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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.

We demonstrate that individual carbon nanotubes (CNTs) can be visualized with fluorescence microscopy through noncovalent labeling with conventional fluorophores. Reversal of contrast in fluorescence imaging of the CNTs was observed when performing labeling procedure in a nonpolar solvent. Our results are consistent with a CNT-fluorophore affinity mediated by hydrophobic interaction. The reverse-contrast images also provide clear indication of nanotube location. © 2003 American Institute of Physics.

Phagocytosis is a phosphatidylinositol-3-OH-kinase (PI(3)K)-dependent process in macrophages. We identified Myo10 (Myosin-X), an unconventional myosin with pleckstrin homology (PH) domains, as a potential downstream target of PI(3)K. Myo10 was recruited to phagocytic cups in a wortmannin-sensitive manner. Expression of a truncation construct of Myo10 (Myo10 tail) in a macrophage cell line or cytosolic loading of anti-Myo10 antibodies in bovine alveolar macrophages inhibited phagocytosis. In contrast, expression of a Myo10 tail construct containing a point mutation in one of its PH domains failed to inhibit phagocytosis. Expression of Myo10 tail inhibited spreading, but not adhesion, on IgG-coated substrates, consistent with a function for Myo10 in pseudopod extension. We propose that Myo10 provides a molecular link between PI(3)K and pseudopod extension during phagocytosis.

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.

Interactions between microtubules and filamentous actin (F-actin) are crucial for many cellular processes, including cell locomotion and cytokinesis, but are poorly understood. To define the basic principles governing microtubule/F-actin interactions, we used dual-wavelength digital fluorescence and fluorescent speckle microscopy to analyze microtubules and F-actin labeled with spectrally distinct fluorophores in interphase Xenopus egg extracts. In the absence of microtubules, networks of F-actin bundles zippered together or exhibited serpentine gliding along the coverslip. When microtubules were nucleated from Xenopus sperm centrosomes, they were released and translocated away from the aster center. In the presence of microtubules, F-actin exhibited two distinct, microtubule-dependent motilities: rapid ( approximately 250-300 nm/s) jerking and slow ( approximately 50 nm/s), straight gliding. Microtubules remodeled the F-actin network, as F-actin jerking caused centrifugal clearing of F-actin from around aster centers. F-actin jerking occurred when F-actin bound to motile microtubules powered by cytoplasmic dynein. F-actin straight gliding occurred when F-actin bundles translocated along the microtubule lattice. These interactions required Xenopus cytosolic factors. Localization of myosin-II to F-actin suggested it may power F-actin zippering, while localization of myosin-V on microtubules suggested it could mediate interactions between microtubules and F-actin. We examine current models for cytokinesis and cell motility in light of these findings.

The molecular mechanisms underlying directed motility of growth cones have not been determined. The role of myosin-V, an unconventional myosin, in growth cone dynamics was examined by chromophore-assisted laser inactivation (CALI). CALI of purified chick brain myosin-V absorbed onto nitrocellulose-coated cover slips inhibited the ability of myosin-V to translocate actin filaments. CALI of myosin-V in growth cones of chick dorsal root ganglion neurons resulted in rapid filopodial retraction. The rate of filopodial extension was significantly decreased, whereas the rate of filopodial retraction was not affected, which suggests a specific role for myosin-V in filopodial extension.

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.

To facilitate functional studies of novel myosins, we have developed a strategy for characterizing the mechanochemical properties of motors isolated by immunoadsorption directly from small amounts of crude tissue extracts. In this initial study, silica beads coated with an antibody that specifically recognizes the tail of myosin-V were used to immunoadsorb this motor protein from brain extracts. The myosin-containing beads were then positioned with optical tweezers onto actin filaments nucleated from Limulus sperm acrosomal processes and observed for motility using high resolution video DIC microscopy. The addition of brush border spectrin to the motility chamber enabled the growth of stable actin filament tracks that were approximately 4-fold longer than filaments grown in the absence of this actin crosslinking protein. The velocity of myosin-V immunoadsorbed from brain extracts was similar to that observed for purified myosin-V that was antibody-linked to beads or assessed using the sliding actin filament assay. Motile beads containing myosin-V immunoadsorbed from brain extracts bound poorly to nucleated actin filaments and were incapable of linear migrations following the addition of a different antibody that specifically recognizes the motor-containing head domain of myosin-V. Myosin-V motility was most robust in the absence of Ca2+. Interestingly, skeletal muscle tropomyosin and brush border spectrin had no detectable effect on myosin-V mechanochemistry. Myosin-V containing beads were also occasionally observed migrating directly on acrosomal processes in the absence of exogenously added actin.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

