Walking to work: roles for class V myosins as cargo transporters

Cells use molecular motors, such as myosins, to move, position and segregate their organelles. Class V myosins possess biochemical and structural properties that should make them ideal actin-based cargo transporters. Indeed, studies show that class V myosins function as cargo transporters in yeast, moving a range of organelles, such as the vacuole, peroxisomes and secretory vesicles. There is also increasing evidence in vertebrate cells that class V myosins not only tether organelles to actin but also can serve as short-range, point-to-point organelle transporters, usually following long-range, microtubule-dependent organelle transport.     

Mouse class III myosins: kinase activity and phosphorylation sites

As class III unconventional myosins are motor proteins with an N-terminal kinase domain, it seems likely they play a role in both signaling and actin based transport. A growing body of evidence indicates that the motor functions of human class IIIA myosin, which has been implicated in progressive hearing loss, are modulated by intermolecular autophosphorylation. However, the phosphorylation sites have not been identified. We studied the kinase activity and phosphorylation sites of mouse class III myosins, mMyo3A and 3B, which are highly similar to their human orthologs. We demonstrate that the kinase domains of mMyo3A and 3B are active kinases, and that they have similar, if not identical, substrate specificities. We show that the kinase domains of these proteins autophosphorylate, and that they can phosphorylate sites within their myosin and tail domains. Using liquid chromatography-mass spectrometry, we identified phosphorylated sites in the kinase, myosin motor and tail domains of both mMyo3A and 3B. Most of the phosphorylated sites we identified and their consensus phosphorylation motifs are highly conserved among vertebrate class III myosins, including human class III myosins. Our findings are a major step toward understanding how the functions of class III myosins are regulated by phosphorylation.     

Myosin Va is required for P body but not stress granule formation

In the present study we demonstrate an association between mammalian myosin Va and cytoplasmic P bodies, microscopic ribonucleoprotein granules that contain components of the 5'-3' mRNA degradation machinery. Myosin Va colocalizes with several P body markers and its RNAi-mediated knockdown results in the disassembly of P bodies. Overexpression of a dominant-negative mutant of myosin Va reduced the motility of P bodies in living cells. Co-immunoprecipitation experiments demonstrate that myosin Va physically associates with eIF4E, an mRNA binding protein that localizes to P bodies. In contrast, we find that myosin Va does not play a role in stress granule formation. Stress granules are ribonucleoprotein structures that are involved in translational silencing and are spatially, functionally, and compositionally linked to P bodies. Myosin Va is found adjacent to stress granules in stressed cells but displays minimal localization within stress granules, and myosin Va knockdown has no effect on stress granule assembly or disassembly. Combined with recently published reports demonstrating a role for Drosophila and mammalian class V myosins in mRNA transport and the involvement of the yeast myosin V orthologue Myo2p in P body assembly, our results provide further evidence that the class V myosins serve an important role in the transport and turnover of mRNA.     

The temporal and spatial expression pattern of myosin Va, Vb and VI in the mouse ovary

There are 16 classes of unconventional myosins. Class V myosins have been shown to be involved in transporting cargo to and from the cell periphery. Class VI myosins have also been shown to transport cargo from the cell periphery, although it seems that these proteins have many roles which include the mediation of cell migration and stereocillia stabilisation. With the requirement of myosin VI for Drosophila oogenesis, the localised expression of Myosin V in the developing egg chamber and recent mounting evidence which links myosin VI to the migration of human ovarian cancer cell lines, we wanted to investigate the expression pattern of these two myosin classes in the normal mouse ovary. Here we show that these myosins are expressed, localised and regulated within the oocyte and granulosa cells of the developing mouse follicle.     

Functions of unconventional myosins

To date, fourteen classes of unconventional myosins have been identified. Recent reports have implicated a number of these myosins in organelle transport, and in the formation, maintenance and/or dynamics of actin-rich structures involved in a variety of cellular processes including endocytosis, cell migration, and sensory transduction. Characterizations of organelle dynamics in pigment cells and neurons have further defined the contributions made by unconventional myosins and microtubule motors to the transport and distribution of organelles. Several studies have provided evidence of complexes through which cooperative organelle transport may be coordinated. Finally, the myosin superfamily has been shown to contain at least one processive motor and one backwards motor.     

