Presence of accessory abductor digiti minimi muscle in two cadavers.

During routine cadaveric dissection, accessory hypothenar muscles were incidentally discovered in two cadavers, both males, aged 86 and 92. Both muscles originated from the palmaris longus tendon in the distal portion of the forearm and were identified as accessory abductor digiti minimi (AADM) muscles, based on their association with abductor digiti minimi. While AADM is a common variant in the antebrachium, it is less typical for them to originate from the palmaris longus tendon. The presence of such an AADM could complicate surgical procedures requiring resection of the palmaris longus tendon. Moreover, the surrounding neurovasculature - namely the ulnar nerve as it passes through the ulnar canal between the pisiform and hook of the hamate - could be compressed by contractions of an AADM with such a proximal origin. This can manifest as ulnar neuropathies resulting in pain, weakness, or protracted flexion of the fourth and fifth digits (ulnar claw). Our description of these muscles adds to previous accounts of variation of the palmaris longus and abductor digiti minimi muscles while considering potential clinical implications.

The molecular motor Myosin Va interacts with the cilia-centrosomal protein RPGRIP1L.

Myosin Va (MyoVa) is an actin-based molecular motor abundantly found at the centrosome. However, the role of MyoVa at this organelle has been elusive due to the lack of evidence on interacting partners or functional data. Herein, we combined yeast two-hybrid screen, biochemical studies and cellular assays to demonstrate that MyoVa interacts with RPGRIP1L, a cilia-centrosomal protein that controls ciliary signaling and positioning. MyoVa binds to the C2 domains of RPGRIP1L via residues located near or in the Rab11a-binding site, a conserved site in the globular tail domain (GTD) from class V myosins. According to proximity ligation assays, MyoVa and RPGRIP1L can interact near the cilium base in ciliated RPE cells. Furthermore, we showed that RPE cells expressing dominant-negative constructs of MyoVa are mostly unciliated, providing the first experimental evidence about a possible link between this molecular motor and cilia-related processes.

A novel SDS-stable dimer of a heterogeneous nuclear ribonucleoprotein at presynaptic terminals of squid neurons.

The presence of mRNAs in synaptic terminals and their regulated translation are important factors in neuronal communication and plasticity. Heterogeneous nuclear ribonucleoprotein (hnRNP) complexes are involved in the translocation, stability, and subcellular localization of mRNA and the regulation of its translation. Defects in these processes and mutations in components of the hnRNP complexes have been related to the formation of cytoplasmic inclusion bodies and neurodegenerative diseases. Despite much data on mRNA localization and evidence for protein synthesis, as well as the presence of translation machinery, in axons and presynaptic terminals, the identity of RNA-binding proteins involved in RNA transport and function in presynaptic regions is lacking. We previously characterized a strongly basic RNA-binding protein (p65), member of the hnRNPA/B subfamily, in squid presynaptic terminals. Intriguingly, in sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), p65 migrated as a 65-kDa protein, whereas members of the hnRNPA/B family typically have molecular masses ranging from 35 to 42kDa. In this report we present further biochemical and molecular characterization that shows endogenous p65 to be an SDS-stable dimer composed of ∼37-kDa hnRNPA/B-like subunits. We cloned and expressed a recombinant protein corresponding to squid hnRNPA/B-like protein and showed its propensity to aggregate and form SDS-stable dimers in vitro. Our data suggest that this unique hnRNPA/B-like protein co-localizes with synaptic vesicle protein 2 and RNA-binding protein ELAV and thus may serve as a link between local mRNA processing and presynaptic function and regulation.

Myosin Va is developmentally regulated and expressed in the human cerebellum from birth to old age.

