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Skeletal muscle fibers are multinucleated cellular giants formed by the fusion of mononuclear myoblasts. Several molecules involved in myoblast fusion have been discovered, and finger-like projections coincident with myoblast fusion have also been implicated in the fusion process. The role of these cellular projections in muscle cell fusion was investigated herein. We demonstrate that these projections are filopodia generated by class X myosin (Myo10), an unconventional myosin motor protein specialized for filopodia. We further show that Myo10 is highly expressed by differentiating myoblasts, and Myo10 ablation inhibits both filopodia formation and myoblast fusion in vitro. In vivo, Myo10 labels regenerating muscle fibers associated with Duchenne muscular dystrophy and acute muscle injury. In mice, conditional loss of Myo10 from muscle-resident stem cells, known as satellite cells, severely impairs postnatal muscle regeneration. Furthermore, the muscle fusion proteins Myomaker and Myomixer are detected in myoblast filopodia. These data demonstrate that Myo10-driven filopodia facilitate multinucleated mammalian muscle formation.

The development of cell-cell junctions was a fundamental step in metazoan evolution, and human health depends on the formation and function of cell junctions. Although it has long been known that actin and conventional myosin have important roles in cell junctions, research has begun to reveal the specific functions of the different forms of conventional myosin. Exciting new data also reveals that a growing number of unconventional myosins have important roles in cell junctions. Experiments showing that cell junctions act as mechanosensors have also provided new impetus to understand the functions of myosins and the forces they exert. In this review we will summarize recent developments on the roles of myosins in cell junctions.

Myosin-X (Myo10) is an unconventional myosin with MyTH4-FERM domains that is best known for its striking localization to the tips of filopodia and its ability to induce filopodia. Although the head domain of Myo10 enables it to function as an actin-based motor, its tail contains binding sites for several molecules with central roles in cell biology, including phosphatidylinositol (3,4,5)-trisphosphate, microtubules and integrins. Myo10 also undergoes fascinating long-range movements within filopodia, which appear to represent a newly recognized system of transport. Myo10 is also unusual in that it is a myosin with important roles in the spindle, a microtubule-based structure. Exciting new studies have begun to reveal the structure and single-molecule properties of this intriguing myosin, as well as its mechanisms of regulation and induction of filopodia. At the cellular and organismal level, growing evidence demonstrates that Myo10 has crucial functions in numerous processes ranging from invadopodia formation to cell migration.

Although many proteins, receptors, and viruses are transported rearward along filopodia by retrograde actin flow, it is less clear how molecules move forward in filopodia. Myosin-X (Myo10) is an actin-based motor hypothesized to use its motor activity to move forward along actin filaments to the tips of filopodia. Here we use a sensitive total internal reflection fluorescence (TIRF) microscopy system to directly visualize the movements of GFP-Myo10. This reveals a novel form of motility at or near the single-molecule level in living cells wherein extremely faint particles of Myo10 move in a rapid and directed fashion toward the filopodial tip. These fast forward movements occur at approximately 600 nm/s over distances of up to approximately 10 microm and require Myo10 motor activity and actin filaments. As expected for imaging at the single-molecule level, the faint particles of GFP-Myo10 are diffraction limited, have an intensity range similar to single GFP molecules, and exhibit stepwise bleaching. Faint particles of GFP-Myo5a can also move toward the filopodial tip, but at a slower characteristic velocity of approximately 250 nm/s. Similar movements were not detected with GFP-Myo1a, indicating that not all myosins are capable of intrafilopodial motility. These data indicate the existence of a novel system of long-range transport based on the rapid movement of myosin molecules along filopodial actin filaments.

