treated with SDS-sample buffer at 60°C for 5 min
(which does not break up the SNARE complex). The 80-kDa protein complex
was analyzed by immunoblotting with antibodies specific to syntaxin-1A,
SNAP-25, and VAMP-2, which are components of the neuronal SNARE
Morphological Studies Using Atomic Force Microscopy
AFM was carried out as described previously (Mizuta et al., 2003). Myosin-Va
was diluted to 5–10 ?g/ml in 10 mM HEPES, pH 7.4, containing 2 mM MgCl2.
Next, 5 ?l of the sample was dripped onto freshly cleaved mica and dried
with compressed air. Two minutes later, Milli-Q water (10 ?l) was dripped
onto the mica surface to remove salts, and the surface was immediately
air-dried. The cantilevers (SI-DF40-AL; Seiko Instruments, Neu Isenburg,
Germany) used were rectangular, the force constant was 40 Nm?1, and the
resonance frequency was 250–390 kHz.
Myosin-Va Associates with Syntaxin in a Ca2?-dependent
In the current studies, we isolated a synaptosomal fraction
from cortex and used it as a source for synaptic vesicle
M. Watanabe et al.
Molecular Biology of the Cell4520
protein complexes (Huttner et al., 1983). After solubilization
with a nonionic detergent, we performed immunoprecipita-
tion with a myosin-Va antibody. We found that myosin-Va
was associated with a 35-kDa protein in the presence of 10?6
M Ca2?and Mg2?/ATP. This 35-kDa protein was recog-
nized by antibody against syntaxin-1A, a membrane protein
that is also known as t-SNARE and is involved in regulated
exocytosis (Li and Chin, 2003). Association of syntaxin-1A
with myosin-Va required Ca2?and Mg2?/ATP (Figure 1A).
After treatment with Ca2?, myosin-Va remained associated
with synaptic vesicles purified from the cortex (Figure 1B).
A GST pull-down study using syntaxin-1A mixed with rat
brain homogenate in (Ohyama et al., 2002) revealed a 190-
kDa protein that bound specifically to syntaxin-1A in the
presence of Ca2?and ATP. Using mass spectroscopy, we
confirmed that this protein was myosin-Va. Furthermore,
binding of brain myosin-Va to syntaxin-1A required the
presence of both Ca2?and ATP (Figure 1C). This association
of syntaxin-1A and myosin-Va required at least 10?6M
Ca2?, corresponding to a physiological elevation of Ca2?,
whereas two other syntaxin-1A-binding proteins, Munc-18,
and tomosyn (Ohyama et al., 2002), bound to syntaxin-1A in
the absence of Ca2?(Figure 1D). Although the interaction
between myosin-Va and syntaxin-1A required ATP (Figure
1C), nonhydrolyzable analogues of ATP and ADP also en-
using plasmon resonance revealed that the stoichiometry of
binding was 0.77 ? 0.12 (mean ? SD; n ? 7), implying a 1:1
interaction between syntaxin-1A and myosin-Va dimer. Rat
brain contained other myosins, such as myosin-I and -IIB, but
these did not bind to syntaxin-1A (Figure 1F).
Syntaxin-1A Binding Alters the ATPase Activity but Not
the Motility of Myosin-Va
We next examined whether the properties of myosin-Va are
altered by F-actin. Syntaxin-1A cosedimented with both ac-
tin and myosin-Va (Figure 2A). Syntaxin-1A could bind to
the myosin-Va–actin complex, and actin could associate
with the myosin-Va–syntaxin-1A complex (Figure 2B), indi-
cating that these three proteins can form a complex. Myo-
sin-Va ATPase is activated by Ca2?and actin (Cheney et al.,
1993), and, interestingly, this enhancement of ATPase was
completely inhibited by syntaxin-1A binding at pCa ? 6
(Figure 2C). In contrast, the F-actin myosin Va-dependent
sliding motility was unchanged under these conditions (Fig-
ure 2D). Thus, at pCa ? 6, the binding of myosin-Va to
syntaxin-1A occurs without a large loss of ATP due to
hydrolysis and without an effect on motility.
Interference with Syntaxin-1A–Myosin-Va Complex
Formation Inhibits Exocytosis from Chromaffin Cells
We found that the binding site of myosin-Va lies between
amino acids 191 and 240 of syntaxin-1A, which comprises
the first two-thirds of its H3 domain (Figure 3; A; Li and
Chin, 2003). We also screened the mutated fragments de-
rived from syntaxin-1A [191-240] without the myosin-Va-
binding activity and found that the L222E mutant (syn-
taxin-1A [191-240 L222E]) lacks myosin-Va binding activity
(Figure 3, A and B). Specifically, silver staining showed that,
in the presence of Ca2?, the only protein that was unique to
syntaxin-1A [191-240] was of 190 kDa (Figure 3A). This
190-kDa protein was identified as myosin-Va using mass
spectrometry. A separate experiment confirmed that myo-
sin-Va bound specifically to syntaxin-1A [191-240] but not to
syntaxin-1A [191-240 L222E] (Figure 3, B and C). Further-
more, complex formation between myosin-Va and syn-
taxin-1A was inhibited by syntaxin-1A [191-240] but not by
Immunoprecipitation of the myosin-Va–syntaxin complex from brain
homogenate using an anti-myosin-V antibody. The immunoprecipita-
tion was carried out in the presence or absence of 10?6M Ca2?/2 mM
Mg2?/0.5 mM ATP. (B) Myosin-Va (MV) is localized and remains on
synaptic vesicles purified from cortex even after addition of 1 ?M
Ca2?. Myosin-Va was immunoprecipitated from brain homogenate in
the presence and absence of 1 ?M Ca2?, and myosin-Va, synaptotag-
min I, and synaptophysin were detected by immunoblotting. Synap-
markers. (C) Confirmation of the 10?6M Ca2?/2 mM Mg2?/0.5 mM
ATP-dependent binding of syntaxin-1A to myosin-Va in brain homog-
enate (Crude) or to purified myosin-Va (Purified). Binding of myo-
sin-Va to GST-Syntaxin-1A was assessed in the presence and absence
of Ca2?and Mg-ATP using a GST pull-down assay, followed by
anti-myosin-Va immunoblotting. (D) Ca2?-dependence of myosin-Va
binding to syntaxin-1A. Brain homogenate was incubated with immo-
bilized GST-syntaxin-1A in the presence or absence of 10?6M Ca2?/2
mM Mg2?/0.5 mM ATP. Bound myosin-Va was detected by immu-
noblotting. Myosin-Va binding to syntaxin-1A required 10?6M Ca2?.
