Cloning and characterization of Myosin VI homologs

The mouse represents an excellent model system for the study of genetic deafness in humans. Many mouse deafness mutants have been identified and the anatomy of the mouse and human ear is similar. A positional cloning approach has been used to identify the gene encoded by the mouse recessive deafness mutation, Snell's waltzer (sv). sv encodes an unconventional myosin heavy chain, myosin VI, which is expressed within the sensory hair cells of the inner ear, and appears to be required for maintaining their structural integrity. The requirement for myosin VI in hearing makes this gene an excellent candidate for a human deafness disorder (Avraham, 1995).

Several forms of sensory deficit have been associated with unconventional myosin defects in humans and other animals. Normal hearing in mammals has been shown to require functional myosin VI and myosin VIIA, and the combined blindness and deafness of Usher syndrome type IB has been shown to be produced by specific defects in myosin VIIA. The cloning and characterization is reported of two distinct myosin VI isoforms (FMVIA and FMVIB) initially identified in a degenerate PCR screen of retinal cDNA from the striped bass, Morone saxatilis. Open reading frames for FMVIA and FMVIB encode predicted proteins of 1304 and 1270 amino acids respectively, that are 83% identical at the amino acid level. Both fish isoforms are likewise approximately 83%-86% identical to mammalian class VI myosins. Northern blot analysis has revealed that FMVIA mRNA is broadly expressed and most abundant in kidney, a pattern similar to that previously reported for mammalian myosin VI. FMVIB expression is dramatically more abundant in retina than in any other tissue examined. Antibodies directed against pig myosin VI detect a doublet at approximately 150 kDa in bass retina and RPE. Since both fish VIA and VIB isoforms share high sequence identity with pig myosin VI within the domain used for antibody production, it seems likely that this antibody crossreacts with both FMVIA and FMVIB. Immunocytochemistry with this same affinity-purified rabbit anti-myosin VI antibody shows that myosin VI isoforms are primarily localized in photoreceptors, horizontal cells and Muller cells in both fish and primate retinas. This report is the first demonstration that two myosin VI genes are expressed in the same organism and the same cell type (RPE). The relatively high abundance of FMVIB expression in retina suggests that it may play an important role in retinal motility events (Breckler, 2000).

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 reveal 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, a similar number of myosin V and myosin VI puncta were observed. 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, no such changes are detected 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 (Suter, 2000).

Myosin VI protein interactions

Myosin VI contains an inserted sequence that is unique among myosin superfamily members and has been suggested to be a determinant of the reverse directionality and unusual motility of the motor. It is thought that each head of a two-headed myosin VI molecule binds one calmodulin (CaM) by means of a single 'IQ motif'. Using truncations of the myosin VI protein and electrospray ionization(ESI)-MS, it has been demonstrated that in fact each myosin VI head binds two CaMs. One CaM binds to a conventional IQ motif either with or without calcium and likely plays a regulatory role when calcium binds to its N-terminal lobe. The second CaM binds to a unique insertion between the converter region and IQ motif. This unusual CaM-binding site normally binds CaM with four Ca2+ and can bind only if the C-terminal lobe of CaM is occupied by calcium. Regions of the MD outside of the insert peptide contribute to the Ca(2+)-CaM binding; truncations that eliminate elements of the MD alter CaM binding and allow calcium dissociation. It is suggested that the Ca(2+)-CaM bound to the unique insert represents a structural CaM, and not a calcium sensor or regulatory component of the motor. This structure is likely an integral part of the myosin VI 'converter' region and repositions the myosin VI 'lever arm' to allow reverse direction (minus-end) motility on actin (Bahloul, 2004).