Myosin Va functions as a processive, actin-based motor molecule highly enriched in the nervous system, which transports and/or tethers organelles, vesicles, and mRNA and protein translation machinery. Mutation of myosin Va leads to Griscelli disease that is associated with severe neurological deficits and a short life span. Despite playing a critical role in development, the expression of myosin Va in the central nervous system throughout the human life span has not been reported. To address this issue, the cerebellar expression of myosin Va from newborns to elderly humans was studied by immunohistochemistry using an affinity-purified anti-myosin Va antibody. Myosin Va was expressed at all ages from the 10th postnatal day to the 98 th year of life, in molecular, Purkinje and granular cerebellar layers. Cerebellar myosin Va expression did not differ essentially in localization or intensity from childhood to old age, except during the postnatal developmental period. Structures resembling granules and climbing fibers in Purkinje cells were deeply stained. In dentate neurons, long processes were deeply stained by anti-myosin Va, as were punctate nuclear structures. During the first postnatal year, myosin Va was differentially expressed in the external granular layer (EGL). In the EGL, proliferating prospective granule cells were not stained by anti-myosin Va antibody. In contrast, premigratory granule cells in the EGL stained moderately. Granule cells exhibiting a migratory profile in the molecular layer were also moderately stained. In conclusion, neuronal myosin Va is developmentally regulated, and appears to be required for cerebellar function from early postnatal life to senescence.

Myosin Va is developmentally regulated and expressed in the human cerebellum from birth to old age

Myosin Va functions as a processive, actin-based motor molecule highly enriched in the nervous system, which transports and/or tethers organelles, vesicles, and mRNA and protein translation machinery. Mutation of myosin Va leads to Griscelli disease that is associated with severe neurological deficits and a short life span. Despite playing a critical role in development, the expression of myosin Va in the central nervous system throughout the human life span has not been reported. To address this issue, the cerebellar expression of myosin Va from newborns to elderly humans was studied by immunohistochemistry using an affinity-purified anti-myosin Va antibody. Myosin Va was expressed at all ages from the 10th postnatal day to the 98th year of life, in molecular, Purkinje and granular cerebellar layers. Cerebellar myosin Va expression did not differ essentially in localization or intensity from childhood to old age, except during the postnatal developmental period. Structures resembling granules and climbing fibers in Purkinje cells were deeply stained. In dentate neurons, long processes were deeply stained by anti-myosin Va, as were punctate nuclear structures. During the first postnatal year, myosin Va was differentially expressed in the external granular layer (EGL). In the EGL, proliferating prospective granule cells were not stained by anti-myosin Va antibody. In contrast, premigratory granule cells in the EGL stained moderately. Granule cells exhibiting a migratory profile in the molecular layer were also moderately stained. In conclusion, neuronal myosin Va is developmentally regulated, and appears to be required for cerebellar function from early postnatal life to senescence.

Differential patterns of myosin Va expression during the ontogenesis of the rat hippocampus

Myosin Va is an actin-based, processive molecular motor protein highly enriched in the nervous tissue of vertebrates. It has been associated with processes of cellular motility, which include organelle transport and neurite outgrowth. The in vivo expression of myosin Va protein in the developing nervous system of mammals has not yet been reported. We describe here the immunolocalization of myosin Va in the developing rat hippocampus. Coronal sections of the embryonic and postnatal rat hippocampus were probed with an affinity-purified, polyclonal anti-myosin Va antibody. Myosin Va was localized in the cytoplasm of granule cells in the dentate gyrus and of pyramidal cells in Ammon's horn formation. Myosin Va expression changed during development, being higher in differentiating rather than already differentiated granule and pyramidal cells. Some of these cells presented a typical migratory profile, while others resembled neurons that were in the process of differentiation. Myosin Va was also transiently expressed in fibers present in the fimbria. Myosin Va was not detected in germinative matrices of the hippocampus proper or of the dentate gyrus. In conclusion, myosin Va expression in both granule and pyramidal cells showed both position and time dependency during hippocampal development, indicating that this motor protein is under developmental regulation.

Differential patterns of myosin Va expression during the ontogenesis of the rat hippocampus.

Myosin Va is an actin-based, processive molecular motor protein highly enriched in the nervous tissue of vertebrates. It has been associated with processes of cellular motility, which include organelle transport and neurite outgrowth. The in vivo expression of myosin Va protein in the developing nervous system of mammals has not yet been reported. We describe here the immunolocalization of myosin Va in the developing rat hippocampus. Coronal sections of the embryonic and postnatal rat hippocampus were probed with an affinity-purified, polyclonal anti-myosin Va antibody. Myosin Va was localized in the cytoplasm of granule cells in the dentate gyrus and of pyramidal cells in Ammon's horn formation. Myosin Va expression changed during development, being higher in differentiating rather than already differentiated granule and pyramidal cells. Some of these cells presented a typical migratory profile, while others resembled neurons that were in the process of differentiation. Myosin Va was also transiently expressed in fibers present in the fimbria. Myosin Va was not detected in germinative matrices of the hippocampus proper or of the dentate gyrus. In conclusion, myosin Va expression in both granule and pyramidal cells showed both position and time dependency during hippocampal development, indicating that this motor protein is under developmental regulation.