In contrast, binding of tomosyn and Munc-18 to syntaxin-1A does not
require Ca2?. (E) Binding of myosin-Va by syntaxin-1A requires ATP
analogues in presence of 10?6M Ca2?. As shown by immunoblotting,
myosin-Va bound to syntaxin-1A only in the presence of 0.5 mM ATP,
ADP, or the nonhydrolyzable ATP analogues adenosine 5?-O-[3-thio-
triphosphate] (ATP?S), 5?-adenylylimidodiphosphate (AMP-PNP), or
0.5 mM ATP with 5 mM 2,3-butanedione monoxime (BDM; a myosin-
syntaxin-1A, even though they are present in the brain homogenate.
Rat brain homogenate was mixed with mixed with GST-syntaxin-1A.
Equal amounts (15 ?l) of the rat brain homogenate (1 ?g/?l; Input)
and of the fraction bound to GST-syntaxin-1A (Bound; see C) were
analyzed by immunoblotting with antibodies against myosin-Va, my-
osin-IIB (gift of T. Shirao, Gunma University Graduate School of Med-
icine, Maebashi, Gunma, Japan), or myr 1A (gift of M. Ba ¨hler, West-
falische Wilhelms University, Mu ¨nster, Germany).
Ca2?-dependent binding of myosin-Va to syntaxin-1A. (A)
Ca2?Interaction of Myosin-V with Syntaxin
Vol. 16, October 20054521
syntaxin-1A [191-240 L222E] (Figure 3D). We confirmed that
syntaxin-1A [191-240] did not affect the SNARE complex
formation (i.e., the SDS-resistant 80-kDa complex; Hayashi et
al., 1995; Figure 3, E and F). Thus, the myosin-Va-binding
fragment [191-240] inhibits the association of myosin-Va and
Chromaffin cells are a typical model system for analyzing
exocytosis, and they are more easily studied than other
motility. (A) In the presence of 10?6M Ca2?, GST-syntaxin-1A [1-262] (52-kDa; indicated by an arrow) cosedimented with myosin-Va (190
kDa) in the presence of F-actin. Brain homogenate was incubated with or without GST-syntaxin-1A [1-262] and in the presence of 10?6M
Ca2?. The bound proteins were separated by SDS-PAGE and stained with Coomassie Brilliant Blue. The molecular masses are shown on the
left. S, supernatant; P, pellet. The thick 43-kDa band consists of monomeric actin (Nascimento et al., 1996). (B) The myosin-Va–actin complex
recruits syntaxin-1A, and the syntaxin-1A–myosin-Va complex recruits actin. Purified brain myosin-Va was first incubated with actin or
recombinant syntaxin-1A, after which the binary protein complexes were immunoprecipitated with an anti-syntaxin-1A antibody (lanes 3 and
4) and an anti-myosin-Va antibody (lanes 1 and 2). In some reactions, recombinant syntaxin-1A was added to the myosin-Va–actin binary
complexes, and in others, actin was added to the myosin-Va-syntaxin-1A binary complexes. Finally, the ternary protein complex, composed
of myosin-Va, syntaxin-1A, and actin, was isolated by immunoprecipitation, and myosin-Va, actin, and syntaxin-1A were detected by
immunoblotting. Lanes 1 and 3, the immunoprecipitated binary complex composed of myosin-Va and either actin (lane 1) or syntaxin-1A
(lane 3); lanes 2 and 4, the immunoprecipitated ternary complex formed by the addition of syntaxin-1A to the myosin-Va-actin binary
complex (lane 2) or by the addition of actin to the myosin-Va-syntaxin-1A binary complex (lane 4). (C) Ca2?/actin-dependent enhancement
of myosin-Va ATPase activity is completely blocked by syntaxin-1A binding. In the presence or absence of 30 ?g/ml syntaxin-1A [1-262], the
ATPase activity was determined in a reaction mixture containing myosin-Va (50 ?g/ml) and F-actin (420 ?g/ml), by calculation of the
released Pi concentration (see Materials and Methods), In the presence of syntaxin-1A (closed circles), actin-activated ATPase activity was
suppressed even at high [Ca2?] (pCa ? 6), whereas in the absence of syntaxin-1A, the actin-activated ATPase activity remained Ca2?
dependent (open circles). (D) The motility of myosin-Va was not altered by binding of syntaxin-1A at pCa ? 6. The assay of myosin-Va
motility was carried out using rhodamine-phalloidin-labeled F-actin as described previously (Rock et al., 2000). Purified myosin-Va (20–30
?g/ml) was added and adsorbed to the cells for 2 min at room temperature. The flow buffer contained Ca2?(pCa ? 6) in the presence or
absence of 1 ?M syntaxin-1A. The average sliding velocities were measured. Each value represents the mean of 70 determinations.
