Myosin IIIB uses an actin-binding motif in its espin-1 cargo to reach the tips of actin protrusions

Myosin IIIA (MYO3A) targets actin protrusion tips using a motility mechanism dependent on both motor and tail actin-binding activity [1]. We show that myosin IIIB (MYO3B) lacks tail actin-binding activity and is unable to target COS7 cell filopodia tips, yet is somehow able to target stereocilia tips. Strikingly, when MYO3B is coexpressed with espin-1 (ESPN1), a MYO3A cargo protein endogenously expressed in stereocilia [2], MYO3B targets and carries ESPN1 to COS7 filopodia tips. We show that this tip localization is lost when we remove the ESPN1 C terminus actin-binding site. We also demonstrate that, like MYO3A [2], MYO3B can elongate filopodia by transporting ESPN1 to the polymerizing end of actin filaments. The mutual dependence of MYO3B and ESPN1 for tip localization reveals a novel mechanism for the cell to regulate myosin tip localization via a reciprocal relationship with cargo that directly participates in actin binding for motility. Our results are consistent with a novel form of motility for class III myosins that requires both motor and tail domain actin-binding activity and show that the actin-binding tail can be replaced by actin-binding cargo. This study also provides a framework to better understand the late-onset hearing loss phenotype in patients with MYO3A mutations.     

Localization of a class III myosin to filopodia tips in transfected HeLa cells requires an actin-binding site in its tail domain

Bass Myo3A, a class III myosin, was expressed in HeLa cells as a GFP fusion in order to study its cellular localization. GFP-Myo3A localized to the cytoplasm and to the tips of F-actin bundles in filopodia, a localization that is consistent with the observed concentration toward the distal ends of F-actin bundles in photoreceptor cells. A mutation in the motor active site resulted in a loss of filopodia localization, suggesting that Myo3A motor activity is required for filopodial tip localization. Deletion analyses showed that the NH2-terminal kinase domain is not required but the CO2H-terminal 22 amino acids of the Myo3A tail are required for filopodial localization. Expression of this tail fragment alone produced fluorescence associated with F-actin throughout the cytoplasm and filopodia and a recombinant tail fragment bound to F-actin in vitro. An actin-binding motif was identified within this tail fragment, and a mutation within this motif abolished both filopodia localization by Myo3A and F-actin binding by the tail fragment alone. Calmodulin localized to filopodial tips when coexpressed with Myo3A but not in the absence of Myo3A, an observation consistent with the previous proposal that class III myosins bind calmodulin and thereby localize it in certain cell types.     

A class III myosin expressed in the retina is a potential candidate for Bardet-Biedl syndrome

Class III myosins are actin-based motors with amino-terminal kinase domains. Expression of these motors is highly enhanced in retinal photoreceptors. As mutations in the gene encoding NINAC, a Drosophila melanogaster class III myosin, cause retinal degeneration, human homologs of this gene are potential candidates for human retinal disease. We have recently reported the cloning of MYO3A, a human myosin III expressed predominantly in the retina and retinal pigmented epithelium [1]. The map locus of MYO3A is close to, but does not overlap, that of human Usher's 1F [2]. Here we introduce a shorter class III myosin isoform, MYO3B, which is expressed in the retina, kidney, and testis. We describe the cDNA sequence, genomic organization, and splice variants of MYO3B expressed in the human retina. A product of 36 exons, MYO3B has several splice variants containing either one or two calmodulin binding (IQ) motifs in the neck domain and one of three predominant tail variations: a short tail ending just past the second IQ motif, or two alternatively spliced longer tails. MYO3B maps to 2q31.1-q31.2, a region that overlaps the locus for a Bardet-Biedl syndrome (BBS5) linked to markers at 2q31 [3].     

A heterologous in-cell assay for investigating intermicrovillar adhesion complex interactions reveals a novel protrusion length-matching mechanism

Solute transporting epithelial cells build arrays of microvilli on their apical surface to increase membrane scaffolding capacity and enhance function potential. In epithelial tissues such as the kidney and gut, microvilli are length-matched and assembled into tightly packed “brush borders,” which are organized by ∼50-nm thread-like links that form between the distal tips of adjacent protrusions. Composed of protocadherins CDHR2 and CDHR5, adhesion links are stabilized at the tips by a cytoplasmic tripartite module containing the scaffolds USH1C and ANKS4B and the actin-based motor MYO7B. Because several questions about the formation and function of this “intermicrovillar adhesion complex” remain open, we devised a system that allows one to study individual binary interactions between specific complex components and MYO7B. Our approach employs a chimeric myosin consisting of the MYO10 motor domain fused to the MYO7B cargo-binding tail domain. When expressed in HeLa cells, which do not normally produce adhesion complex proteins, this chimera trafficked to the tips of filopodia and was also able to transport individual complex components to these sites. Unexpectedly, the MYO10–MYO7B chimera was able to deliver CDHR2 and CDHR5 to distal tips in the absence of USH1C or ANKS4B. Cells engineered to localize high levels of CDHR2 at filopodial tips acquired interfilopodial adhesion and exhibited a striking dynamic length-matching activity that aligned distal tips over time. These findings deepen our understanding of mechanisms that promote the distal tip accumulation of intermicrovillar adhesion complex components and also offer insight on how epithelial cells minimize microvillar length variability.     

