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.     

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.     

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.     

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.     

Ca2+ entry through mechanotransduction channels localizes BAIAP2L2 to stereocilia tips

Brain-specific angiogenesis inhibitor 1-associated protein 2-like protein 2 (BAIAP2L2), a membrane-binding protein required for the maintenance of mechanotransduction in hair cells, is selectively retained at the tips of transducing stereocilia. BAIAP2L2 trafficked to stereocilia tips in the absence of EPS8, but EPS8 increased the efficiency of localization. A tripartite complex of BAIAP2L2, EPS8, and MYO15A formed efficiently in vitro, and these three proteins robustly targeted to filopodia tips when coexpressed in cultured cells. Mice lacking functional transduction channels no longer concentrated BAIAP2L2 at row 2 stereocilia tips, a result that was phenocopied by blocking channels with tubocurarine in cochlear explants. Transduction channels permit Ca²⁺ entry into stereocilia, and we found that membrane localization of BAIAP2L2 was enhanced in the presence of Ca²⁺. Finally, reduction of intracellular Ca²⁺ in hair cells using BAPTA-AM led to a loss of BAIAP2L2 at stereocilia tips. Taken together, our results show that a MYO15A-EPS8 complex transports BAIAP2L2 to stereocilia tips, and Ca²⁺ entry through open channels at row 2 tips retains BAIAP2L2 there.     

Role of myosin VI in the differentiation of cochlear hair cells.

The mouse mutant Snell's waltzer (sv) has an intragenic deletion of the Myo6 gene, which encodes the unconventional myosin molecule myosin VI (K. B. Avraham et al., 1995, Nat. Genet. 11, 369-375). Snell's waltzer mutants exhibit behavioural abnormalities suggestive of an inner ear defect, including lack of responsiveness to sound, hyperactivity, head tossing, and circling. We have investigated the effects of a lack of myosin VI on the development of the sensory hair cells of the cochlea in these mutants. In normal mice, the hair cells sprout microvilli on their upper surface, and some of these grow to form a crescent or V-shaped array of modified microvilli, the stereocilia. In the mutants, early stages of stereocilia development appear to proceed normally because at birth many stereocilia bundles have a normal appearance, but in places there are signs of disorganisation of the bundles. Over the next few days, the stereocilia become progressively more disorganised and fuse together. Practically all hair cells show fused stereocilia by 3 days after birth, and there is extensive stereocilia fusion by 7 days. By 20 days, giant stereocilia are observed on top of the hair cells. At 1 and 3 days after birth, hair cells of mutants and controls take up the membrane dye FM1-43, suggesting that endocytosis occurs in mutant hair cells. One possible model for the fusion is that myosin VI may be involved in anchoring the apical hair cell membrane to the underlying actin-rich cuticular plate, and in the absence of normal myosin VI this apical membrane will tend to pull up between stereocilia, leading to fusion.     

The MYO6 interactome reveals adaptor complexes coordinating early endosome and cytoskeletal dynamics

The intracellular functions of myosin motors requires a number of adaptor molecules, which control cargo attachment, but also fine‐tune motor activity in time and space. These motor–adaptor–cargo interactions are often weak, transient or highly regulated. To overcome these problems, we use a proximity labelling‐based proteomics strategy to map the interactome of the unique minus end‐directed actin motor MYO6. Detailed biochemical and functional analysis identified several distinct MYO6‐adaptor modules including two complexes containing RhoGEFs: the LIFT (LARG‐Induced F‐actin for Tethering) complex that controls endosome positioning and motility through RHO‐driven actin polymerisation; and the DISP (DOCK7‐Induced Septin disPlacement) complex, a novel regulator of the septin cytoskeleton. These complexes emphasise the role of MYO6 in coordinating endosome dynamics and cytoskeletal architecture. This study provides the first in vivo interactome of a myosin motor protein and highlights the power of this approach in uncovering dynamic and functionally diverse myosin motor complexes.     

Mosaic complementation demonstrates a regulatory role for myosin VIIa in actin dynamics of stereocilia

We have developed a bacterial artificial chromosome transgenesis approach that allowed the expression of myosin VIIa from the mouse X chromosome. We demonstrated the complementation of the Myo7a null mutant phenotype producing a fine mosaic of two types of sensory hair cells within inner ear epithelia of hemizygous transgenic females due to X inactivation. Direct comparisons between neighboring auditory hair cells that were different only with respect to myosin VIIa expression revealed that mutant stereocilia are significantly longer than those of their complemented counterparts. Myosin VIIa-deficient hair cells showed an abnormally persistent tip localization of whirlin, a protein directly linked to elongation of stereocilia, in stereocilia. Furthermore, myosin VIIa localized at the tips of all abnormally short stereocilia of mice deficient for either myosin XVa or whirlin. Our results strongly suggest that myosin VIIa regulates the establishment of a setpoint for stereocilium heights, and this novel role may influence their normal staircase-like arrangement within a bundle.     

