Calcium-Induced Quenching of Intrinsic Fluorescence in Brain Myosin V Is Linked to Dissociation of Calmodulin Light Chains

Cameron, Luiz-Cláudio; Carvalho, R. N.; Araújo, José Ricardo; Santos, Ana C. Vicente; Tauhata, Sinji Borges Ferreira; Larson, Roy Edward; Sorenson, Martha M.

doi:10.1006/abbi.1998.0700

Cited by 15 publications

(12 citation statements)

References 37 publications

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“…CaM is known to be released from the neck of myosin-Va in the presence of micromolar Ca 2ϩ (Cameron et al, 1998;Homma et al, 2000). We confirmed that preincubation of myosin-Va with Ca 2ϩ released the bound CaM ( Figure 5F).…”

Section: Syntaxin-1a Binds To the Neck Domain Of Myosin-va After Ca 2supporting

confidence: 76%

“…Further studies in a bacterial two-hybrid assay, which is a modification 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 Ca 2ϩ (Cameron et al, 1998;Homma et al, 2000). We confirmed that preincubation of myosin-Va with Ca 2ϩ released the bound CaM ( Figure 5F).…”

supporting

confidence: 76%

“…Thus, we suspected that the apparent Ca 2ϩ dependence of myosin Va-syntaxin-1A binding is due to Ca 2ϩ -dependent release of CaM from the myosin-Va neck, and exposure of an otherwise concealed syntaxin-1A binding site. Our results further indicate that CaM can sense submicromolar Ca 2ϩ 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 Ca 2ϩ (Figure 4B), which corresponds to the level of Ca 2ϩ when secretory granules enter the readily releasable pool (Burgoyne and Morgan, 2003).…”

Section: Cam-dependent Regulation Of Exocytosis Through Ca 2؉ -Dependsupporting

confidence: 53%

“…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 Ca 2ϩ rises to micromolar concentrations (Cameron et al, 1998;Homma et al, 2000). We found that, after Ca 2ϩ -dependent release of CaM, syntaxin-1A can bind myosin-Va even in the absence of Ca 2ϩ .…”

Section: Cam-dependent Regulation Of Exocytosis Through Ca 2؉ -Dependmentioning

confidence: 81%

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

Watanabe

Nomura

Ohyama

et al. 2005

MBoC

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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 Ca 2؉ . 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 Ca 2؉ 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 Ca 2؉ . INTRODUCTIONMyosin-V, a processive molecular motor, conveys vesicles and other organelles along F-actin (Mercer et al., 1991;Espreafico 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 interacts with membrane-bound vesicles. In these ways, myosin-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 vertebrates, 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 Ca 2ϩ -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 h...

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Section: Syntaxin-1a Binds To the Neck Domain Of Myosin-va After Ca 2supporting

confidence: 76%

supporting

confidence: 76%

Section: Cam-dependent Regulation Of Exocytosis Through Ca 2؉ -Dependsupporting

confidence: 53%

Section: Cam-dependent Regulation Of Exocytosis Through Ca 2؉ -Dependmentioning

confidence: 81%

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

Watanabe

Nomura

Ohyama

et al. 2005

MBoC

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“…Scale bars, 50 nm. Bar in panel e is for panels a, c, d, and e. may result in a mechanically weakened protein, since some CaM molecules dissociate from myosin V in the presence of Ca 2ϩ (15,28). Myosin II stripped of one of its light chains retains its actin activated MgATPase activity but performs poorly in mechanical assays such as the in vitro motility assay or step size measurements using the optical trap (29 -31).…”