Syntaxin-1A interacts with myosin-Va independently of F-actin and regulates myosin-Va ATPase activity without affecting its
M. Watanabe et al.
Molecular Biology of the Cell4522
taxin-1A on catecholamine release from chromaffin cells as de-
termined by amperometry. Chromaffin cells were microinjected
with syntaxin-1A fragments with potent (syntaxin-1A [191-240])
or lacking potent (syntaxin-1A [191-240 L222E]) myosin-Va-bind-
ing activity and then stimulated for 4 s with 60 mM KCl. As
controls, cells also were microinjected with distilled water (DW)
or not injected (none). (A) Amperometric wave patterns. (B)
Number of spikes of catecholamine release. The values represent
the means ? SEM, and the numbers of determinations (n) were as
follows: None, 15; DW, 11; syntaxin-1A [191-240], 18; and L222E
syntaxin-1A, 13. The asterisk (*) represents a significant difference
between the results using the two syntaxin-1A fragments (p ? 0.05).
Effects of the myosin-Va binding fragment of syn-
by the myosin-V-binding fragment (syntaxin-1A [191-240]) reduces
the frequency of exocytosis from chromaffin cells. (A) The myo-
sin-Va binding site on syntaxin-1A is localized in the first two-thirds
(amino acids 191–240) of its H3 domain. Various GST-syntaxin-1A
constructs were incubated with purified brain myosin-Va, and my-
osin-Va binding was detected by immunoblotting. The numbers
indicate the residue numbers encoded by the GST–syntaxin-1A
constructs. (B) Ca2?-dependent binding of brain proteins to syn-
taxin-1A [191-240] and syntaxin-1A [191-240 L222E]. The immobi-
lized GST-syntaxin-1A [191-240] or GST-syntaxin-1A [191-240
L222E] was incubated with the brain homogenate, and bound pro-
teins were eluted by PreScission protease (GE Healthcare), and
detected by SDS-PAGE and silver staining. The molecular masses
are shown in kilodaltons on the left. Only binding of the 190-kDa
protein, identified as myosin-Va, was different between the two
forms of syntaxin-1A. (C) Syntaxin-1A [191-240] but not syn-
taxin-1A [191-240 L222E] binds myosin-Va. Syntaxin-1A [191-240]
or syntaxin-1A [191-240 L222E] were incubated with brain homog-
enate, after which syntaxin-1A was immunoprecipitated, and
bound myosin-Va was detected by immunoblotting. (D) Competi-
tive inhibition of myosin-Va binding to syntaxin-1A by syntaxin-1A
[191-240]. Immobilized GST-syntaxin-1A [1-262] was incubated
with chromaffin cell lysate, 0.1 mM Ca2?, and no addition (Control),
10 ?M syntaxin-1A [191-240] (?191-240), or 10 ?M syntaxin-1A
[191-240 L222E] (?L222E). Bound myosin-Va was detected by im-
munoblotting. (E) Syntaxin-1A [191-240] does not inhibit formation
of the SNARE complex. GST-syntaxin-1A [1-262] (0.2 ?M) was
incubated for 2 h at 4°C with SNAP-25 (0.2 ?M) or VAMP-2 (0.2
?M) in the absence (Control) or presence of 10 ?M syntaxin-1A
[191-240] (?191-240). The formed SNARE complex was immobi-
lized on glutathione-Sepharose and analyzed by SDS-PAGE, fol-
lowed by silver staining. (F) Syntaxin-1A [191-240] does not disrupt
preformed SNARE complexes. Isolated SNARE complex was incu-
bated with and without syntaxin-1A [191-240] and then analyzed by
immunoblotting for syntaxin-1A (?-Syntaxin), SNAP-25 (?-SNAP-
25), or VAMP-2 (?-VAMP-2).
Inhibition of myosin-Va–syntaxin-1A complex formation
Ca2?Interaction of Myosin-V with Syntaxin
Vol. 16, October 20054523
systems such as central neurons (Burgoyne and Morgan,
2003). Moreover, it is the most suitable system for examining
whether a biochemical interaction plays a physiological role
in exocytosis (Fisher et al., 2001; Ohyama et al., 2002; Quet-
glas et al., 2002). For these reasons, we used chromaffin cell
exocytosis to investigate the function of the Ca2?-dependent
interaction between syntaxin-1A and myosin-Va. Ampero-
metric measurements were used to examine the physiolog-
ical role of myosin-Va–syntaxin-1A binding because it is a
powerful method not only for quantitative measurement of
exocytosis but also for characterizing the mechanism of exo-
cytosis (Segre et al., 2000; Fisher et al., 2001). We therefore
performed an amperometric assay of catecholamine release
for dense-core vesicles in chromaffin cells (Ohyama et al.,
2002; Quetglas et al., 2002), which are known to possess
myosin-Va (Rose ´ et al., 2003). We found that the syntaxin-1A
[191-240] specifically reduced the exocytotic frequency,
whereas syntaxin-1A [191-240 L222E] fragment had no effect
(Figure 4, A and B).
