The growing family of myosin motors and their role in neurons and sensory cells

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The growing family of myosin motors and their role in neurons and sensory cells Tama Hasson* and Mark S Mooseker Biochemical and physiological evidence has suggested that myosins, both conventional and unconventional, are critical for neurosensory activities. In the past few years, this premise has been supported by genetic evidence that has shown that unconventional myosins are essential for the proper functioning of neurons, retina and the sensory cells of the inner ear.

Addresses

Departments of Biorogy, Pathology and Cell Biology, Yale University, 266 Whitney Avenue, Room 342, Kline Biology Tower, New Haven, Connecticut 06520, USA re-mail: [email protected] re-mail: [email protected] Current Opinion in Neurobiology 1997, 7:615-623

http://biomednet.com/elecref/0959438800700615 © Current Biology Ltd ISSN 0959-4388 Abbreviations chromophore-assisted laser inactivation CALl electron microscopy EM electroretinogram ERG light microscopy LM N-ethyl maleimide NEM neither inactivation nor afterpotential C NinaC PCR polymerase chain reaction retinal pigmented epithelium RPE superior cervical ganglion SCG

Introduction T h e myosin superfamily consists of over a dozen structurally distinct classes of actin-based molecular motors [1-4]. Myosins are found in a wide range of tissues and cell types, including neurons and sensory cells. T h e functions of myosins both in cells, in general, and in cells of the nervous system, in particular, are largely unknown. In this short review, we will highlight recent studies that have begun to examine the expression and function on myosins in neurons and sensory cells, including members of class I, II, III, V, VI, and VII myosins (Figure 1). Genetic evidence has directly implicated four of these myosins (III, V, VI, and Vlla) as performing essential functions for cells in the nervous system. Other available evidence suggests a wide range of potential functions for these molecular motors, including cytoplasmic contractility, intracellular transport, organelle movement, endocytosis, exocytosis, growth cone motility, dynamic rearrangements of the cortical actin cytoskeleton and mechanoregulation of membrane ion channels. The purpose of this review is not to discuss the properties of conventional or unconventional myosins (for reviews, see [1-3]), but to comment on recent evidence for the roles

played by myosins in these neurosensory processes. This review aims to introduce the reader to both the unique aspects of the actin cytoskeleton found within neurons and sensory cells and the myosins traveling their actin highways.

Myosins in neurons T h e actin-based cytoskeleton of neurons is complex; only the organization and assembly dynamics of the growth cone are well understood. Even though the focus of this review is on myosins, it is obvious that future studies that further define the organization of actin in the neuron will be essential for better understanding of myosin function. As with most cells, ultrastructural studies and F-actin staining have revealed the presence of an actin cytoskeleton throughout the various cellular domains of neurons, including the cell body and along the length of dendrites and axons (reviewed in [5]). Although it is clear that the bulk of axonal transport is mediated by microtubules [5], the early studies examining the effects of disrupting actin polymers in the axon provide striking evidence that an intact actin cytoskeleton is critical for axonal transport (reviewed in [6]). T h e precise functions of actin in axonal transport remain unclear, although both structural and mechanochemical roles are likely. A recent series of studies on axoplasmic transport in the squid giant axon and on mitochondrial transport in mammalian neurons (reviewed in [6,7]), do provide direct evidence that neuronal organelles can carry (and be carried by) motors for both microtubules and actin. In contrast, the organization of actin within the growth cone is relatively well understood, largely because this region of the neuron is thin enough for high-resolution microscopic analysis. T h e organization and assembly dynamics of actin in the growth cone have been the topic of numerous reviews (e.g. [5,8]). T h e leading margin of the growth cone, with its actin-filament-rich filopodial and lamellar extensions, participates in tension generation, as well as protrusive and retractile cytoplasmic movements that are critical for growth cone motility and guidance. Moreover, as noted below, the actin within this region is continuously moved rearward toward the microtubule-rich central domain of the growth cone, a process termed retrograde actin flow [9,10]. Much of the recent progress summarized below regarding our understanding of myosin expression and function in neurons has stemmed from studies examining the dynamic phenomena associated with this region of the neuron. Myosin II

Myosin II (conventional myosin) is capable of forming the bipolar filaments that are responsible for the majority of

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Figure 1 Motor

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The myosins that have been characterized in neuronal and sensory cells. The schematic diagrams depict the heavy chains of the six classes of myosins discussed in the text. Each myosin has a 'motor' or head domain, a 'neck' domain that has repeats of the light chain binding 'IQ' motif, and a distinct carboxy-terminal 'tail' domain. Myosin tails include both coiled-coil (CC) regions and regions that target myosins to membranes and other subcellular domains. For a detailed description of the structural subdomains of these myosins, as well as the known biochemical and functional properties of these molecular motors, see [1-4].