Myosin 3A Kinase Activity Is Regulated by Phosphorylation of the Kinase Domain Activation Loop

Class III myosins are unique members of the myosin superfamily in that they contain both a motor and kinase domain. We have found that motor activity is decreased by autophosphorylation, although little is known about the regulation of the kinase domain. We demonstrate by mass spectrometry that Thr-178 and Thr-184 in the kinase domain activation loop and two threonines in the loop 2 region of the motor domain are autophosphorylated (Thr-908 and Thr-919). The kinase activity of MYO3A 2IQ with the phosphomimic (T184E) or phosphoblock (T184A) mutations demonstrates that kinase activity is reduced 30-fold as a result of the T184A mutation, although the Thr-178 site only had a minor impact on kinase activity. Interestingly, the actin-activated ATPase activity of MYO3A 2IQ is slightly reduced as a result of the T178A and T184A mutations suggesting coupling between motor and kinase domains. Full-length GFP-tagged T184A and T184E MYO3A constructs transfected into COS7 cells do not disrupt the ability of MYO3A to localize to filopodia structures. In addition, we demonstrate that T184E MYO3A reduces filopodia elongation in the presence of espin-1, whereas T184A enhances filopodia elongation in a similar fashion to kinase-dead MYO3A. Our results suggest that as MYO3A accumulates at the tips of actin protrusions, autophosphorylation of Thr-184 enhances kinase activity resulting in phosphorylation of the MYO3A motor and reducing motor activity. The differential regulation of the kinase and motor activities allows for MYO3A to precisely self-regulate its concentration in the actin bundle-based structures of cells.     

Deafness mutation in the MYO3A motor domain impairs actin protrusion elongation mechanism

Class III myosins are actin-based motors proposed to transport cargo to the distal tips of stereocilia in the inner ear hair cells and/or to participate in stereocilia length regulation, which is especially important during development. Mutations in the MYO3A gene are associated with delayed onset deafness. A previous study demonstrated that L697W, a dominant deafness mutation, disrupts MYO3A ATPase and motor properties but does not impair its ability to localize to the tips of actin protrusions. In the current study, we characterized the transient kinetic mechanism of the L697W motor ATPase cycle. Our kinetic analysis demonstrates that the mutation slows the ADP release and ATP hydrolysis steps, which results in a slight reduction in the duty ratio and slows detachment kinetics. Fluorescence recovery after photobleaching (FRAP) of filopodia tip localized L697W and WT MYO3A in COS-7 cells revealed that the mutant does not alter turnover or average intensity at the actin protrusion tips. We demonstrate that the mutation slows filopodia extension velocity in COS-7 cells which correlates with its twofold slower in vitro actin gliding velocity. Overall, this work allowed us to propose a model for how the motor properties of MYO3A are crucial for facilitating actin protrusion length regulation.     

The Drosophila melanogaster retinophilin gene encodes the peripheral membrane protein in photoreceptor cells

The Drosophila melanogaster retinophilin (rtp) gene encoding the protein containing MORN (Membrane Occupation and Recognition Nexus) motifs that are known to interact with the plasma membrane has been identified to be expressed predominantly in adult eyes by several independent studies. Isolation and characterization of rtp mutant flies showed that the gene is involved in the phototransduction process by interacting with NINAC (neither inactivation nor afterpotential C) protein in adult photoreceptor cells. The gene was also reported to be involved in phagocytosis in embryos. We examined rtp gene expression during D. melanogaster development and in adult head tissues. The results showed that the gene is expressed at detectable levels only in adult photoreceptor cells but not in other developmental stages and other adult tissues, confirming its phototransduction functions. The RTP protein contains only 4 MORN motifs, whereas 8 MORN motifs are reported to be required for interactions with the plasma membrane. We found that RTP protein is present free in the cytosol and also is bound peripherally to the plasma membrane; this binding ability was found to be modulated by light. Our results suggest that the D. melanogaster RTP protein is a light-regulated peripheral membrane protein of photoreceptor cells.     

