Identification and phylogenetic analysis of Drosophila melanogaster myosins

Myosins constitute a superfamily of motor proteins that convert energy from ATP hydrolysis into mechanical movement along the actin filaments. Phylogenetic analysis currently places myosins into 17 classes based on class-specific features of their conserved motor domain. Traditionally, the myosins have been divided into two classes depending on whether they form monomers or dimers. The conventional myosin of muscle and nonmuscle cells forms class II myosins. They are complex molecules of four light chains bound to two heavy chains that form bipolar filaments via interactions between their coiled-coil tails (type II). Class I myosins are smaller monomeric myosins referred to as unconventional myosins. Now, at least 15 other classes of unconventional myosins are known. How many myosins are needed to ensure the proper development and function of eukaryotic organisms? Thus far, three types of myosins were found in budding yeast, six in the nematode Caenorhabditis elegans, and at least 12 in human. Here, we report on the identification and classification of Drosophila melanogaster myosins. Analysis of the Drosophila genome sequence identified 13 myosin genes. Phylogenetic analysis based on the sequence comparison of the myosin motor domains, as well as the presence of the class-specific domains, suggests that Drosophila myosins can be divided into nine major classes. Myosins belonging to previously described classes I, II, III, V, VI, and VII are present. Molecular and phylogenetic analysis indicates that the fruitfly genome contains at least five new myosins. Three of them fall into previously described myosin classes I, VII, and XV. Another myosin is a homolog of the mouse and human PDZ-containing myosins, forming the recently defined class XVIII myosins. PDZ domains are named after the postsynaptic density, disc-large, ZO-1 proteins in which they were first described. The fifth myosin shows a unique domain composition and a low homology to any of the existing classes. We propose that this is classified when similar myosins are identified in other species.     

The functional, comparative and evolutionary anatomy of myosins

Actins and myosins are generally present in all animal, plant and fungal cells, and in some, if not all, prokaryotes as well. It is proposed that, in general, myosins can carry specific loads as they move along actin filaments, thus mediating a form of active transport. Myosins exert their mechanical forces by a lever action (moklokinesis) of a part of the molecule: the S-1 "head" in the case of muscle myosins. Various portions of myosin molecules can be assigned specialized functions. Each such part can be designated by an appropriate functional name. Examples are: the enzyme portion (zymomere), the motor portion (dynamere), the lever portion (moklomere), a connecting portion (desmomere), a coupling or binding portion (haptomere), and one or more flexible portions (kamptomeres). These parts can be recognized in highly evolved and specialized muscle myosins and can be postulated in simpler, single headed myosins. A primitive myosin, represented principally by a moklomere equipped with a zymomere and a dynamere, is envisioned as an evolutionary ancestor of all myosins. This primitive myosin resembles the S-1 head of muscle myosin. I suggest that from such a primitive myosin, more elaborate single-headed myosins have evolved, equipped with specific haptomeres coupled to the moklomere by desmomeres and kamptomeres. From such general single-headed myosins have arisen the highly specialized two-headed myosins represented in muscle. It is suggested that the two-headed feature is favored in myosins capable of forming bipolar filaments.     

Myosin surface loop 4 modulates inhibition of actomyosin 1b ATPase activity by tropomyosin

Structural studies of the class I myosin, MyoE, led to the predictions that loop 4, a surface loop near the actin-binding region that is longer in class I myosins than in other myosin subclasses, might limit binding of myosins I to actin when actin-binding proteins, like tropomyosin, are present, and might account for the exclusion of myosin I from stress fibers. To test these hypotheses, mutant molecules of the related mammalian class I myosin, Myo1b, in which loop 4 was truncated (from an amino acid sequence of RMNGLDES to NGLD) or replaced with the shorter and distinct loop 4 found in Dictyostelium myosin II (GAGEGA), were expressed in vitro and their interaction with actin and with actin-tropomyosin was tested. Saturating amounts of expressed fibroblast tropomyosin-2 resulted in a decrease in the maximum actin-activated Mg2+-ATPase activity of wild-type Myo1b but had little or no effect on the actin-activated Mg2+-ATPase activity of the two mutants. In motility assays, few actin filaments bound tightly to Myo1b-WT-coated cover slips when tropomyosin-2 was present, whereas actin filaments both bound and were translocated by Myo1b-NGLD or Myo1b-GAGEGA in both the presence and absence of tropomyosin-2. When expressed in mammalian cells, like the wild type, the mutant myosins were largely excluded from tropomyosin-containing actin filaments, indicating that in the cell additional factors besides loop 4 determine targeting of myosins I to specific subpopulations of actin filaments.     

