Mutations in the myosin VIIA gene cause non-syndromic recessive deafness.

Genetic hearing impairment affects around 1 in every 2,000 births. The bulk (approximately 70%) of genetic deafness is non-syndromic, in which hearing impairment is not associated with any other abnormalities. Over 25 loci involved in non-syndromic deafness have been mapped and mutations in connexin 26 have been identified as a cause of non-sydromic deafness. One locus for non-syndromic recessive deafness, DFNB2 (ref. 4), has been localized to the same chromosomal region, 11q14, as one of the loci, USH1B, underlying the recessive deaf-blind syndrome. Usher syndrome type 1b, which is characterized by profound congenital sensorineural deafness, constant vestibular dysfunction and prepubertal onset of retinitis pigmentosa. Recently, it has been shown that a gene encoding an unconventional myosin, myosin VIIA, underlies the mouse recessive deafness mutation, shaker-1 (ref. 5) as well as Usher syndrome type 1b. Mice with shaker-1 demonstrate typical neuroepithelial defects manifested by hearing loss and vestibular dysfunction but no retinal pathology. Differences in retinal patterns of expression may account for the variance in phenotype between shaker-1 mice and Usher type 1 syndrome. Nevertheless, the expression of MYO7A in the neuroepithelium suggests that it should be considered a candidate for non-syndromic deafness in the human population. By screening families with non-syndromic deafness from China, we have identified two families carrying MYO7A mutations.

Structural changes accompanying phosphorylation of tarantula muscle myosin filaments.

Electron microscopy has been used to study the structural changes that occur in the myosin filaments of tarantula striated muscle when they are phosphorylated. Myosin filaments in muscle homogenates maintained in relaxing conditions (ATP, EGTA) are found to have nonphosphorylated regulatory light chains as shown by urea/glycerol gel electrophoresis and [32P]phosphate autoradiography. Negative staining reveals an ordered, helical arrangement of crossbridges in these filaments, in which the heads from axially neighboring myosin molecules appear to interact with each other. When the free Ca2+ concentration in a homogenate is raised to 10(-4) M, or when a Ca2+-insensitive myosin light chain kinase is added at low Ca2+ (10(-8) M), the regulatory light chains of myosin become rapidly phosphorylated. Phosphorylation is accompanied by potentiation of the actin activation of the myosin Mg-ATPase activity and by loss of order of the helical crossbridge arrangement characteristic of the relaxed filament. We suggest that in the relaxed state, when the regulatory light chains are not phosphorylated, the myosin heads are held down on the filament backbone by head-head interactions or by interactions of the heads with the filament backbone. Phosphorylation of the light chains may alter these interactions so that the crossbridges become more loosely associated with the filament backbone giving rise to the observed changes and facilitating crossbridge interaction with actin.

Role of the COOH-terminal nonhelical tailpiece in the assembly of a vertebrate nonmuscle myosin rod.

A short nonhelical sequence at the COOH-terminus of vertebrate nonmuscle myosin has been shown to enhance myosin filament assembly. We have analyzed the role of this sequence in chicken intestinal epithelial brush border myosin, using protein engineering/site-directed mutagenesis. Clones encoding the rod region of this myosin were isolated and sequenced. They were truncated at various restriction sites and expressed in Escherichia coli, yielding a series of mutant myosin rods with or without the COOH-terminal tailpiece and with serial deletions from their NH2-termini. Deletion of the 35 residue COOH-terminal nonhelical tailpiece was sufficient to increase the critical concentration for myosin rod assembly by 50-fold (at 150 mM NaCl, pH 7.5), whereas NH2-terminal deletions had only minor effects. The only exception was the longest NH2-terminal deletion, which reduced the rod to 119 amino acids and rendered it assembly incompetent. The COOH-terminal tailpiece could be reduced by 15 amino acids and it still efficiently promoted assembly. We also found that the tailpiece promoted assembly of both filaments and segments; assemblies which have different molecular overlaps. Rod fragments carrying the COOH-terminal tailpiece did not promote the assembly of COOH-terminally deleted material when the two were mixed together. The tailpiece sequence thus has profound effects on assembly, yet it is apparently unstructured and can be bisected without affecting its function. Taken together these observations suggest that the nonhelical tailpiece may act sterically to block an otherwise dominant but unproductive molecular interaction in the self assembly process and does not, as has been previously thought, bind to a specific target site(s) on a neighboring molecule.

Regulation of contraction and thick filament assembly-disassembly in glycerinated vertebrate smooth muscle cells.