Myosin-VIIb, a novel unconventional myosin, is a constituent of microvilli in transporting epithelia

Mouse myosin-VIIb, a novel unconventional myosin, was cloned from the inner ear and kidney. The human myosin-VIIb (HGMW-approved symbol MYO7B) sequence and exon structure were then deduced from a human BAC clone. The mouse gene was mapped to chromosome 18, approximately 0.5 cM proximal to D18Mit12. The human gene location at 2q21.1 was deduced from the map location of the BAC and confirmed by fluorescence in situ hybridization. Myosin-VIIb has a conserved myosin head domain, five IQ domains, two MyTH4 domains coupled to two FERM domains, and an SH3 domain. A phylogenetic analysis based on the MyTH4 domains suggests that the coupled MyTH and FERM domains were duplicated in myosin evolution before separation into different classes. Myosin-VIIb is expressed primarily in kidney and intestine, as shown by Northern and immunoblot analyses. An antibody to myosin-VIIb labeled proximal tubule cells of the kidney and enterocytes of the intestine, specifically the distal tips of apical microvilli on these transporting epithelial cells.     

Structural basis of cargo recognition by the myosin-X MyTH4-FERM domain

Myosin-X is an important unconventional myosin that is critical for cargo transportation to filopodia tips and is also utilized in spindle assembly by interacting with microtubules. We present a series of structural and biochemical studies of the myosin-X tail domain cassette, consisting of myosin tail homology 4 (MyTH4) and FERM domains in complex with its specific cargo, a netrin receptor DCC (deleted in colorectal cancer). The MyTH4 domain is folded into a helical VHS-like structure and is associated with the FERM domain. We found an unexpected binding mode of the DCC peptide to the subdomain C groove of the FERM domain, which is distinct from previously reported β-β associations found in radixin-adhesion molecule complexes. We also revealed direct interactions between the MyTH4-FERM cassette and tubulin C-terminal acidic tails, and identified a positively charged patch of the MyTH4 domain, which is involved in tubulin binding. We demonstrated that both DCC and integrin bindings interfere with microtubule binding and that DCC binding interferes with integrin binding. Our results provide the molecular basis by which myosin-X facilitates alternative dual binding to cargos and microtubules.     

The tail of a yeast class V myosin, myo2p, functions as a localization domain

Myo2p is a yeast class V myosin that functions in membrane trafficking. To investigate the function of the carboxyl-terminal-tail domain of Myo2p, we have overexpressed this domain behind the regulatable GAL1 promoter (MYO2DN). Overexpression of the tail domain of Myo2p results in a dominant-negative phenotype that is phenotypically similar to a temperature-sensitive allele of myo2, myo2-66. The tail domain of Myo2p is sufficient for localization at low- expression levels and causes mislocalization of the endogenous Myo2p from sites of polarized cell growth. Subcellular fractionation of polarized, mechanically lysed yeast cells reveals that Myo2p is present predominantly in a 100,000 x g pellet. The Myo2p in this pellet is not solubilized by Mg++-ATP or Triton X-100, but is solubilized by high salt. Tail overexpression does not disrupt this fractionation pattern, nor do mutations in sec4, sec3, sec9, cdc42, or myo2. These results show that overexpression of the tail domain of Myo2p does not compete with the endogenous Myo2p for assembly into a pelletable structure, but does compete with the endogenous Myo2p for a factor that is necessary for localization to the bud tip.     

Synthetic lethality screen identifies a novel yeast myosin I gene (MYO5): myosin I proteins are required for polarization of the actin cytoskeleton

The organization of the actin cytoskeleton plays a critical role in cell physiology in motile and nonmotile organisms. Nonetheless, the function of the actin based motor molecules, members of the myosin superfamily, is not well understood. Deletion of MYO3, a yeast gene encoding a "classic" myosin I, has no detectable phenotype. We used a synthetic lethality screen to uncover genes whose functions might overlap with those of MYO3 and identified a second yeast myosin 1 gene, MYO5. MYO5 shows 86 and 62% identity to MYO3 across the motor and non- motor regions. Both genes contain an amino terminal motor domain, a neck region containing two IQ motifs, and a tail domain consisting of a positively charged region, a proline-rich region containing sequences implicated in ATP-insensitive actin binding, and an SH3 domain. Although myo5 deletion mutants have no detectable phenotype, yeast strains deleted for both MYO3 and MYO5 have severe defects in growth and actin cytoskeletal organization. Double deletion mutants also display phenotypes associated with actin disorganization including accumulation of intracellular membranes and vesicles, cell rounding, random bud site selection, sensitivity to high osmotic strength, and low pH as well as defects in chitin and cell wall deposition, invertase secretion, and fluid phase endocytosis. Indirect immunofluorescence studies using epitope-tagged Myo5p indicate that Myo5p is localized at actin patches. These results indicate that MYO3 and MYO5 encode classical myosin I proteins with overlapping functions and suggest a role for Myo3p and Myo5p in organization of the actin cytoskeleton of Saccharomyces cerevisiae.     