actin-based contractile events. T h e two major nonmuscle myosins II, myosin IIA and myosin liB, are expressed in vertebrate brain [11]. Several neural-specific and developmentally regulated splice variants of myosin liB have been identified [12]. Rochlin etal. [13] have utilized isoform-specific antibodies to examine the distribution of myosins IIA and liB in cultured neurons, focusing primarily on their localization in the growth cone. T h e y employed fixation protocols to optimize preservation of the actin cytoskeleton, and they confirmed earlier studies reporting that myosin IIB is the predominant form of myosin II expressed in cultured neurons. In their paper, they also provide striking localization data indicating that myosin liB is concentrated within the central domain of the growth cone, the region that lies at the base of the actin-rich peripheral zone. This location for myosin liB is compared by the authors to the sites of active growth cone protusion and retraction. On the basis of overlapping locations, the authors proposed a role for myosin liB in both filopodial retraction and tension generation, but

do not see a role for myosin lIB in growth cone protrusion [13]. T h e localization of myosin II within the central domain of the growth cone also places it in an ideal location to provide the driving force for the bulk retrograde flow of actin from the peripheral to the central d o m a i n - - a s best documented in studies of the growth cones of Aplysia bag cell neurons (see [9]). Lin et al. [14"] have provided evidence that a myosin--although not necessarily myosin I I - - i s involved in this phenomenon. To test the potential involvement of myosin in retrograde actin flow, they microinjected Aplysia bag cell neurons with N-ethyl maleimide (NEM)-modified myosin subfragment 1 (S1), which binds to actin with high affinity in an ATP-independent manner [14°]. T h e y hypothesized that NEM-S1 would bind to actin and block sites for interaction with endogenous myosin. Consistent with this idea, they found that retrograde flow was inhibited, with a concomitant increase in protrusive activity at the leading

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edge of the growth cone [14°]. Similar effects have been observed using 2,3-butanedione monoxime (BDM) [14°], an inhibitor of myosin II ATPase. Taken together, these findings provide indirect evidence for the involvement of myosin in retrograde actin flow. T h e y also suggest that there is a balance between actin-assembly-driven protrusive activity and myosin-driven rearward movement of actin filaments.

Myosin I Of the 16 unconventional myosin genes identified and mapped in the mouse genome [15°], half are members of the myosin I class. There are at least four structurally distinct subclasses of myosin I, and members of all four classes are expressed in tissues of the nervous system [3]. However, remarkably little is known about the subcellular distribution (let alone function) of myosin I in neurons. T h e best myosin I study to date is that of Lewis and Bridgman [16°], who examined the subcellular localization of myosin lot in cultured rat neurons. Myosin lot together with brush border (BB) myosin I comprise one of the four myosin lot subfamilies. Like BB myosin I, myosin lot contains multiple calmodulin light chains; however, unlike BB myosin I, myosin lot is expressed in a wide range of tissues, including brain (reviewed in [3]). In cultured rat superior cervical ganglion (SCG) neurons, myosin Iot exhibits a punctate distribution throughout the cell. Immunoelectron microscopy has revealed the presence of myosin lot on tubulovesicular structures within the cell body. However, within the growth cone, where it is concentrated, myosin lot is associated with the plasma membrane and actin cytoskeleton, but not with organelles [16"]. T h e steady-state distribution of myosin Icx in the growth cone suggests that it may be delivered on vesicles, but that once it is delivered (either passively or by driving its own transport), it functions in association with the plasma membrane. T h e passive transport model is favored by a recent kinetic study of myosin I [17"], which indicates that myosin I is a poor candidate for an organelle motor. In this study, it was shown that myosin I purified from Acanthamoeba is kinetically similar to myosin II in that during most of the mechanochemical cycle, myosin I exhibits a low affinity for actin. Thus, for myosin I to move organelles, there would have to be a relatively high density of motors on the organelle surface to prevent the organelle from diffusing away from the actin substrate. Myosin V

Several lines of evidence suggest that myosin V, in contrast to myosin I, is an excellent candidate for an organelle motor in neurons (reviewed in [18]). Two different class V myosin heavy chains have been identified, myosin V and myr6 [19,20]. Myosin V is encoded by the well-characterized dilute locus and is the predominant form in brain. T h e melanocytes of dilute mice fail to

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properly transfer pigment granules to the keratinocytes of the hair shaft, leading to a diminution or dilution of coat color (relative to wild type). This transfer is a complex process that first involves the movement of pigment granules into the dendritic processes of the melanocyte, followed by phagocytosis of the dendritic tips by the keratinocytes of the hair shaft. Recent studies have shown that myosin V is localized to the pigment granules [21",22"], suggesting a role in the transport process. Mice with lethal mutations in the dilute gene also have a neurological phenotype. Within a few weeks after birth, these mice develop seizures and die. Recent ultrastructural studies of brain tissue from dilute-lethal mice [23 °'] and dilute-opisthotonus rats [24"] indicate that the actin-rich spines on the dendrites of Purkinje neurons in these animals are devoid of endoplasmic reticulum. This organelle is thought to play a role in regulating dendritic intracellular Ca 2+. These exciting findings strongly suggest that myosin V is required for either transport or retention of the endoplasmic reticulum within these spines, and the mislocalization of the endoplasmic reticulum may be the basis for the neurological defects observed. It is important to note, however, that expression of myosin V is not restricted to dendritic spines. In cultured neurons, myosin V exhibits a complex subcellular distribution. Initial localization studies revealed myosin V to be associated with both punctate structures within the cell body and neurite processes, with the highest levels of staining in the cell body and growth cones [3]. Recent high-resolution light microscopy (LM) and electron microscopy (EM) localization studies of myosin V in growth cones of rat SCG revealed that myosin V is localized to organelles that are associated with both microtubules and actin bundles, as well as with dense structures on the plasma membrane [25"]. These same workers had previously documented actin-based organelle movements in SCG growth cones [26]. Although these studies suggest a role for myosin V in organelle transport within the growth cone, Evans et al. [25"] have also demonstrated that growth cone formation and morphology in neurons cultured from dilute-lethal (a null mutation) mice are comparable to that in wild-type neurons. Thus, myosin V does not appear to be essential for growth-cone formation or motility. A strikingly different conclusion was suggested by the studies of Wang et al. [27°], who used the technique of chromophore-assisted laser inactivation (CALI) to assess the function of myosin V in the growth cones of chick dorsal root ganglion neurons. These neurons were injected with malachite-green-labeled antibodies directed against the tail domain of chick brain myosin V. Laser treatment of the growth cones of these injected neurons resulted in rapid retraction of filopodia within the laser-treated portion of the growth cone [27"]. One interpretation of this result is that myosin V participates in filopodial extension and that CALI results in filopodial collapse as a result