Isolated smooth muscle cells and cell fragments prepared by glycerination and subsequent homogenization will contract to one-third their normal length, provided Ca++ and ATP are present. Ca++-independent contraction was obtained by preincubation in Ca++ and ATP gamma S, or by addition of trypsin-treated myosin light chain kinase (MLCK) that no longer requires Ca++ for activation. In the absence of Ca++, myosin was rapidly lost from the cells upon addition of ATP. Glycerol-urea-PAGE gels showed that none of this myosin is phosphorylated. The extent of myosin loss was ATP- and pH-dependent and occurred under conditions similar to those previously reported for the in vitro disassembly of gizzard myosin filaments. Ca++-dependent contraction was restored to extracted cells by addition of gizzard myosin under rigor conditions (i.e., no ATP), followed by addition of MLCK, calmodulin, Ca++, and ATP. Function could also be restored by adding all these proteins in relaxing conditions (i.e., in EGTA and ATP) and then initiating contraction by Ca++ addition. Incubation with skeletal myosin will restore contraction, but this was not Ca++-dependent unless the cells were first incubated in troponin and tropomyosin. These results strengthen the idea that contraction in glycerinated cells and presumably also in intact cells is primarily thick filament regulated via MLCK, that the myosin filaments are unstable in relaxing conditions, and that the spatial information required for cell length change is present in the thin filament-intermediate filament organization.

Modulation of cellular morphology and locomotory activity by antibodies against myosin.

Three monoclonal antibodies directed against chicken brush border myosin were used to study the possible function of myosin in microfilament organization and locomotion of chicken fibroblasts. These antibodies bind to distinct and separate epitopes on the heavy chain of chicken nonmuscle myosin and display differential effects of myosin filament formation and actin-myosin interaction (Citi, S., and J. Kendrick-Jones. 1988. J. Musc. Res. Cell Motil. 9: 306-319). When injected into chicken fibroblasts, all antibodies induced breakdown of stress fibers. Concomitantly, a large proportion of the cells developed extensive lamellae which altered their morphology drastically. These cells showed also increased locomotory activity. All effects were concentration dependent and reversible. The most drastic alterations were observed with cells injected with antibody quantities exceeding the quantity of cellular myosin (molar ratios of antibody to myosin greater than 3:1). The finding that antibodies with different effects on myosin filament formation in vitro all induce similar intracellular processes suggests that it is the antibody-induced decrease in functional myosin that triggers an increase in plasma membrane dynamics and locomotory activity, rather than differences in myosin filament length or conformation.

Spatial pattern of myosin phosphorylation in contracting smooth muscle cells: evidence for contractile zones.

We have purified a polyclonal antibody by affinity chromatography which binds specifically to the phosphorylated form of the regulatory light chain (Mr = 20,000) of smooth muscle myosin. This antibody does not stain relaxed, permeabilized smooth muscle cells isolated from guinea pig taenia coli. However, when these cells were stimulated to contract with CaCl2 (100 microM) and ATP (1 mM), the immunofluorescence staining was localized in a series of transverse bands. This distribution of activated myosin appears to reflect an underlying structural organization of the smooth muscle cell cytoskeleton into mechanically coupled contractile zones.

Parallel modulation of brush border myosin conformation and enzyme activity induced by monoclonal antibodies.

Monoclonal antibodies binding to distinct epitopes on the tail of brush border myosin were used to modulate the conformation and state of assembly of this myosin. BM1 binds 1:3 of the distance from the tip of the tail to the head and prevents the extended-tail (6S) monomer from folding into the assembly-incompetent folded-tail (10S) state, whereas BM4 binds to the tip of the myosin tail, and induces the myosin to fold into the 10S state. Thus, at physiological ionic strength BM1 promotes and BM4 blocks the assembly of the myosin into filaments. Using BM1 and BM4 together, we were able to prevent both folding and filament assembly, thus locking myosin molecules in the extended-tail 6S monomer conformation at low ionic strength where they normally assemble into filaments. Using these myosin-antibody complexes, we were able to investigate independently the effects of folding of the myosin tail and assembly into filaments on the myosin MgATPase. The enzymatic activities were measured from the fluorescent profiles during the turnover of the ATP analogue formycin triphosphate (FTP). Extended-tail (6S) myosin molecules had an FTPase activity of 1-5 X 10(-3) s-1, either at high ionic strength as a monomer alone or when complexed with antibody, or at low ionic strength as filaments or when maintained as extended-tail monomers by the binding of BM1 and BM4. Folding of the molecules into the 10S state reduced this rate by an order of magnitude, effectively trapping the products of FTP hydrolysis in the active sites.