Rat myr 4 defines a novel subclass of myosin I: identification,distribution, localization,and mapping of calmodulin-binding sites with differential calcium sensitivity

We report the identification and characterization of myr 4 (myosin from rat), the first mammalian myosin I that is not closely related to brush border myosin I. Myr 4 contains a myosin head (motor) domain, a regulatory domain with light chain binding sites and a tail domain. Sequence analysis of myosin I head (motor) domains suggested that myr 4 defines a novel subclass of myosin I''s. This subclass is clearly different from the vertebrate brush border myosin I subclass (which includes myr 1) and the myosin I subclass(es) identified from Acanthamoeba castellanii and Dictyostelium discoideum. In accordance with this notion, a detailed sequence analysis of all myosin I tail domains revealed that the myr 4 tail is unique, except for a newly identified myosin I tail homology motif detected in all myosin I tail sequences. The Ca(2+)-binding protein calmodulin was demonstrated to be associated with myr 4. Calmodulin binding activity of myr 4 was mapped by gel overlay assays to the two consecutive light chain binding motifs (IQ motifs) present in the regulatory domain. These two binding sites differed in their Ca2+ requirements for optimal calmodulin binding. The NH2-terminal IQ motif bound calmodulin in the absence of free Ca2+, whereas the COOH-terminal IQ motif bound calmodulin in the presence of free Ca2+. A further Ca(2+)-dependent calmodulin binding site was mapped to amino acids 776-874 in the myr 4 tail domain. These results demonstrate a differential Ca2+ sensitivity for calmodulin binding by IQ motifs, and they suggest that myr 4 activity might be regulated by Ca2+/calmodulin. Myr 4 was demonstrated to be expressed in many cell lines and rat tissues with the highest level of expression in adult brain tissue. Its expression was developmentally regulated during rat brain ontogeny, rising 2-3 wk postnatally, and being maximal in adult brain. Immunofluorescence localization demonstrated that myr 4 is expressed in subpopulations of neurons. In these neurons, prominent punctate staining was detected in cell bodies and apical dendrites. A punctate staining that did not obviously colocalize with the bulk of F- actin was also observed in C6 rat glioma cells. The observed punctate staining for myr 4 is reminiscent of a membranous localization.     

Localization of Myosin 1b to Actin Protrusions Requires Phosphoinositide Binding

Myosin 1b (Myo1b), a class I myosin, is a widely expressed, single-headed, actin-associated molecular motor. Transient kinetic and single-molecule studies indicate that it is kinetically slow and responds to tension. Localization and subcellular fractionation studies indicate that Myo1b associates with the plasma membrane and certain subcellular organelles such as endosomes and lysosomes. Whether Myo1b directly associates with membranes is unknown. We demonstrate here that full-length rat Myo1b binds specifically and with high affinity to phosphatidylinositol 4,5-bisphosphate (PIP₂) and phosphatidylinositol 3,4,5-triphosphate (PIP₃), two phosphoinositides that play important roles in cell signaling. Binding is not Ca²⁺-dependent and does not involve the calmodulin-binding IQ region in the neck domain of Myo1b. Furthermore, the binding site is contained entirely within the C-terminal tail region, which contains a putative pleckstrin homology domain. Single mutations in the putative pleckstrin homology domain abolish binding of the tail domain of Myo1b to PIP₂ and PIP₃ in vitro. These same mutations alter the distribution of Myc-tagged Myo1b at membrane protrusions in HeLa cells where PIP₂ localizes. In addition, we found that motor activity is required for Myo1b localization in filopodia. These results suggest that binding of Myo1b to phosphoinositides plays an important role in vivo by regulating localization to actin-enriched membrane projections.     