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of inactivation of the motor or disruption of the tail tethering to the membrane. However, the antibody used was directed to the tail domain. Thus, it is possible that filopodial retraction results either from damage to as yet uncharacterized membrane components with which the tail domain of myosin V interacts or, conversely, from nonspecific membrane damage. T h e biochemical properties of myosin V purified from chick brain have been characterized extensively (reviewed in [3]). It is a two-headed motor with multiple calmodulin light chains and several additional other light chains. Recent studies by Nascimento et al. [28"] have provided biochemical evidence that myosin V has properties that are consistent with its function as an organelle motor. In contrast to the ATPase activity of myosin II, the MgATPase of myosin V is activated at very low concentrations of F-actin. Moreover, it has been shown that myosin V binds to actin with relatively high affinity, even in the presence of ATP [28"]; thus, unlike myosins II and I, myosin V may remain bound to the actin filament throughout much of its mechanochemical cycle.

M y o s i n s in h e a r i n g a n d b a l a n c e T h e primary sensory cell of the inner ear, the hair cell, has actin-based apical projections called stereocilia that serve to convert mechanical forces such as sound and gravity into electrical signals (for a review, see [29]). Each stereocilium is a membrane-enclosed rigid structure that contains hundreds of bundled actin filaments. At the bottom of each stereocilia, the number of actin filaments decreases to only a few dozen and, as a result, the stereocilium pivots at the point of insertion into the apical domain of the hair cell (Figure 2). T h e stereocilia are arranged in rows of increasing height and are glued together into a bundle by an array of extracellular linkages. Therefore, the entire bundle moves as a unit when it is deflected by mechanical forces. Upon deflection, the stereocilia slide past each other, stretching the extracellular linkages that join them. A unique linkage, termed the tip link, joins the tip of each stereocilium to its next highest neighbor. When the tip links are stretched, transduction channels, located at either end of the tip link, are opened by the increased tension on this linkage. It is in the resetting of these channels (after stimulus) that myosins have been implicated as players. This resetting process, termed adaptation, occurs within a few tens of milliseconds after stimulus. In the case of a positive deflection (movement of the bundle towards the tallest stereocilium), adaptation leads to a release of tension on the tip link and results in the closing of the transduction channels. Conversely, in the case of a negative deflection, tension is increased on the tip link, resulting in channel reopening. As an actin-based motor, myosins are ideal candidates for mediating this adaptation process (for a review, see [30]).

Myosins VI and Vlla Recent genetic evidence has shown that two different unconventional myosins are essential for hair cell function. T h e genes responsible for the mouse inner ear mutant shaker-1 and the corresponding human deafness syndrome, Usher syndrome type 1B, have both been found to encode myosin VIIa [31,32]. In addition, the gene responsible for Snell's waltzer, a different mouse inner ear mutant, was found to encode myosin VI [33]. Both myosin VI and myosin VIIa defects lead to profound sensorineural hearing loss and vestibular dysfunction. In the case of the mouse mutants, it has been observed that the entire sensory epithelium of the cochlea and vestibular apparatus degenerate. T h e primary defect appears to lie within the hair cells themselves as studies have shown that both myosins are expressed exclusively by the hair cells in these tissues [33,34]. Surprisingly, neither myosin VI nor myosin VIIa is enriched at the tips of stereocilia, the site of adaptation. In studies in both rodents and frogs, it has been shown that myosin VI is located within a different actin-based structure in the hair cell termed the cuticular plate [35"] (Figure 2), which is an actin meshwork that anchors the stereocilia into the apical cytoplasm. Myosin VI is tightly associated with this meshwork and is also found associated with the rootlet actin filaments of the stereocilia that penetrate this domain [35"]. These results have led to the hypothesis that myosin VI is important for the maintenance of the attachment, or the tension, between the stereocilia and the actin cytoskeleton of the hair cell. This localization of myosin VI to the bases of polarized actin-bundles is also seen in the intestine [36] and kidney [37]. Perhaps myosin VI has a conserved role in this domain for all these tissues. Myosin VIIa, unlike myosin VI, is found along the length of the stereocilia in mammals [34,35"] (Figure 2), although this localization is not observed in all species. In the frog, myosin VIIa is enriched in a band towards the bottom of the stereocilia at the position of the basal tapers (Figure 2). This difference in localization--all along the length of the stereocilium versus at the basal t a p e r s - - i s consistent with an association of myosin VIIa with an intracellular component of the extracellular linkages [35°], because these linkages are enriched at the basal tapers in frog stereocilia but are found all along the length of the stereocilium in mammals. Myosin VIIa would therefore provide a 'regulatable' association between the actin cytoskeleton (within the stereocilium) and the extracellular linkages that join each stereocilium to its n e i g h b o r - - p e r h a p s allowing for maintenance of rigidity in this dynamic structure. On the basis of PCR screens [38] and direct cloning of cochlear myosin cDNAs [39], it has been estimated that hair cell epithelia express upwards of 10 different myosins. Earlier photoaffinity labeling studies had already identified