Syntaxin-1A Binds to the Neck Domain of myosin-Va
after Ca2?-dependent CaM Release
All of the known membrane-associated myosin-Va-binding
proteins bind to its globular tail (Reck-Peterson et al., 2000).
Therefore, to investigate where on myosin-Va syntaxin-1A
binds, we first examined the binding of syntaxin-1A to
cated in the neck domain of myosin-Va. (A)
Recombinant myosin-Va without the globular
tail (DHM5) can bind syntaxin-1A in a Ca2?-
dependent manner in the presence of and
MgATP. DHM5 was incubated with GST-syn-
taxin-1A in the presence and absence of 10?6
M Ca2?and with or without MgATP. DHM5
binding was detected by immunoblotting
with an antibody against anti-myosin-Va
head. The binding specificity of the recombi-
nant tailless myosin-Va (DHM5) is similar to
the full-length brain myosin-Va (see Figure
1D). (B) Syntaxin-1A binds to immobilized
[1-262] (30-kDa) was incubated with Ni2?-
NTA-immobilized His6-DHM5 in the pres-
ence of 10?6M Ca2?(see Figure 1C) and
eluted with SDS-sample buffer. The eluted
sample was separated by SDS-PAGE and sil-
ver stained. The 150-kDa band is DHM5. (C)
The binding of truncated myosin-Va (DHM5)
by syntaxin-1A requires a pCa of 6.6. Syn-
taxin-1A [1-262] was incubated with DHM5
and the purified myosin-Va from brain in the
presence of various concentrations of Ca2?.
Binding of myosin-Va and DHM5 was de-
tected by immunoblotting with an antibody
against myosin-Va. Top and middle, dose re-
sponse of Ca2?for binding of syntaxin-1A by
DHM5 between pCa 4 and 8. Brain myo-
sin-Va (Myosin-V) and DHM5 show a similar
dependence on Ca2?for the binding of syn-
taxin-1A. Bottom, dose response of Ca2?for
binding of syntaxin-1A by DHM5 between
pCa 6 and 7. (D) The neck domain of myo-
sin-Va is necessary for syntaxin-1A binding.
Truncated forms of myosin-Va were pro-
duced by biotin-labeled in vitro translation of
mouse myosin-Va cDNA (dilute): H, head do-
main only (amino acids 1–755); HN-ATPBS,
The syntaxin-1A-binding site is lo-
head and neck domains (amino acids 1–911) lacking the ATP-binding site (amino acids 164–171); HN-AcBS, head and neck domain lacking
the actin-binding site (amino acids 643–666); and HN, head and neck domains (Espreafico et al., 1992). Top, in vitro-translated proteins.
Bottom, binding of these constructs to syntaxin-1A in the presence of 10?6M Ca2?and 0.5 mM ATP. Unlike the head domain alone, the
head-and-neck portion can bind to syntaxin-1A. Note that the ATP-binding site but not the actin-binding site is also necessary for the binding.
The biotin-labeled in vitro-translated proteins were visualized by the streptavidin-conjugated alkaline phosphatase. (E) A bacterial two-
hybrid assay (BacterioMatch) reveals that the neck domain of myosin-Va contains the syntaxin-1A-binding site. Reporter strains of Escherichia
coli (Stratagene) were cotransfected with myosin-Va and syntaxin-1A constructs and incubated at 30°C for 24 h. Lane 1, pBT?pTRG
(manufacturer’s negative control); lane 2, pBT-LGF2 ? pTRG-GAL11p (manufacturer’s positive control); lane 3, pBT-6IQ (amino acids
764–908 of mouse myosin-Va) ? pTRG-CaM; lane 4, pBT-6IQ ? pTRG-syntaxin-1A [1-262]; lane 5, pBT-1IQ (amino acids 764–787 of mouse
myosin Va) ? pTRG-syntaxin-1A [1-262]. Lanes 2–4 were judged to be the binding-positive plates. (F) Ca2?requirement for binding of
syntaxin-1A to myosin-V is due to Ca2?-dependent release of CaM from the neck domain of myosin-Va. Immobilized His6-DHM5, which
copurified with CaM from Sf9 cells (Homma et al., 2000), was first preincubated for 1 h with phosphate-buffered saline (PBS) containing 10?6
M Ca2?(Ca2?-phosphate-buffered saline; ?) or with Ca2?-free PBS (?). The His6-DHM5-CaM complex was then incubated for another 1 h
with syntaxin-1A in Ca2?-phosphate-buffered saline (?) or Ca2?-free PBS (?). Top, detection of syntaxin-1A binding by immunoblotting.
After the preincubation with Ca2?, which released CaM from DHM5, Ca2?was no longer necessary for the binding of syntaxin-1A by DHM5.
Immunoblotting for CaM confirmed that, after the preincubation with Ca2?, CaM was released into the supernatant (middle) and no longer
bound to DHM5 (bottom).
