John Kendrick-Jones

Optineurin links myosin VI to the Golgi complex and is involved in Golgi organization and exocytosis

Myosin VI plays a role in the maintenance of Golgi morphology and in exocytosis. In a yeast 2-hybrid screen we identified optineurin as a binding partner for myosin VI at the Golgi complex and confirmed this interaction in a range of protein interaction studies. Both proteins colocalize at the Golgi complex and in vesicles at the plasma membrane. When optineurin is depleted from cells using RNA interference, myosin VI is lost from the Golgi complex, the Golgi is fragmented and exocytosis of vesicular stomatitis virus G-protein to the plasma membrane is dramatically reduced. Two further binding partners for optineurin have been identified: huntingtin and Rab8. We show that myosin VI and Rab8 colocalize around the Golgi complex and in vesicles at the plasma membrane and overexpression of constitutively active Rab8-Q67L recruits myosin VI onto Rab8-positive structures. These results show that optineurin links myosin VI to the Golgi complex and plays a central role in Golgi ribbon formation and exocytosis.

Localized mutations in the gene encoding the cytoskeletal protein filamin A cause diverse malformations in humans

Remodeling of the cytoskeleton is central to the modulation of cell shape and migration. Filamin A, encoded by the gene FLNA, is a widely expressed protein that regulates re-organization of the actin cytoskeleton by interacting with integrins, transmembrane receptor complexes and second messengers. We identified localized mutations in FLNA that conserve the reading frame and lead to a broad range of congenital malformations, affecting craniofacial structures, skeleton, brain, viscera and urogenital tract, in four X-linked human disorders: otopalatodigital syndrome types 1 (OPD1; OMIM 311300) and 2 (OPD2; OMIM 304120), frontometaphyseal dysplasia (FMD; OMIM 305620) and Melnick–Needles syndrome (MNS; OMIM 309350). Several mutations are recurrent, and all are clustered into four regions of the gene: the actin-binding domain and rod domain repeats 3, 10 and 14/15. Our findings contrast with previous observations that loss of function of FLNA is embryonic lethal in males but manifests in females as a localized neuronal migration disorder, called periventricular nodular heterotopia (PVNH; refs. 3–6). The patterns of mutation, X-chromosome inactivation and phenotypic manifestations in the newly described mutations indicate that they have gain-of-function effects, implicating filamin A in signaling pathways that mediate organogenesis in multiple systems during embryonic development.

Regulation in molluscan muscles

In molluscan muscles the factors which regulate contraction by interacting with calcium are associated with myosin. Purified myosin preparations from the striated and smooth muscles of Aequipecten and from the smooth red adductor muscle of Mercenaria bind calcium with a great affinity and the ATPase activity of this myosin combined with purified actin requires calcium. The actin-containing thin filaments of these muscles do not bind calcium and although they activate the ATPase of rabbit myosin this activity is not calcium dependent. The thin filaments of molluscs, however, combine readily in vitro with the relaxing proteins of rabbit and behave, then, like rabbit preparations. The components responsible for calcium binding and for the calcium dependence of ATPase cannot readily be removed from molluscan myosin and are not obtained from molluscan muscles or actomyosins by procedures successfully applied to rabbit preparations. Tropomyosin does not appear to be necessary for the regulation of molluscan actomyosin by calcium. It is likely that in molluscs the calcium-binding component interacts directly with myosin to prevent cross-bridge formation. The possibility that in vertebrate muscles cross-bridge formation may also be controlled by a direct interaction of some of the regulatory proteins with-myosin is discussed.

Paramyosin and the filaments of molluscan "catch" muscles. II. Native filaments: isolation and characterization.