Structure and Regulation of the Movement of Human Myosin VIIA

Human myosin VIIA (HM7A) is responsible for human Usher syndrome type 1B, which causes hearing and visual loss in humans. Here we studied the regulation of HM7A. The actin-activated ATPase activity of full-length HM7A (HM7AFull) was lower than that of tail-truncated HM7A (HM7AΔTail). Deletion of the C-terminal 40 amino acids and mutation of the basic residues in this region (R2176A or K2179A) abolished the inhibition. Electron microscopy revealed that HM7AFull is a monomer in which the tail domain bends back toward the head-neck domain to form a compact structure. This compact structure is extended at high ionic strength or in the presence of Ca²⁺. Although myosin VIIA has five isoleucine-glutamine (IQ) motifs, the neck length seems to be shorter than the expected length of five bound calmodulins. Supporting this observation, the IQ domain bound only three calmodulins in Ca²⁺, and the first IQ motif failed to bind calmodulin in EGTA. These results suggest that the unique IQ domain of HM7A is important for the tail-neck interaction and, therefore, regulation. Cellular studies revealed that dimer formation of HM7A is critical for its translocation to filopodial tips and that the tail domain (HM7ATail) markedly reduced the filopodial tip localization of the HM7AΔTail dimer, suggesting that the tail-inhibition mechanism is operating in vivo. The translocation of the HM7AFull dimer was significantly less than that of the HM7AΔTail dimer, and R2176A/R2179A mutation rescued the filopodial tip translocation. These results suggest that HM7A can transport its cargo molecules, such as USH1 proteins, upon release of the tail-dependent inhibition.     