M. Watanabe et al.
Molecular Biology of the Cell 4524
DHM5, a recombinant myosin-Va protein the lacks the glob-
ular tail (Homma et al., 2000). DHM5 did not bind VAMP-2
(our unpublished data) because it lacks a VAMP-2-binding
site (Ohyama et al., 2001). Surprisingly, DHM5 bound syn-
taxin-1A in the presence of Ca2?(Figure 5A). The Ca2?
dependence of DHM5 binding by syntaxin-1A was similar
to that of native brain myosin-Va (Figures 1C and 5B), and
the minimal requirement of Ca2?for binding was pCa ? 6.6
(Figure 5C). In addition, using truncated myosin-Va pro-
duced by in vitro translation, we confirmed that the whole
head-and-neck portion could bind to syntaxin-1A in the
presence of Ca2?and ATP. Removal of the neck region
resulted in a substantial loss of syntaxin-1A binding. In
addition, a head-and-neck fragment lacking the actin bind-
ing site still bound syntaxin-1A, but binding was absent
when the ATP binding site was deleted (Figure 5D). Further
studies in a bacterial two-hybrid assay, which is a modifi-
cation of the yeast two-hybrid method, indicated that the six
IQ-motifs of the neck bind to syntaxin-1A as well as CaM,
although the first IQ alone does not mediate binding (Figure
5E). Collectively, these results demonstrate that the binding
site for syntaxin-1A is in the neck of myosin-Va.
CaM is known to be released from the neck of myosin-Va
in the presence of micromolar Ca2?(Cameron et al., 1998;
Homma et al., 2000). We confirmed that preincubation of
myosin-Va with Ca2?released the bound CaM (Figure 5F).
In addition, after this treatment, syntaxin-1A could bind
myosin-Va in the absence of Ca2?(Figure 5F). Thus, we
suspected that the Ca2?-dependent syntaxin-1A-myosin-Va
binding is due to the Ca2?-dependent release of CaM from
the myosin-Va neck; in other words, the binding of syn-
taxin-1A occurs at the same site as CaM.
Next, we directly visualized syntaxin-1A-myosin-Va
binding by AFM, a new technology for imaging biological
molecules at nanometer resolution (Horber and Miles, 2003).
The AFM studies show that syntaxin-1A binding occurs
between two heads of the myosin dimer and not in the head
or tail (Figure 6, A and B; Cheney et al., 1993). Similar to
these AFM findings, rotary shadowing views of this com-
plex reveal that the binding site was between the two heads
and distinct from the head or the tail (Katayama, Watanabe,
and Igarashi, unpublished observations). This is also the first
report that the IQ-motif binds proteins other than the myo-
sin light chains or CaM family proteins (Cheney et al., 1993;
Vale, 2003). Homma et al. (2000) suggested that Ca2?-depen-
dent CaM release most likely occurs at the sixth IQ motif.
Our AFM results provide further support for this possibility
because they showed that syntaxin-1A binds close to bifur-
cation of the neck region of myosin-Va (Figure 6, A and B).
Myosin-Va Can Bind to the SNARE Complex via
showed that the myosin-Va–syntaxin-1A complex bound
SNAP-25 and VAMP-2, two neuronal SNAREs involved in
exocytosis (Figure 7A). Because VAMP-2 binds to the tail of
myosin-V (Prekeris and Terrian, 1997; Ohyama et al., 2001),
we examined whether the SNARE complex can be bound by
a complex between syntaxin-1A and DHM5, the truncated
form of myosin-Va lacking a tail (Figure 5, B and C). We first
confirmed that the binding of syntaxin-1A to DHM5 satu-
rated at a 1:1 ratio. At concentrations below saturation,
VAMP-2 and SNAP-25 bound to the DHM5–syntaxin-1A
complex quantitatively (Figure 7B). Immunoprecipitation
further showed that the myosin-Va–syntaxin-1A complex
did not associate with NSF or ?-SNAP, proteins that disso-
ciate the SNARE complex (Duman and Forte, 2003; Figure
the top row of images) and syntaxin-1A-bound myosin-Va (14
views in the next four rows). The 14 views of the complex show that
the bound syntaxin-1A is located around the bifurcation of the two
necks, forming a knot-like structure (arrowheads) that can be clearly
distinguished from the heads and the globular tails. Illustrations
depicting the arrangement of syntaxin-1A and the heads and tails of
myosin-Va are shown under each micrograph. Bars, 50 nm. (B)
Putative model of the complex between myosin-Va and syntaxin-
1A. The neck region of myosin-Va senses the Ca2?elevation its neck
and exchanges one CaM molecule for one syntaxin-1A molecule.
(A) AFM views of native myosin-Va alone (four views in
Ca2?Interaction of Myosin-V with Syntaxin
Vol. 16, October 2005 4525
5A), and reconstitution studies revealed that, in the presence
of VAMP and SNAP-25, syntaxin-1A associates with either
?-SNAP/NSF or DHM5 (Figure 7C). Similarly, we found
that the SNARE complex interacts with either ?-SNAP/NSF
or DHM5 (Figure 7D). These results demonstrate that myo-
sin-Va can bind the SNARE complex including VAMP-2 and
SNAP-25 and that NSF/?-SNAP can release myosin-Va
from the SNARE complex.