Myosin Vc is the product of one of the three genes of the class V myosin found in vertebrates. It is widely found in secretory and glandular tissues, with a possible involvement in transferrin trafficking. Transient and steady-state kinetic studies of human myosin Vc were performed using a truncated, single-headed construct. Steady-state actin-activated ATPase measurements revealed a V(max) of 1.8 +/- 0.3 s(-1) and a K(ATPase) of 43 +/- 11 microm. Unlike previously studied vertebrate myosin Vs, the rate-limiting step in the actomyosin Vc ATPase pathway is the release of inorganic phosphate (~1.5 s(-1)), rather than the ADP release step (~12.0-16.0 s(-1)). Nevertheless, the ADP affinity of actomyosin Vc (K(d) = 0.25 +/- 0.02 microm) reflects a higher ADP affinity than seen in other myosin V isoforms. Using the measured kinetic rates, the calculated duty ratio of myosin Vc was approximately 10%, indicating that myosin Vc spends the majority of the actomyosin ATPase cycle in weak actin-binding states, unlike the other vertebrate myosin V isoforms. Consistent with this, a fluorescently labeled double-headed heavy meromyosin form showed no processive movements along actin filaments in a single molecule assay, but it did move actin filaments at a velocity of approximately 24 nm/s in ensemble assays. Kinetic simulations reveal that the high ADP affinity of actomyosin Vc may lead to elevations of the duty ratio of myosin Vc to as high as 64% under possible physiological ADP concentrations. This, in turn, may possibly imply a regulatory mechanism that may be sensitive to moderate changes in ADP concentration.

Cystic fibrosis transmembrane conductance regulator (CFTR)-mediated Cl(-) secretion across fluid-transporting epithelia is regulated, in part, by modulating the number of CFTR Cl(-) channels in the plasma membrane by adjusting CFTR endocytosis and recycling. However, the mechanisms that regulate CFTR recycling in airway epithelial cells remain unknown, at least in part, because the recycling itineraries of CFTR in these cells are incompletely understood. In a previous study, we demonstrated that CFTR undergoes trafficking in Rab11a-specific apical recycling endosomes in human airway epithelial cells. Myosin Vb is a plus-end-directed, actin-based mechanoenzyme that facilitates protein trafficking in Rab11a-specific recycling vesicles in several cell model systems. There are no published studies examining the role of myosin Vb in airway epithelial cells. Thus, the goal of this study was to determine whether myosin Vb facilitates CFTR recycling in polarized human airway epithelial cells. Endogenous CFTR formed a complex with endogenous myosin Vb and Rab11a. Silencing myosin Vb by RNA-mediated interference decreased the expression of wild-type CFTR and DeltaF508-CFTR in the apical membrane and decreased CFTR-mediated Cl(-) secretion across polarized human airway epithelial cells. A recombinant tail domain fragment of myosin Vb attenuated the plasma membrane expression of CFTR by arresting CFTR recycling. The dominant-negative effect was dependent on the ability of the myosin Vb tail fragment to interact with Rab11a. Taken together, these data indicate that myosin Vb is required for CFTR recycling in Rab11a-specific apical recycling endosomes in polarized human airway epithelial cells.

Research in several areas, including unconventional myosins and deafness genes, has converged recently on a group of myosins whose tails contain myosin tail homology 4 (MyTH4) and band 4.1, ezrin, radixin, moesin (FERM) domains. Although these 'MyTH-FERM' myosins are not present in yeast and plants, they are present in slime molds, worms, flies and mammals, where they mediate interactions between the cytoskeleton and the plasma membrane. The most broadly distributed MyTH-FERM myosin in vertebrate cells appears to be myosin-X (Myo10). This myosin can act as a link to integrins and microtubules, stimulate the formation of filopodia and undergo a novel form of motility within filopodia.

Unconventional myosins are actin-based motors with a growing number of attributed functions. Interestingly, it has been proposed that integrins are transported by unidentified myosins to facilitate cellular remodelling. Here we present an interaction between the unconventional myosin-X (Myo10) FERM (band 4.1/ezrin/radixin/moesin) domain and an NPXY motif within beta-integrin cytoplasmic domains. Importantly, knock-down of Myo10 by short interfering RNA impaired integrin function in cell adhesion, whereas overexpression of Myo10 stimulated the formation and elongation of filopodia in an integrin-dependent manner and relocalized integrins together with Myo10 to the tips of filopodia. This integrin relocalization and filopodia elongation did not occur with Myo10 mutants deficient in integrin binding or with a beta(1)-integrin point mutant deficient in Myo10 binding. Taken together, these results indicate that Myo10-mediated relocalization of integrins might serve to form adhesive structures and thereby promote filopodial extension.