Myosin functions in Xenopus retinal ganglion cell growth cone motility in vivo

The role of myosins in Xenopus retinal ganglion cell growth cone motility in the optic tract was studied using two pharmacologic inhibitors with different specificities. 2,3-Butanedione monoxime (BDM) disrupts myosin—actin interactions of all myosins, and ML-7 specifically inhibits activation of myosin II. Both inhibitors caused growth cones to assume a collapsed morphology and decreased growth cone speed. Similar effects were observed in vitro. Interestingly, the effects of the two inhibitors, while similar, were clearly distinguishable, raising the possibility that different myosins may have different functional roles in growth cone motility. BDM caused growth cones to withdraw lamellipodia and some filopodia and eventually to freeze, whereas ML-7 caused total collapse and retraction. Concentrations of BDM and ML-7 that had no effect when applied independently stopped growth cones when applied simultaneously, suggesting that these inhibitors act synergistically on myosin function, thus providing evidence of specificity. These results imply that normal growth cone motility in the molecularly and spatially complex environment of the living brain requires myosin function. © 1997 John Wiley & Sons, Inc. J Neurobiol 32: 567–578, 1997     

Polarized actin bundles formed by human fascin-1: their sliding and disassembly on myosin II and myosin V in vitro

Fascin-1 is a putative bundling factor of actin filaments in the filopodia of neuronal growth cones. Here, we examined the structure of the actin bundle formed by human fascin-1 (actin/fascin bundle), and its mode of interaction with myosin in vitro. The distance between cross-linked filaments in the actin/bundle was 8-9 nm, and the bundle showed the transverse periodicity of 36 nm perpendicular to the bundle axis, which was confirmed by electron microscopy. Decoration of the actin/fascin bundle with heavy meromyosin revealed that the arrowheads of filaments in the bundle pointed in the same direction, indicating that the bundle has polarity. This result suggested that fascin-1 plays an essential role in polarity of actin bundles in filopodia. In the in vitro motility assay, actin/fascin bundles slid as fast as single actin filaments on myosin II and myosin V. When myosin was attached to the surface at high density, the actin/fascin bundle disassembled to single filaments at the pointed end of the bundle during sliding. These results suggest that myosins may drive filopodial actin bundles backward by interacting with actin filaments on the surface, and may induce disassembly of the bundle at the basal region of filopodia.     

Novel myosins

The traditional view of myosin, drawn from studies of myosins from striated muscles, is that of an elongated two-headed molecule that assembles into filaments. However, biochemical, molecular genetic and genetic studies have uncovered a host of ubiquitous single-headed nonfilamentous myosins known collectively as myosins I. All of the myosins I possess the myosin head domain, the motor portion of muscle myosins they have tail the filament-forming tail domain of muscle myosins they have tail domains that interact variously with membranes, actin and calmodulin. These alternative molecular interactions confer novel motile properties on myosins I, such as the ability to move membranes relative to actin and to move actin relative to actin without having to assemble into filaments. The numerous actin-based movements retained by cells lacking myosin II, the two-headed filamentous form of nonmuscle myosin, may be supported by myosins I.     

Myosin-X provides a motor-based link between integrins and the cytoskeleton

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.     

The organization of myosin and actin in rapid frozen nerve growth cones

Rapid freezing and freeze substitution were used in conjunction with immunofluorescence, whole mount EM, and immunoelectron microscopy to study the organization of myosin and actin in growth cones of cultured rat superior cervical ganglion neurons. The general cytoplasmic organization was determined by whole mount EM; tight microfilament bundles formed the core of filopodia while a dense meshwork formed the underlying structure of lamellipodia. Although the central microtubule and organelle-rich region of the growth cone had fewer microfilaments, dense foci and bundles of microfilaments were usually observed. Anti-actin immunofluorescence and rhodamine phalloidin staining of f-actin both showed intense staining of filopodia and lamellipodia. In addition, staining of bundles and foci were observed in central regions suggesting that the majority of the microfilaments seen by whole mount EM are actin filaments. Anti-myosin immunofluorescence was brightest in the central region and usually had a punctate pattern. Although less intense, anti-myosin staining was also seen in peripheral regions; it was most prominent at the border with the central region, in portions of lamellipodia undergoing ruffling, and in spots along the shaft and at the base of filopodia. Immunoelectron microscopy of myosin using postembedment labeling with colloidal gold showed a similar distribution to that seen by immunofluorescence. Label was scattered throughout the growth cone, but present as distinct aggregates in the peripheral region mainly along the border with the central region. Less frequently, aggregates were also seen centrally and along the shaft and at the base of filopodia. This distribution is consistent with myosins involvement in the production of tension and movements of growth cone filopodia and lamellipodia that occur during active neurite elongation.     