Abstract A method has been developed for the partial separation of the filaments of molluscan muscles. A fraction has been obtained which consisted mainly of actin filaments many of which were attached to dense bodies. When complexed with rabbit heavy meromyosin, the actin filaments showed arrowhead structures which appeared to have opposite polarity at either end of the dense bodies. The other fraction consisted of very long and thick filaments containing essentially all of the paramyosin and the myosin in the muscle. Some actin filaments were also present in this fraction. When myosin solubilization was prevented, myosin filaments similar to those of vertebrate striated muscle were rarely found in either of the two fractions. Myosin could be selectively extracted from the thick filaments without the solubilization of paramyosin; extraction of paramyosin, however, was always accompanied by the solubilization of myosin. The removal of myosin changed the surface appearance of the thick filaments: a characteristic pattern of darkly staining nodes or gap regions in the paramyosin filament became visible in negatively stained preparations. The nodes have a roughly triangular shape defining the polarity of the structure. A number of filaments were found which showed a reversal of polarity along their length. These results lead to the conclusion that paramyosin forms a bipolar core of the thick filament which is covered by a surface layer of myosin. The specific interactions between paramyosin and myosin could be demonstrated structurally and enzymically. Molluscan myosin formed filaments resembling those of rabbit myosin when precipitated in vitro . Its assembly in the molluscan thick filament is therefore determined by the underlying paramyosin core. Paramyosin greatly inhibited the actin activated ATPase of myosin in a selective manner when these proteins were mixed in about a mole to mole ratio. The long bipolar thick filaments with myosin on the surface can account for the tension development in these molluscan muscles according to the sliding filament theory. These results also suggest that paramyosin may have a specific regulatory role in tension maintenance: in the catch mechanism a phase change in paramyosin may be coupled to the movement of cross-bridges formed between myosin and actin.

The light chains of scallop myosin as regulatory subunits

Abstract In molluscan muscles contraction is regulated by the interaction of calcium with myosin. The calcium dependence of the aotin-activated ATPase activity of scallop myosin requires the presence of a specific light chain. This light chain is released from myosin by EDTA treatment (EDTA-light chains) and its removal desensitizes the myosin, i.e. abolishes the calcium requirement for the actin-activated ATPase activity, and reduces the amount of calcium the myosin binds; the isolated light chain, however, does not bind calcium and has no ATPase activity. Calcium regulation and calcium binding is restored when the EDTA-light chain is recombined with desensitized myosin preparations. Dissociation of the EDTA-light chain from myosin depends on the concentration of divalent cations; half dissociation is reached at about 10−5 M-magnesium or 10−7 M-calcium concentrations. The EDTA-light chain and the residual myosin are fairly stable and the components may be kept separated for a day or so before recombination. Additional light chains containing half cystine residues (SH-light chains) are detached from desensitized myosin by sodium dodecyl sulfate. The EDTA-light chains and the SH-light chains have a similar chain weight of about 18,000 daltons; however, they differ in several amino acid residues and the EDTA-light chains contain no half cystine. The SH-light chains and EDTA-light chains have different tryptic fingerprints. Both light chains can be prepared from washed myofibrils. Densitometry of dodecyl sulfate gel electrophoresis bands and Sephadex chromatography in sodium dodecyl sulfate indicate that there are three moles of light chains in a mole of purified myosin, but only two in myosin treated with EDTA. The ratio of the SH-light chains to EDTA-light chains was found to be two to one in experiments where the total light-chain complements of myosin or myofibril preparations were carboxymethylated. A similar ratio was obtained from the densitometry of urea-acrylamide gel electrophoresis bands. We conclude that a myosin molecule contains two moles of SH-light chain and one mole of EDTA-light chain, and that the removal of a single EDTA-light chain completely desensitizes scallop myosin. Heavy meromyosin and S-1 subfragment can be prepared from scallop myosin. Both of these preparations bind calcium and contain light chains in significant amounts. The heavy meromyosin of scallop is extensively degraded; the S-1 preparation, however, is remarkably intact. Significantly, heavy meromyosin has a calcium-dependent actin-activated ATPase while the S-1 does not require calcium and shows high ATPase activity in its absence. These results suggest that regulation involves a co-operativity between the two globular ends of the myosin. Desensitized scallop myosin and scallop S-1 preparations can be made calcium sensitive when mixed with rabbit actin containing the rabbit regulatory proteins. This result makes it unlikely that specific light chains of myosin are involved in the regulation of the vertebrate system. The fundamental similarity in the contractile regulation of molluscs and vertebrates is that interaction between actin and myosin in both systems requires a critical level of calcium. We propose that the difference in regulation of these systems is that the interaction between myosin and actin is prevented by blocking sites on actin in the case of vertebrate muscles, whereas in the case of molluscan muscles it is the sites on myosin which are blocked in the absence of calcium.