The localization of myosin VI at the golgi complex and leading edge of fibroblasts and its phosphorylation and recruitment into membrane ruffles of A431 cells after growth factor stimulation.

Myosin VI is an unconventional myosin that may play a role in vesicular membrane traffic through actin rich regions of the cytoplasm in eukaryotic cells. In this study we have cloned and sequenced a cDNA encoding a chicken intestinal brush border myosin VI. Polyclonal antisera were raised to bacterially expressed fragments of this myosin VI. The affinity purified antibodies were highly specific for myosin VI by immunoblotting and immunoprecipitation and 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.

Cingulin contains globular and coiled-coil domains and interacts with ZO-1, ZO-2, ZO-3, and myosin.

We characterized the sequence and protein interactions of cingulin, an M(r) 140-160-kD phosphoprotein localized on the cytoplasmic surface of epithelial tight junctions (TJ). The derived amino acid sequence of a full-length Xenopus laevis cingulin cDNA shows globular head (residues 1-439) and tail (1,326-1,368) domains and a central alpha-helical rod domain (440-1,325). Sequence analysis, electron microscopy, and pull-down assays indicate that the cingulin rod is responsible for the formation of coiled-coil parallel dimers, which can further aggregate through intermolecular interactions. Pull-down assays from epithelial, insect cell, and reticulocyte lysates show that an NH(2)-terminal fragment of cingulin (1-378) interacts in vitro with ZO-1 (K(d) approximately 5 nM), ZO-2, ZO-3, myosin, and AF-6, but not with symplekin, and a COOH-terminal fragment (377-1,368) interacts with myosin and ZO-3. ZO-1 and ZO-2 immunoprecipitates contain cingulin, suggesting in vivo interactions. Full-length cingulin, but not NH(2)-terminal and COOH-terminal fragments, colocalizes with endogenous cingulin in transfected MDCK cells, indicating that sequences within both head and rod domains are required for TJ localization. We propose that cingulin is a functionally important component of TJ, linking the submembrane plaque domain of TJ to the actomyosin cytoskeleton.

Myosin-I nomenclature.

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.

Paramyosin: molecular length and assembly.

Paratnyosin paracrystails formned with divalent cations have a 725-angstrom axial period and show simple negative staining patterns in the electron microscope. The structure of the aggregates is interpreted in terms of an array of polar molecules about 1275 angstroms long, with "gaps" and "overlaps" in the molecular assembly. Antiparallel relatiotns between molecules lead to the assembly of paracrystals with opposite polarity at either end. Implications of the in vitro structure for filaments containing paramyosin in muscle are discussed.

Dystrophin and related proteins.

During the past year significant progress has been made in understanding how dystrophin deficiency leads to muscle cell necrosis in Duchenne muscular dystrophy and Becker muscular dystrophy. Dystrophin interacts with a glycoprotein complex spanning the muscle sarcolemma, effectively linking the actin cytoskeleton to the extracellular matrix. The carboxyl terminus of dystrophin is required for glycoprotein binding. Interestingly, at least three mRNAs transcribed from the distal end of the DMD gene in tissues other than muscle have been shown to encode this domain. Deficiency of a second component of the dystrophin-associated glycoprotein complex has been shown to occur in another muscle-wasting disorder, severe childhood autosomal recessive muscular dystrophy. Sequence analysis of the entire cDNA for the autosomal dystrophin-related protein utrophin has shown that dystrophin and utrophin are closely related. Furthermore, both of these proteins have been shown to bind to the same or a similar glycoprotein complex in muscle.

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.

Crystal structure of the N-terminal domain of MukB: a protein involved in chromosome partitioning.

BACKGROUND: The 170 kDa protein MukB has been implicated in ATP-dependent chromosome partitioning during cell division in Escherichia coli. MukB shares its dimeric structure and domain architecture with the ubiquitous family of SMC (structural maintenance of chromosomes) proteins that facilitate similar functions. The N-terminal domain of MukB carries a putative Walker A nucleotide-binding region and the C-terminal domain has been shown to bind to DNA. Mutant phenotypes and a domain arrangement similar to motor proteins that move on microtubules led to the suggestion that MukB might be a motor protein acting on DNA. RESULTS: We have cloned, overexpressed and crystallized a 26 kDa protein consisting of 227 N-terminal residues of MukB from E. coli. The structure has been solved using multiple anomalous dispersion and has been refined to 2.2 A resolution. The N-terminal domain of MukB has a mixed alpha/beta fold with a central six-stranded antiparallel beta sheet. The putative nucleotide-binding loop, which is part of an unexpected helix-loop-helix motif, is exposed on the surface and no nucleotide-binding pocket could be detected. CONCLUSIONS: The N-terminal domain of MukB has no similarity to the kinesin family of motor proteins or to any other nucleotide-binding protein. Together with the finding of the exposed Walker A motif this observation supports a model in which the N- and C-terminal domains come together in the dimer of MukB to form the active site. Conserved residues on one side of the molecule delineate a region of the N-terminal domain that is likely to interact with the C-terminal domain.