Myosin VI must dimerize and deploy its unusual lever arm in order to perform its cellular roles

It is unclear whether the reverse-direction myosin (myosin VI) functions as a monomer or dimer in cells and how it generates large movements on actin. This study deleted a stable, single-alpha-helix (SAH) domain that has been proposed to function as part of a lever arm to amplify movements without impact on in vitro movement or in vivo functions. A myosin VI construct that used this SAH domain as part of its lever arm was able to take large steps in vitro but did not rescue in vivo functions. It was necessary for myosin VI to internally dimerize, triggering unfolding of a three-helix bundle and calmodulin binding in order to step normally in vitro and rescue endocytosis and Golgi morphology in myosin VI-null fibroblasts. A model for myosin VI emerges in which cargo binding triggers dimerization and unfolds the three-helix bundle to create a lever arm essential for in vivo functions (Mukherjea, 2014).

Myosin VI expression and function in the inner ear

To understand how cells differentially use the dozens of myosin isozymes present in each genome, the distributions were examined of four unconventional myosin isozymes in the inner ear, a tissue that is particularly reliant on actin-rich structures and unconventional myosin isozymes. Of the four isozymes, each from a different class, three are expressed in the hair cells of amphibia and mammals. In stereocilia, constructed of cross-linked F-actin filaments, myosin-Ibeta is found mostly near stereociliary tips; myosin-VI is largely absent, and myosin-VIIa colocalizes with crosslinks that connect adjacent stereocilia. In the cuticular plate, a meshwork of actin filaments, myosin-Ibeta is excluded, myosin-VI is concentrated, and modest amounts of myosin-VIIa are present. These three myosin isozymes are excluded from other actin-rich domains, including the circumferential actin belt and the cortical actin network. A member of a fourth class, myosin-V, is not expressed in hair cells but is present at high levels in afferent nerve cells that innervate hair cells. Substantial amounts of myosins-Ibeta, -VI, and -VIIa are located in a pericuticular necklace that is largely free of F-actin, squeezed between (but not associated with) actin of the cuticular plate and the circumferential belt. These localization results suggest specific functions for three hair-cell myosin isozymes. Myosin-Ibeta probably plays a role in adaptation; concentration of myosin-VI in cuticular plates and association with stereociliary rootlets suggest that this isozyme participates in rigidly anchoring stereocilia, and finally, colocalization with cross-links between adjacent stereocilia indicates that myosin-VIIa is required for the structural integrity of hair bundles (Hasson, 1997).

The mouse mutant Snell's waltzer (sv) has an intragenic deletion of the Myo6 gene, which encodes the unconventional myosin molecule myosin VI. Snell's waltzer mutants exhibit behavioral abnormalities suggestive of an inner ear defect, including lack of responsiveness to sound, hyperactivity, head tossing, and circling. The effects of a lack of myosin VI on the development of the sensory hair cells of the cochlea has been investigated in these mutants. In normal mice, the hair cells sprout microvilli on their upper surface, and some of these grow to form a crescent or V-shaped array of modified microvilli, the stereocilia. In the mutants, early stages of stereocilia development appear to proceed normally because at birth many stereocilia bundles have a normal appearance, but in places there are signs of disorganization of the bundles. Over the next few days, the stereocilia become progressively more disorganized and fuse together. Practically all hair cells show fused stereocilia by 3 days after birth, and there is extensive stereocilia fusion by 7 days. By 20 days, giant stereocilia are observed on top of the hair cells. At 1 and 3 days after birth, hair cells of mutants and controls take up the membrane dye FM1-43, suggesting that endocytosis occurs in mutant hair cells. One possible model for the fusion is that myosin VI may be involved in anchoring the apical hair cell membrane to the underlying actin-rich cuticular plate, and in the absence of normal myosin VI this apical membrane will tend to pull up between stereocilia, leading to fusion (Self, 1999).