The UCS domain protein She4p binds to myosin motor domains and is essential for class I and class V myosin function

BACKGROUND: Myosins are motor proteins involved in processes like cell motility, vesicle transport, or cytokinesis. In a variety of organisms, a novel group of proteins forming the UCS (UNC-45/CRO1/SHE4) domain-containing family are essential for proper myosin function. The Saccharomyces cerevisae UCS domain protein She4p is involved in two myosin-requiring events, endocytosis and mRNA localization. RESULTS: In contrast to UCS domain proteins from other organisms that interact with class II myosins, we demonstrate that She4p associates with yeast class I and class V myosins. She4p binds to motor domains of class V myosin Myo4p and class I myosin Myo5p, and this binding depends on She4p's UCS domain. In vivo, She4p is essential for the function and localization of Myo3p, Myo4p, and Myo5p (but not of Myo2p) and for colocalization of class I myosins with cortical actin patches. In vitro, She4p stimulates binding of Myo5p to filamentous actin. Wild-type She4p, but not a mutant lacking the UCS domain, accumulates in a cap-like structure at the bud tip. This localization requires Myo2p and actin, suggesting a Myo2-dependent mechanism by which She4p is targeted to the bud cap. Localization of She4p is essential for proper positioning and myosin-actin association of cortical Myo5p. CONCLUSIONS: Our results suggest that She4p is a novel myosin motor domain binding protein and operates as a localized regulator of myosin function of class I and likely class V myosins.     

The yeast class V myosins, Myo2p and Myo4p, are nonprocessive actin-based motors

The motor properties of the two yeast class V myosins, Myo2p and Myo4p, were examined using in vitro motility assays. Both myosins are active motors with maximum velocities of 4.5 microm/s for Myo2p and 1.1 microm/s for Myo4p. Myo2p motility is Ca(2+) insensitive. Both myosins have properties of a nonprocessive motor, unlike chick myosin-Va (M5a), which behaves as a processive motor when assayed under identical conditions. Additional support for the idea that Myo2p is a nonprocessive motor comes from actin cosedimentation assays, which show that Myo2p has a low affinity for F-actin in the presence of ATP and Ca(2+), unlike chick brain M5a. These studies suggest that if Myo2p functions in organelle transport, at least five molecules of Myo2p must be present per organelle to promote directed movement.     

Unique sequences and predicted functions of myosins in Tetrahymena thermophila

Myosins are eukaryotic actin-dependent molecular motors that play important roles in many cellular events. The function of each myosin is determined by a variety of functional domains in its tail region. In some major model organisms, the functions and properties of myosins have been investigated based on their amino acid sequences. However, in protists, myosins have been little studied beyond the level of genome sequences. We therefore investigated the mRNA expression levels and amino acid sequences of 13 myosin genes in the ciliate Tetrahymena thermophila. This study is an overview of myosins in T. thermophila, which has no typical myosins, such as class I, II, or V myosins. We showed that all 13 myosins were expressed in vegetative cells. Furthermore, these myosins could be divided into 3 subclasses based on four functional domains in their tail regions. Subclass 1 comprised of 8 myosins has both MyTH4 and FERM domains, and has a potential to function in vesicle transport or anchoring between membrane and actin filaments. Subclass 2 comprised of 4 myosins has RCC1 (regulator of chromosome condensation 1) domains, which are found only in some protists, and may have unconventional features. Subclass 3 is comprised of one myosin, which has a long coiled-coil domain like class II myosin. In addition, phylogenetic analysis on the basis of motor domains showed that T. thermophila myosins are separated into two clusters: one consists of subclasses 1 and 2, and the other consists of subclass 3.     