My oh my(osin): Insights into how auditory hair cells count,measure, and shape

The mechanisms underlying mechanosensory hair bundle formation in auditory sensory cells are largely mysterious. In this issue, Lelli et al. (2016. J. Cell Biol. http://dx.doi.org/10.1083/jcb.201509017) reveal that a pair of molecular motors, myosin IIIa and myosin IIIb, is involved in the hair bundle’s morphology and hearing.The mammalian cochleae are lined with hair cells, each with a precisely arranged, morphologically polar hair bundle that captures energy produced by sound stimuli. A fascinating problem in cell biology is how this elaborately structured mechanosensory hair bundle forms with precision to enable hearing. Each hair bundle is comprised of up to a few hundred actin-filled stereocilia, arrayed in a staircase-like pattern of increasing heights. Deflection of a hair bundle in the direction of the tallest stereocilium opens mechanically gated ion channels at the stereociliary tips, allowing an influx of cations from the endolymph bathing the stereocilia and thus depolarizing the cell. The staircase array of stereocilia forms by elongation of microvilli at the apical surface of the developing hair cell (Tilney et al., 1992). Over 20 years ago, Tilney et al. (1992) detailed the steps of stereociliogenesis as observed by electron microscopy. In this issue, Lelli et al. provide molecular insight into how hair cells count, measure, and shape stereocilia.Loss of hair cells is a major cause of human hearing loss, which is often the result of genetic mutations affecting the development of stereocilia (Raphael, 2002). Mutations in genes encoding several different myosin motor proteins in stereocilia have been associated with human hearing loss, including myosin VIIA (Weil et al., 1995), myosin VI (Avraham et al., 1995; Melchionda et al., 2001), and myosin XVA (Wang et al., 1998). Additionally, loss-of-function mutations in MYO3A, encoding myosin IIIA, are responsible for hereditary progressive hearing loss DFNB30 (Walsh et al., 2002).Myosins, the ATP-dependent motors that move along actin-based filaments, are typically composed of three functional domains: the head, the neck, and the tail domains (Krendel and Mooseker, 2005). Class III myosins are unconventional myosins that each have a kinase domain at their N terminus regulated by PKA phosphorylation and autophosphorylation (Kempler et al., 2007). The kinase activity of myosin III was shown to act on its own motor domain to reduce the motor activity. Myosin III proteins concentrate at actin-based cellular protrusions, such as stereocilia of inner ear hair cells (Dosé et al., 2003; Schneider et al., 2006). The two class III isoforms found in vertebrates, myosin IIIa and IIIb, differ toward their C termini: myosin IIIa is longer than myosin IIIb and has one additional actin-binding domain, myosin III tail homology domain II (3THDII). In cultured cells, whereas myosin IIIa localizes to the tips of filopodia on its own, previous work showed that myosin IIIb requires its interaction partner espin-1, an actin-binding protein, for localization to filopodia tips (Merritt et al., 2012). Mutagenesis studies performed on myosin IIIa revealed a relationship between the phosphorylation state of myosin IIIa and the length and density of filopodia (Quintero et al., 2013). How myosin IIIa and IIIb work individually or together to regulate stereociliogenesis remains to be investigated.In this issue, Lelli et al. (2016) use Myo3a (Myo3a^−/−), Myo3b (Myo3b^−/−), and double (Myo3a^−/−Myo3b^−/−) knockout mice to dissect the complex roles of myosin IIIa and IIIb in hearing. Mice null for myosin IIIa, Myo3a^−/−, were initially normal but showed defects in hearing quality from 2 to 4 mo of age, as detected by auditory brain stem response measurements, which monitor the electrical response of the auditory pathway to short sound stimuli, but not by distortion product otoacoustic emissions, which test outer hair cell (OHC) function. These results indicated that inner hair cells (IHCs) are deleteriously impacted but that OHCs are not, and that these mice had progressive hearing loss. Myo3b^−/− mutants had normal hearing, indicating that redundancy with myosin IIIa may obscure the role of this protein in single knockouts. Investigation of double knockouts, however, clarified the redundant functions of the two isoforms, as the mice were profoundly deaf at 1 mo of age.Interestingly, Lelli et al. (2016) investigated the potential requirement for myosin IIIa in the maintenance of hearing. They generated conditional knockout (cKO) mice, Myo3a-cKO, in which myosin IIIa is inactivated postnatally in a Myo3b^−/− background. These animals hear normally up to at least 6 mo of age. Therefore, myosin IIIa is required during a critical period in auditory system development but is not required to maintain hearing. The researchers further studied the relationship between myosin IIIa and myosin IIIb in the Myo3a-cKO mice and noted an increase in auditory brain stem response thresholds. This fascinating result likely indicates that inactivation of myosin IIIa elicits a pernicious effect of myosin IIIb at mature stages. Determining the mechanism by which myosin IIIb damages hearing in the absence of myosin IIIa and whether it is due to its lack of a single 3THDII domain may yield insight into the roles of class III myosins in hearing.Given that myosin III proteins localize to the tips of stereocilia and are required for hearing, Lelli et al. (2016) explored the morphology of hair bundles lacking these proteins. Although double knockout Myo3a^−/−Myo3b^−/− mice displayed normal positioning of the kinocilium, the cilium reflecting the initial position and polarity of the developing hair bundle, and of asymmetric cell division proteins Par-6 and Gαi₃, which constrain the shape and positioning of the hair bundle, many hair cells from these mice exhibited hair bundle abnormalities. These abnormalities were first described during embryonic auditory hair bundle morphogenesis, at which time 19% of IHCs and 81% of OHCs displayed misshapen bundles. Several of these misshapen IHC and OHC bundles also contained abnormally long, ungraded protrusions, which Lelli et al. (2016) referred to as “long amorphous” bundles. By birth, most IHCs displayed long amorphous bundles. Although most OHC hair bundles were abnormally shaped, they had developed normal staircase organization of their stereocilia heights, and the long amorphous protrusions were no longer present. Presumably, another molecular mechanism comes into play and “corrects” the protrusions to establish the staircase organization. Knockout of candidate proteins could be an interesting strategy to uncover this mechanism in the future. Furthermore, other stereociliary phenotypes of Myo3a^−/−Myo3b^−/− mutant mice are also intriguing. Do myosin III proteins serve different mechanistic roles in IHCs and OHCs to give rise to different phenotypes? Or, are there other key factors that drive stereocilia into various degrees of hair bundle defects in the absence of myosin III proteins?In Myo3a^−/−Myo3b^−/− mice, many of the OHC bundles had abnormal side rows of extra stereocilia, which sometimes closed the bundle off into a circular shape. Interestingly, the stereocilia in these abnormal bundles were also significantly taller than in controls. Although the height of the tallest row of stereocilia in OHCs normally decreases after birth (Sekerková et al., 2011), the height of these stereocilia did not change in the Myo3a^−/−Myo3b^−/− mice, leading to an increased height difference 9 d after birth. The increased height and number of stereocilia in Myo3a^−/−Myo3b^−/− hair bundles is suggestive of unstable actin dynamics. The authors suggest that class III myosins stabilize the F-actin cores of the stereocilia, thus controlling their selective elongation by limiting their growth (Fig. 1). Surprisingly, class III myosins, which presumably climb the full lengths of the stereocilia to perch themselves near the tips, act to restrict their growth. This result seems to contrast with previous work in which myosin IIIa had been proposed to promote elongation of the stereocilia by transporting espin-1 to the stereocilia tips (Salles et al., 2009). Interestingly, Lelli et al. (2016) found that espin-1 was still properly targeted to the tips of stereocilia in Myo3a^−/−Myo3b^−/− mutant mice. The researchers extended the study to another binding partner of myosin IIIa, retinophilin/MORN4. Surprisingly, MORN4 was normally targeted to stereocilia tips in Myo3a^−/−Myo3b^−/− mutant mice. This suggests that there may be redundancy in the mechanisms of transport of stereocilia tip proteins. Whether or not distinct myosin isoforms interchange cargoes so that these cargoes are properly localized is a relevant avenue of future investigation. Furthermore, identification and knockout of class III myosin binding partners could help define the mechanism by which these proteins control elongation of stereocilia.Open in a separate windowFigure 1.Myosin IIIa and myosin IIIb regulate stereociliary length. (top) Schematics of myosin IIIa and myosin IIIb. The stop signs indicate the role these proteins have in limiting the heights of stereocilia. (bottom) A single stereocilium with wild-type copies of myosin IIIa and myosin IIIb is shorter than a stereocilium from a Myo3a^−/−Myo3b^−/− mutant when stereocilia from equivalent positions between hair bundles are compared. In addition to characterizing the lengths of stereocilia, Lelli et al. (2016) noted transient defects in the numbers of stereocilia during development of hair cells within genetically altered mice. At birth, Myo3a^−/−Myo3b^−/− mice had an increased number and density of stereocilia in both IHCs and OHCs as compared with controls. However, 9 d after birth, the number and density of the stereocilia had decreased to levels similar to those seen in control animals. Their results indicate that class III myosins help to control the initial selection of microvilli to become stereocilia, but a different, dominant molecular mechanism overrides this step and is likely involved in refining the number of stereocilia at later, postnatal stages. Identification of this mechanism by knockout of candidate molecules in the Myo3a^−/−Myo3b^−/− mice would be of interest for future work. The defects, though transient, indicate that myosins are key molecular components that establish how cells count the number of protrusions to be produced.In this work, Lelli et al. (2016) determined that myosins IIIa and IIIb are required for normal hair bundle development and hearing. These motor proteins influence the number and lengths of stereocilia to be produced and the overarching shape of the hair bundle. Evaluating the importance of the kinase domains of myosin IIIa and IIIb in hair bundle development is an important next step. The phosphorylation states of proteins in cellular protrusions, such as filopodia and stereocilia, can be regulatory. For example, phosphorylation of the actin cross-linking protein fascin 2b reduces this protein’s capacity to lengthen filopodia (Chou et al., 2011). Moreover, phosphorylation of fascin 2b increases the exchange rate of this protein within zebrafish stereocilia (Hwang et al., 2015). Additionally, in other systems, PKA modulates a signaling cascade that activates the actin-severing protein cofilin to control actin-filament dynamics (Nadella et al., 2009). Future work will be needed to test whether signal transduction stemming from the kinase domains of class III myosins are consequential for hair bundle development.Lelli et al. (2016) observed that the constitutive absence of both class III myosins leads to deafness as a result of hair bundle developmental defects. However, mice deficient for only one of the isoforms did not display such a striking hearing phenotype, indicating that myosins IIIa and IIIb compensate for the loss of one another in the developing cochlea. A role for myosin IIIa in the maintenance of stereocilia had been suggested by the discovery of loss-of-function mutations in MYO3A in patients with hereditary progressive hearing loss DFNB30 (Walsh et al., 2002). Remarkably, the analyses of Myo3a^−/−Myo3b^−/− and Myo3a-cKO Myo3b^−/− mice showed that the redundancy between myosins IIIa and IIIb is critical during hair bundle formation, but not during later, mature stages. Furthermore, the hearing impairment seen in Myo3a-cKO mice suggests that myosin IIIb exerts deleterious effects on hearing past the developmental stages of hair bundle morphogenesis. Although the mechanisms at work are unclear, these findings suggest that the down-regulation of MYO3B expression might be an effective way of preventing late-onset hearing loss in patients with MYO3A mutations.     