Section: Camentioning

confidence: 99%

Regulated Conformation of Myosin V

Wang

Thirumurugan

Stafford

et al. 2004

Journal of Biological Chemistry

160

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We have found that myosin V, an important actinbased vesicle transporter, has a folded conformation that is coupled to inhibition of its enzymatic activity in the absence of cargo and Ca 2؉ . In the absence of Ca 2؉where the actin-activated MgATPase activity is low, purified brain myosin V sediments in the analytical ultracentrifuge at 14 S as opposed to 11 S in the presence of Ca 2؉ where the activity is high. At high ionic strength it sediments at 10 S independent of Ca 2؉ , and its regulation is poor. These data are consistent with myosin V having a compact, inactive conformation in the absence of Ca 2؉ and an extended conformation in the presence of Ca 2؉ or high ionic strength. Electron microscopy reveals that in the absence of Ca 2؉ the heads and tail are both folded to give a triangular shape, very different from the extended appearance of myosin V at high ionic strength. A recombinant myosin V heavy meromyosin fragment that is missing the distal portion of the tail domain is not regulated by calcium and has only a small change in sedimentation coefficient, which is in the opposite direction to that seen with intact myosin V. Electron microscopy shows that its heads are extended even in the absence of calcium. These data suggest that interaction between the motor and cargo binding domains may be a general mechanism for shutting down motor protein activity and thereby regulating the active movement of vesicles in cells.Mammalian myosin Va is involved in the transport of melanosomes, the pigment granules found in melanocytes, and secretory granules in neuronal cells (1-4). Its enzymatic and mechanical properties demonstrate that it is a processive motor, capable of taking many 35-nm steps per encounter with actin filaments (5-8). The 35-nm step coincides with the halfhelical repeat of the actin filament and allows myosin V to effectively walk along one side of an actin filament (9).The myosin V heavy chain is composed of four structural domains. The N-terminal motor domain possesses the actin and nucleotide binding sites of the molecule and is followed by a neck domain containing six IQ motifs which bind calmodulin (CaM) 1 (10). The proximal portion of the tail contains several segments of coiled-coil, driving the dimerization of two heavy chains, and the distal portion comprises a globular cargo binding domain. Electron micrographs of myosin V confirm that the molecule contains two elongated heads, a short rod and a bifurcated globular domain (11).Tissue-purified myosin V requires micromolar Ca 2ϩ for full MgATPase yet moves actin independent of the Ca 2ϩ concentration in the in vitro assay (5, 11-13). Ca 2ϩ also regulates the binding of myosin V to actin in the presence of ATP (14). However, recombinant myosin V heavy meromyosin-like fragments (HMM), which are missing the globular tail domain and the distal portion of the coiled-coil domain, have high actinactivated MgATPase rates in the absence of Ca 2ϩ and are partially inhibited by Ca 2ϩ if excess CaM is not present (10, 15), suggesting that the globul...

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Myosin Va is locally synthesized following nerve injury

Calliari

Sotelo‐Silveira

Costa

et al. 2002

Cell Motil. Cytoskeleton

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The presence of Myosin Va (an actin-based molecular motor) in the peripheral nervous system was examined and its subcellular distribution within the axons of the sciatic nerve was demonstrated via immunocytochemistry. Myosin Va (M-Va) in the nerve was detected by using SDS-PAGE and Western blot techniques with a polyclonal antibody specifically raised against the M-Va globular tail domain. In addition, purification of M-Va from the rat sciatic nerve prior to immunoblotting yielded a M-Va standard band. Likewise, optical immunocytochemical procedures revealed the presence of M-Va, particularly in the cortical axoplasmic territory, but also in the Schwann cell soma. The above experiments were carried out both on intact as well as on severed sciatic nerves with similar results. The proximal stumps of severed sciatic nerves (from 0 to 72 h after injury) were labelled in vivo with (35)S-methionine. SDS-PAGE autoradiography of the immunoabsorbed M-Va from the radiolabelled homogenized nerve tissue showed a significant increment of the radioactive intensity of M-Va heavy chain band through time. Moreover, a significant increment of transcripts coding for M-Va heavy chain was detected through time using RT-PCR after nerve injury and compared to intact nerves. This data suggest that M-Va is up-regulated in a time-dependent manner. The latter suggests a possible involvement of M-Va in nerve regeneration processes.

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Calcium-Induced Quenching of Intrinsic Fluorescence in Brain Myosin V Is Linked to Dissociation of Calmodulin Light Chains

Cited by 15 publications

References 37 publications

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

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

Regulated Conformation of Myosin V

Myosin Va is locally synthesized following nerve injury

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Calcium-Induced Quenching of Intrinsic Fluorescence in Brain Myosin V Is Linked to Dissociation of Calmodulin Light Chains

Cited by 15 publications

References 37 publications

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

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

Regulated Conformation of Myosin V

Myosin Va is locally synthesized following nerve injury

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

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