Localization of unconventional myosins V and VI in neuronal growth cones

Class V and VI myosins, two of the six known classes of actin-based motor genes expressed in vertebrate brain (Class I, II, V, VI, IX, and XV), have been suggested to be organelle motors. In this report, the neuronal expression and subcellular localization of chicken brain myosin V and myosin VI is examined. Both myosins are expressed in brain during embryogenesis. In cultured dorsal root ganglion (DRG) neurons, immunolocalization of myosin V and myosin VI revealed a similar distribution for these two myosins. Both are present within cell bodies, neurites and growth cones. Both of these myosins exhibit punctate labeling patterns that are found in the same subcellular region as microtubules in growth cone central domains. In peripheral growth cone domains, where individual puncta are more readily resolved, we observe a similar number of myosin V and myosin VI puncta. However, less than 20% of myosin V and myosin VI puncta colocalize with each other in the peripheral domains. After live cell extraction, punctate staining of myosin V and myosin VI is reduced in peripheral domains. However, we do not detect such changes in the central domains, suggesting that these myosins are associated with cytoskeletal/organelle structures. In peripheral growth cone domains myosin VI exhibits a higher extractability than myosin V. This difference between myosin V and VI was also found in a biochemical growth cone particle preparation from brain, suggesting that a significant portion of these two motors has a distinct subcellular distribution.     

Myosin function in nervous and sensory systems

Development of the nervous system requires remarkable changes in cell structure that are dependent upon the cytoskeleton. The importance of specific components of the neuronal cytoskeleton, such as microtubules and neurofilaments, to neuronal function and development has been well established. Recently, increasing focus has been put on understanding the functional role of the actin cytoskeleton in neurons. Important modulators of the actin cytoskeleton are the large family of myosins, many of which (classes I, II, III, V, VI, VII, IX, and XV; Fig. 1) are expressed in developing neurons or sensory cells. Myosins are force-producing proteins that have been implicated in a wide variety of cellular functions in the developing nervous system, including neuronal migration, process outgrowth, and growth cone motility, as well as other aspects of morphogenesis, axonal transport, and synaptic and sensory functions. We review the roles that neuronal myosins play in these functions with particular focus on the first three events listed above, as well as sensory function.     

Walking with myosin V

The cytoplasm of cells is teaming with vesicles and other cargo that are moving along tracks of microtubules or actin filaments, powered by myosins, kinesins and dyneins. Myosin V has been implicated in several types of intracellular transport. The mechanism by which myosin V moves processively along actin filaments has been the subject of many biophysical and biochemical studies and a consensus is starting to emerge about how this minute molecular motor operates.     

Myosin genes in Tetrahymena

This report presents an initial comparison of motor, neck, and tail domains of myosin genes in Tetrahymena thermophila. An unrooted phylogenetic tree drawn from alignment of predicted amino acid translations determined the relationship among 13 myosins in Tetrahymena and their relationship to the myosin superfamily. The myosins in Tetrahymena did not align with any of the previously named myosin classes. Twelve of the Tetrahymena myosins form a new class designated as XX. The other Tetrahymena myosin is divergent from the twelve. Surprisingly, none of the myosins in Tetrahymena aligned with either class I, class II, or class V myosins. Apparent absence of a class II myosin is an indication that cytokinesis in Tetrahymena either utilizes an unconventional myosin or does not require a myosin motor.     

‘Should I stay or should I go?’: myosin V function in organelle trafficking

Actin- and microtubule-based motors can propel different cargos along filaments. Within cells, they control the distribution of membrane-bound compartments by performing complementary tasks. Organelles make long journeys along microtubules, with class V myosins ensuring their capture and their dispersal in actin-rich regions. Myosin Va is recruited on to diverse organelles, such as melanosomes and secretory vesicles, by a mechanism involving Rab GTPases. The role of myosin Va in the recruitment of secretory vesicles at the plasma membrane reveals that the cortical actin network cannot merely be seen as a physical barrier hindering vesicle access to release sites. In neurons, myosin Va controls the targeting of IP(3) (inositol 1,4,5-trisphosphate)-sensitive Ca(2+) stores to dendritic spines and the transport of mRNAs. These defects probably account for the severe neurological symptoms observed in Griscelli syndrome due to mutations in the MYO5A gene.     