The kinase domain alters the kinetic properties of the myosin IIIA motor

Myosin IIIA is unique among myosin proteins in that it contains an N-terminal kinase domain capable of autophosphorylating sites on the motor domain. A construct of myosin IIIA lacking the kinase domain localizes more efficiently to the stereocilia tips and alters the morphology of the tips in inner ear hair cells. Therefore, we performed a kinetic analysis of myosin IIIA without the kinase domain (MIII DeltaK) and compared these results with our reported analysis of myosin IIIA containing the kinase domain (MIII). The steady-state kinetic properties of MIII DeltaK indicate that it has a 2-fold higher maximum actin-activated ATPase rate (kcat = 1.5 +/- 0.1 s-1) and a 5-fold tighter actin affinity (KATPase = 6.0 +/- 1.4 microM, and KActin = 1.4 +/- 0.4 microM) compared to MIII. The rate of ATP binding to the motor domain is enhanced in MIII DeltaK (K1k+2 approximately 0.10 +/- 0.01 microM-1.s-1) to a level similar to the rate of binding to MIII in the presence of actin. The rate of ATP hydrolysis in the absence of actin is slow and may be rate limiting. Actin-activated phosphate release is identical with and without the kinase domain. The transition between actomyosin.ADP states, which is rate limiting in MIII, is enhanced in MIII DeltaK. MIII DeltaK accumulates more efficiently at the tips of filopodia in HeLa cells. Our results suggest a model in which the activity and concentration of myosin IIIA localized to the tips of actin bundles mediates the morphology of the tips in sensory cells.     