Involvement of Myosin VI in membrane trafficking

A cDNA encoding a chicken intestinal brush border myosin VI has been cloned and characterized. Polyclonal antisera were raised to bacterially expressed fragments of this myosin VI. Affinity purified antibodies were used to study the localization of the protein by immunofluorescence and immunoelectron microscopy. It was found that in NRK and A431 cells, myosin VI was associated with both the Golgi complex and the leading, ruffling edge of the cell as well as being present in a cytosolic pool. In A431 cells in which cell surface ruffling was stimulated by EGF, myosin VI was phosphorylated and recruited into the newly formed ruffles along with ezrin and myosin V. In vitro experiments suggested that a p21-activated kinase (PAK) might be the kinase responsible for phosphorylation in the motor domain. These results strongly support a role for myosin VI in membrane traffic on secretory and endocytic pathways (Buss, 1998).

Myosin VI is involved in membrane traffic and dynamics and is the only myosin known to move towards the minus end of actin filaments. Splice variants of myosin VI with a large insert in the tail domain are specifically expressed in polarized cells containing microvilli. In these polarized cells, endogenous myosin VI containing the large insert is concentrated at the apical domain co-localizing with clathrin-coated pits/vesicles. Using full-length myosin VI and deletion mutants tagged with green fluorescent protein (GFP) it has been shown that myosin VI associates and co-localizes with clathrin-coated pits/vesicles by its C-terminal tail. Myosin VI, precipitated from whole cytosol, is present in a protein complex containing adaptor protein (AP)-2 and clathrin, and enriched in purified clathrin-coated vesicles. Over-expression of the tail domain of myosin VI containing the large insert in fibroblasts reduces transferrin uptake in transiently and stably transfected cells by >50%. Myosin VI is the first motor protein to be identified associated with clathrin-coated pits/vesicles and shown to modulate clathrin-mediated endocytosis (Buss, 2001).

Golgi morphology and function are dependent on an intact microtubule and actin cytoskeleton. Myosin VI, an unusual actin-based motor protein moving towards the minus ends of actin filaments, has been localized to the Golgi complex at the light and electron microscopic level. Myosin VI is present in purified Golgi membranes as a peripheral membrane protein, targeted by its globular tail domain. To investigate the function of myosin VI at the Golgi complex, immortal fibroblastic cell lines of Snell's waltzer mice lacking myosin VI were established. In these cell lines, where myosin VI is absent, the Golgi complex is reduced in size by approximately 40% compared with wild-type cells. Furthermore, protein secretion of a reporter protein from Snell's waltzer cells is also reduced by 40% compared with wild-type cells. Rescue experiments show that fully functional myosin VI is able to restore Golgi complex morphology and protein secretion in Snell's waltzer cells to the same level as that observed in wild-type cells (Warner, 2003).

Kinetic mechanism and regulation of myosin VI

Myosins and kinesins are molecular motors that hydrolyse ATP to track along actin filaments and microtubules, respectively. Although the kinesin family includes motors that move towards either the plus or minus ends of microtubules, all characterized myosin motors move towards the barbed (+) end of actin filaments. Crystal structures of myosin II have shown that small movements within the myosin motor core are transmitted through the 'converter domain' to a 'lever arm' consisting of a light-chain-binding helix and associated light chains. The lever arm further amplifies the motions of the converter domain into large directed movements. Myosin VI, an unconventional myosin, moves towards the pointed (-) end of actin. The myosin VI construct bound to actin was visualized using cryo-electron microscopy and image analysis; an ADP-mediated conformational change in the domain distal to the motor, a structure likely to be the effective lever arm, was found to be in the opposite direction to that observed for other myosins. Thus, it appears that myosin VI achieves reverse-direction movement by rotating its lever arm in the opposite direction to conventional myosin lever arm movement (Wells, 1999).

Myosin VI is a molecular motor involved in intracellular vesicle and organelle transport. To carry out its cellular functions myosin VI moves toward the pointed end of actin, backward in relation to all other characterized myosins. Myosin V, a motor that moves toward the barbed end of actin, is processive, undergoing multiple catalytic cycles and mechanical advances before it releases from actin. Myosin VI is also shown to be processive as revealed by using single molecule motility and optical trapping experiments. Remarkably, myosin VI takes much larger steps than expected, based on a simple lever-arm mechanism, for a myosin with only one light chain in the lever-arm domain. Unlike other characterized myosins, myosin VI stepping is highly irregular with a broad distribution of step sizes (Rock, 2001).