A novel 65 kDa RNA-binding protein in squid presynaptic terminals.

A polyclonal antibody (C4), raised against the head domain of chicken myosin Va, reacted strongly towards a 65 kDa polypeptide (p65) on Western blots of extracts from squid optic lobes but did not recognize the heavy chain of squid myosin V. This peptide was not recognized by other myosin Va antibodies, nor by an antibody specific for squid myosin V. In an attempt to identify it, p65 was purified from optic lobes of Loligo plei by cationic exchange and reverse phase chromatography. Several peptide sequences were obtained by mass spectroscopy from p65 cut from sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) gels. BLAST analysis and partial matching with expressed sequence tags (ESTs) from a Loligo pealei data bank indicated that p65 contains consensus signatures for the heterogeneous nuclear ribonucleoprotein (hnRNP) A/B family of RNA-binding proteins. Centrifugation of post mitochondrial extracts from optic lobes on sucrose gradients after treatment with RNase gave biochemical evidence that p65 associates with cytoplasmic RNP complexes in an RNA-dependent manner. Immunohistochemistry and immunofluorescence studies using the C4 antibody showed partial co-labeling with an antibody against squid synaptotagmin in bands within the outer plexiform layer of the optic lobes and at the presynaptic zone of the stellate ganglion. Also, punctate labeling by the C4 antibody was observed within isolated optic lobe synaptosomes. The data indicate that p65 is a novel RNA-binding protein located to the presynaptic terminal within squid neurons and may have a role in synaptic localization of RNA and its translation or processing.

Immunohistochemical localization of myosin Va in the adult rat brain.

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=14), 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.

High affinity binding of brain myosin-Va to F-actin induced by calcium in the presence of ATP.

Brain myosin-Va consists of two heavy chains, each containing a neck domain with six tandem IQ motifs that bind four to five calmodulins and one to two essential light chains. Previous studies demonstrated that myosin-Va exhibits an unusually high affinity for F-actin in the presence of ATP and that its MgATPase activity is stimulated by micromolar Ca(2+) in a highly cooperative manner. We demonstrate here that Ca(2+) also induces myosin-Va binding to and cosedimentation with F-actin in the presence of ATP in a similar cooperative manner and calcium concentration range as that observed for the ATPase activity. Neither hydrolysis of ATP nor buildup of ADP was required for Ca(2+)-induced cosedimentation. The Ca(2+)-induced binding was inhibited by low temperature or by 0.6 m NaCl, but not by 1% Triton X-100. Tight binding between myosin-Va and pyrene-labeled F-actin in the presence of ATP and Ca(2+) was also detected by quenching of the pyrene fluorescence. Negatively stained preparations of actomyosin-Va under Ca(2+)-induced binding conditions showed tightly packed F-actin bundles cross-linked by myosin-Va. Our data demonstrate that high affinity binding of myosin-Va and F-actin in the presence of ATP or 5'-O-(thiotriphosphate) is induced by micromolar concentrations of Ca(2+). Since Ca(2+) regulates both the actin binding properties and actin-activated ATPase of myosin-Va over the same concentration range, we suggest that the calcium signal may regulate the mechanism of processivity of myosin Va.

Immunolocalisation of myosin-V in the enteric nervous system of the rat.

We show here the localisation of myosin-V in whole mount preparations of the mucous-submucous and the muscular layers of rat small intestine by using an affinity purified antibody specific to the tail domain of myosin-V. Myosin-V immunostaining was intense in the submucous and myenteric nervous plexuses, allowing the visualisation of neuronal cell bodies and fibres. Western blots of total muscle layers homogenates detected with the same antibody revealed a single band of the expected size for myosin-V. Understanding the cellular localisation and function of this class of myosin is an important challenge and the accessibility and simplicity of the enteric nervous system as compared to the central nervous system, makes the digestive tract an attractive model for studying possible functional roles of myosin-V in neurotransmission and neuroplasticity.

