High Affinity Binding of Brain Myosin-Va to F-actin Induced by Calcium in the Presence of ATP

Tauhata, Sinji Borges Ferreira; Santos, D Vital dos; Taylor, E. W.; Mooseker, Mark S.; Larson, Roy Edward

doi:10.1074/jbc.m102583200

Cited by 29 publications

(28 citation statements)

References 35 publications

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“…When Myo5a was incubated with fluorescein-labeled MTs, there was no apparent change to the distribution or appearance of the filaments (Figure 3c). As expected from previous studies (Cheney et al, 1993;Tauhata et al, 2001), when Myo5a was incubated with Texas-Red-labeled F-actin under rigor conditions, discrete bundled networks of actin filaments could be seen throughout the preparation (Figure 3d). The incubation of MTs and F-actin together did not affect the distribution or appearance of either filament (Figure 3, e-g).…”

Section: Myo5a Mediates Cross-linking Of Mts To Actin Filamentssupporting

confidence: 60%

“…Another indication that there is little actin-filamentonly-associated Myo5a in these regions of the pellet is that the actin is present as single filaments rather than the bundles that are formed when Myo5a is added to actin filaments under these rigor conditions (Cheney et al, 1993;Tauhata et al, 2001). Unfortunately, there are no obvious cross-linking structures discernible between MTs and actin filaments, presumably because of the low resolution of such thin-sectioned preparations.…”

Section: Tt Cao Et Almentioning

confidence: 88%

“…Under the low Ca 2ϩ conditions used for these binding studies, Myo5a in solution dissociates from actin in the presence of ATP (Cheney et al, 1993;Nascimento et al, 1996;Tauhata et al, 2001). In contrast, Myo5a absorbed to the surface of a bead or coverglass in in vitro motility assays exhibits high-affinity actin binding in the absence of Ca 2ϩ (Cheney et al, 1993) suggesting that surface binding conformationally regulates the motor properties of this myosin in either a specific or nonspecific manner.…”

Section: Myo5a Mediates Cross-linking Of Mts To Actin Filamentsmentioning

confidence: 99%

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Myosin-Va Binds to and Mechanochemically Couples Microtubules to Actin Filaments

Cao

Chang

Masters

et al. 2004

MBoC

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Myosin-Va was identified as a microtubule binding protein by cosedimentation analysis in the presence of microtubules. Native myosin-Va purified from chick brain, as well as the expressed globular tail domain of this myosin, but not head domain bound to microtubule-associated protein-free microtubules. Binding of myosin-Va to microtubules was saturable and of moderately high affinity (ϳ1:24 Myosin-Va:tubulin; K d ‫؍‬ 70 nM). Myosin-Va may bind to microtubules via its tail domain because microtubule-bound myosin-Va retained the ability to bind actin filaments resulting in the formation of cross-linked gels of microtubules and actin, as assessed by fluorescence and electron microscopy. In low Ca 2؉ , ATP addition induced dissolution of these gels, but not release of myosin-Va from MTs. However, in 10 M Ca 2؉ , ATP addition resulted in the contraction of the gels into aster-like arrays. These results demonstrate that myosin-Va is a microtubule binding protein that cross-links and mechanochemically couples microtubules to actin filaments. INTRODUCTIONMyosin-Va (Myo5a) is the best characterized of three class V myosins identified in vertebrates (Berg et al., 2001). Myo5a is a two-headed motor, consisting of two heavy chains and multiple light chains (reviewed in Reck-Peterson et al., 2000). The N-terminal half of each heavy chain consists of a head or motor domain followed by a neck domain composed of six IQ motifs, each of which binds a calmodulin light chain although chicken Myo5a also contains a pair of myosin-II essential light chains . The C-terminal tail domain consists of a proximal dimerization stalk of largely coiled-coil forming alpha helix followed by a globular domain that has been shown to interact with numerous presumed "cargo" molecules (Reck-Peterson et al., 2000;Karcher et al., 2002;Langford, 2002). The tail domain also contains at least a pair of the low-molecular-weight light chain, LC8 or PIN, first identified as a light chain of the microtuble (MT) motor, dynein, but subsequently shown to interact with numerous other proteins . Biochemical studies on both tissue-purified and baculovirus-expressed Myo5a (reviewed in Reck-Peterson et al., 2000;Mehta, 2001) have shown that it is a Ca 2ϩ -regulated, barbed-end directed, processive motor that takes large, ϳ36-nm steps in a hand-overhand manner (Forkey et al., 2003; Yildiz et al., 2003) along the actin filament.Insights into the potential functions for Myo5a have come from several lines of investigation, including identification of organelles and proteins that interact with Myo5a (ReckPeterson et al., 2000;Karcher et al., 2002;Langford, 2002). The most powerful strategy has been the phenotypic characterization of lethal alleles of the dilute (Myo5a lethal mutant) mouse and cells derived from them. The dilute mice develop severe seizures and die within 3 wk after birth. The primary neuronal defect thought to be responsible is the absence of smooth endoplasmic reticulum (SER) within the dendritic spines of Purkinje neurons (Takagishi et al., 1996). Anal...