Two types of in vitro motility assays are currently used for examining the mechanochemical properties of purified myosins. The Nitella bead movement assay (Sheetz and Spudich: Nature 303:31–35, 1983) allows determination of both velocity and directionality of movement, but is of limited utility because of the fragile nature of the dissected Nitella internodal cells. On the other hand, the sliding actin filament assay (Kron and Spudich: Proc. Natl. Acad. Sci. U.S.A. 83:6272–6276, 1986) is technically much simpler to perform than the Nitella assay, and is suitable for the study of numerous physiological parameters. As it is currently used, however, the sliding actin filament assay does not indicate the directionality of motor movement. Previous studies have demonstrated that the velocities of filament-forming conventional myosins-II from either muscle or nonmuscle cells are comparable in both motility assays (Umemoto and Sellers: J. Biol. Chem. 265:14864–14869, 1990). However, similar studies using unconventional myosins are lacking. In the present report we have compared the rates of two structurally distinct unconventional myosins: brush border (BB) myosin-I and chick brain (CB) myosin-V (p190-calmodulin), using the sliding actin filament and Nitella-based in vitro motility assays. These two unconventional myosins differ from conventional myosins in that they appear unable to associate in to bipolar filaments, and have extended rod-like neck domains which bind multiple calmodulin light chains in a Ca2+-sensitive manner. Unlike conventional myosins, both of these unconventional myosins exhibit motility rates using the Nitella assay (BB myosin-I:∼10 nm/s; CB myosin-V:∼30 nm/s) that are 5-to 10-fold slower than that determined for these myosins using the sliding filament assay (BB myosin-I: 40–67 nm/s; CB myosin-V: 260–340 nm/s). © 1993 Wiley-Liss, Inc.

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.

Most organelle transport ceases when cells begin mitosis. But how is this accomplished? As Cheney and Rodriguez explain in their Perspective, cell cycle-dependent phosphorylation of myosin-V--a motor protein that moves organelles along actin filaments--results in release of myosin-V from the organelle and cessation of actin-based organelle transport ( Karcher et al .).

Endothelial cell migration is an important step during angiogenesis, and its dysregulation contributes to aberrant neovascularization. The bone morphogenetic proteins (BMPs) are potent stimulators of cell migration and angiogenesis. Using microarray analyses, we find that myosin-X (Myo10) is a BMP target gene. In endothelial cells, BMP6-induced Myo10 localizes in filopodia, and BMP-dependent filopodial assembly decreases when Myo10 expression is reduced. Likewise, cellular alignment and directional migration induced by BMP6 are Myo10 dependent. Surprisingly, we find that Myo10 and BMP6 receptor ALK6 colocalize in a BMP6-dependent fashion. ALK6 translocates into filopodia after BMP6 stimulation, and both ALK6 and Myo10 possess intrafilopodial motility. Additionally, Myo10 is required for BMP6-dependent Smad activation, indicating that in addition to its function in filopodial assembly, Myo10 also participates in a requisite amplification loop for BMP signaling. Our data indicate that Myo10 is required to guide endothelial migration toward BMP6 gradients via the regulation of filopodial function and amplification of BMP signals.

Publisher Summary The only vertebrate myosin I that has been thoroughly characterized is termed brush border (BB) myosin I and is expressed within the microvilli of the intestinal epithelial cell. This chapter discusses the structural and functional properties of brush border myosin I— the subunit composition, the protein domain structure, and the primary structure of its heavy chain— with compliment of multiple calmodulin (CM) light chains. The chapter also discusses the acid binding properties— that is, stoichiometry and Ca 2+ dependence and actin filament cross-linking by brush border Myosin I. It describes the adenosine triphosphate (ATP)ase properties of the brush border myosin I, its mechanochemistry, the function of brush border myosin I calmodulin light chains, the role of calmodulin light chains as repressors rather than activators of brush border myosin I Mg 2+ -ATPase. The chapter discusses the interaction of brush border myosin I, with the microvillar membrane, with acidic phospholipids, the evidence for a microvillar membrane “Docking” protein for brush border myosin I and notions regarding brush border myosin I function. A ∼140-kDa glycoprotein (GP-140) binds to the heavy chain of BB myosin I. GP-140 is the linker protein that tethers BB myosin I to the microvillus (MV) membrane. The disk-bound BB myosin I molecules are tethered to the membrane by their tails. The bound BB myosin I retain all the activities, associated with the free molecule, including actin binding and mechanochemical activity. BB myosin I might participate in the biogenesis and recycling of the apical membrane, by transporting vesicles, up through the terminal web. BB myosin I could cause a relative downward or perhaps rotational movement of the core within the MV membrane and could promote bulk movement of the membrane upward along the MV axis, as a part of an active vesicular shedding mechanism.