Microwave-stimulated recovery of myosin-V immunoreactivity from formalin-fixed, paraffin-embedded human CNS.

The lability of brain myosin-V (BM-V) to aldehyde-fixation has hindered immunohistochemical (IH) studies of this actin-based motor. We show here that BM-V immunoreactivity (IR) can be retrieved from formalin-fixed, paraffin-embedded human tissue. BM-V IR was optimally retrieved by boiling 5 microm cerebellar tissue sections in 10 mM sodium citrate buffer, pH 6, for 15 min, using a microwave oven set at 900 W and 2.45 GHz. A polyclonal, affinity purified anti-BM-V antibody, raised in rabbits against the tail domain of chicken BM-V, was shown here to recognize a single band in Western blots of human cortical homogenates. The combined use of this monospecific antibody and of the antigen retrieval (AR) method above allowed us to verify that BM-V IR is strongly expressed in human Purkinje cell bodies and dendrites, and in granule cells. The same pattern of BM-V IR expression was consistently and maximally detected in tissues stored in 10% formalin from 1 week to 2.5 months. The AR protocol for BM-V described here permits its IH study in formaldehyde-fixed tissues. It is a valuable tool to study BM-V in well fixed tissues, as occurs with the large collection of human archival tissue available.

Characterization of myosin V from PC12 cells.

PC12 cell line is a cellular model to study neurite outgrowth and neurotransmitter release mechanisms. Molecular motors may be involved in these responses and myosin V could be a candidate to mediate these effects. Overlay experiments using [(125)I]-calmodulin showed that PC12 cells possess several calmodulin-binding proteins, some of them around 190-210 kDa. Western blots using affinity purified polyclonal antibodies raised against chicken brain myosin V revealed a component of 190 kDa, a molecular mass typical of myosin V. Furthermore, Northern blots using a myosin V probe also detected a transcript of around 12 kbp. Immunofluorescence cytochemistry demonstrated the localization of myosin V throughout the cytoplasm, in the neurites, growth cone tips, and with an intense asymmetrical perinuclear labeling. Western blot analyses of PC12 cellular extracts after FGF-2 and/or dibutyryl cAMP treatment revealed variations between myosin V and myosin II expression during neuronal differentiation. These results demonstrated the presence of myosin V in PC12 cells and also suggest a role for this motor molecule in the neuronal differentiation response in PC12 cells.

Subcellular localization of GFP-myosin-V in live mouse melanocytes.

Class-V myosins are two-headed actin-based mechanoenzymes that function in the transport and subcellular localization of organelles and possibly in the outgrowth of cellular processes. To determine which domains of myosin-V are involved in intracellular localization of this motor protein, we have expressed fusions of the green fluorescent protein with segments from two distinct myosin-V heavy chains. The expression patterns of constructs encoding four different domains of chick brain myosin-Va were compared to a single construct encoding the globular tail region of mouse myosin-Vb. In transfected mouse melanocytes, expression of the NH(2)-terminal head (catalytic domain) of chick brain myosin-Va codistributed with actin filaments throughout the cytoplasm. A similar construct encoding the myosin-Va head with the associated neck (light chain binding sites), also codistributed with actin filaments. The GFP-head-neck peptide was also highly concentrated in the tips of filopodia in B16 melanocytes wild type for myosin-Va (MYO5a gene), but was concentrated throughout the entire filopodia of S91-6 melanocytes derived from dilute mice with mutations in the MYO5a gene. Evidence is also presented that the globular tail of myosin-Va, but not myosin-Vb, targets this motor molecule to the centrosome as confirmed by colocalization in cells stained with antibodies to (gamma)-tubulin. Expression of the GFP-myosin-Va globular tail causes displacement of endogenous myosin-V from centrosomes as visualized by immunolabeling with antibodies to the head domain of myosin-V. Treatment with the microtubule-disrupting drug nocodazole markedly reduces myosin-V staining at the centrosome. In contrast, there was no detectable diminution of myosin-V staining at the centrosome in cells treated with the actin filament-disrupting drug cytochalasin D. Thus, while localization of the myosin-V motor domain to actin-rich regions is consistent with conventional models of actomyosin-based motility, localization to the centrosome occurs in the complete absence of the myosin-V motor domain and is dependent on intact microtubules.