Class VIII myosins are required for plasmodesmatal localization of a closterovirus Hsp70 homolog

The Hsp70 homolog (Hsp70h) of Beet yellows virus (BYV) functions in virion assembly and cell-to-cell movement and is autonomously targeted to plasmodesmata in association with the actomyosin motility system (A. I. Prokhnevsky, V. V. Peremyslov, and V. V. Dolja, J. Virol. 79:14421-14428, 2005). Myosins are a diverse category of molecular motors that possess a motor domain and a tail domain involved in cargo binding. Plants have two classes of myosins, VIII and XI, whose specific functions are poorly understood. We used dominant negative inhibition to identify myosins required for Hsp70h localization to plasmodesmata. Six full-length myosin cDNAs from the BYV host plant Nicotiana benthamiana were sequenced and shown to encode apparent orthologs of the Arabidopsis thaliana myosins VIII-1, VIII-2, VIII-B, XI-2, XI-F, and XI-K. We found that the ectopic expression of the tail domains of each of the class VIII, but not the class XI, myosins inhibited the plasmodesmatal localization of Hsp70h. In contrast, the overexpression of the motor domains or the entire molecules of the class VIII myosins did not affect Hsp70h targeting. Further mapping revealed that the minimal cargo-binding part of the myosin VIII tails was both essential and sufficient for the inhibition of the proper Hsp70h localization. Interestingly, plasmodesmatal localization of the Tobacco mosaic virus movement protein and Arabidopsis protein RGP2 was not affected by myosin VIII tail overexpression. Collectively, our data implicate class VIII myosins in protein delivery to plasmodesmata and suggest that more than one mechanism of such delivery exist in plants.     

Tropomyosin is essential for processive movement of a class v Myosin from budding yeast

Myosin V is an actin-based motor protein involved in intracellular cargo transport [1]. Given this physiological role, it was widely assumed that all class V myosins are processive, able to take multiple steps along actin filaments without dissociating. This notion was challenged when several class?V myosins were characterized as nonprocessive in?vitro, including Myo2p, the essential class V myosin from S.?cerevisiae [2-6]. Myo2p moves cargo including secretory vesicles and other organelles for several microns along actin cables in?vivo. This demonstrated cargo transporter must therefore either operate in small ensembles or?behave processively in the cellular context. Here we show?that Myo2p moves processively in?vitro as a single motor when it walks on an actin track that more closely resembles the actin cables found in?vivo. The key to processivity is tropomyosin: Myo2p is not processive on bare actin?but highly processive on actin-tropomyosin. The major yeast tropomyosin isoform, Tpm1p, supports the most robust processivity. Tropomyosin slows the rate of MgADP release, thus increasing the time the motor spends strongly attached to actin. This is the first example of tropomyosin switching a motor from nonprocessive to processive motion on actin.     

Myosins: a diverse superfamily

Myosins constitute a large superfamily of actin-dependent molecular motors. Phylogenetic analysis currently places myosins into 15 classes. The conventional myosins which form filaments in muscle and non-muscle cells form class II. There has been extensive characterization of these myosins and much is known about their function. With the exception of class I and class V myosins, little is known about the structure, enzymatic properties, intracellular localization and physiology of most unconventional myosin classes. This review will focus on myosins from class IV, VI, VII, VIII, X, XI, XII, XIII, XIV and XV. In addition, the function of myosin II in non-muscle cells will also be discussed.     

Transport of Myosin II to the Equatorial Region without Its Own Motor Activity in Mitotic Dictyostelium Cells

Fluorescently labeled myosin moved and accumulated circumferentially in the equatorial region of dividing Dictyostelium cells within a time course of 4 min, followed by contraction of the contractile ring. To investigate the mechanism of this transport process, we have expressed three mutant myosins that cannot hydrolyze ATP in myosin null cells. Immunofluorescence staining showed that these mutant myosins were also correctly transported to the equatorial region, although no contraction followed. The rates of transport, measured using green fluorescent protein-fused myosins, were indistinguishable between wild-type and mutant myosins. These observations demonstrate that myosin is passively transported toward the equatorial region and incorporated into the forming contractile ring without its own motor activity.     