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Figure 2

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CurrentOpinionin Neurobiology Myosin isozyme location in the sensory epithelium of the inner ear. The hair cells, supporting cells and nerve cells that make up the sensory epithelium exhibit an array of actin domains that are delineated on the right. As described in the text, these cells express a number of myosins, and the subcellular locations of these myosins are delineated on the left. For a more detailed description of actin and myosin domains of the sensory hair cell, see [30,35°].

three potential adaptation motor myosins of different molecular weights (120 kDa, 160 kDa and 230 kDa) within the frog stereocilia [40]. T h e 160kDa and 230kDa myosins may well be myosins VI and VIIa, respectively, both on the basis of their SDS-PAGE mobilities and

on the fact that myosins VI and Vlla are present in isolated frog hair bundles [35"]. T h e adaptation process is affected by the presence of ADP analogs and tight binding phosphate analogs (e.g. vanadate), again suggesting a myosin ATPase is involved [41,42°°[. Adaptation is

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also affected by the presence of calmodulin inhibitors, suggesting that the adaptation motor is regulated by this CaZ+-binding protein [43°']. Of the three myosins identified by photoaffinity labeling, only the 120 kDa form appears to be sensitive to the presence of phosphate analogs and calmodulin inhibitors [42",43°']. Adaptation is insensitive to the presence of NEM; of the three myosins identified, only the 120kDa myosin isozyme appears to be NEM-insensitive during photoaffinity labeling [44°°]. Therefore, the 120kDa myosin isoform is currently the most popular candidate for the adaptation motor. This motor may well be myosin I[3. Myosin 113 Myosin I13 is the correct size for the adaptation motor (-120kDa) and is associated with multiple calmodulin light chains. Both indirect immunofluorescence and immunoelectron microscopy have confirmed that myosin II3 is enriched at the osmiophilic plaques at the ends of tip links, the putative sites of the adaptation apparatus [35",40] (Figure 2). Myosin II3 does indeed exhibit a number of features characteristic of the adaptation motor, including Ca2+-sensitive motility and an in vitro motility rate similar to the climbing rate of the adaptation motor [45"]. T h e NEM-insensitive ATPase present within the hair bundles is sensitive both to changes in the concentration of Ca 2+ and to calmodulin inhibitors [44"'], consistent again with the adaptation motor being myosin I[3. It remains to be seen, however, whether purified frog myosin II3 exhibits similar sensitivities to calmodulin inhibitors, N E M and nucleotide analogs, as observed for the adaptation process. Three myosins differentially located in the stereocilium? We can only speculate as to how these three different unconventional myosins (VI, VIIa and I[3) locate to unique subdomains within the stereocilium. Given the polarized orientation of the actin filaments, it would be predicted that all the myosins would move towards the barbed ends of the actin filaments at the tip of each stereocilium. Instead, only one myosin is at the tips, myosin I[3, whereas myosin VIIa is found along the length of the stereocilium and myosin VI at the base. Targeting specific myosins to these regions may be facilitated by the differential assembly of actin isozymes: for example, in chicken auditory hair cells, [3-actin is limited to the stereocilia, whereas 7-actin is found in all the actin domains of the hair cell [46]. Perhaps these myosins have different affinities for different actins. Direct binding of the myosins via their unique tail domains may also be involved in anchoring each myosin to its proper domain. Indeed, in Usher syndrome patients and shaker-1 mice, mutations have been found in both the motor and tail domains of myosin VIIa [31,47,48].

One surprising result with regard to the shaker-1 mice is that all seven alleles of shaker-1 express normal myosin VIIa mRNA levels but maintain very different levels of myosin VIIa protein [49"]. Expression of

myosin VIIa protein varied from wild-type levels to less than 1% of normal levels, all with the same phenotype of degeneration of the inner ear sensory epithelium [49°]. T h e shaker-1 allele with wild-type levels of myosin VIIa protein, shaker-1 (original), has a single amino acid change within the motor domain [32,49°]. This mutation does not affect protein targeting, as the localization of the mutated myosin VIIa protein in both the testis and the retina is comparable to wild-type myosin VIIa [49"]. Thus, there are two general types of myosin VIIa mutations that lead to deafness: those that presumably result in motor dysfunction and those that result in destabilization of the protein. This destabilization may be attributable to misfolding or, perhaps, to disruption of sites on the molecule required for proper targeting to the cytoskeleton. Other myosins in the inner ear