Anti-Myosin-V Neck Antibody, Which Blocks the
Interaction between Myosin-V and Syntaxin-1A, Affects
the Late Step of Exocytosis
We generated an antibody specific to the neck domain of
myosin-V (Figure 8A). This antibody inhibits the myosin-
Va–syntaxin-1A interaction as effectively as syntaxin-1A
[191-240] (Figure 8B). The antibody did not affect formation
of the SNARE complex (Figure 8C) nor did it significantly
reduce the sliding velocity of myosin-Va (0.24 ? 0.15 ?m/s;
n ? 70; p ? 0.1 based on Student’s t test; Figure 8D; see also
Like syntaxin-1A [191-240], the neck-specific antibody re-
duced the exocytotic frequency as measured by amperom-
etry (Figure 9, A–C). In this particular experiment, we stim-
ulated the cells for 5 min and analyzed the exocytotic
frequency in the initial (0–1 min) and sustained phases (1–5
min) to detect in which step the syntaxin-1A–myosin-Va
interaction is involved. The anti-myosin-Va neck antibody
reduced the total number events during the full 5 min of
stimulation (Figure 9B). Interestingly, the reduction of the
event frequency was predominant not in the initial phase
but in the sustained phase (Figure 9C).
To further define at which step this interaction is involved,
chromaffin cells were injected with the anti-myosin-Va neck
antibody and stimulated with high K?for 5 min. This stim-
ulation was for a much longer time than we used for studies
of inhibition by syntaxin-1A [191-240] (4 s; Figure 4). The
wave patterns reveal that the anti-neck antibody inhibited
exocytosis (Figure 9A). Also, the sum of events over the
entire 5-min period shows that anti-myosin-Va neck anti-
body significantly reduced exocytosis compared with the
normal IgG (Figure 9B). Interestingly, exocytotic release dur-
ing the first minute (0–1 min; initial phase) was not affected
by the anti-myosin-Va neck antibody, but this antibody se-
verely attenuated exocytosis during the next 4 min (1–5 min;
sustained phase) (Figure 9C).
taxin-1A. (A) The myosin-Va–syntaxin-1A complex contains other
SNAREs. The myosin-Va–syntaxin-1A complex was immunopre-
cipitated with an anti-syntaxin-1A antibody (?-syntaxin monoclonal
antibody), an anti-myosin-Va antibody (?-Myosin-V pAb), or a
control IgG from a pool of myosin-Va and syntaxin-1A-enriched
fractions, which were obtained by 5–40% sucrose density gradient
fractionation of hypotonically treated and Triton X-100-solubilized
brain P2fraction. The immunoprecipitated complexes were ana-
lyzed by immunoblotting. (B) Reconstitution study using recombi-
nant SNAREs. Protein binding was assessed by immunoblotting.
Top, His6-DHM5 (0.2 ?M of dimer) immobilized on Ni2?-NTA resin
was incubated with 0.05–1.6 ?M of recombinant syntaxin-1A in the
presence of 10?6M Ca2?, and syntaxin-1A binding was assessed by
immunoblotting DHM5 binding saturated at 0.2 ?M syntaxin-1A.
Bottom four, His6-DHM5-syntaxin-1A complex was formed with
0.05, 0.1, or 0.2 ?M of syntaxin-1A and then incubated with the
equal concentrations of SNAP-25 or VAMP-2. Controls contained no
Myosin-Va interacts with the SNARE complex via syn-
syntaxin-1A. Bound SNAP-25 and VAMP-2 were eluted with SDS-
sample buffer and detected by immunoblotting. Note that SNAP-25
and VAMP-2 did not bind to myosin-Va in the absence of syntaxin-
1A. (C) Reconstitution study using recombinant SNAREs, NSF, and
?-SNAP. Immobilized GST-syntaxin-1A (0.2 ?M) was mixed with
(lane 1) or without (lane 2) DHM5 (0.2 ?M dimer), and in the
presence of 10?6M Ca2?. NSF was incubated with ?-SNAP for 2 h
at 4°C to form a complex. This NSF–?-SNAP complex was mixed
with VAMP-2 and SNAP-25 and then incubated for 2 h with GST-
syntaxin-1A-DHM5 in the presence of 10?6M Ca2?. Proteins bound
to syntaxin-1A were visualized by immunoblotting. DHM5 bound
to syntaxin-1A in the presence of SNAP-25 and VAMP-2 but not in
the presence of NSF/?-SNAP. (D) The SNARE complex interacts
with either ?-SNAP/NSF or DHM5. The SNARE complex (0.2 ?M)
was formed as in B and C and then incubated for 1 h with ?-SNAP/
NSF (0.2 ?M). This complex was then incubated for 1 h with DHM5
(1 ?M) in the presence of 10?6M Ca2?(lane 1). Alternatively,
DHM5 (0.2 ?M) was first incubated with the SNARE complex
followed by ?-SNAP/NSF (1 ?M) (lane 2). Bound proteins were
detected by immunoblotting.
M. Watanabe et al.
Molecular Biology of the Cell 4526
The current model of exocytosis, based on the SNARE mech-
anism (Duman and Forte, 2003), does not completely ac-
count for the fact that Ca2?is required at several steps of
vesicular recycling (Burgoyne and Morgan, 2003). In this
study, we found a submicromolar Ca2?-dependent interac-
tion between myosin-Va, a putative molecular motor for
synaptic vesicles, and syntaxin-1A, a neuronal membrane
t-SNARE. We also presented evidence that this interaction
contributes to the regulation of exocytosis in chromaffin
cells. Our current study is the first clear and detailed dem-
onstration that the Ca2?-dependent binding site for syn-
taxin-1A is neck rather than its tail of myosin-Va.