Myosins constitute a superfamily of at least 18 known classes of molecular motors that move along actin filaments. Myosins move towards the plus end of F-actin filaments; however, class VI myosin moves towards the opposite end of F-actin, that is, in the minus direction. Since there is a large, unique insertion in the myosin VI head domain between the motor domain and the light-chain-binding domain (the lever arm), it was thought that this insertion alters the angle of the lever-arm switch movement, thereby changing the direction of motility. The direction of motility has been determined for chimaeric myosins that comprise the motor domain and the lever-arm domain (containing an insert) from myosins that have movement in the opposite direction. The results show that the motor core domain, but neither the large insert nor the converter domain, determines the direction of myosin motility (Homma, 2001).

Myosin VI is the only pointed end-directed myosin identified and is likely regulated by heavy chain phosphorylation (HCP) at the actin-binding site in vivo. A detailed kinetic analysis of the actomyosin VI ATPase cycle was undertaken to determine whether there are unique adaptations to support reverse directionality and to determine the molecular basis of regulation by HCP. ADP release is the rate-limiting step in the cycle. ATP binds slowly and with low affinity. At physiological nucleotide concentrations, myosin VI is strongly bound to actin and populates the nucleotide-free (rigor) and ADP-bound states. Therefore, myosin VI is a high duty ratio motor adapted for maintaining tension and has potential to be processive. A mutant mimicking HCP increases the rate of P(i) release, which lowers the K(ATPase) but does not affect ADP release. These measurements are the first to directly measure the steps regulated by HCP for any myosin. Measurements with double-headed myosin VI demonstrate that the heads are not independent, and the native dimer hydrolyzes multiple ATPs per diffusional encounter with an actin filament. An alternating site model is proposed for the stepping and processivity of two-headed high duty ratio myosins (De La Cruz, 2001).

Myosin VI is expressed in a variety of cell types and is thought to play a role in membrane trafficking and endocytosis, yet its motor function and regulation are not understood. The present study clarified mammalian myosin VI motor function and regulation at a molecular level. Myosin VI ATPase activity is highly activated by actin with K(actin) of 9 microm. A predominant amount of myosin VI binds to actin in the presence of ATP unlike conventional myosins. K(ATP) is much higher than those of other known myosins, suggesting that myosin VI has a weak affinity or slow binding for ATP. In contrast, ADP markedly inhibits the actin-activated ATPase activity, suggesting a high affinity for ADP. These results suggest that myosin VI is predominantly in a strong actin binding state during the ATPase cycle. p21-activated kinase 3 phosphorylates myosin VI, and the site was identified as threonine 406. The phosphorylation of myosin VI significantly facilitates the actin-translocating activity of myosin VI. In contrast, Ca(2+) diminishes the actin-translocating activity of myosin VI although the actin-activated ATPase activity is not affected by Ca(2+). Calmodulin does not dissociate from the heavy chain at high Ca(2+), suggesting that a conformational change of calmodulin upon Ca(2+) binding, but not its physical dissociation, determines the inhibition of the motility activity. The present results reveal the dual regulation of myosin VI by phosphorylation and Ca(2+) binding to calmodulin light chain (Yoshimura, 2001).

Among a superfamily of myosin, class VI myosin moves actin filaments backwards. Myosin VI moves processively on actin filaments backwards with large (approximately 36 nm) steps, nevertheless it has an extremely short neck domain. Myosin V also moves processively with large (approximately 36 nm) steps and it is believed that myosin V strides along the actin helical repeat with its elongated neck domain that is critical for its processive movement with large steps. Myosin VI having a short neck cannot follow this scenario. It has been found by electron microscopy that myosin VI cooperatively binds to an actin filament at approximately 36 nm intervals in the presence of ATP, raising a hypothesis that the binding of myosin VI evokes 'hot spots' on actin filaments that attract myosin heads. Myosin VI may step on these 'hot spots' on actin filaments in every helical pitch, thus producing processive movement with 36 nm steps (Nishikawa, 2002).

