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1.
Figure 6.

Figure 6. From: Myosin-Va Regulates Exocytosis through the Submicromolar Ca2+-dependent Binding of Syntaxin-1A.

(A) AFM views of native myosin-Va alone (four views in 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.

Michitoshi Watanabe, et al. Mol Biol Cell. 2005 October;16(10):4519-4530.
2.
Figure 10.

Figure 10. From: Myosin-Va Regulates Exocytosis through the Submicromolar Ca2+-dependent Binding of Syntaxin-1A.

Putative model summarizing the results. Myosin-Va, which mediates F-actin-dependent conveyance to the plasma membrane (cf. Giner et al., 2005), is on the surface of the vesicle and binds to syntaxin-1A when the intracellular [Ca2+] increases to 0.3–0.4 μM. After the complex between myosin-Va and syntaxin-1A tethers the vesicle to the membrane, it recruits other SNARE proteins to form the SNARE complex. A second larger elevation in [Ca2+] induces vesicular fusion (Rettig and Neher, 2002) and stimulates the exchange of myosin-Va for NSF/α-SNAP (Our unpublished data; see Figure 5) in the SNARE complex. Myosin-VI, a reverse motor, mediates endocytosis, whereas myosin-Va, an orthotropic motor, probably participates in exocytosis.

Michitoshi Watanabe, et al. Mol Biol Cell. 2005 October;16(10):4519-4530.
3.
Figure 4.

Figure 4. From: Myosin-Va Regulates Exocytosis through the Submicromolar Ca2+-dependent Binding of Syntaxin-1A.

Effects of the myosin-Va binding fragment of syntaxin-1A on catecholamine release from chromaffin cells as determined 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-binding 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).

Michitoshi Watanabe, et al. Mol Biol Cell. 2005 October;16(10):4519-4530.
4.
Figure 9.

Figure 9. From: Myosin-Va Regulates Exocytosis through the Submicromolar Ca2+-dependent Binding of Syntaxin-1A.

Effect of the anti-myosin-Va neck antibody on amperometric measurements. Amperometric analysis of the exocytotic response of chromaffin cells to a 5-min stimulation with 60 mM KCl in the absence (Control) and presence of anti-myosin-Va neck antibody (α-MV neck) or normal mouse IgG (Normal IgG). (A) Amperometric patterns and the (B) total frequency of exocytotic events in response to the 5-min stimulation with 60 mM KCl. (C) Amperometric 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 [α-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).

Michitoshi Watanabe, et al. Mol Biol Cell. 2005 October;16(10):4519-4530.
5.
Figure 8.

Figure 8. From: Myosin-Va Regulates Exocytosis through the Submicromolar Ca2+-dependent Binding of Syntaxin-1A.

Inhibition of myosin-Va–syntaxin-1A complex formation by anti-myosin-Va neck antiserum reduces the frequency of exocytosis from chromaffin cells. (A) Characterization of the myosin-V-neck antiserum 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 inhibits 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 (+α-MV neck antibody), normal rabbit serum (+Normal serum), or no serum (No addition) in the presence of 1 μM Ca2+. Bound myosin-Va was detected by immunoblotting using an anti-myosin-Va globular tail antibody. (C) SNARE complex formation is not affected by the anti-myosin-Va neck antibody. Experiments were carried out in the absence (Control) or presence of 1:200 anti-myosin-Va neck antibody (+α-MV neck antibody) as described in B. (D) The anti-myosin-Va neck antibody does not alter myosin-Va-based sliding velocity. Velocity measurements were carried 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).

Michitoshi Watanabe, et al. Mol Biol Cell. 2005 October;16(10):4519-4530.
6.
Figure 3.

Figure 3. From: Myosin-Va Regulates Exocytosis through the Submicromolar Ca2+-dependent Binding of Syntaxin-1A.