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Section: Myo5a Mediates Cross-linking Of Mts To Actin Filamentssupporting

confidence: 60%

Section: Tt Cao Et Almentioning

confidence: 88%

Section: Myo5a Mediates Cross-linking Of Mts To Actin Filamentsmentioning

confidence: 99%

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Myosin-Va Binds to and Mechanochemically Couples Microtubules to Actin Filaments

Cao

Chang

Masters

et al. 2004

MBoC

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“…The class V myosins have been implicated in organelle and RNA transport in several organisms (for review see Reck-Peterson et al, 2000). Mammalian brain is a rich source of MVa, from which it has been purified and biochemically characterized (Espíndola et al, 1992;Espreafico et al, 1992;Cheney et al, 1993;Nascimento et al, 1996;Tauhata et al, 2001). The expression of MVa is deficient in dilute mice (Mercer et al, 1991) and diluteopisthotonus rats (Futaki et al, 2000), both of which present seizures soon after birth and die within the third post-natal week.…”

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confidence: 99%

Immunohistochemical localization of myosin va in the adult rat brain

et al. 2003

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Abstract-Brain myosin Va (MVa) is a molecular motor associated with plastic changes during development. MVa has previously been detected in the cell body and in dendrites of neuronal cells in culture, in cells of the guinea-pig cochlea, as well as in cerebellar cells. Adult Wistar rats (n‫,)41؍‬ 250 -300 g, were perfused with standard methods for immunohistochemistry, using a polyclonal, affinity-purified rabbit antibody against MVa tail domain. Anti-MVa antibody specifically stained neuronal nuclei from forebrain to cerebellar regions, and more intensely sensory nuclei. Differences in MVa immunoreactivity were detected between brain nuclei, ranging from very intense to weak staining. The analysis of MVa and glial fibrillary acidic protein staining in adjacent brain sections demonstrated a clear-cut neuronal labeling rather than an astroglial staining. The studies presented here represent a comprehensive map of MVa regional distribution in the CNS of the adult rat and may contribute to the basic understanding of its role in brain function and plasticity, particularly in relationship to phenomena that involve molecular motors, such as neurite outgrowth, organelle transport and neurotransmitter-vesicle cycling. It is important to highlight that this is a pioneer immunohistochemical study on the distribution of MVa on the whole brain of adult rats, a first step toward the understanding of its function in the CNS.

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“…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 globular tail domain may be involved in down-regulation of enzymatic activity in the absence of Ca 2ϩ .…”

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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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High Affinity Binding of Brain Myosin-Va to F-actin Induced by Calcium in the Presence of ATP

Cited by 29 publications

References 35 publications

Myosin-Va Binds to and Mechanochemically Couples Microtubules to Actin Filaments

Myosin-Va Binds to and Mechanochemically Couples Microtubules to Actin Filaments

Immunohistochemical localization of myosin va in the adult rat brain

Regulated Conformation of Myosin V

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