Myosin VI is thought to function as both a transporter and an anchor. While in vitro studies suggest possible mechanisms for processive stepping, a biochemical basis for anchoring has not been demonstrated. Using optical trapping, myosin VI has been observed stepping against applied forces. Step size is not strongly affected by such loads. At saturating ATP, myosin VI kinetics show little dependence on load until, at forces near stall, stepping slows dramatically as load increases. At subsaturating ATP or in the presence of ADP, stepping kinetics is significantly inhibited by load. From these results, a mechanism of myosin VI stepping is proposed that predicts a regulation through load of the motor's roles as transporter and anchor (Altman, 2004).

Myosin VI is hypothesized to perform both roles as a transporter and an anchor. As a transporter, the motor is implicated in carrying proteins to the leading edge of a migrating cell (Buss, 2002) and moving endocytic vesicles into a cell. As an anchor, it may link actin-regulatory proteins to an actin complex during Drosophila spermatogenesis and moor stereocilia to the hair cell of inner ear sensory epithelia. The motor may also behave as a dynamic tension sensor, translocating along actin to establish tension in a system and then anchoring to the filament to maintain this tension. To behave as an anchor, the motor must remain bound to an actin filament through its catalytic domains, anchoring to actin whatever is associated to its tail. The mechanisms by which myosin VI functions as a processive motor and an anchor are not understood, nor how the motor regulates which function is exhibited. These questions are addressed using single molecule assays that probe effects of applied external forces, or loads, on motor stepping (Altman, 2004).

Studies of motor proteins have been aided by tools such as optical traps and glass micro-needles , which allow for observation of individual motors. Whereas information about intermediate states is averaged out in bulk protein studies, single molecule assays allow for observation of microscopic rates within a motor's kinetic cycle. An especially useful approach has been to apply a load to an active motor. Applied forces selectively perturb mechanical transitions, and so observed effects on activity single out these steps in the chemomechanical cycle (Altman, 2004).

The stepping mechanism for processive myosins has been most thoroughly studied with myosin V. A popular scheme to explain the stepping of this motor is the lever arm model, which predicts that small changes in the catalytic head of the motor are amplified to large, directed displacements through a relatively rigid lever arm. For myosin V, each catalytic domain is followed by six light chain binding motifs that serve as the motor's lever arm, enabling it to span its 36 nm step (Altman, 2004 and references therein).

Myosin VI, also a processive motor, is the only characterized myosin to move predominantly toward the pointed end of actin. This directionality is hypothesized to arise from a rotation of the motor's putative lever arm. Following the myosin VI catalytic head is a 50 amino acid insert recently shown to bind calmodulin (Bahloul, 2004), and following this insert is an IQ domain that also binds a calmodulin light chain. Together, these two regions may form a lever arm consisting of two bound light chains that, according to the lever arm model, could account for a step size of 10 nm. In agreement with this, laser trap analysis has been used to measure the stroke size of a single-headed myosin VI construct to be 12 nm (Altman, 2004 and references therein).

Recent studies, however, show that myosin VI takes a step of 30 nm, too large to be explained simply by a lever arm mechanism. Myosin VI stepping also differs from myosin V in the variability of its step size. The standard deviation of a myosin VI step distribution is twice that of myosin V, suggesting that it may have a larger diffusive character to its stepping relative to myosin V. Myosin VI also undergoes a considerable number of backward steps, behavior not frequently observed with myosin V (Altman, 2004 and references therein).