Inhibition of myosin-Va–syntaxin-1A complex formation by the myosin-V-binding fragment (syntaxin-1A [191-240]) reduces the frequency of exocytosis from chromaffin cells. (A) The myosin-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 myosin-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 syntaxin-1A [191-240] and syntaxin-1A [191-240 L222E]. The immobilized GST-syntaxin-1A [191-240] or GST-syntaxin-1A [191-240 L222E] was incubated with the brain homogenate, and bound proteins 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 syntaxin-1A [191-240 L222E] binds myosin-Va. Syntaxin-1A [191-240] or syntaxin-1A [191-240 L222E] were incubated with brain homogenate, after which syntaxin-1A was immunoprecipitated, and bound myosin-Va was detected by immunoblotting. (D) Competitive 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 μM syntaxin-1A [191-240] (+191-240), or 10 μM syntaxin-1A [191-240 L222E] (+L222E). Bound myosin-Va was detected by immunoblotting. (E) Syntaxin-1A [191-240] does not inhibit formation of the SNARE complex. GST-syntaxin-1A [1-262] (0.2 μM) was incubated for 2 h at 4°C with SNAP-25 (0.2 μM) or VAMP-2 (0.2 μM) in the absence (Control) or presence of 10 μM syntaxin-1A [191-240] (+191-240). The formed SNARE complex was immobilized on glutathione-Sepharose and analyzed by SDS-PAGE, followed by silver staining. (F) Syntaxin-1A [191-240] does not disrupt preformed SNARE complexes. Isolated SNARE complex was incubated with and without syntaxin-1A [191-240] and then analyzed by immunoblotting for syntaxin-1A (α-Syntaxin), SNAP-25 (α-SNAP-25), or VAMP-2 (α-VAMP-2).

Michitoshi Watanabe, et al. Mol Biol Cell. 2005 October;16(10):4519-4530.
7.
Figure 7.

Figure 7. From: Myosin-Va Regulates Exocytosis through the Submicromolar Ca2+-dependent Binding of Syntaxin-1A.

Myosin-Va interacts with the SNARE complex via syntaxin-1A. (A) The myosin-Va–syntaxin-1A complex contains other SNAREs. The myosin-Va–syntaxin-1A complex was immunoprecipitated with an anti-syntaxin-1A antibody (α-syntaxin monoclonal antibody), an anti-myosin-Va antibody (α-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 P2 fraction. The immunoprecipitated complexes were analyzed by immunoblotting. (B) Reconstitution study using recombinant SNAREs. Protein binding was assessed by immunoblotting. Top, His6-DHM5 (0.2 μM of dimer) immobilized on Ni2+-NTA resin was incubated with 0.05–1.6 μM of recombinant syntaxin-1A in the presence of 10–6 M Ca2+, and syntaxin-1A binding was assessed by immunoblotting DHM5 binding saturated at 0.2 μM syntaxin-1A. Bottom four, His6-DHM5-syntaxin-1A complex was formed with 0.05, 0.1, or 0.2 μM of syntaxin-1A and then incubated with the equal concentrations of SNAP-25 or VAMP-2. Controls contained no 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 α-SNAP. Immobilized GST-syntaxin-1A (0.2 μM) was mixed with (lane 1) or without (lane 2) DHM5 (0.2 μM dimer), and in the presence of 10–6 M Ca2+. NSF was incubated with α-SNAP for 2 h at 4°C to form a complex. This NSF–α-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 10–6 M 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/α-SNAP. (D) The SNARE complex interacts with either α-SNAP/NSF or DHM5. The SNARE complex (0.2 μM) was formed as in B and C and then incubated for 1 h with α-SNAP/NSF (0.2 μM). This complex was then incubated for 1 h with DHM5 (1 μM) in the presence of 10–6 M Ca2+ (lane 1). Alternatively, DHM5 (0.2 μM) was first incubated with the SNARE complex followed by α-SNAP/NSF (1 μM) (lane 2). Bound proteins were detected by immunoblotting.

Michitoshi Watanabe, et al. Mol Biol Cell. 2005 October;16(10):4519-4530.
8.
Figure 1.

Figure 1. From: Myosin-Va Regulates Exocytosis through the Submicromolar Ca2+-dependent Binding of Syntaxin-1A.