Cellular distribution of myosin-V in the guinea pig cochlea

The importance of unconventional myosins to hearing has recently been revealed by the identification of myosins-VI and -VII as the defective genes in mouse mutations and in a human syndrome which lead to profound hearing loss. Another class of novel myosins (V) has been implicated in the trafficking of intracellular vesicles in neurons and other secretory cells. We used affinity-purified antibodies to determine the localization of myosin-V in the guinea pig inner ear. In the sensory epithelium of the cochlea, myosin-V epitopes were recognized in neuronal and supporting cells. Neuronal labelling was most intense in the afferent innervation of inner and outer hair cells. Supporting cells labelled were cells of Hensen and Deiters, and inner border, inner phalangeal, inner sulcus and interdental cells. In the vascular tissue of the cochlea, we observed staining of intermediate cells of the stria vascularis and of border cells between the stria and the spiral prominence. Staining of afferent chalice nerve endings was observed on type I vestibular hair cells. The results suggest that, like myosins VI and VII, myosin-V is localized in positions that may be critical to auditory function.     

Myosins from angiosperms, ferns, and algae amplification of gene fragments with versatile PCR primers and detection of protein products with a monoclonal antibody to a conserved head epitope

Summary Myosins providing the motors for the actin-based motility that occurs in diverse plants have proved difficult to study. To facilitate those studies, we describe polymerase chain reaction primers that reliably amplify part of the myosin head from diverse plants, consensus sequences that characterise the amplified product as encoding a class V or class VIII myosin, and a monoclonal antibody that recognises an epitope conserved in the head of most plant, fungal, and animal myosins. A pair of stringent oligonucleotide primers was designed that, when used in the polymerase chain reaction, amplified at least eleven different myosins from five species of angiosperms and one sequence from each of the fernAzolla and the algaeNitella andPhaeodactylum. The amplified products, comprising 126 to 135 nucleotides encoding part of the myosin head domain, can be used as myosin-specific probes to screen genomic and cDNA libraries. To identify the products of plant myosin genes, we raised a monoclonal antibody (anti-CHE) to a nine amino acid peptide matching a conserved head epitope showing not more than single amino acid substitutions in most published myosin genes. This antibody recognises rabbit skeletal myosin and multiple polypeptides of >100 kDa in four angiosperms and in the algaNitella. Relating the M_r values of immunoreactive bands inArabidopsis extracts to the predicted M_r values of the products of five myosin genes supports the view that the antibody recognises both myosins V and VIII together with the products of some as yet unsequenced genes. The previously described MB170 antibodies may, in contrast, be specific for one or more type V myosins. Together, the polymerase chain reaction primers and the antibody represent versatile tools for identifying and categorising myosins in diverse plants.     

Sequence and phylogenetic analysis of squid myosin-V: a vesicle motor in nerve cells

We have shown that vesicles in the axoplasm of the squid giant axon move on actin filaments and that movement is inhibited by myosin V-specific antibodies [Tabb et al., 1998]. In the study reported in this article, experiments were performed to clone and sequence the cDNA for squid brain myosin V. Five proteolytic fragments of purified squid brain myosin V were analyzed by direct protein sequencing [Tabb et al., 1998]. Based on this sequence information, degenerate primers were constructed and used to isolate cDNA clones by PCR. Five clones, representing overlapping segments of the gene, were sequenced. The sequence data and the previous biochemical characterization of the molecule support the classification of this vesicle-associated myosin as a member of the class V myosins. Motif analysis of the head, neck, and tail domains revealed that squid MyoV has consensus sequences for all the motifs found in vertebrate members of the myosin V family of motor proteins. A phylogenetic tree was constructed from a sequence alignment by the neighbor-joining method, using Megalign (DNAStar, Madison, WI); the resulting phylogenetic tree showed that squid MyoV is more closely related to vertebrate MyoV (mouse dilute, chicken dilute, rat myr6, and human myo5a) than Drosophila and yeast (myo2, and myo4) myosins V. These new data on the phylogenetic relationships of squid myosin V to vertebrate myosin V strengthens the argument that myosin V functions as a vesicle motor in vertebrate neurons.