Are other myosins involved in hair cell function? Class II myosins have been shown to be present in the circumferential actin I~and at the zona adherens of hair cells and supporting cells (reviewed in [30]; see also Figure 2), although the hair cell myosin II subtypes have yet to be characterized in detail. Myosin V has been shown to expressed in the neurons associated with cochlear and vestibular hair cells [35"]. In particular, this myosin is enriched in afferent nerve cell bodies and dendritic calyxeal and bouton terminals [35"] (see Figure 2). It is not known whether dilute mice, which lack functional myosin V, are hearing impaired in addition to having altered coat colors and the various neurological phenotypes described above. Given the recent successes in identifying human and mouse deafness genes (reviewed in [50]), it will be interesting to see whether other unconventional myosin genes are related to deafness. Presently, 16 unconventional myosin genes have been mapped on the mouse genome [15"]; of these, myosin I13 (locus name: Myolc) and myosin VIIb (locus: Myo7b), as well as myosin VI (locus: Myo6), are considered potential human deafness loci. Given the variety of actin-based motilities in hair cells, it is not surprising that several different classes of myosins are involved. M y o s i n s in v i s i o n Genetic studies have identified two different myosins as essential for vision, NinaC myosins in Drosophila and myosin Vlla in humans. In both cases, lack of normal myosin function results in abnormal electroretinograms (ERGs) and retinal degeneration. In contrast, the subcellular locations and potential functions of these two very different unconventional myosins are quite distinct. Myosin III/NinaC

T h e site of phototransduction in Drosophila photoreceptors is the rhabdomere, a series of closely packed microvilli containing rhodopsin. NinaC (neither inactivation nor

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aftetpotential C) is a Drosophila mutant that exhibits a defect in the electrophysiological responses to light. T h e NinaC gene encodes an unconventional myosin with an amino-terminal kinase domain, followed by a myosin motor domain, a calmodulin light chain binding domain, and a distinct tail domain (reviewed in [3]). This unique myosin is the founding member of class III of the myosin superfamily. There are two forms of NinaC: one form, p174, localizes to the rhabdomere, whereas the other form, p132, remains in the cytoplasm of the photoreceptor. Genetic studies have confirmed that p174 is the only NinaC protein essential for the electrophysiology of the rhabdomere (reviewed in [3]). Interestingly, both the kinase and myosin domains appear essential for this process. Mutations in the kinase domain result in abnormal ERGs, but no degeneration, whereas mutations in the myosin motor domain result in both ERG and retinal degeneration phenotypes and to changes in the location of the mutant protein [51]. Clearly, the enzymatic activities of both the kinase and motor domains are important for NinaC function. Surprisingly, one of the major functions of NinaC appears to be to target the calmodulin light chain to the rhabdomere [52].

Myosin Vlla As mentioned previously, mutations in myosin VIIa are the basis for human Usher syndrome type lb [31]. In addition to the inner ear defects mentioned above, Usher patients exhibit a progressive retinal degeneration, termed retinitis pigmentosa, which leads to blindness during adolescence. Preliminary immunolocalization studies have shown that myosin Vlla expression is restricted to the retinal pigmented epithelium (RPE) in rodents [34]. In this cell type, myosin Vlla is enriched in the actin-rich apical villi. These villi serve in phagocytosis of photoreceptor outer segments, a role essential for the viability of the photoreccptors. T h e visual impairment characteristic of Usher patients may therefore be attributable to a defect in the phagocytosis of photoreceptors by the RPE [34]. Shaker-1 mice do not exhibit any retinal degeneration throughout their lifetime [49"], however. More recent studies have shown that although myosin VIIa is restricted to the RPE in rodents, it is also expressed in the inner segment of photoreceptors in humans [56°]. Therefore, it has been proposed that the primary defect in Usher patients may be in the photoreceptors themselves and not the RPE.

More recently, Hicks eta/. [53] examined the development of the rhabdomere at the EM level in a variety of ninaC mutants and identified a defect in the axial microvillar cytoskeleton of the animals lacking either p174 or the NinaC myosin domain. One interpretation of these findings is that NinaC serves to anchor this set of actin filaments to the microvillar membrane as NinaC proteins are capable of binding F-actin, presumably via the myosin motor domain [53]. A perhaps more compelling interpretation, given the importance of the motor domain for proper NinaC subcellular localization, is that NinaC serves a role in transport along the actin filaments of the axial cytoskeleton, delivering important components (such as calmodulin) or enzymatic activities (such as its kinase activity) essential for the maintenance of the rhabdomere. Indeed, the NinaC proteins exhibit kinase activity [54"]. NinaC kinase domains were expressed and found capable of phosphorylation on serine and threonine residues. Interestingly, one substrate for the kinase activity is p132, suggesting that phosphorylation of p132 may be important for this protein's function [54°].

Other myosins in the retina Historically, studies on myosin expression in the retina have focused on myosin II and its potential location within the connecting cilium of the photoreceptor cell [57,58]. With the genetic data described above, and the advent of reverse transcription (RT)-PCR techniques, the number of myosins identified as being expressed in this tissue has grown to include myosins of all vertebrate classes. Of these myosins, recent studies have located myosin V in the retina. Myosin V is present in the photoreceptor synapses, as well as throughout the inner nuclear layer and the ganglion cell layer [59]. These locations are consistent with its locations in other neural tissues (mentioned above). In addition, myosin II3 and related myosin I immunogens have been detected in RPE cells [60]. These studies are the tip of the iceberg, as more more probes for unconventional myosins become available to investigate this sensory tissue.