Ca2+-dependent binding of myosin-Va to syntaxin-1A. (A) Immunoprecipitation of the myosin-Va–syntaxin complex from brain homogenate using an anti-myosin-V antibody. The immunoprecipitation was carried out in the presence or absence of 10–6 MCa2+/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 μM Ca2+. Myosin-Va was immunoprecipitated from brain homogenate in the presence and absence of 1 μM Ca2+, and myosin-Va, synaptotagmin I, and synaptophysin were detected by immunoblotting. Synaptotagmin I (Stg) and synaptophysin (Syp) are shown as synaptic vesicle markers. (C) Confirmation of the 10–6 M Ca2+/2 mM Mg2+/0.5 mM ATP-dependent binding of syntaxin-1A to myosin-Va in brain homogenate (Crude) or to purified myosin-Va (Purified). Binding of myosin-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 immobilized GST-syntaxin-1A in the presence or absence of 10–6 M Ca2+/2 mM Mg2+/0.5 mM ATP. Bound myosin-Va was detected by immunoblotting. Myosin-Va binding to syntaxin-1A required 10–6 M 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 10–6 M 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-thiotriphosphate] (ATPγS), 5′-adenylylimidodiphosphate (AMP-PNP), or 0.5 mM ATP with 5 mM 2,3-butanedione monoxime (BDM; a myosin-ATPase inhibitor). (F) Myosin-I (myr1A) and myosin-IIB do not bind to 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 μl) of the rat brain homogenate (1 μg/μl; Input) and of the fraction bound to GST-syntaxin-1A (Bound; see C) were analyzed by immunoblotting with antibodies against myosin-Va, myosin-IIB (gift of T. Shirao, Gunma University Graduate School of Medicine, Maebashi, Gunma, Japan), or myr 1A (gift of M. Bähler, Westfalische Wilhelms University, Münster, Germany).

Michitoshi Watanabe, et al. Mol Biol Cell. 2005 October;16(10):4519-4530.
9.
Figure 2.

Figure 2. From: Myosin-Va Regulates Exocytosis through the Submicromolar Ca2+-dependent Binding of Syntaxin-1A.

Syntaxin-1A interacts with myosin-Va independently of F-actin and regulates myosin-Va ATPase activity without affecting its motility. (A) In the presence of 10–6 M 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 10–6 M 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 μg/ml syntaxin-1A [1-262], the ATPase activity was determined in a reaction mixture containing myosin-Va (50 μg/ml) and F-actin (420 μg/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 μg/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 μM syntaxin-1A. The average sliding velocities were measured. Each value represents the mean of 70 determinations.

Michitoshi Watanabe, et al. Mol Biol Cell. 2005 October;16(10):4519-4530.
10.
Figure 5.

Figure 5. From: Myosin-Va Regulates Exocytosis through the Submicromolar Ca2+-dependent Binding of Syntaxin-1A.

The syntaxin-1A-binding site is located 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-syntaxin-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 recombinant tailless myosin-Va (DHM5) is similar to the full-length brain myosin-Va (see Figure 1D). (B) Syntaxin-1A binds to immobilized tailless myosin-Va (DHM5). Syntaxin-1A [1-262] (30-kDa) was incubated with Ni2+-NTA-immobilized His6-DHM5 in the presence of 10–6 M Ca2+ (see Figure 1C) and eluted with SDS-sample buffer. The eluted sample was separated by SDS-PAGE and silver stained. The 150-kDa band is DHM5. (C) The binding of truncated myosin-Va (DHM5) by syntaxin-1A requires a pCa of 6.6. Syntaxin-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 detected by immunoblotting with an antibody against myosin-Va. Top and middle, dose response of Ca2+ for binding of syntaxin-1A by DHM5 between pCa 4 and 8. Brain myosin-Va (Myosin-V) and DHM5 show a similar dependence on Ca2+ for the binding of syntaxin-1A. Bottom, dose response of Ca2+ for binding of syntaxin-1A by DHM5 between pCa 6 and 7. (D) The neck domain of myosin-Va is necessary for syntaxin-1A binding. Truncated forms of myosin-Va were produced by biotin-labeled in vitro translation of mouse myosin-Va cDNA (dilute): H, head domain only (amino acids 1–755); HN-ATPBS, 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 10–6 M 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).

Michitoshi Watanabe, et al. Mol Biol Cell. 2005 October;16(10):4519-4530.

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