Crystal structure of the actin-binding region of utrophin reveals a head-to-tail dimer.

BACKGROUND: Utrophin is a large multidomain protein that belongs to a superfamily of actin-binding proteins, which includes dystrophin, alpha-actinin, beta-spectrin, fimbrin, filamin and plectin. All the members of this family contain a common actin-binding region at their N termini and perform a wide variety of roles associated with the actin cytoskeleton. Utrophin is the autosomal homologue of dystrophin, the protein defective in the X-linked Duchenne and Becker muscular dystrophies, and upregulation of utrophin has been suggested as a potential therapy for muscular dystrophy patients. RESULTS: The structure of the actin-binding region of utrophin, consisting of two calponin-homology (CH) domains, has been solved at 3.0 A resolution. It is composed of an antiparallel dimer with each of the monomers being present in an extended dumbell shape and the two CH domains being separated by a long central helix. This extended conformation is in sharp contrast to the compact monomer structure of the N-terminal actin-binding region of fimbrin. CONCLUSIONS: The crystal structure of the actin-binding region of utrophin suggests that these actin-binding domains may be more flexible than was previously thought and that this flexibility may allow domain reorganisation and play a role in the actin-binding mechanism. Thus utrophin could possibly bind to actin in an extended conformation so that the sites previously identified as being important for actin binding may be directly involved in this interaction.

The structure of the N-terminal actin-binding domain of human dystrophin and how mutations in this domain may cause Duchenne or Becker muscular dystrophy.

BACKGROUND: Dystrophin is an essential component of skeletal muscle cells. Its N-terminal domain binds to F-actin and its C terminus binds to the dystrophin-associated glycoprotein (DAG) complex in the membrane. Dystrophin is therefore thought to serve as a link from the actin-based cytoskeleton of the muscle cell through the plasma membrane to the extracellular matrix. Pathogenic mutations in dystrophin result in Duchenne or Becker muscular dystrophy. RESULTS: The crystal structure of the dystrophin actin-binding domain (ABD) has been determined at 2.6 A resolution. The structure is an antiparallel dimer of two ABDs each comprising two calponin homology domains (CH1 and CH2) that are linked by a central alpha helix. The CH domains are both alpha-helical globular folds. Comparisons with the structures of utrophin and fimbrin ABDs reveal that the conformations of the individual CH domains are very similar to those of dystrophin but that the arrangement of the two CH domains within the ABD is altered. The dystrophin dimer reveals a change of 72 degrees in the orientation of one pair of CH1 and CH2 domains (from different monomers) relative to the other pair when compared with the utrophin dimer. The dystrophin monomer is more elongated than the fimbrin ABD. CONCLUSIONS: The dystrophin ABD structure reveals a previously uncharacterised arrangement of the CH domains within the ABD. This observation has implications for the mechanism of actin binding by dystrophin and related proteins. Examining the position of three pathogenic missense mutations within the structure suggests that they exert their effects through misfolding of the ABD, rather than through disruption of the binding to F-actin.

Chimaeric myosin regulatory light chains: sub-domain switching experiments to analyse the function of the N-terminal EF hand.