The Myosin IXb motor activity targets the myosin IXb RhoGAP domain as cargo to sites of actin polymerization

Myosin IXb (Myo9b) is a single-headed processive myosin that exhibits Rho GTPase-activating protein (RhoGAP) activity in its tail region. Using live cell imaging, we determined that Myo9b is recruited to extending lamellipodia, ruffles, and filopodia, the regions of active actin polymerization. A functional motor domain was both necessary and sufficient for targeting Myo9b to these regions. The head domains of class IX myosins comprise a large insertion in loop2. Deletion of the large Myo9b head loop 2 insertion abrogated the enrichment in extending lamellipodia and ruffles, but enhanced significantly the enrichment at the tips of filopodia and retraction fibers. The enrichment in the tips of filopodia and retraction fibers depended on four lysine residues C-terminal to the loop 2 insertion and the tail region. Fluorescence recovery after photobleaching and photoactivation experiments in lamellipodia revealed that the dynamics of Myo9b was comparable to that of actin. The exchange rates depended on the Myo9b motor region and motor activity, and they were also dependent on the turnover of F-actin. These results demonstrate that Myo9b functions as a motorized RhoGAP molecule in regions of actin polymerization and identify Myo9b head sequences important for in vivo motor properties.     

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.     

Are class III and class IX myosins motorized signalling molecules?

Myosins exist that are fused to domains that harbour signalling activities. Class III myosins (NINAC) are protein kinases that play important roles in phototransduction. Class IX myosins inactivate the small G-protein Rho that acts as molecular switch. Because these myosins interact via their myosin head domain with actin filaments, they link signal transduction to the actin cytoskeleton. The exact motor properties of these myosins, however, remain to be determined.     

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.     

Polymerization of vertebrate non-muscle and smooth muscle myosins

We investigated how light chain phosphorylation controls the stability of filaments of vertebrate non-muscle myosins (from bovine thymocytes and chicken intestine epithelial brush border cells) and smooth muscle myosin (from chicken gizzard) in vitro. Using a sedimentation assay, the solubilities of the myosins were determined by measuring the amounts of myosin monomers (Cm) and filaments (Cp) present under a given set of conditions as a function of the total myosin concentration (Ct). Below 200 mM-NaCl, each myosin displayed distinct "critical monomer concentrations" (Cc) for polymerization, which were dependent on the salt concentration, the state of light chain phosphorylation and the presence of MgATP. At 150 mM-NaCl, MgATP increased the Cc of non-phosphorylated brush border myosin approximately five to tenfold, thymus myosin approximately 10 to 15-fold, and gizzard myosin approximately 25 to 50-fold. When these myosins were phosphorylated, MgATP had little effect on their solubilities, and their Cc values remained low. Analytical ultracentrifugation and electron microscopy demonstrated that the myosins were present in three different conformational states under the conditions used in the sedimentation assays, i.e. filaments, extended monomer (6 S) and folded monomer (10 S). Since at equilibrium only filaments and monomers were observed, we suggest that the polymerization pathway for these myosins can be analysed in terms of a dynamic monomer-polymer equilibrium (polymer in equilibrium 6 S monomer in equilibrium 10 S monomer). At roughly physiological ionic strength, light chain dephosphorylation (in the presence of MgATP) promotes the folded state (10 S), whereas phosphorylation promotes the extended state (6 S), and thereby favours filament assembly. The relevance of the monomer-polymer equilibrium to the state of organization of the myosin in vivo is discussed.     

The calcium binding properties of phosphorylated and unphosphorylated cardiac and skeletal myosins.

Porcine left ventricular cardiac myosin and rabbit white skeletal myosin were phosphorylated by rabbit skeletal myosin light chain kinase and their Ca2+ binding properties were examined by equilibrium dialysis techniques. No significant effect of phosphorylation on the Ca2+ binding properties of these myosins was observed. Both types of striated muscle myosins bound approximately 2 mol of Ca2+/mol of myosin with similar affinities of 3 x 10(7) M-1. In the presence of 3 x 10(-4) M Mg2+ the myosins bound Ca2+ with a reduced affinity of 3 to 4 x 10(5) M-1. Assuming competition between Mg2+ and Ca2+ for the binding sites on myosin, the changes in Ca2+ binding can be accounted for by a Mg2+ affinity of 2.5 to 3.0 x 10(5) M-1.     