Myosin-X (Myo10) is a motor protein best known for its role in filopodia formation. New research implicates Myo10 in a number of disease states including cancer metastasis and pathogen infection. This review focuses on these developments with emphasis on the emerging roles of Myo10 in formation of cancer cell protrusions and metastasis. A number of aggressive cancers show high levels of Myo10 expression and knockdown of Myo10 has been shown to dramatically limit cancer cell motility in 2D and 3D systems. Myo10 knockdown also limits spread of intracellular pathogens marburgvirus and Shigella flexneri. Consideration is given to how these properties might arise and potential paths of future research.

Class V myosins are one of the most ancient and widely distributed groups of the myosin superfamily and are hypothesized to function as motors for actin-dependent organelle transport. We report the discovery and initial characterization of a novel member of this family, human myosin-Vc (Myo5c). The Myo5c protein sequence shares ∼50% overall identity with the two other class V myosins in vertebrates, myosin-Va (Myo5a) and myosin-Vb (Myo5b). Systematic analysis of the mRNA and protein distribution of these myosins indicates that Myo5a is most abundant in brain, whereas Myo5b and Myo5c are expressed chiefly in non-neuronal tissues. Myo5c is particularly abundant in epithelial and glandular tissues including pancreas, prostate, mammary,stomach, colon and lung. Immunolocalization in colon and exocrine pancreas indicates that Myo5c is expressed chiefly in epithelial cells. A dominant negative approach using a GFP-Myo5c tail construct in HeLa cells reveals that the Myo5c tail selectively colocalizes with and perturbs a membrane compartment containing the transferrin receptor and rab8. Transferrin also accumulates in this compartment, suggesting that Myo5c is involved in transferrin trafficking. As a class V myosin of epithelial cells, Myo5c is likely to power actin-based membrane trafficking in many physiologically crucial tissues of the human body.

Filopodia are thin cellular protrusions that are important in cell motility and neuronal growth cone guidance. The actin filaments that make up the core of a filopodium undergo continuous retrograde flow towards the cell body1,2. Surface receptors or particles can couple to this retrograde flow3,4 and can also move forward to the tips of filopodia4,5, although the molecular basis of forward transport is unknown. We report here that myosin-X (Myo10 or M10), the founding member of a novel class of myosins6, localizes to the tips of filopodia and undergoes striking forward and rearward movements within filopodia, which we term intrafilopodial motility. The movements of the GFP–M10 puncta correspond to forward and rearward movements of phase-dense granules along the filopodia. Finally, overexpressing full-length M10 (but not truncated forms of M10) causes an increase in the number and length of filopodia, indicating that M10 or its cargo may function in filopodial dynamics. The localization and movements of M10 strongly suggest that it functions as a motor for intrafilopodial motility.

Class V myosins are actin-based motors with conserved functions in vesicle and organelle trafficking. Herein we report the discovery of a function for Myosin Vc in melanosome biogenesis as an effector of melanosome-associated Rab GTPases. We isolated Myosin Vc in a yeast two-hybrid screening for proteins that interact with Rab38, a Rab protein involved in the biogenesis of melanosomes and other lysosome-related organelles. Rab38 and its close homolog Rab32 bind to Myosin Vc but not to Myosin Va or Myosin Vb. Binding depends on residues in the switch II region of Rab32 and Rab38 and regions of the Myosin Vc coiled-coil tail domain. Myosin Vc also interacts with Rab7a and Rab8a but not with Rab11, Rab17, and Rab27. Although Myosin Vc is not particularly abundant on pigmented melanosomes, its knockdown in MNT-1 melanocytes caused defects in the trafficking of integral membrane proteins to melanosomes with substantially increased surface expression of Tyrp1, nearly complete loss of Tyrp2, and significant Vamp7 mislocalization. Knockdown of Myosin Vc in MNT-1 cells more than doubled the abundance of pigmented melanosomes but did not change the number of unpigmented melanosomes. Together the data demonstrate a novel role for Myosin Vc in melanosome biogenesis and secretion.