The regulatory light chains (RLCs) located on the myosin head, regulate the interaction of myosin with actin in response to either Ca2+ or phosphorylation signals. The RLCs belong to a family of calcium binding proteins and are composed of four "EF hand" ancestral calcium binding motifs (numbered I to IV). To determine the role of the first EF hand (EF hand I) in the regulatory process, chimaeric light chains were constructed by protein engineering, by switching this region between smooth muscle and skeletal muscle myosin RLCs. For example, chimaera G(I)S consisted of EF hand I of the smooth muscle (gizzard) RLC and EF hands II to IV of the skeletal muscle RLC, whereas chimaera S(I)G consisted of EF hand I of the skeletal muscle RLC and EF hands II to IV of the smooth muscle RLC. The chimaeric RLCs were expressed in Escherichia coli using the pLcII expression system, and after isolation and purification their regulatory properties were compared with those of wild-type smooth and skeletal muscle myosin RLCs. The chimaeric RLCs bound to the myosin heads in scallop striated muscle myofibrils from which the endogenous RLCs had been removed ("desensitized" myofibrils) with similar affinities to those of the wild-type smooth and skeletal muscle RLCs. Both chimaeric RLCs were able to regulate the actin-activated Mg(2+)-ATPase activity of scallop myosin: G(I)S inhibited the ATPase in the presence and absence of Ca2+, like the wild-type skeletal muscle RLC, while S(I)G inhibited the myosin ATPase in the absence of Ca2+, and this inhibition was relieved on Ca2+ addition, in the same way as the wild-type smooth muscle RLC. Thus the type of regulation that the RLCs confer on the myosin is determined by the source of EF hands II to IV rather than that of EF hand I.

Regulation in vitro of brush border myosin by light chain phosphorylation.

Myosin was purified from chicken brush border cells to greater than 95% homogeneity and in a predominantly non-phosphorylated state. The effects of light chain phosphorylation by a Ca2+-calmodulin-dependent myosin light chain kinase on the conformational, enzymatic and filament assembly properties of this myosin were investigated. The actin-activated MgATPase activity of the non-phosphorylated myosin was low, and upon light chain phosphorylation an eight- to ninefold increase in this activity was observed, which was further potentiated by tropomyosin. Light chain phosphorylation was shown to control the assembly and disassembly of brush border myosin filaments. For example, turbidity measurements and electron microscopy demonstrated that MgATP disassembled non-phosphorylated myosin filaments; the disassembled myosin could reassemble when the light chains were phosphorylated, and could be disassembled again by dephosphorylating the light chains with phosphatase. In the electron microscope, the disassembled non-phosphorylated myosin molecules appeared in a folded conformation, and they were extended when phosphorylated. Proteolytic digestion was used to probe further the conformation of these folded and extended molecules, and their subunit organizations were characterized by a gel overlay technique. Quantitative analysis further demonstrated that light chain phosphorylation alters dramatically the monomer/polymer equilibrium of brush border myosin, shifting it towards filament formation. Comparison of analogous data for myosin from gizzard and thymus shows that each myosin has distinct solubility properties.

A myosin-like protein from a higher plant.

As part of a study of the diversity of myosins, we have cloned a cDNA encoding a myosin-like protein from Arabidopsis thaliana. This is the first molecular motor of any kind to be cloned from a higher plant. The predicted polypeptide (molecular weight 131 kDa) has a motor domain (head) very similar to those of other myosins, but the remainder of the sequence is unusual. The tail contains four potential calmodulin binding sites ("IQ-motifs"), but no sequence motifs suggestive of actin or phospholipid binding, like those found in other myosins. There is also a small region of probable alpha-helical coiled-coil, which suggests that the molecule could be dimeric, though unlikely to form filaments. The N-terminal and C-terminal regions of the molecule are unique. We present a phylogenetic analysis of myosin head sequences, which suggests that this is a new type of myosin.

Effects of light chain phosphorylation and skeletal myosin on the stability of non-muscle myosin filaments.

The effect of light chain phosphorylation and the presence of skeletal muscle myosin on the stability of non-phosphorylated non-muscle myosin filaments was investigated. Purified skeletal, brush border and thymus myosins were assembled in vitro into hybrid filaments consisting of varying proportions of (1) non-muscle and skeletal myosins, or (2) phosphorylated and non-phosphorylated non-muscle myosins. The stability of these hetero- and homopolymers in the presence of MgATP was determined using sedimentation, gel electrophoresis and immunochemical techniques. In addition, the effect of a monoclonal antibody, binding to the tip of brush border myosin tail, on the assembly of the homo- and heteropolymers, was tested. Filamentous non-phosphorylated non-muscle myosin was disassembled by MgATP to the same extent whether in homo- or heteropolymers, indicating that skeletal myosin has no stabilising effect on the hybrid filaments. The presence of small amounts of phosphorylated non-muscle myosin was, however, found to prevent the complete disassembly by MgATP of non-phosphorylated non-muscle myosin filaments, indicating that light chain phosphorylation stabilizes co-operatively non-muscle myosin filaments. The monoclonal antibody prevented the assembly of brush border myosin into both homo- and heteropolymers, and its effect on the filaments was compared with that of MgATP.