A multitude of myosins

MARK MOOSEKER

MYOSIN SUPERFAMILY

A multitude As more

and

more

myosins

of myosins are being

discovered

they

are seen to fall into distinct classes of ancient lineage, but their functions are only just beginning to be elucidated. This yrdr marks the twentieth anniversary [ 1,2] of the disCOWLS of the first ‘unconventional’ actin-based molecular motor, Acar2thavnorbn myosin-I - unconventional because this single-headed myosin lacks the filamentforming tail of the conventional two-headed (now known as class II) myosins of muscle and non-muscle cells. Ironically, Acc~ntbanno~~amy&n-I was once dismissed by skeptics as a probable proteolytic fragment of a conventional myosin. Two decades later, &nntlJnrno&a myosin-1 stands as the ‘founding’ member of a rapidly growing SW perfamily of StrUCtUEl~~)~ diverse, actin-based motors, manyof which made their debut within the past year, These include myosins from a broad range of organisms for example, flowering plants, yeast, fruit Ilies, chickens, sea urchins and a variety of mammals - expressed in an equally diverse set of tissues and cell types, as would be evident from perusing the abstracts of last year’s American Society for Cell Biology Meeting ( ;2101GUNRio1 3 (suppl) ). lInti recently, all myosins that lack the filament-forming tail of CklSS II myosins were considered, by default, to be class I myosins. Although all myosins share a structurally conserved head or motor domain, a detailed comparison of the sequences of the head domains of most known myosins has led to the surprising conclusion that they fall into seven, not just two, distinct classes [ 3-51 ( Fig. 1). The additional classes III-VII are numbered in order of their discovery. Each of these classes is evolutionarily ancient and thus likely to be expressed throughout phylogeny, ;I conclusion borne out by the divers@ of species in which members of these various classes have been found (with the exception of the ‘orphan’ classes III and IV. for both of which only one example has been characterized so far ). It seems likely that the family of myosins expressed within a given orgzznism will grow. not onlv by the addition of more members to each class but als&, by the addition of new classes. This is despite the fact that the number of known myosin genes, based on available published and unpilh~ished data for vertebrates, is already greater than twenty. Most importantly, as was first shown for amoeboid cells [2,6], the potentially awesome complexiv of myosin expression holds, not only for organisms as a Whole, but also for a given cell type within an orgdiiism (for example, see [7-111). Additional complexiry can arise from alternative pathwdys of processing the primaT RNA transcripts of a single myosin gene (for example, see [ 12-14 JJ. In multicellular organisms, some myosin genes may exhibit broad patterns of expression ( for example, See [3,7,8.1&16] ). whereas others may be expressed in @

Current

Biology

only a single cell type (for example, the nindC myosins of Drosophila photoreceptor cells [ 171 and brush border myosin-I of intestinal epithelial cells [ 21 1. A ‘consensus’ plan of the domain arrangement in the heavy chains of known myosins is shown in Figure 2. Mykns are operationally defined by the presence of an amino-terminal head domain ( the only exceptions being the ninaC myosins, which have a kinase domain at the amino terminus beyond the head domain [ 171 ), the sequence of which is moderately conserved among all known myosins. The hcdd domain is the myosin motor, responsible for converting the ener
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ctyostelium

Drosophila

M-II

Drosophila nonmuscle M-II Chicken nonmuscle M-IIA Human nonmuscle M-IIA Chicken nonmuscle M-IIB Chicken smooth muscle M-II Rabbit smooth muscle M-II

M-l

C. e/egans uric-54 body wall M-II Drosophila muscle M-II Scallop adductor muscle M-II Rat alpha-cardiac muscle M-It muscle M-II Rat beta-cardiac ryonic skeletal muscle M-II Chicken skeletal muscle M-II Chicken embryonic skeletal muscle M-II