Myosin-X is the founding member of a novel class of unconventional myosins characterized by a tail domain containing multiple pleckstrin homology domains. We report here the full-length cDNA sequences of human and bovine myosin-X as well as the first characterization of this protein's distribution and biochemical properties. The 235 kDa myosin-X contains a head domain with

Members of the myosin superfamily of actin-based motor proteins were previously thought to move only towards the barbed end of the actin filament. In an extraordinary reversal of this dogma, an abundant and widespread unconventional myosin known as myosin VI has recently been shown to move towards the pointed end of the actin filament – the opposite direction of all other characterized myosins. This discovery raises novel and intriguing questions about the molecular mechanisms of reversal and the biological roles of this ‘backwards' myosin.

Myosin X (Myo10) is an unconventional myosin with two known isoforms: full-length (FL)-Myo10 that has motor activity, and a recently identified brain-expressed isoform, headless (Hdl)-Myo10, which lacks most of the motor domain. FL-Myo10 is involved in the regulation of filopodia formation in non-neuronal cells; however, the biological function of Hdl-Myo10 remains largely unknown. Here, we show that FL- and Hdl-Myo10 have important, but distinct, roles in the development of dendritic spines and synapses in hippocampal neurons. FL-Myo10 induces dendritic filopodia formation and modulates filopodia dynamics by trafficking the actin-binding protein vasodilator-stimulated phosphoprotein (VASP) to the tips of filopodia. In contrast, Hdl-Myo10 acts on dendritic spines to enhance spine and synaptic density as well as spine head expansion by increasing the retention of VASP in spines. Thus, this study shows a novel biological function for Hdl-Myo10, and an important new role for both Myo10 isoforms in the development of dendritic spines and synapses.

Dipeptidyl-peptidase 6 is an auxiliary subunit of Kv4-mediated A-type K(+) channels that, in addition to enhancing channel surface expression, potently accelerates their kinetics. The dipeptidyl-peptidase 6 gene has been associated with a number of human central nervous system disorders including autism spectrum disorders and schizophrenia. Here we employ knockdown and genetic deletion of dipeptidyl-peptidase 6 to reveal its importance for the formation and stability of dendritic filopodia during early neuronal development. We find that the hippocampal neurons lacking dipeptidyl-peptidase 6 show a sparser dendritic branching pattern along with fewer spines throughout development and into adulthood. In electrophysiological and imaging experiments, we show that these deficits lead to fewer functional synapses and occur independently of the potassium channel subunit Kv4.2. We report that dipeptidyl-peptidase 6 interacts with a filopodia-associated myosin as well as with fibronectin in the extracellular matrix. dipeptidyl-peptidase 6 therefore has an unexpected but important role in cell adhesion and motility, impacting the hippocampal synaptic development and function.

Myosins are molecular motors that upon interaction with actin filaments convert energy from ATP hydrolysis into mechanical force. Evidence has emerged for the existence of a large, widely expressed and evolutionarily ancient superfamily of myosin genes. In addition to the well-catheterized conventional, filament-forming, two-headed myosin-II of muscle and nonmuscle cells, at least ten additional classes of myosins have been identified. In vertebrates, at least seven of the eleven classes are expressed, and many myosins can be expressed in a single cell type. This review summarizes known structural and functional features of these novel unconventional myosins.

