The Experts below are selected from a list of 1530 Experts worldwide ranked by ideXlab platform
Christine Petit - One of the best experts on this subject based on the ideXlab platform.
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Cadherin-23, Myosin VIIa and harmonin, encoded by Usher syndrome type I genes, form a ternary complex and interact with membrane phospholipids
Human molecular genetics, 2010Co-Authors: Amel Bahloul, Jean-pierre Hardelin, Sylvie Nouaille, Vincent Michel, Sylviane Hoos, Anne Houdusse, Patrick England, Christine PetitAbstract:Cadherin-23 is a component of early transient lateral links of the auditory sensory cells' hair bundle, the mechanoreceptive structure to sound. This protein also makes up the upper part of the tip links that control gating of the mechanoelectrical transduction channels. We addressed the issue of the molecular complex that anchors these links to the hair bundle F-actin core. By using surface plasmon resonance assays, we show that the cytoplasmic regions of the two cadherin-23 isoforms that do or do not contain the exon68-encoded peptide directly interact with harmonin, a submembrane PDZ (post-synaptic density, disc large, zonula occludens) domain-containing protein, with unusually high affinity. This interaction involves the harmonin Nter-PDZ1 supramodule, but not the C-terminal PDZ-binding motif of cadherin-23. We establish that cadherin-23 directly binds to the tail of Myosin VIIa. Moreover, cadherin-23, harmonin and Myosin VIIa can form a ternary complex, which suggests that Myosin VIIa applies tension forces on hair bundle links. We also show that the cadherin-23 cytoplasmic region, harmonin and Myosin VIIa interact with phospholipids on synthetic liposomes. Harmonin and the cytoplasmic region of cadherin-23, both independently and as a binary complex, can bind specifically to phosphatidylinositol 4,5-bisphosphate (PI(4,5)P(2)), which may account for the role of this phospholipid in the adaptation of mechanoelectrical transduction in the hair bundle. The distributions of cadherin-23, harmonin, Myosin VIIa and PI(4,5)P(2) in the growing and mature auditory hair bundles as well as the abnormal locations of harmonin and Myosin VIIa in cadherin-23 null mutant mice strongly support the functional relevance of these interactions.
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Shroom2, a Myosin-VIIa- and actin-binding protein, directly interacts with ZO-1 at tight junctions
Journal of Cell Science, 2007Co-Authors: Raphael Etournay, Pierre Legrain, Christine Petit, Isabelle Perfettini, Ingrid Zwaenepoel, Aziz El-amraouiAbstract:International audienceDefects in Myosin VIIa lead to developmental anomalies of the auditory and visual sensory cells. We sought proteins interacting with the Myosin VIIa tail by using the yeast two-hybrid system. Here, we report on shroom2, a submembranous PDZ domain-containing protein that is associated with the tight junctions in multiple embryonic and adult epithelia. Shroom2 directly interacts with the C-terminal MyTH4-FERM domain of Myosin VIIa and with F-actin. In addition, a shroom2 fragment containing the region of interaction with F-actin was able to protect actin filaments from cytochalasin-D-induced disruption in MDCK cells. Transfection experiments in MDCK and LE (L fibroblasts that express E-cadherin) cells led us to conclude that shroom2 is targeted to the cell-cell junctions in the presence of tight junctions only. In Ca(2+)-switch experiments on MDCK cells, ZO-1 (also known as TJP1) preceded GFP-tagged shroom2 at the differentiating tight junctions. ZO-1 directly interacts with the serine- and proline-rich region of shroom2 in vitro. Moreover, the two proteins colocalize in vivo at mature tight junctions, and could be coimmunoprecipitated from brain and cochlear extracts. We suggest that shroom2 and ZO-1 form a tight-junction-associated scaffolding complex, possibly linked to Myosin VIIa, that bridges the junctional membrane to the underlying cytoskeleton, thereby contributing to the stabilization of these junctions
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Shroom2, a Myosin-VIIa- and actin-binding protein, directly interacts with ZO-1 at tight junctions.
Journal of cell science, 2007Co-Authors: Raphael Etournay, Pierre Legrain, Christine Petit, Isabelle Perfettini, Ingrid Zwaenepoel, Aziz El-amraouiAbstract:Defects in Myosin VIIa lead to developmental anomalies of the auditory and visual sensory cells. We sought proteins interacting with the Myosin VIIa tail by using the yeast two-hybrid system. Here, we report on shroom2, a submembranous PDZ domain-containing protein that is associated with the tight junctions in multiple embryonic and adult epithelia. Shroom2 directly interacts with the C-terminal MyTH4-FERM domain of Myosin VIIa and with F-actin. In addition, a shroom2 fragment containing the region of interaction with F-actin was able to protect actin filaments from cytochalasin-D-induced disruption in MDCK cells. Transfection experiments in MDCK and LE (L fibroblasts that express E-cadherin) cells led us to conclude that shroom2 is targeted to the cell-cell junctions in the presence of tight junctions only. In Ca(2+)-switch experiments on MDCK cells, ZO-1 (also known as TJP1) preceded GFP-tagged shroom2 at the differentiating tight junctions. ZO-1 directly interacts with the serine- and proline-rich region of shroom2 in vitro. Moreover, the two proteins colocalize in vivo at mature tight junctions, and could be coimmunoprecipitated from brain and cochlear extracts. We suggest that shroom2 and ZO-1 form a tight-junction-associated scaffolding complex, possibly linked to Myosin VIIa, that bridges the junctional membrane to the underlying cytoskeleton, thereby contributing to the stabilization of these junctions.