Brain myosin-V, a calmodulin-carrying myosin, binds to calmodulin-dependent protein kinase II and activates its kinase activity.

Myosin-V, an unconventional myosin, has two notable structural features: (i) a regulatory neck domain having six IQ motifs that bind calmodulin and light chains, and (ii) a structurally distinct tail domain likely responsible for its specific intracellular interactions. Myosin-V copurifies with synaptic vesicles via its tail domain, which also is a substrate for calmodulin-dependent protein kinase II. We demonstrate here that myosin-V coimmunoprecipitates with CaM-kinase II from a Triton X-100-solubilized fraction of isolated nerve terminals. The purified proteins also coimmunoprecipitate from dilute solutions and bind in overlay experiments on Western blots. The binding region on myosin-V was mapped to its proximal and medial tail domains. Autophosphorylated CaM-kinase II binds to the tail domain of myosin-V with an apparent Kd of 7.7 nM. Surprisingly, myosin-V activates CaM-kinase II activity in a Ca2+-dependent manner, without the need for additional CaM. The apparent activation constants for the autophosphorylation of CaM-kinase II were 10 and 26 nM, respectively, for myosin-V versus CaM. The maximum incorporation of 32P into CaM-kinase II activated by myosin-V was twice that for CaM, suggesting that myosin-V binding to CaM-kinase II entails alterations in kinetic and/or phosphorylation site parameters. These data suggest that myosin-V, a calmodulin-carrying myosin, binds to and delivers CaM to CaM-kinase II, a calmodulin-dependent enzyme.

Calcium-induced quenching of intrinsic fluorescence in brain myosin V is linked to dissociation of calmodulin light chains.

Myosin V isolated from chick brain (BM V) is a multimeric protein of about 640 kDa consisting of two intertwined heavy chains of 212 kDa and multiple light chains of 10 to 20 kDa. A distinctive feature of the heavy chain is an extended neck region with six consensus IQ sites for the binding of calmodulin (CaM) and myosin light chains. The actin-activated MgATPase has been shown to require >/=1 microM Ca2+ for full activity, and evidence points to a myosin-linked regulatory system where the CaM light chains participate as modulators for the Ca2+ signal. Still, the precise mechanism of Ca2+ regulation remains unknown. In the present study we have used the intrinsic tryptophan fluorescence of native BM V to monitor conformational changes of BM V induced by Ca2+, and we relate these changes to CaM dissociation from the BM V molecule. The fluorescence intensity decreases approximately 17% upon addition of sub-micromolar concentrations of Ca2+ (K0.5 = 0.038 microM). This decrease in fluorescence, which is dominated by a conformational change in the heavy chain, can be reversed by addition of 1, 2-di(2-aminoethoxy)ethane-N,N,N',N'tetraacetic acid (EGTA) followed by an excess of CaM, but not by addition of EGTA alone. Gel filtration of native BM V using HPLC shows that CaM is partially dissociated from the heavy chain in EGTA and dissociates further upon addition of sub-micromolar concentrations of Ca2+. These observations suggest that the affinity of CaM for at least one of the IQ sites on the BM V heavy chain decreases with Ca2+ and that the Ca2+ concentration required for this effect is lower than that needed to activate acto-BM V. Using a cosedimentation assay in the presence of actin, we also observe partial dissociation of CaM when Ca2+ is absent, but now the addition of Ca2+ has a biphasic effect: sub-micromolar Ca2+ concentrations lead to reassociation of CaM with the heavy chain, followed by dissociation when Ca2+ exceeds 5-10 microM. Thus, the binding of CaM to BM V is affected by both actin and Ca2+.

Subcellular localization of myosin-V in the B16 melanoma cells, a wild-type cell line for the dilute gene.

The discovery that the dilute gene encodes a class V myosin led to the hypothesis that this molecular motor is involved in melanosome transport and/or dendrite outgrowth in mammalian melanocytes. The present studies were undertaken to gain insight into the subcellular distribution of myosin-V in the melanoma cell line B16-F10, which is wild-type for the dilute gene. Immunofluorescence studies showed some degree of superimposed labeling of myosin-V with melanosomes that predominated at the cell periphery. A subcellular fraction highly enriched in melanosomes was also enriched in myosin-V based on Western blot analysis. Immunoelectron microscopy showed myosin-V labeling associated with melanosomes and other organelles. The stimulation of B16 cells with the alpha-melanocyte-stimulating hormone led to a significant increase in myosin-V expression. This is the first evidence that a cAMP signaling pathway might regulate the dilute gene expression. Immunofluorescence also showed an intense labeling of myosin-V independent of melanosomes that was observed within the dendrites and at the perinuclear region. Although the results presented herein are consistent with the hypothesis that myosin-V might act as a motor for melanosome translocation, they also suggest a broader cytoplasmic function for myosin-V, acting on other types of organelles or in cytoskeletal dynamics.