Though a stroke involving its short lever arm may influence the predominant direction of myosin VI stepping, an alternative stepping model is required to explain its large diffusive steps. One proposed model is that an interaction between the motor and actin causes a conformational change in the actin track. This change allows the motor to slide along the track in a manner not envisaged by conventional models of stepping. Another model is that myosin VI undergoes multiple small steps for each ATP hydrolysis. This suggestion would be unique from the usual relationship observed for myosins in which a single step is tightly coupled with a single ATP hydrolysis (Altman, 2004 and references therein).

If it is assumed the actin is relatively unaffected and the motor exhibits a one-to-one ratio between stepping and ATP hydrolysis, a conformational change in myosin VI must occur that allows it to span 30 nm. A mechanism in which some part of the motor extends and becomes flexible is a plausible explanation for the observed behavior. An extended, flexible element would allow the motor to span a large step and diffusively reach numerous actin monomers surrounding its usual binding site, leading to a wide distribution in step size and occasional backward stepping. A motor whose stepping mechanism involves a large diffusive search may also be more likely to bind different actin filaments to each head. Thus, this unique stepping mechanism may dictate specific in vivo functions of myosin VI (Altman, 2004).

If the mechanical transition for myosin VI involves an extended, flexible element, load effects on kinetics should differentiate its stepping scheme from that of myosin V, which undergoes its mechanical transition with relatively rigid arms. This study describes effects of applied load on myosin VI and proposes a mechanism for its stepping (Altman, 2004).

Studying load-affected kinetics can also yield information about the mechanism of anchoring by myosin VI. A load applied against a motor's motion forces the motor to do additional work to undergo mechanical transitions. This results in a slowing of the rates describing these transitions and thus can also slow overall stepping. A processive motor experiencing a load could thus function as an anchor if, under sufficient backward force, its stepping kinetics slows to a halt while it remains bound to actin. Effects of backward forces on the stepping of myosin VI have been explored to test whether this is the biochemical basis for its anchoring (Altman, 2004).

According to a current model for the myosin VI stepping cycle, the trailing head of the myosin VI dimer is strongly bound to actin in an ADP state, releases its ADP to allow ATP to bind, and releases from actin upon ATP association. The head then traverses 30 nm and rebinds to actin after hydrolyzing ATP and releasing phosphate, positioning itself as the new lead head. The new trailing head then repeats the cycle identically (Altman, 2004).

This stepping cycle is represented in a model showing the nucleotide and actin bound states of the motor. According to this model, there are three likely ways in which load could affect the nucleotide-dependent kinetics and cause the motor to anchor to an actin filament: load could slow release of ADP from the trailing myosin VI head, slow association of ATP to this head, or increase the rate of ADP binding to this head and thus compete with binding of ATP. The effects of load on these rates has been characterized; a reduction was observed in the ATP on rate and an increase in the ADP on rate as load increases. At physiological conditions, however, the latter effect is most relevant to motor function. Thus, a load-induced increase in ADP association to the trailing head is the likely mechanism for switching myosin VI function from that of a translocator to an anchor (Altman, 2004).

Myosin VI is a reverse direction actin-based motor capable of taking large steps (30-36 nm) when dimerized. However, all dimeric myosin VI molecules so far examined have included non-native coiled-coil sequences, and reports on full-length myosin VI have failed to demonstrate the existence of dimers. This study demonstrates that full-length myosin VI is capable of forming stable, processive dimers when monomers are clustered; these dimers move up to 1-2 mum in approximately 30 nm, hand-over-hand steps. Furthermore, data is presented consistent with the monomers being prevented from dimerizing unless they are held in close proximity and that dimerization is somewhat inhibited by the cargo binding tail. A model thus emerges that cargo binding likely clusters and initiates dimerization of full-length myosin VI molecules. Although this mechanism has not been previously described for members of the myosin superfamily, it is somewhat analogous to the proposed mechanism of dimerization for the kinesin Unc104 (Park, 2006).