Development of the hair bundle and mechanotransduction

This review focuses on the cellular and molecular mechanisms underlying the development of the sensory hair bundle, an apical specialisation of the hair cell that is essential for mechanotransduction. The structure, function and development of the hair bundle is described, with an emphasis on the properties and possible roles played by the different link types that interconnect the individual elements of the hair bundle - the multiple stereocilia and the single kinocilium. Studies of mouse and zebrafish mutants have revealed that several classes of molecule are required for the genesis and maintenance of hair-bundle structure. These include cell surface molecules that are associated with the different hair-bundle links, along with myosin motors, scaffolding proteins and an actin cross-linker. Finally we consider how differences in the form and shape of hair bundles within and between different sensory organs are generated.     

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].     

Myosin VIIa,harmonin and cadherin 23, three Usher I gene products that cooperate to shape the sensory hair cell bundle

Deaf-blindness in three distinct genetic forms of Usher type I syndrome (USH1) is caused by defects in myosin VIIa, harmonin and cadherin 23. Despite being critical for hearing, the functions of these proteins in the inner ear remain elusive. Here we show that harmonin, a PDZ domain-containing protein, and cadherin 23 are both present in the growing stereocilia and that they bind to each other. Moreover, we demonstrate that harmonin b is an F-actin-bundling protein, which is thus likely to anchor cadherin 23 to the stereocilia microfilaments, thereby identifying a novel anchorage mode of the cadherins to the actin cytoskeleton. Moreover, harmonin b interacts directly with myosin VIIa, and is absent from the disorganized hair bundles of myosin VIIa mutant mice, suggesting that myosin VIIa conveys harmonin b along the actin core of the developing stereocilia. We propose that the shaping of the hair bundle relies on a functional unit composed of myosin VIIa, harmonin b and cadherin 23 that is essential to ensure the cohesion of the stereocilia.     

Characterization of a myosin VII MyTH/FERM domain

A group of closely related myosins is characterized by the presence of at least one MyTH/FERM (myosin tail homology; band 4.1, ezrin, radixin, moesin) domain in their C-terminal tails. This domain interacts with a variety of binding partners, and mutations in either the MyTH4 or the FERM domain of myosin VII and myosin XV result in deafness, highlighting the functional importance of each domain. The N-terminal MyTH/FERM region of Dictyostelium myosin VII (M7) has been isolated as a first step toward gaining insight into the function of this domain and its interaction with binding partners. The M7 MyTH4/FERM domain (MF1) binds to both actin and microtubules in vitro, with dissociation constants of 13.7 and 1.7 μM, respectively. Gel filtration and UV spectroscopy reveal that MF1 exists as a monomer in solution and forms a well-folded, compact conformation with a high degree of secondary structure. These results indicate that MF1 forms an integrated structural domain that serves to couple actin filaments and microtubules in specific regions of the cytoskeleton.     

A Myo6 mutation destroys coordination between the myosin heads, revealing new functions of myosin VI in the stereocilia of mammalian inner ear hair cells

Myosin VI, found in organisms from Caenorhabditis elegans to humans, is essential for auditory and vestibular function in mammals, since genetic mutations lead to hearing impairment and vestibular dysfunction in both humans and mice. Here, we show that a missense mutation in this molecular motor in an ENU-generated mouse model, Tailchaser, disrupts myosin VI function. Structural changes in the Tailchaser hair bundles include mislocalization of the kinocilia and branching of stereocilia. Transfection of GFP-labeled myosin VI into epithelial cells and delivery of endocytic vesicles to the early endosome revealed that the mutant phenotype displays disrupted motor function. The actin-activated ATPase rates measured for the D179Y mutation are decreased, and indicate loss of coordination of the myosin VI heads or ‘gating’ in the dimer form. Proper coordination is required for walking processively along, or anchoring to, actin filaments, and is apparently destroyed by the proximity of the mutation to the nucleotide-binding pocket. This loss of myosin VI function may not allow myosin VI to transport its cargoes appropriately at the base and within the stereocilia, or to anchor the membrane of stereocilia to actin filaments via its cargos, both of which lead to structural changes in the stereocilia of myosin VI–impaired hair cells, and ultimately leading to deafness.     