Functional characterization of myosin I tail regions in Candida albicans

The molecular motor myosin I is required for hyphal growth in the pathogenic yeast Candida albicans. Specific myosin I functions were investigated by a deletion analysis of five neck and tail regions. Hyphal formation requires both the TH1 region and the IQ motifs. The TH2 region is important for optimal hyphal growth. All of the regions, except for the SH3 and acidic (A) regions that were examined individually, were required for the localization of myosin I at the hyphal tip. Similarly, all of the domains were required for the association of myosin I with pelletable actin-bound complexes. Moreover, the hyphal tip localization of cortical actin patches, identified by both rhodamine-phalloidin staining and Arp3-green fluorescent protein signals, was dependent on myosin I. Double deletion of the A and SH3 domains depolarized the distribution of the cortical actin patches without affecting the ability of the mutant to form hyphae, suggesting that myosin I has distinct functions in these processes. Among the six myosin I tail domain mutants, the ability to form hyphae was strictly correlated with endocytosis. We propose that the uptake of cell wall remodeling enzymes and excess plasma membrane is critical for hyphal formation.     

Direct localization of monoclonal antibody-binding sites on Acanthamoeba myosin-II and inhibition of filament formation by antibodies that bind to specific sites on the myosin-II tail

Electron microscopy of myosin-II molecules and filaments reacted with monoclonal antibodies demonstrates directly where the antibodies bind and shows that certain antibodies can inhibit the polymerization of myosin-II into filaments. The binding sites of seven of 23 different monoclonal antibodies were localized by platinum shadowing of myosin monomer-antibody complexes. The antibodies bind to a variety of sites on the myosin-II molecule, including the heads, the proximal end of the tail near the junction of the heads and tail, and the tip of the tail. The binding sites of eight of the 23 antibodies were also localized on myosin filaments by negative staining. Antibodies that bind to either the myosin heads or to the proximal end of the tail decorate the ends of the bipolar filaments. Some of the antibodies that bind to the tip of the myosin-II tail decorate the bare zone of the myosin-II thin filament with 14-nm periodicity. By combining the data from these electron microscope studies and the peptide mapping and competitive binding studies we have established the binding sites of 16 of 23 monoclonal antibodies. Two of the 23 antibodies block the formation of myosin-II filaments and given sufficient time, disassemble preformed myosin-II filaments. Both antibodies bind near one another at the tip of the myosin-II tail and are those that decorate the bare zone of preformed bipolar filaments with 14-nm periodicity. None of the other antibodies affect myosin filament formation, including one that binds to another site near the tip of the myosin-II tail. This demonstrates that antibodies can inhibit polymerization of myosin-II, but only when they bind to key sites on the tail of the molecule.     

The Myosin IXb motor activity targets the myosin IXb RhoGAP domain as cargo to sites of actin polymerization

Myosin IXb (Myo9b) is a single-headed processive myosin that exhibits Rho GTPase-activating protein (RhoGAP) activity in its tail region. Using live cell imaging, we determined that Myo9b is recruited to extending lamellipodia, ruffles, and filopodia, the regions of active actin polymerization. A functional motor domain was both necessary and sufficient for targeting Myo9b to these regions. The head domains of class IX myosins comprise a large insertion in loop2. Deletion of the large Myo9b head loop 2 insertion abrogated the enrichment in extending lamellipodia and ruffles, but enhanced significantly the enrichment at the tips of filopodia and retraction fibers. The enrichment in the tips of filopodia and retraction fibers depended on four lysine residues C-terminal to the loop 2 insertion and the tail region. Fluorescence recovery after photobleaching and photoactivation experiments in lamellipodia revealed that the dynamics of Myo9b was comparable to that of actin. The exchange rates depended on the Myo9b motor region and motor activity, and they were also dependent on the turnover of F-actin. These results demonstrate that Myo9b functions as a motorized RhoGAP molecule in regions of actin polymerization and identify Myo9b head sequences important for in vivo motor properties.