The intracellular pathogen Shigella flexneri forms membrane protrusions to spread from cell to cell. As protrusions form, myosin-X (Myo10) localizes to Shigella. Electron micrographs of immunogold-labelled Shigella-infected HeLa cells reveal that Myo10 concentrates at the bases and along the sides of bacteria within membrane protrusions. Time-lapse video microscopy shows that a full-length Myo10 GFP-construct cycles along the sides of Shigella within the membrane protrusions as these structures progressively lengthen. RNAi knock-down of Myo10 is associated with shorter protrusions with thicker stalks, and causes a >80% decrease in confluent cell plaque formation. Myo10 also concentrates in membrane protrusions formed by another intracellular bacteria, Listeria, and knock-down of Myo10 also impairs Listeria plaque formation. In Cos7 cells (contain low concentrations of Myo10), the expression of full-length Myo10 nearly doubles Shigella-induced protrusion length, and lengthening requires the head domain, as well as the tail-PH domain, but not the FERM domain. The GFP-Myo10-HMM domain localizes to the sides of Shigella within membrane protrusions and the GFP-Myo10-PH domain localizes to host cell membranes. We conclude that Myo10 generates the force to enhance bacterial-induced protrusions by binding its head region to actin filaments and its PH tail domain to the peripheral membrane.

Class V myosins are actin-based motor proteins that have critical functions in organelle trafficking. Of the three class V myosins expressed in mammals, relatively little is known about Myo5c except that it is abundant in exocrine tissues. Here we use MCF-7 cells to identify the organelles that Myo5c associates with, image the dynamics of Myo5c in living cells, and test the functions of Myo5c. Endogenous Myo5c localizes to two distinct compartments: small puncta and slender tubules. Myo5c often exhibits a highly polarized distribution toward the leading edge in migrating cells and is clearly distinct from the Myo5a or Myo5b compartments. Imaging with GFP-Myo5c reveals that Myo5c puncta move slowly (approximately 30 nm/s) and microtubule independently, whereas tubules move rapidly (approximately 440 nm/s) and microtubule dependently. Myo5c puncta colocalize with secretory granule markers such as chromogranin A and Rab27b, whereas Myo5c tubules are labeled by Rab8a. TIRF imaging indicates that the granules can be triggered to undergo secretion. To test if Myo5c functions in granule trafficking, we used the Myo5c tail as a dominant negative and found that it dramatically perturbs the distribution of granule markers. These results provide the first live-cell imaging of Myo5c and indicate that Myo5c functions in secretory granule trafficking.

Summary Mitochondria are pleomorphic organelles [1, 2] that have central roles in cell physiology. Defects in their localization and dynamics lead to human disease [3–5]. Myosins are actin-based motors that power processes such as muscle contraction, cytokinesis, and organelle transport [6]. Here we report the initial characterization of myosin-XIX (Myo19), the founding member of a novel class of myosin that associates with mitochondria. The 970 aa heavy chain consists of a motor domain, three IQ motifs, and a short tail. Myo19 mRNA is expressed in multiple tissues, and antibodies to human Myo19 detect an ∼109 kDa band in multiple cell lines. Both endogenous Myo19 and GFP-Myo19 exhibit striking localization to mitochondria. Deletion analysis reveals that the Myo19 tail is necessary and sufficient for mitochondrial localization. Expressing full-length GFP-Myo19 in A549 cells reveals a remarkable gain of function where the majority of the mitochondria move continuously. Moving mitochondria travel for many micrometers with an obvious leading end and distorted shape. The motility and shape change are sensitive to latrunculin B, indicating that both are actin dependent. Expressing the GFP-Myo19 tail in CAD cells resulted in decreased mitochondrial run lengths in neurites. These results suggest that this novel myosin functions as an actin-based motor for mitochondrial movement in vertebrate cells.

Filopodia are dynamic cell surface protrusions that are required for proper cellular development and function. We report that the integral membrane protein lipid-phosphatase-related protein 1 (LPR1) localizes to and promotes the formation of actin-rich, dynamic filopodia, both along the cell periphery and the dorsal cell surface. Regulation of filopodia by LPR1 was not mediated by cdc42 or Rif, and is independent of the Arp2/3 complex. We found that LPR1 can induce filopodia formation in the absence of the Ena/Vasp family of proteins, suggesting that these molecules are not essential for the development of the protrusions. Mutagenesis experiments identified residues and regions of LPR1 that are important for the induction of filopodia. RNA interference experiments in an ovarian epithelial cancer cell line demonstrated a role for LPR1 in the maintenance of filopodia-like membrane protrusions. These observations, and our finding that LPR1 is a not an active lipid phosphatase, suggest that LPR1 may be a novel integral membrane protein link between the actin core and the surrounding lipid layer of a nascent filopodium.