The many different cellular functions of MYO7A in the retina

Mutations in MYO7A (myosin VIIa) cause Usher syndrome type?1B, a disorder involving profound congenital deafness and progressive blindness. In the retina, most MYO7A is localized in the apical region of the RPE (retinal pigmented epithelial) cells, and a small amount is associated with the ciliary and periciliary membranes of the photoreceptor cells. Its roles appear to be quite varied. Studies with MYO7A-null mice indicate that MYO7A participates in the apical localization of RPE melanosomes and in the removal of phagosomes from the apical RPE for their delivery to lysosomes in the basal RPE. In the first role, MYO7A competes with microtubule motors, but in the second one, it may function co-operatively. An additional role of MYO7A in the RPE is indicated by the requirement for it in the light-dependent translocation of the ER (endoplasmic reticulum)-associated visual cycle enzyme RPE65 and normal functioning of the visual retinoid cycle. In photoreceptor cells lacking MYO7A, opsin accumulates abnormally in the transition zone of the cilium, suggesting that MYO7A functions as a selective barrier for membrane proteins at the distal end of the transition zone. It is likely that the progressive retinal degeneration that occurs in Usher syndrome 1B patients results from a combination of cellular defects in the RPE and photoreceptor cells.     

Protein localization by actin treadmilling and molecular motors regulates stereocilia shape and treadmilling rate

We present a physical model that describes the active localization of actin-regulating proteins inside stereocilia during steady-state conditions. The mechanism of localization is through the interplay of free diffusion and directed motion, which is driven by coupling to the treadmilling actin filaments and to myosin motors that move along the actin filaments. The resulting localization of both the molecular motors and their cargo is calculated, and is found to have an exponential (or steeper) profile. This localization can be at the base (driven by actin retrograde flow and minus-end myosin motors), or at the stereocilia tip (driven by plus-end myosin motors). The localization of proteins that influence the actin depolymerization and polymerization rates allow us to describe the narrow shape of the stereocilia base, and the observed increase of the actin polymerization rate with the stereocilia height.     

Correction: Talin tension sensor reveals novel features of focal adhesion force transmission and mechanosensitivity

The precise architecture of hair bundles, the arrays of mechanosensitive microvilli-like stereocilia crowning the auditory hair cells, is essential to hearing. Myosin IIIa, defective in the late-onset deafness form DFNB30, has been proposed to transport espin-1 to the tips of stereocilia, thereby promoting their elongation. We show that Myo3a^−/−Myo3b^−/− mice lacking myosin IIIa and myosin IIIb are profoundly deaf, whereas Myo3a-cKO Myo3b^−/− mice lacking myosin IIIb and losing myosin IIIa postnatally have normal hearing. Myo3a^−/−Myo3b^−/− cochlear hair bundles display robust mechanoelectrical transduction currents with normal kinetics but show severe embryonic abnormalities whose features rapidly change. These include abnormally tall and numerous microvilli or stereocilia, ungraded stereocilia bundles, and bundle rounding and closure. Surprisingly, espin-1 is properly targeted to Myo3a^−/−Myo3b^−/− stereocilia tips. Our results uncover the critical role that class III myosins play redundantly in hair-bundle morphogenesis; they unexpectedly limit the elongation of stereocilia and of subsequently regressing microvilli, thus contributing to the early hair bundle shaping.     

Invertebrate and Vertebrate Class III Myosins Interact with MORN Repeat-Containing Adaptor Proteins

In Drosophila photoreceptors, the NINAC-encoded myosin III is found in a complex with a small, MORN-repeat containing, protein Retinophilin (RTP). Expression of these two proteins in other cell types showed NINAC myosin III behavior is altered by RTP. NINAC deletion constructs were used to map the RTP binding site within the proximal tail domain of NINAC. In vertebrates, the RTP ortholog is MORN4. Co-precipitation experiments demonstrated that human MORN4 binds to human myosin IIIA (MYO3A). In COS7 cells, MORN4 and MYO3A, but not MORN4 and MYO3B, co-localize to actin rich filopodia extensions. Deletion analysis mapped the MORN4 binding to the proximal region of the MYO3A tail domain. MYO3A dependent MORN4 tip localization suggests that MYO3A functions as a motor that transports MORN4 to the filopodia tips and MORN4 may enhance MYO3A tip localization by tethering it to the plasma membrane at the protrusion tips. These results establish conserved features of the RTP/MORN4 family: they bind within the tail domain of myosin IIIs to control their behavior.