Protein interactions of Myosin VI

Myosin VI is a molecular motor that moves processively along actin filaments and is believed to play a role in cargo movement in cells. DOC-2/DAB2, a signaling molecule inhibiting the Ras cascade, binds to myosin VI at the globular tail domain. DOC-2/DAB2 binds stoichiometrically to myosin VI with one molecule per one myosin VI heavy chain. The C-terminal 122 amino acid residues of DOC-2/DAB2, containing the Grb2 binding site has been found to be critical for the binding to myosin VI. Actin gliding assay revealed that the binding of DOC-2/DAB2 to myosin VI can support the actin filament gliding by myosin VI, suggesting that it can function as a myosin VI anchoring molecule. The C-terminal domain but not the N-terminal domain of DOC-2/DAB2 functions as a myosin VI anchoring site. The present findings suggest that myosin VI plays a role in transporting DOC-2/DAB2, a Ras cascade signaling molecule, thus involved in Ras signaling pathways (Inoue, 2002).

Myosin VI, an actin-based motor protein, and Disabled 2 (Dab2), a molecule involved in endocytosis and cell signalling, have been found to bind together using yeast and mammalian two-hybrid screens. In polarized epithelial cells, myosin VI is known to be associated with apical clathrin-coated vesicles and is believed to move them towards the minus end of actin filaments, away from the plasma membrane and into the cell. Dab2 belongs to a group of signal transduction proteins that bind in vitro to the FXNPXY sequence found in the cytosolic tails of members of the low-density lipoprotein receptor family. The central region of Dab2, containing two DPF motifs, binds to the clathrin adaptor protein AP-2, whereas a C-terminal region contains the binding site for myosin VI. This site is conserved in Dab1, the neuronal counterpart of Dab2. The interaction between Dab2 and myosin VI was confirmed by in vitro binding assays and coimmunoprecipitation and by their colocalization in clathrin-coated pits/vesicles concentrated at the apical domain of polarized cells. These results suggest that the myosin VI-Dab2 interaction may be one link between the actin cytoskeleton and receptors undergoing endocytosis (Morris, 2002).

Myosin VI undergoes cargo-mediated dimerization

Myosin VI is the only known molecular motor that moves toward the minus ends of actin filaments; thus, it plays unique roles in diverse cellular processes. The processive walking of myosin VI on actin filaments requires dimerization of the motor, but the protein can also function as a nonprocessive monomer. The molecular mechanism governing the monomer-dimer conversion is not clear. This study reports the high-resolution NMR structure of the cargo-free myosin VI cargo-binding domain (CBD) and shows that it is a stable monomer in solution. The myosin VI CBD binds to a fragment of the clathrin-coated vesicle adaptor Dab2 with a high affinity, and the X-ray structure of the myosin VI CBD in complex with Dab2 reveals that the motor undergoes a cargo-binding-mediated dimerization. The cargo-binding-induced dimerization may represent a general paradigm for the regulation of processivity for myosin VI as well as other myosins, including myosin VII and myosin X (Yu, 2009).

Myosin VI and spermatogenesis

Asymmetric division of cells and unequal allocation of cell contents is essential for correct development. This process of active segregation is poorly understood but in many instances has been shown to depend on the cytoskeleton. Motor proteins moving along actin filaments and microtubules are logical candidates to provide the motive force for asymmetric sorting of cell contents. The role of myosins in such processes has been suggested, but few examples of their involvement are known. Analysis of a Caenorhabditis elegans class VI myosin deletion mutant reveals a role for this motor protein in the segregation of cell components during spermatogenesis. Mutant spermatocytes cannot efficiently deliver mitochondria and endoplasmic reticulum/Golgi-derived fibrous-body membranous organelle complexes to budding spermatids, and fail to remove actin filaments and microtubules from the spermatids. The segregation defects are not due to a global sorting failure since nuclear inheritance is unaffected. It is concluded that C. elegans myosin VI has an important role in the unequal partitioning of both organelles and cytoskeletal components, a novel role for this class of motor proteins (Kelleher, 2000).

jaguar: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | References

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