Is the Interaction between Myosin-Va and Syntaxin-1A
Physiologically Important for Exocytotic Regulation?
We applied two probes to inhibit this interaction specifically:
the myosin-Va-binding fragment (syntaxin-1A [191-240]),
and an anti-myosin-Va neck antibody. We found that the
exocytotic frequency was reduced by both probes, indicating
that they inhibited the association of myosin-Va and syn-
taxin-1A. Furthermore, these results confirmed this interac-
tion participates in the regulation of exocytosis.
We next asked in which step of exocytosis this interaction
functions. Exocytotic vesicles are classified into readily re-
leasable and reserve pools. The readily released pool is
released first, and the reserve pool is released after the
former is depleted (Rettig and Neher, 2002). In amperomet-
ric analysis, the frequency of the exocytotic response in the
initial phase corresponds to the number of docked or readily
releasable vesicles, and the frequency in the sustained phase
represents the release of the newly recruited vesicles (Ku-
makura et al., 2004). The pronounced inhibition of the fre-
quency in the sustained phase by the anti-myosin-Va neck
antibody indicates that the interaction between myosin-Va
and syntaxin-1A affects the recruitment of vesicles to the
readily releasable pool. Therefore, these results, together
with the fact that myosin-Va is a cargo-conveying motor
molecule (Reck-Peterson et al., 2000), suggest that the inter-
action between myosin-Va and syntaxin-1A affects the pro-
cess of vesicle mobilization from the reserve pool (i.e., re-
plenishment of the docked vesicle pool).
As in trafficking via the Golgi apparatus, we anticipate
that the exocytotic vesicle tethering process is mediated by a
long coiled-coil protein that regulates the vesicle-target
membrane distance at a point before fusion (Li and Chin,
2003; Gillingham and Munro, 2003). Myosin-V, which has a
long coiled-coil shaft, is likely involved in this process
(Cheney et al., 1993). Myosin-Va on the vesicles binds to
syntaxin-1A at the plasma membrane, and, along with other
putative tethering molecules (i.e., Rab proteins and/or the
exocyst complex), induces vesicular tethering and exocyto-
sis. It also is thought that myosin-VI, a minus-end motor,
plays a role in endocytosis (Hasson, 2003). Thus, as depicted
in Figure 10, it is plausible that myosin-Va, a plus-directed
motor (Cheney et al., 1993), is involved in exocytotic events.
This possibility is strongly supported by a very recent report
that movements of insulin-containing dense-core secretory
vesicles along the cortical actin network depend on myo-
sin-Va and are essential for regulated exocytosis (Varadi et
al., 2005). In addition, syntaxin-1 is localized close to the site
of exocytosis (Stanley et al., 2003; Ohara-Imaizumi et al.,
2004), where it could participate in the process of exocytosis
by interacting with myosin-Va.
A previous report indicated that hippocampal slices of
dilute lethal mice, which lack myosin-Va, do not show a
by anti-myosin-Va neck antiserum reduces the frequency of exocytosis
from chromaffin cells. (A) Characterization of the myosin-V-neck an-
tiserum by immunoblotting. The antiserum recognized the 190-kDa
myosin-V and weakly detected (?10% of the 190-kDa protein), its
130-kDa proteolytic fragment. (B) Anti-myosin-V neck antiserum in-
hibits the binding of myosin-Va by syntaxin-1A. Immobilized GST-
syntaxin-1A [1-262] was incubated with chromaffin cell lysate and
1:200 anti-myosin-Va neck antiserum (??-MV neck antibody), normal
rabbit serum (?Normal serum), or no serum (No addition) in the
presence of 1 ?M Ca2?. Bound myosin-Va was detected by immuno-
blotting using an anti-myosin-Va globular tail antibody. (C) SNARE
complex formation is not affected by the anti-myosin-Va neck anti-
body. Experiments were carried out in the absence (Control) or pres-
ence of 1:200 anti-myosin-Va neck antibody (??-MV neck antibody) as
described in B. (D) The anti-myosin-Va neck antibody does not alter
out as described in Figure 2D in the presence of 1:200 anti-myosin-Va
neck antibody (bottom; n ? 80) or normal IgG (middle; n ? 66) at
pCa ? 6. These results were similar to those obtained in the presence
of the Ca2?chelator EGTA and no added antibody (top; n ? 85).
Inhibition of myosin-Va–syntaxin-1A complex formation
Ca2?Interaction of Myosin-V with Syntaxin
Vol. 16, October 20054527
significant defect in glutamate release (Schnell and Nicoll,
2001), which seems to contradict our current results. This
may be due to differences in the myosin-Va dependence of
glutamate and catecholamine release from dense-core vesi-
cles. Despite this contradictory data, our findings are con-
sistent with recent reports that retinal neurotransmitter re-
lease is abnormal in dilute lethal mice (Libby et al., 2004) and
that dilute lethal mutant mice have a defect in basal neuro-
transmitter release and presynaptic plasticity (Trinchese et
al., 2003). This latter report used cultured neurons derived
from 1-d-old mice, whereas Schnell and Nicoll (2001) used
tissue slices obtained from 12- to 19-d-old mice, suggesting
that the differences may be due to the age of the mice used
in the studies. Furthermore, according to a recent detailed
immunohistochemical study the brain (Tilelli et al., 2003), the
hippocampus does not express much myosin-Va, and most
of the myosin-Va immunoreactivity is associated with the
neuronal cell bodies rather than the neuropila, which are
enriched with synaptic terminals. Thus, differences in the
levels of myosin-Va in hippocampal synapses mutant be-
tween wild-type and the dilute lethal may be subtle. Finally,
other proteins, such as additional myosin-V isoforms, may
compensate for the loss of myosin-Va function in the dilute
lethal hippocampal slices (Schnell and Nicoll, 2001; Vale,
2003), but not in cell culture.