Viruses exploit the cytoskeleton of host cells to transport their components and spread to neighbouring cells. Here we show that the actin cytoskeleton is involved in the release of Marburgvirus (MARV) particles. We found that peripherally located nucleocapsids and envelope precursors of MARV are located either at the tip or at the side of filopodial actin bundles. Importantly, viral budding was almost exclusively detected at filopodia. Inhibiting actin polymerization in MARV-infected cells significantly diminished the amount of viral particles released into the medium. This suggested that dynamic polymerization of actin in filopodia is essential for efficient release of MARV. The viral matrix protein VP40 plays a key role in the release of MARV particles and we found that the intracellular localization of recombinant VP40 and its release in form of virus-like particles were strongly influenced by overexpression or inhibition of myosin 10 and Cdc42, proteins important in filopodia formation and function. We suggest that VP40, which is capable of interacting with viral nucleocapsids, provides an interface of MARV subviral particles and filopodia. As filopodia are in close contact with neighbouring cells, usurpation of these structures may facilitate spread of MARV to adjacent cells.

Despite recent progress in understanding lamellipodia extension, the molecular mechanisms regulating filopodia formation remain largely unknown. Myo10 is a MyTH4-FERM myosin that localizes to the tips of filopodia and is hypothesized to function in filopodia formation. To determine whether endogenous Myo10 is required for filopodia formation, we have used scanning EM to assay the numerous filopodia normally present on the dorsal surfaces of HeLa cells. We show here that siRNA-mediated knockdown of Myo10 in HeLa cells leads to a dramatic loss of dorsal filopodia. Overexpressing the coiled coil region from Myo10 as a dominant- negative also leads to a loss of dorsal filopodia, thus providing independent evidence that Myo10 functions in filopodia formation. We also show that expressing Myo10 in COS-7 cells, a cell line that normally lacks dorsal filopodia, leads to a massive induction of dorsal filopodia. Because the dorsal filopodia induced by Myo10 are not attached to the substrate, Myo10 can promote filopodia by a mechanism that is independent of substrate attachment. Consistent with this observation, a Myo10 construct that lacks the FERM domain, the region that binds to integrin, retains the ability to induce dorsal filopodia. Deletion of the MyTH4-FERM region, however, completely abolishes Myo10's filopodia-promoting activity, as does deletion of the motor domain. Additional experiments on the mechanism of Myo10 action indicate that it acts downstream of Cdc42 and can promote filopodia in the absence of VASP proteins. Together, these data demonstrate that Myo10 is a molecular motor that functions in filopodia formation.

Although Myo10 (myosin-X) is an unconventional myosin associated with filopodia, little is known about its isoforms and roles in the nervous system. We report here that, in addition to full-length Myo10, brain expresses a shorter form of Myo10 that lacks a myosin head domain. This `headless' Myo10 is thus unable to function as a molecular motor, but is otherwise identical to full-length Myo10 and, like it, contains three pleckstrin homology (PH) domains, a myosin-tail homology 4 (MyTH4) domain, and a band-4.1/ezrin/radixin/moesin (FERM) domain. Immunoblotting demonstrates that both full-length and headless Myo10 exhibit dramatic developmental regulation in mouse brain. Immunofluorescence with an antibody that detects both isoforms demonstrates that Myo10 is expressed in neurons, such as Purkinje cells, as well as non-neuronal cells, such as astrocytes and ependymal cells. CAD cells, a neuronal cell line, express both full-length and headless Myo10, and this endogenous Myo10 is present in cell bodies, neurites, growth cones and the tips of filopodia. To investigate the dynamics of the two forms of Myo10 in neurons, CAD cells were transfected with GFP constructs corresponding to full-length or headless Myo10. Only full-length Myo10 localizes to filopodial tips and undergoes intrafilopodial motility, demonstrating that the motor domain is necessary for these activities. Live cell imaging also reveals that full-length Myo10 localizes to the tips of neuronal filopodia as they explore and interact with their surroundings, suggesting that this myosin has a role in neuronal actin dynamics.