Fodrin, a spectrin-like protein, is localized in gametes, zygotes, and embryos from sea urchins and mice. Mammalian fodrin comprises two polypeptides with molecular weights of approximately 240 kDa (alpha) and 235 kDa (beta). An antibody specific for mammalian α-fodrin cross-reacted with a 240-kDa polypeptide from sea urchin egg extracts. This indicates that sea urchins contain a protein of similar electrophoretic mobility and immunological properties to mammalian α-fodrin. When this antibody was used to stain the sea urchin gametes with indirect immunofluorescence, fodrinspecific fluorescence was localized to the acrosome of the sperm and was distributed over the entire egg near the surface in a punctate pattern similar to the distribution of polymeric actin. During sperm incorporation, the fodrin-specific fluorescence is found at the site of sperm incorporation, in the fertilization cone. After fertilization, the intensity of fodrin fluorescence increases. During mitosis and cytokinesis in sea urchins, the entire surface of the egg remains stained; the cleavage furrow also was stained but no more intensely than was the rest of the egg surface. Antibody labeling with colloidal gold followed by electron microscopy showed that fodrin was loated in the cytoplasm immediately beneath the plasma membrane. In unfertilized mouse oocytes, both actin and fodrin were stained most intensely beneath the membrane adjacent to the meiotic spindle. After insemination, the cell surfaces of the pronucleate egg and the second polar body were stained; however, the actin matrix surrounding the apposed pronuclei did not bind the fodrin antibody. During cytokinesis in the mouse, the cleavage furrow stained more intensely than did the rest of the egg cortex, and in embryos the cell borders were delineated. These results indicate that organisms as unrelated to mammals as sea urchins have fodrin-like proteins; the rearrangements of such proteins suggest that they participate in the actin-mediated events at the cell surface during fertilization and early development in both mice and sea urchins.

Fodrin is an actin/calmodulin-binding protein with similarities to spectrin (erythrocytes) and TW 260/240 (brush border). It is concentrated beneath the plasma membranes of neurons and other cells. We have observed translocations of fodrin in both neurons and lymphocytes. Newly synthesized, radiolabeled fodrin moves down axons at a maximum velocity (about 50 mm/day) that is slower than the most rapidly axonally transported proteins (group I). A portion of fodrin appears to move more slowly at velocities (1–10 mm/day) resembling those of actin and myosin (group IV) and tubulin and neurofilament proteins (group V). In lymphocytes, when certain surface antigens are induced by cross-linking agents to migrate to one pole of the cell and form a cap, fodrin redistributes beneath the membrane and forms a subcap. The movements of fodrin in lympohocyte capping and in the axonal transport of group IV polypeptides have certain similarities. In both cases, the redistribution of fodrin is accompanied by concomitant redistributions of actin, myosin, and calmodulin, and both processes proceed at similar velocities. We consider the possibilities that these two processes are related, both being driven by a submembrane force-generating system comprising in part actin, myosin, and fodrin, and that fodrin serves to link various organelles or proteins to this system.

We have determined that a protein of the fodrin-spectrin-TW260/240 (FST) family is a component of the thin fibrils (approximately 5 nm wide, 100-200 nm long) that cross-link bundles of actin filaments to adjacent actin bundles and to the plasma membrane in the terminal web of the brush border of the intestinal epithelium. When isolated brush borders were incubated with anti-fodrin antibodies and prepared for electron microscopy by the quick-freeze, deep-etch technique, these approximately 5 nm fibrils were specifically decorated with the antibody. In addition, these cross-linking fibrils disappeared when the anti-fodrin-reactive proteins were extracted from the brush border. We conclude that FST is a component of a cross-linking system composed of approximately 5 nm fibrils that are morphologically distinct from the approximately 8 nm myosin-containing fibrils which were identified by anti-myosin decoration. In addition to linking actin bundles to adjacent actin bundles and to the plasma membrane, these FST fibrils may mediate actin-vesicle, actin-intermediate filament and vesicle-plasma membrane linkages.

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.