Ca2+/calmodulin-binding proteins in yeast. Catabolite repression and induction by carbon sources.

Soluble calmodulin-binding proteins from Saccharomyces carlsbergensis were analyzed in cells grown on glucose, maltose and galactose as carbon source. A large number of polypeptide chains showed affinity for calmodulin by affinity chromatography and overlay techniques. Amongst these, polypeptides of 115, 67 and 45 kDa were only detected during the second exponential phase of growth on glucose or non-fermentative carbon sources, suggesting that they might be subjected to catabolite repression. Polypeptides of 195 and 22 kDa were only observed in cells grown on maltose, whereas 88 kDa polypeptide was only observed in galactose-grown cells. Among the calmodulin -binding polypeptides, eight were phosphorylated in a Ca2+/calmodulin -dependent manner (220, 200, 175, 100, 62, 55, 31 and 16 kDa). Ca2+/calmodulin dependent [gamma-32P] incorporation was dramatically decreased in yeast cells submitted to a heat treatment.

Enzymatic characterization and functional domain mapping of brain myosin-V.

The actin binding and ATPase properties, as well as the functional domain structure of chick brain myosin-V, a two-headed, unconventional myosin, is reported here. Compared to conventional myosin from skeletal muscle, brain myosin-V exhibits low K-EDTA- and Ca-ATPase activities (1.8 and 0.8 ATP/s per head). The physiologically relevant Mg-ATPase is also low (approximately 0.3 ATP/s), unless activated by the presence of both F-actin and Ca2+ (Vmax of 27 ATP/s). Ca2+ stimulates the actin-activated Mg-ATPase over a narrow concentration range between 1 and 3 microM. In the presence of saturating Ca2+ and 75 mM KCl, surprisingly low concentrations of F-actin activate the Mg-ATPase in a hyperbolic manner (KATPase of 1.3 microM). Brain myosin-V also binds with relatively high affinity (compared to other known myosins) to F-actin in the presence of ATP, as assayed by cosedimentation. Digestion of brain myosin-V with calpain yielded a 65-kDa head domain fragment that cosediments with actin in an ATP-sensitive manner and a 80-kDa tail fragment that does not interact with F-actin. The 80-kDa fragment results from cleavage one residue beyond the proline-, glutamate-, serine-, threonine-rich region. Our data indicate that the Mg-ATPase cycle of brain myosin-V is tightly regulated by Ca2+, probably via direct binding to the calmodulin light chains in the neck domain, which like brush border myosin-I, results in partial (approximately 30%) dissociation of the calmodulin associated with brain myosin-V. The effect of Ca2+ binding, which appears to relieve suppression by the neck domain, can be mimicked by calpain cleavage near the head/neck junction.

Myosin-V: a class of unconventional molecular motors.

In this review we focus on the biochemical and structural properties of the myosin-V class of unconventional myosins as an example of the diversity of molecular motors within the myosin superfamily. A member of this class was first identified as a novel calmodulin-binding protein in mammalian brain (Larson RE, Pitta DE and Ferro JA (1988). Brazilian Journal of Medical and Biological Research, 21: 213-217). To date, the myosin-V class is represented by two molecules from yeast, one from nematodes, several from vertebrates (chickens, rats, mice and humans) and possibly one from plants. The domain structure of these myosins features a highly conserved head containing the ATP-hydrolysis and actin-binding sites, an extended neck composed of six tandem IQ-motifs which are sites for calmodulin binding and a large tail which has coiled-coil segments intercalated with globular regions of as yet unknown function. Biochemical studies on purified myosin-V from vertebrate brains and the description of myosin-V mutants in yeast and mice have made myosin-V one of the best characterized, unconventional myosin classes at the present time, surpassed only by the well-studied myosin-I class.