Bcl-2 family members bearing only the BH3 domain are essential inducers of apoptosis. We identified a BH3-only protein, Bmf, and show that its BH3 domain is required both for binding to prosurvival Bcl-2 proteins and for triggering apoptosis. In healthy cells, Bmf is sequestered to myosin V motors by association with dynein light chain 2. Certain damage signals, such as loss of cell attachment (anoikis), unleash Bmf, allowing it to translocate and bind prosurvival Bcl-2 proteins. Thus, at least two mammalian BH3-only proteins, Bmf and Bim, function to sense intracellular damage by their localization to distinct cytoskeletal structures.

The past decade has seen a remarkable explosion in our knowledge of the size and diversity of the myosin superfamily. Since these actin-based motors are candidates to provide the molecular basis for many cellular movements, it is essential that motility researchers be aware of the complete set of myosins in a given organism. The availability of cDNA and/or draft genomic sequences from humans, Drosophila melanogaster, Caenorhabditis elegans, Arabidopsis thaliana, Saccharomyces cerevisiae, Schizosaccharomyces pombe, and Dictyostelium discoideum has allowed us to tentatively define and compare the sets of myosin genes in these organisms. This analysis has also led to the identification of several putative myosin genes that may be of general interest. In humans, for example, we find a total of 40 known or predicted myosin genes including two new myosins-I, three new class II (conventional) myosins, a second member of the class III/ninaC myosins, a gene similar to the class XV deafness myosin, and a novel myosin sharing at most 33% identity with other members of the superfamily. These myosins are in addition to the recently discovered class XVI myosin with N-terminal ankyrin repeats and two human genes with similarity to the class XVIII PDZ-myosin from mouse. We briefly describe these newly recognized myosins and extend our previous phylogenetic analysis of the myosin superfamily to include a comparison of the complete or nearly complete inventories of myosin genes from several experimentally important organisms.

Myosin diversity in the human epithelial cell line Caco-2BBe, the porcine epithelial cell line LLC-PK1 (CL-4), human peripheral blood leukocytes, and human liver was analyzed. PCR amplification yielded 8-11 putative myosins (depending on the cDNA source) representing six distinct myosin classes. Analysis of clones obtained by hybridization screening demonstrated that the original PCR products correspond to bona fide myosins, based on the presence of sequences highly conserved in other myosins. RNase protection analysis confirmed mRNA expression of 11 myosins in Caco-2BBe cells. Immunoblot analysis showed that at least 6 myosin immunogens are expressed in Caco-2BBe cells. The results reveal the existence of at least 11 unconventional human myosin genes, most of which are expressed in an overlapping fashion in different cell types. The abundance of myosins suggests that the myosin I vs. myosin II paradigm is inadequate to explain actin-based cellular motility.

We suggest that the vertebrate myosin-I field adopt a common nomenclature system based on the names adopted by the Human Genome Organization (HUGO). At present, the myosin-I nomenclature is very confusing; not only are several systems in use, but several different genes have been given the same name. Despite their faults, we believe that the names adopted by the HUGO nomenclature group for genome annotation are the best compromise, and we recommend universal adoption.

Fodrin, a protein related to erythrocyte spectrin, redistributes within the cell in certain situations. We compare such movements of fodrin and several fodrin binding proteins during the processes of axonal transport in neurons, and capping of surface proteins in lymphocytes. In neurons, three different populations of newly synthesized fodrin appear to be transported down the axons at different velocities corresponding to those of groups of transported proteins designated II, IV, and V. Actin, which can interact with fodrin, is transported at the velocity of group IV. Synapsin, a component of synaptic vesicles, is also reported to bind to fodrin. One population of synapsin is transported more rapidly than fodrin, at the velocity of group I; two additional populations of transported synapsin may overlap fodrin in groups II and IV. We consider possible functional associations of these different populations of fodrin and fodrin binding proteins. We note that the transport of group IV proteins resembles in certain respects the process of capping in lymphocytes, suggesting the possibility of a common mechanism. We outline one of several possible mechanisms.