Burnside's group ([55]; D Hillman, LM Bost-Usingcr, B Burnside, abstract in Invest Ophthalmol Vis Sci 1995, 36S:$598) has recently identified the fish homolog of NinaC in their screen for myosins expressed in the retina. Using degenerate PCR primers, they detected expression of myosins from seven different classes, including two myosin IIl-like molecules. It will be exciting to see whether myosin III is also expressed in humans and whether it too is a retinal degeneration gene.

Conclusions T h e identification of myosins V, VI, and VIla as targets for mutations that affect neuronal and neurosensory function underscores the fundamental importance of actin-based movements in the nervous system. As described in this review, a great deal of progress has been made in the analysis of the role of myosins in hair cell ion channel adaptation. Future studies that utilize a combination of traditional cell biological and physiological techniques, coupled with myosin-isoform-specific probes, should help characterize the role of other myosins in the actin-based movements of neurosensory cells.

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Acknowledgements

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The authors" work presented in this review was supported by National Institutes of Health (NIH) grants DK38979 and GM25387, a basic research grant from the Muscular Dystrophy Association and a grant from the Deafness Research Foundation.

Titus MA: Motor proteins: myosin V - t h e multi-purpose transport motor. Curr Biol 1997, 7:R301 -R304.

19.

Zhao L-P, Koslovsky JS, Reinhard J, Bahler M, Witt AE, Provance WD Jr, Mercer JA: Cloning and characterization of myr 6, an unconventional myosin of the dilute/myosin V family. Proc Nat/Acad Sci USA 1996, 93:10826-10831.

20.

Mercer JA, Seperack PK, Strobel MC, Copeland NG, Jenkins NA: Novel myosin heavy chain encoded by murine dilute coat colour locus. Nature 1991,349:709-713.

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Welch MD, Mallavarapu A, Rosenblatt J, Mitchison TJ: Actin dynamics in vivo. Curr Bio/1997, 9:54-61.

11.

Simons M, Wang M, McBride OW, Kawamoto S, Yamakawa K, Gdula D, Adelstein RS, Weir L: Human nonmuscle myosin heavy chains are encoded by two genes located on different chromosomes. Circ Res 1991,69:530-539,

12.

Itoh K, Adelstein RS: Neuronal cell expression of inserted isoforms of vertebrate nonmuscle myosin heavy chain II-B. J Bio/Chem 1995, 270:14533-14540.

26.

13.

Rochlin MW, Itoh K, Adelstein RS, Bridgman PC: Localization of myosin II A and B isoforms in cultured neurons. J Cell Sci 1995, 108:3661-3670.

2Z •

14. •

Lin CH, Espreafico EM, Mooseker MS, Forscher P: Myosin drives retrograde F-actin flow in neuronal growth cones. Neuron 1996, 16:769-782. The authors utilize two different inhibitors of myosin, both of which block rearward flow of actin in the growth cone and simultaneously increase protrusion at the leading edge. 15. •

Hasson T, Skowron JF, Gilbert DJ, Avraham KB, Perry WL, Bement WM, Anderson BL, Sherr EH, Greene LA, Ward DC et aL: Mapping of unconventional myosins in mouse and human. Genomics 1996, 36:431-439. In this study, the authors identify the genes of the myosin superfamily and correlate a number of the new unconventional myosin genes with disease loci. LewisAK, Bridgman PC: Mammalian myosin la is concentrated near the plasma membrane in nerve growth cones. Ceil Motil Cytoske/eton 1996, 33:130-150. A thorough LM and EM localization analysis of the distribution of myosin la within the growth cone. 16. •

17. •.

Ostap EM, Pollard TD: Biochemical kinetic characterization of the Acanthamoeba myosin I ATPase. J Cell Biol 1996, 132:1053-1060. An in depth analysis of the kinetics of Acanthamoeba myosin I ATPase. This key study demonstrates that the ATPase cycle of this myosin is similar to that of myosin II, and indicates that class I myosins are not good candidates for organelSe motors, at least at low motor density.

21. •

Takagishi Y, Oda S-I, Hayasaka S, Dekker-Ohno K, Shikata T, Inouye M, Yamamura H: The dilute-lethal (or/) gene attacks a Ca 2+ store in the dendritic spine of Purkinje cells in mice. Neurosci Lett 1996, 215:169-172. This electron microscope study provides evidence for the absence of smooth endoplasmic reticulum within the dendritic spines of dilute mice (myosin V mutation). See also annotation [24°']. 24. ••

Dekker-Ohno K, Hayasaka S, Takagishi Y, Oda S-I, Wakasugi N, Mikoshiba K, Inouye M, Yamamura H: Endoplasmic reticulum is missing in dendritic spines of Purkinje cells of the ataxic mutant raL Brain Res 1996, 714:226-230. This electron microscope study provides evidence for the absence of smooth endoplasmic reticulum within the dendritic spines of the dilute rat (presumed myosin V mutation). See also annotation [23"']. 25. •

Evans LL, Hammer J, Bridgman PC: Subcellular localization of myosin V in nerve growth cones and outgrowth from dilutelethal neurons. J Ceil Sci 1997, 110:439-449. Another thorough LM/EM localization study from the Bridgman laboratory demonstrating that myosin-V-containing organelles co-localize within the growth cone to both actin and microtubule arrays. This study also demonstrates that cultured neurons from dilute-lethal mice exhibit normal outgrowth of growth cones. Evans LL, Bridgman PC: Particles move along actin filament bundles in nerve growth cones. Proc Nat/Acad Sci USA 1995, 92:10954-10958.