Mammalian M-t alpha Bovine brush board Chicken brush boar

Ill

Drosophila

ninaC

I Fig. 1. Unrooted phylogenetic tree of myosins, based on the amino-acid sequences connecting two sequences are proportional to their divergence. Myosin classes II-VII Models of the different myosins are shown, with hypothetical or unknown structural

state of a given myosin. For example, myosins of classes II and V are two-headed by virtue of the coiled-coil-forming E-helical domains within their tails; class V myosins, however, do not polymerize into filaments [IS]. Class I myosins, which lack cl-helical domains in their tails, exist as single-headed molecules. The quarternary structures of class III, IV, VI and VII myosins have not yet been determined, though class VI myosins do contain a short segment predicted to form a coiled-coil and thus they may be two-headed [ 11,131. One is left pondering the questions: When are two heads better than one? And vice versa? A common organizational feature of the a&n-based cytoskeleton is its association with membranes although, with few exceptions, little is known about the molecular

5% sequence

divergence

of their head domains. The lengths of the branches have been numbered in the order of their discovery. features indicated by ‘z’. (Adapted from L4l.j

basis or functional significance of such interactions, Elucidation of the various functions of unconventional myosins may provide crucial insights into this fundamental aspect of cellular organization, as many of the newlydiscovered myosins appear to be membrane-associated. Iocalization studies have shown that certain class I myosins are concentrated at the leading edge, or lamellipodia, of migrating amoeboid [ 2,6] and mammalian cells [ 151. In the case of amoeboid cells, class I myosins are localized to the phagocytic cups and on the cytoplasmic surface of the contractile vacuole. Perhaps the most striking exam ple is brush border myosin-I, molecules of which form the spiral links between the actin core within the intestinal microvillus and the plasma membrane [2]. Recent studies of the localization of chick brain myosin-V in cultured neurons have shown this two-headed myosin

DISPATCH

Fig. 2. Consensus arrangment of domains in the heavy chains of known myosins (top) and a speculative model of brush border myosin-I (bottom). All myosins have a motor or head domain linked to a tail domain by a neck segment consisting of I-6 tandemly repeated copies of the IQ motif. In both parts of the figure light chains are shown clamped around the neck domain - this speculative model is based on the recentlv determined three-dimensional structure of calmodulin-target peptide complexes [I81 and arose from discussions with Joe Wolenski a;d Dick Cheney.

to be associated with both cytoplasmic organelles within the cell body and the plasma membrane of growth cones [ 31. Finally, the class VI myosin, Drosophila 95F has been shown to be associated with unidentified organelles that redistribute during embryogenesis [ 131. One attractive hypothesis is that each myosin expressed in a cell line has a specific function. In the case of membrane-associated myosins, that function could be dictated by the interaction of the myosin, presumably through its tail domain, with a ‘docking site’ in or on the membrane. Once on the membrane, the myosin’s function, whether it be the generation of motility (vesicle movement, for example) or the ‘mechanoregulation’ of the activity of the myosin’s docking complex (which, for example, might be a membrane channel), would depend on the molecular identity of its docking site. However, the results of disrupting genes for Dictyostelium class I myosins (five of which have been identified so far [G] ) indicate that this ‘one myosin-one function’ hypothesis does not hold for this organism. Unlike the dramatic phenotype of amoebae lacking the single class II myosin gene (which includes, for example, a blockage of cytokinesis [2,6] 1, disruption of two distinct class I myosin genes [6] gives rise to viable cells with only a mild impairment of membrane-associated motile functions, including the extension of lamellapodia, cell locomotion and phagocy tosis. One wonders, however, whether such cells would survive if returned to the dirt. This result suggests that the class I myosins of Dic;tyostelium have overlapping functions, so that the loss of one is at least partially complemented by the remaining class I myosins.

There are, however, striking examples of unconventional myosins with membrane-associated functions that are highly specialized and/or essential [6,18]. The larger of the two Drosophila ninaC myosins (products of a single gene generated by differential RNA splicing) is essential for both visual functions and the maintenance of rhabdomere structure. In yeast, a mutation of the My02 gene, which encodes a class V myosin, results in cells that accumulate cytoplasmic vesicles and fail to form a bud [20]. The mouse dilute gene encodes a class V myosin that is a close homolog of chick brain myosin-V (Fig.1). Although the dilute gene is apparently expressed in a wide range of tissues [ 211, mice with null dilute mutations appear quite normal up to several weeks after birth; death then occurs from convulsive seizures. Although the dilute myosin is widely expressed, its essential functions appear to be manifested only in specific cell types, presumably a subset within the nervous system. Such model systems provide a powerful means by which to address questions about myosin function. For example, mutations of the yeast MY02 gene have recently been shown to be suppressed by overexpression of a gene encoding a member of the kinesin superfamily of microtubule motors [22], raising the exciting possibility of interplay between microtubules and the actin-based cytoskeleton. Many other surprises surely await us as we continue to explore the myosin superfamily.