Myosin IIIB uses an actin-binding motif in its espin-1 cargo to reach the tips of actin protrusions

Myosin IIIA (MYO3A) targets actin protrusion tips using a motility mechanism dependent on both motor and tail actin-binding activity [1]. We show that myosin IIIB (MYO3B) lacks tail actin-binding activity and is unable to target COS7 cell filopodia tips, yet is somehow able to target stereocilia tips. Strikingly, when MYO3B is coexpressed with espin-1 (ESPN1), a MYO3A cargo protein endogenously expressed in stereocilia [2], MYO3B targets and carries ESPN1 to COS7 filopodia tips. We show that this tip localization is lost when we remove the ESPN1 C terminus actin-binding site. We also demonstrate that, like MYO3A [2], MYO3B can elongate filopodia by transporting ESPN1 to the polymerizing end of actin filaments. The mutual dependence of MYO3B and ESPN1 for tip localization reveals a novel mechanism for the cell to regulate myosin tip localization via a reciprocal relationship with cargo that directly participates in actin binding for motility. Our results are consistent with a novel form of motility for class III myosins that requires both motor and tail domain actin-binding activity and show that the actin-binding tail can be replaced by actin-binding cargo. This study also provides a framework to better understand the late-onset hearing loss phenotype in patients with MYO3A mutations.     

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.     

Two class XI myosins function in organelle trafficking and root hair development in Arabidopsis

Multigene families encoding class XI myosins are conserved in higher plants, however, little information is available on specific functions of these ubiquitous molecular motors. We isolated gene knockout mutants for all 13 class XI myosins present in Arabidopsis (Arabidopsis thaliana) genome. Inactivation of 11 myosin genes resulted in no discernible phenotypes under the normal growth conditions. In contrast, the knockouts of the remaining two myosin genes, XI-2 (formerly MYA2) and XI-K, exhibited similar defects in root hair elongation suggesting that the myosin-driven motility plays a significant role in a polar tip growth. Strikingly, inactivation of each of these myosins also reduced trafficking of Golgi stacks, peroxisomes, and mitochondria in root hairs and in leaf epidermal cells. These results indicate that myosins XI-K and XI-2 play major and overlapping roles in the cell dynamics in Arabidopsis and highlight the redundant nature of myosin function in plants.     

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.     

Regulation of myosin 5a and myosin 7a

The myosin superfamily is diverse in its structure, kinetic mechanisms and cellular function. The enzymatic activities of most myosins are regulated by some means such as Ca2+ ion binding, phosphorylation or binding of other proteins. In the present review, we discuss the structural basis for the regulation of mammalian myosin 5a and Drosophila myosin 7a. We show that, although both myosins have a folded inactive state in which domains in the myosin tail interact with the motor domain, the details of the regulation of these two myosins differ greatly.     

Order-disorder structural transitions in synthetic filaments of fast and slow skeletal muscle myosins under relaxing and activating conditions

In the previous study (Podlubnaya et al., 1999, J. Struc. Biol. 127, 1-15) Ca2+-induced reversible structural transitions in synthetic filaments of pure fast skeletal and cardiac muscle myosins were observed under rigor conditions (-Ca2+/+Ca2+). In the present work these studies have been extended to new more order-producing conditions (presence of ATP in the absence of Ca2+) aimed at arresting the relaxed structure in synthetic filaments of both fast and slow skeletal muscle myosin. Filaments were formed from column-purified myosins (rabbit fast skeletal muscle and rabbit slow skeletal semimebranosusproprius muscle). In the presence of 0.1 mM free Ca2+, 3 mM Mg2+ and 2 mM ATP (activating conditions) these filaments had a spread structure with a random arrangement of myosin heads and subfragments 2 protruding from the filament backbone. Such a structure is indistinguishable from the filament structures observed previously for fast skeletal, cardiac (see reference cited above) and smooth (Podlubnaya et al., 1999, J. Muscle Res. Cell Motil. 20, 547-554) muscle myosins in the presence of 0.1 mM free Ca2+. In the absence of Ca2+ and in the presence of ATP (relaxing conditions) the filaments of both studied myosins revealed a compact ordered structure. The fast skeletal muscle myosin filaments exhibited an axial periodicity of about 14.5 nm and which was much more pronounced than under rigor conditions in the absence of Ca2+ (see the first reference cited). The slow skeletal muscle myosin filaments differ slightly in their appearance from those of fast muscle as they exhibit mainly an axial repeat of about 43 nm while the 14.5 nm repeat is visible only in some regions. This may be a result of a slightly different structural properties of slow skeletal muscle myosin. We conclude that, like other filaments of vertebrate myosins, slow skeletal muscle myosin filaments also undergo the Ca2+-induced structural order-disorder transitions. It is very likely that all vertebrate muscle myosins possess such a property.