Wang F-S, Wolenski JS, Cheney RE, Mooseker MS, Jay DG: Function of myosin V in filopodial extension of neuronal growth cones. Science 1996, 273:660-663. This study utilizes chromophore-assisted laser inactivation to target the function of myosin V in the growth cone. Laser treatment of growth cones of neutons injected with chromophore-tagged, tail-directed antibodies to myosin V results in rapid retraction of filopodial extensions. 28. •

Nascimento AAC, Cheney RE, Tauhata SBF, Larson RE, Mooseker MS: Enzymatic characterization and functional domain mapping of brain myosin V. J Bio/Chem 1996, 271:17561-17569. A detailed biochemical analysis of the enzymatic properties and domain structure of chick brain myosin V. In contrast to other known myosins, myosin V appears to bind to actin with very high affinity in the presence of ATP, making it a good candidate for an organelle motor. 29.

Garcia-Anoveros 3, Corey DP: The molecules of mechanosensation. Annu Rev Neurosci 1997, 20:567-594.

30.

Gillespie PG, Hasson T, Garcia JA, Corey DP: Multiple myosin isozymes and hair-cell function. Cold Spring Herb Syrup Quant Bio/1996, 61:309-318.

31.

Well D, Blanchard S, Kaplan J, Guilford P, Gibson I=, Walsh J, Mburu P, Valera A, Levilliers J, Weston MD et el.: Defective myosin VIIA gene responsible for Usher syndrome type lB. Nature 1995, 374:60-61.

32.

Gibson F, Welsh J, Mburu P, Varela A, Brown KA, Antonio M, Beisel K, Steel KP, Brown SDM: The shaker-1 mouse deafness mutation encodes a myosin VII motor. Nature 1995, 374:62-64.

Myosin motors in neurons and sensory cells Hasson and Mooseker

33.

34.

AvrahamKB, Hasson T, Steel KP, Kingsley DM, Russell LB, Mooseker MS, Copeland NG, Jenkins NA: The mouse Snell's waltzer deafness gene encodes an unconventional myosin required for structural integrity of inner hair cells. Nat Genet 1995, 11:369-374. Hasson T, Heintzelman MB, Santos-Sacchi J, Corey DP, Mooseker MS: Expression in cochlea and retina of myosin Vlla, the gene product defective in Usher syndrome type lB. Proc Nat/Acad Sci USA 1995, 92:9815-9819.

35. •

Hasson T, Gillespie PG, Garcia JA, MacDonald RB, Zhao Y-D, Yee AG, Mooseker MS, Corey DP: Unconventional myosins in inner-ear sensory epithelia. J Ce//Bio/1997, 137:1287-1307. Previous work (described in [41,42"-44"']) had implicated myosins VI, Vlla, and 113as components of the hair bundle. In this study, the authors locate these myosins, as well as myosin V, using isoform-specffic probes in the various sensory epithelia of the inner ear of mouse, bullfrog and guinea pig. 36.

Heintzelman MB, Hasson T, Mooseker MS: Multiple unconventional myosin domains in the intestinal brush border cytoskeleton. J Cell Sci 1994, 107:3535-3543.

37.

Hasson T, Mooseker MS: Porcine myosin Vh characterization of a new mammalian unconventional myosin. J Cell Biol 1994, 127:425-440.

38.

Solc CF, Defiler BH, Duyk GM, Corey DP: Molecular cloning of myosins from the bullfrog saccular macula: a candidate for the hair-cell adaptation motor. Auditory Neurosci 1994, 1:63-75.

39.

Crozet F, EI-Amraoui A, Blanchard S, Lenoir M, Ripoll C, Vago P, Hamel C, Fizames C, Levi-Acobas I=, Depetris D eta/.: Cloning of the genes encoding two murine and human cochlear unconventional type I myosins. Genomics 1997, 40:332-341.

40.

Gillespie PG, Wagner MC, Hudspeth AJ: Identification of a 120kd hair-bundle myosin located near stereociliary tips. Neuron 1993, 11:581-594.

41.

Gillespie P, Hudspeth A: Adenine nucleoside diphosphate block adaptation of mechanoelectrical transduction in hair cells. Proc Natl Acad Sci USA 1993, 90:2710-2713.