CaM-dependent Regulation of Exocytosis through Ca2?-
dependent Interaction between Myosin-Va and Syntaxin-1A
CaM binds to the myosin-Va neck as a light chain via its
IQ-motifs (Cheney et al., 1993) but is released when the
intracellular Ca2?rises to micromolar concentrations (Cam-
eron et al., 1998; Homma et al., 2000). We found that, after
Ca2?-dependent release of CaM, syntaxin-1A can bind my-
osin-Va even in the absence of Ca2?. Thus, we suspected
that the apparent Ca2?dependence of myosin Va-syn-
taxin-1A binding is due to Ca2?-dependent release of CaM
from the myosin-Va neck, and exposure of an otherwise
concealed syntaxin-1A binding site. Our results further in-
dicate that CaM can sense submicromolar Ca2?through the
release of CaM from myosin-Va (Cameron et al., 1998;
Homma et al., 2000); the binding between myosin-Va and
syntaxin-1A requires at least 0.3 ?M intracellular Ca2?(Fig-
ure 4B), which corresponds to the level of Ca2?when secre-
tory granules enter the readily releasable pool (Burgoyne
and Morgan, 2003).
CaM, the most abundant Ca2?-sensitive protein, may be
widely responsible for micromolar Ca2?sensitivity (Bur-
goyne and Clague, 2003). We demonstrated previously that
exocytosis is regulated by Ca2?/CaM-dependent protein
kinase II (CaMKII), which binds to syntaxin-1A in a Ca2?-
metric measurements. Amperometric analysis of the exocytotic re-
sponse of chromaffin cells to a 5-min stimulation with 60 mM KCl in
the absence (Control) and presence of anti-myosin-Va neck anti-
body (?-MV neck) or normal mouse IgG (Normal IgG). (A) Am-
perometric patterns and the (B) total frequency of exocytotic events
in response to the 5-min stimulation with 60 mM KCl. (C) Ampero-
metric analysis of the event frequency in the initial and sustained
phase of the response reveals that anti-myosin-Va neck antibody
predominantly affects the sustained phase. The values represent the
means ? SEM (n ? 11 [Control]; n ? 13 [?-MV neck]; and n ? 16
[Normal IgG]). Open bars, control; closed bars, anti-myosin-Va neck
antibody; hatched bars, no addition. A significant difference was
obtained only for the sustained phase (1–5 min). Asterisks (*) in B
and C indicate significant differences between the results with the
control and anti-myosin-Va neck antibodies (p ? 0.05).
Effect of the anti-myosin-Va neck antibody on ampero-
M. Watanabe et al.
Molecular Biology of the Cell 4528
dependent manner when it is autophosphorylated (Ohyama
et al., 2002). Although the CaM-binding sites of myosin-Va
and CaMKII are distinct (Ba ¨hler and Rhoads, 2002), their
Ca2?-dependencies for syntaxin-1A binding are very simi-
lar. Our current studies also could explain the involvement
of CaM in exocytosis (Sakaba and Neher, 2001) and other
CaM-dependent interactions (Junge et al., 2004).
Our results demonstrate that submicromolar Ca2?concen-
trations induce the 1:1 binding of syntaxin-1A to the myo-
sin-Va neck. This further suggests that syntaxin-1A might
mimic CaM for binding at the vacant IQ-motifs. Our results
suggest that this complex modulates SNARE-dependent in-
teractions and regulates at least the exocytosis of dense-core
vesicles by forming a link between vesicles and their targets;
in other words, the complex regulates the recruitment of the
vesicles to the readily releasable pool during sustained se-
cretion. Recently, the Ca2?/CaM-dependent interaction be-
tween the cargo-conveying tail and the head of myosin-Va
was shown to be an important factor regulating its confor-
mation (Krementsov et al., 2004; Li et al., 2004, 2005; Wang et
al., 2004). Ca2?/CaM is an important regulator of both exo-
cytosis and the cargo-conveying activity of myosin-Va, and
further studies of this novel interaction should help eluci-
date the roles of myosin-Va in Ca2?-regulated exocytosis.
We thank all of the donors for the cDNAs and the antibodies; E. Akaishi-
Onodera and M. Sato-Igarashi for technical assistance; and T. Abe, T. Ando,
O. Arancio, P. C. Bridgman, E. Katayama, K. Hoshino, M. Norita, and M.
Takahashi for helpful discussions. This work was supported by Grants-in-Aid
from the Ministry of Education, Sciences, Culture, Sports, and Technology of
Japan (#15029218, #16015240, #16044216, and #17023019 to M. I.); the Japan
Society for the Promotion of Sciences (#15300123 to M. I.); the Life Science
Foundation (to M. I.); the Brain Science Foundation (to M. I.); the Yujin
Memorial Foundation (to M. I.); the project-promoting grant from Niigata
University (to M. I.); and the Tsukada Milk Foundation for Medical Research
(to M. W.).
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Putative model summarizing the
Ca2?Interaction of Myosin-V with Syntaxin
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