Myosin VI isoform localized to clathrin‐coated vesicles with a role in clathrin‐mediated endocytosis

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 were specifically expressed in polarized cells containing microvilli. In these polarized cells, endogenous myosin VI containing the large insert was 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) we have shown that myosin VI associates and co‐localizes with clathrin‐coated pits/vesicles by its C‐terminal tail. Myosin VI, precipitated from whole cytosol, was 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 reduced 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.

Regulatory light chains in myosins.

Abstract Scallop myosin molecules contain two moles of regulatory light chains and two moles of light chains with unknown function. Removal of one of the regulatory light chains by treatment with EDTA is accompanied by the complete loss of the calcium dependence of the actin-activated ATPase activity and by the loss of one of the two calcium binding sites on the intact molecule. Such desensitized preparations recombine with one mole of regulatory light chain and regain calcium regulation and calcium binding. The second regulatory light chain may be selectively obtained from EDTA-treated scallop muscles by treatment with the Ellman reagent (5,5′-dithiobis(2-nitrobenzoic acid)): treatment with this reagent, however, leads to an irreversible loss of ATPase activity. The light chains obtained by treatment with EDTA and then DTNB are identical in composition and function. A different light chain fraction obtained by subsequent treatment with guanidine-HCl does not bind to desensitized or intact myoflbrils and has no effect on ATPase activity. Regulatory light chains which bind to desensitized scallop myofibrils with high affinity and restore calcium control were found in a number of molluscan and vertebrate myosins, including Mercenaria, Spisula, squid, lobster tail, beef heart, chicken gizzard, frog and rabbit. Although these myosins all have a similar subunit structure and contain about two moles of regulatory light chain, only scallop myosin or myofibrils can be desensitized by treatment with EDTA. There appear to be two classes of regulatory light chains. The regulatory light chains of molluscs and of vertebrate smooth muscles restore full calcium binding and also resensitize purified scallop myosin. The regulatory light chains from vertebrate striated, cardiac, and the fast decapod muscles, on the other hand, have no effect on calcium binding and do not resensitize purified scallop myosin unless the myosin is complexed with actin. The latter class of light chains is found in muscles where in vitro functional tests failed to detect myosin-linked regulation.

Myosin VI Binds to and Localises with Dab2, Potentially Linking Receptor‐Mediated Endocytosis and the Actin Cytoskeleton

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 polarised 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 colocalisation in clathrin‐coated pits/vesicles concentrated at the apical domain of polarised cells. These results suggest that the myosin VI–Dab2 interaction may be one link between the actin cytoskeleton and receptors undergoing endocytosis.

Conservation within the myosin motor domain: implications for structure and function.

BACKGROUND Myosins are motors that use energy supplied by ATP to travel along actin filaments. The structure of myosin is known, but the actin-binding site is not well defined, and the mechanisms by which actin activates ATP hydrolysis by myosin, and myosin moves relative to the actin filament, developing force, are not fully understood. Previous phylogenetic analyses of the motor domain of myosins have identified up to twelve classes. We set out to analyse the positions of conserved residues within this domain in detail, and relate the conserved residues to the myosin structure. RESULTS Our analysis indicates that there are at least thirteen myosin classes. Conserved residues in the motor domain have been positioned within the framework provided by the recent crystal structures, thus helping to define those residues involved in actin and ATP binding, in hydrolysis and in conformational change. This has revealed remarkably poor overall conservation at the site thought to be involved in actin binding, but several highly conserved residues have been identified that may be functionally important. CONCLUSIONS Information from such a sequence analysis is a useful tool in the further interpretation of X-ray structures. It allows the position of crucial residues from other members of a superfamily to be determined within the framework provided by the known structures and the functional significance of conserved or mutated residues to be assessed.

Dynamic measurement of myosin light-chain-domain tilt and twist in muscle contraction

A new method is described for measuring motions of protein domains in their native environment on the physiological timescale. Pairs of cysteines are introduced into the domain at sites chosen from its static structure and are crosslinked by a bifunctional rhodamine. Domain orientation in a reconstituted macromolecular complex is determined by combining fluorescence polarization data from a small number of such labelled cysteine pairs. This approach bridges the gap between in vitro studies of protein structure and cellular studies of protein function and is used here to measure the tilt and twist of the myosin light-chain domain with respect to actin filaments in single muscle cells. The results reveal the structural basis for the lever-arm action of the light-chain domain of the myosin motor during force generation in muscle.