References 1.

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CAMILLI PV, IARSON RE, MOOSEKER MS: Priiaty structure and cellular localization of chicken brain myosin-V @190), an unconventional myosin with calmodulin light chains. J Cell Biol 1992, 119:1541-1558. CHENEY R!3, RIIX~ w MOOSEUR MS: A phylogenetic analysis of the myosin superfamily. Cell Motil Cytoskeletorz, In press. C;OODSON HV, SPUDICH JA: Molecular evolution of the myosin family: relationships derived from comparisons of amino acid sequences. Proc Nat1 Acad Sci USA 1993, 90.659-663. ENDOW S. Tm~s M: Genetic approaches to molecular motors. Annzt Hell Cell Biol 1992, 8:2’+66 SHEIU? EH, JOYCE MP, GREENE IA: Mammaliah myosin I-a, 1-p and I-y: new widely expressed genes of the myosin-I family. J Cell RioI, in press. SHERR EIl, Joyce MP, GKEENE LA: Cloning and characterization of mammalian myosin la, a new neuronally expressed member of the myosin I family. J Ceil Biol in press. BAHI.ER M, BEHRMANN T, KROWSCHWECKI R, RUPPERT C: Mammalian myosin-I motor molecules, myr 1 and myr 4. A4ol CeN Biol 1992. 3(suppl):lGOa. BEMEN~‘ WB, WIRIII J, MOOSIXER M: Identification of multiple unconventional myosins in the human intestinal ceU line Caco-2 BBe. Mel Biol Cell 1992, 3 (suppl):156. H.&SON TB, MOOSEKER MS: Two novel unconventional myosins identified from a kidney proximal tubule cell line. Mel Biol Ce:eN 1992. 3 (suppl):158 HALSALL DJ, HAMMER JA 111: A second isoform of chicken brush border myosin-I contains a 29.residue inserted sequence that binds calmodulin. FEBS 1990. 267:126130.

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JENIG

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MERCER JA,

SEPliRAcK

PK.

STROBEI.

MC,

COPEIAN~

dilucc by a

Mark Mooseker, Department of Biology, Yale University, P.O.Box 6666, New Haven, Connecticut 0651 l-81 12. IJSA.

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ON CYTOPLASM AND CELL MOTILITY READ THE FEBRtJARY 1993 ISSUE OF CUBRENT OPINION IN CELL BIOLOGY

Which included the following reviews, edited by Thomas Pollard and Robert Goldman: Myosins by Margaret A Titus Caldesmon by Fumio Matsumura and Shigeko Yamashiro Phenotypes of cytoskeletal mutants by Susan S. Brown Non-motor microtubule-associated proteins by Gloria Lee Intermediate filament structure and assembly by Murray Stewart Small actin-binding proteins: the P-thymosin family by Vivianne T. Nachmias Dystrophin and the membrane skeleton by James M. Ervasti and Kevin P. Campbell Cytoplasmic microtubule-based motor proteins by Dimitrios A. Skoufias and Jonathan M. Scholq The cellular and molecular biology of keratins: beginning a new era by Pierre A. Coulombe Molecular biology of neuronal intermediate filaments by Ronald K.H. Liem Mitosis: spindle assembly and chromosome motion by Patricia Wadsworth Microtubule-organizing centers in higher plants by Anne-Marie Lambert F-actin capping proteins by Alan Weeds and Sutherland Maciver Unravelling the tangled web at the microtubule-organizing center by Mark D. Rose, Sue Biggins and Lisa L. Satterwhite Actin molecular structure and function by Emil Reisler Desmosomes and hemidesmosomes by David R. Garrod Actin isoforms by Ira M. Herman