42. ••

Yamoah EN, Gillespie PG: Phosphate analogs block adaptation in hair cells by inhibiting adaptation-motor force production. Neuron 1996, 17:523-533. The Gillespie lab has provided further evidence that the adaptation motor is probably a myosin by showing that inhibitors of myosin ATPase also inhibit adaptation. It is exciting to see that the candidate for the adaptation motor, the 120 kDa myosin isozyme, exhibits similar sensitivities to these inhibitors in photoaffinity labeling studies. 43. °•

Walker RG, Hudspeth AJ: Calmodulin controls adaptation of mechanoelectrical transduction by hair cells of the bullfrog's sacculus. Proc Nat/Acad Sci USA 1996, 93:2203-2207. This work confirms that adaptation is regulated by Ca2+-calmodulin. As myosins are associated with calmodulin light chains, this evidence suggests that the sensitivity of adaptation to calmodulin inhibitors is mediated by a myosin. 44. ••

Burlacu S, Tap WD, Lumpkin EA, Hudspeth A: ATPase activity of myosin in hair bundles of the bullfrog's sacculus. Biophys J 1997, 72:263-271. This manuscript analyzes the ATPase activity present in isolated hair bundles and finds that adaptation is insensitive to NEM, a potent inhibitor of many myosins. In this exciting manuscript, the authors observe that the candidate for the adaptation motor, the 120 kDa myosin isozyme, appears to be NEM-insensitive during photoaffinity labeling, providing a further connection between this myosin and the adaptation motor. 45. •

Zhu T, Sata M, Ikebe M: Functional expression of mammalian myosin I~: analysis of its motor activity. Biochemistry 1996, 35:513-522. This important biochemical analysis of myosin I~ provides the baseline for a direct comparison of the properties of the adaptation motor and the properties of this unconventional myosin.

623

46.

Hofer D, Ness W, Drenckhahn D: Sorting of actin isoforms in chicken auditory hair cells. J Ceil Sci 1997, 110:765-770.

47,

Levy G, Levi-Acobas B, Blanchard S, Gerber S, Larget-Piet D, Chenal V, Liu XZ, Newton V, Steel K, Brown S e t aL: Myosin VIIA gene: heterogeneity of the mutations responsible for Usher syndrome type lB. Hum Mo/Genet 1997, 6:111-117.

48.

Weston MD, Kelley PM, Overbeck LD, Wagenaar M, Often DJ, Hasson T, Chen Z-Y, Corey DP, Mooseker MS, Sumegi Jet al.: Myosin VIIA mutation screening in 189 Usher syndrome type 1 patients. Am J Hum Genet 1996, 59:1074-1083.

49. •

Hasson T, Walsh J, Cable J, Mooseker MS, Brown SDM, Steel KP: Effects of shaker-1 mutations on myosin Vlla protein and mRNA expression. Cell Motil Cytoskeleton 1997, 37:127-138. As a first step towards understanding the role of myosin Vlla in disease, these authors have characterized myosin expression in the mouse mutant shaker-1. Here, they present data that the retinal and testicular morphology of these mutants is similar to wild-type, an unexpected result given the high level of expression of myosin Vlla in these tissues. Also, the expression profile of myosin Vlla is found to vary widely among the various shaker-1 alleles, suggesting that small mutations may have drastic effects on myosin Vlla protein stability. 50.

Petit C: Genes responsible for human hereditary deafness: symphony of a thousand. Nat Genet 1996, 14:385-391.

51.

Porter JA, Montell C: Distinct roles of the Drosophila ninaC kinase and myosin domains revealed by systematic mutagenesis. J CeU Biol 1993, 122:601-612.

52.

Porter JA, Yu M, Doberstein SK, Pollard TD, Montell C: Dependence of calmodulin localization in the retina on the NINAC unconventional myosin. Science 1993, 262:1038-1042.

53.

Hicks JL, Liu X, Williams DS: Role of the NinaC proteins in photoreceptor cell structure: ultrastructure of NinaC deletion mutants and binding to actin filaments. Cell Motil Cytoskeleton 1996, 35:367-379.

54. •

Ng KP, Kambara T, Matsuura M, Burke M, Ikebe M: Identification of myosin III as a protein kinase. Biochemistry 1996, 35:93929399. The first evidence that the kinase domain found on the amino terminus of NinaC is an active enzyme capable of phosphorylating a number of substrates. This enzymatic analysis confirms the genetic data [51] showing that the kinase serves an essential role in the function of the protein in the retina of Drosophila. 55.

Burnside B, Bost-Usinger L: The RPE cytoskeleton. In The Retina/ Pigment Epithehum: Current Aspects of Function and Disease. Edited by Marmor MF, Wolfensberger TJ. Oxford: Oxford University Press; 1997:in press.

56. •

EI-AmraouiA, Sahly I, Picaud S, Sahet J, Abitbol M, Petit C: Human Usher 1b/mouse shaker-l: the retinal phenotype discrepancy explained by the presence/absence of myosin VIIA in the photoreceptor cells. Hum Mo/Genet 1996, 5:11711178. This important immunolocalization study shows that myosin Vlla is expressed in both the pigmented epithelium and the photoreceptors of the human eye. This contrasts with earlier work, which had located myosin Vlla only to the pigmented epithelium in rodents [34]. The authors suggest that this expression profile explains the phenotypic differences observed between shaker-1 mice and Usher patients. 57.

Chaitin MH, Ooelho N: Immunogold localization of myosin in the photoreceptor cilium. Invest Ophthalmol Vis Sci 1992, 33:3103-3108.

58.

Williams DS, Hallett MA, Arikawa K: Association of myosin with the connecting cilium of rod photoreceptors. J Cell Sci 1992, 103:183-190.

59.

Schlamp CL, Williams DS: Myosin V in the retina: localization in the rod photoreceptor synapse. Exp Eye Res 1996, 63:613-619.

60.

Breckler J, Burnside B: Myosin-I in retinal pigmented epithelial cells. Invest Ophtha/mol Vis Sci 1994, 35:2489-2499.