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Unconventional Myosin VIIa and vezatin, two proteins crucial for Listeria entry into epithelial cells.
Journal of cell science, 2004Co-Authors: Sandra Sousa, Aziz El-amraoui, Christine Petit, Marc Lecuit, Didier Cabanes, Pascale CossartAbstract:Listeria monocytogenes is a bacterial pathogen with the capacity to invade non-phagocytic cells. This dynamic process involves coordinated membrane remodelling and actin cytoskeleton rearrangements. Although some of the molecular factors promoting these events have been identified, the driving force allowing internalization is unknown. One of the receptors for L. monocytogenes on epithelial cells is E-cadherin, a transmembrane protein normally involved in homophilic interactions that allow cell-cell contacts at the adherens junctions. E-cadherin has to be connected to the actin cytoskeleton to mediate strong cell-cell adhesion and to trigger Listeria entry; alpha- and beta-catenins play key roles in these processes. We have recently identified an unconventional Myosin, Myosin VIIa and its ligand vezatin, at the adherens junctions of polarized epithelial cells. Here, we demonstrate by pharmacological and genetic approaches that both Myosin VIIa and vezatin are crucial for Listeria internalization. These results provide the first evidence for the role of an unconventional Myosin in bacterial internalization and a novel example of the exploitation of mammalian proteins, by a pathogen, to establish a successful infection.
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unconventional Myosin VIIa and vezatin two proteins crucial for listeria entry into epithelial cells
Journal of Cell Science, 2004Co-Authors: Sandra Maria Zakia Lian Sousa, Christine Petit, Marc Lecuit, Aziz Elamraoui, Didier Cabanes, Pascale CossartAbstract:Listeria monocytogenes is a bacterial pathogen with the capacity to invade non-phagocytic cells. This dynamic process involves coordinated membrane remodelling and actin cytoskeleton rearrangements. Although some of the molecular factors promoting these events have been identified, the driving force allowing internalization is unknown. One of the receptors for L. monocytogenes on epithelial cells is E-cadherin, a transmembrane protein normally involved in homophilic interactions that allow cell-cell contacts at the adherens junctions. E-cadherin has to be connected to the actin cytoskeleton to mediate strong cell-cell adhesion and to trigger Listeria entry; α- and β-catenins play key roles in these processes. We have recently identified an unconventional Myosin, Myosin VIIa and its ligand vezatin, at the adherens junctions of polarized epithelial cells. Here, we demonstrate by pharmacological and genetic approaches that both Myosin VIIa and vezatin are crucial for Listeria internalization. These results provide the first evidence for the role of an unconventional Myosin in bacterial internalization and a novel example of the exploitation of mammalian proteins, by a pathogen, to establish a successful infection.
Aziz El-amraoui - One of the best experts on this subject based on the ideXlab platform.
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Shroom2, a Myosin-VIIa- and actin-binding protein, directly interacts with ZO-1 at tight junctions
Journal of Cell Science, 2007Co-Authors: Raphael Etournay, Pierre Legrain, Christine Petit, Isabelle Perfettini, Ingrid Zwaenepoel, Aziz El-amraouiAbstract:International audienceDefects in Myosin VIIa lead to developmental anomalies of the auditory and visual sensory cells. We sought proteins interacting with the Myosin VIIa tail by using the yeast two-hybrid system. Here, we report on shroom2, a submembranous PDZ domain-containing protein that is associated with the tight junctions in multiple embryonic and adult epithelia. Shroom2 directly interacts with the C-terminal MyTH4-FERM domain of Myosin VIIa and with F-actin. In addition, a shroom2 fragment containing the region of interaction with F-actin was able to protect actin filaments from cytochalasin-D-induced disruption in MDCK cells. Transfection experiments in MDCK and LE (L fibroblasts that express E-cadherin) cells led us to conclude that shroom2 is targeted to the cell-cell junctions in the presence of tight junctions only. In Ca(2+)-switch experiments on MDCK cells, ZO-1 (also known as TJP1) preceded GFP-tagged shroom2 at the differentiating tight junctions. ZO-1 directly interacts with the serine- and proline-rich region of shroom2 in vitro. Moreover, the two proteins colocalize in vivo at mature tight junctions, and could be coimmunoprecipitated from brain and cochlear extracts. We suggest that shroom2 and ZO-1 form a tight-junction-associated scaffolding complex, possibly linked to Myosin VIIa, that bridges the junctional membrane to the underlying cytoskeleton, thereby contributing to the stabilization of these junctions
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Shroom2, a Myosin-VIIa- and actin-binding protein, directly interacts with ZO-1 at tight junctions.
Journal of cell science, 2007Co-Authors: Raphael Etournay, Pierre Legrain, Christine Petit, Isabelle Perfettini, Ingrid Zwaenepoel, Aziz El-amraouiAbstract:Defects in Myosin VIIa lead to developmental anomalies of the auditory and visual sensory cells. We sought proteins interacting with the Myosin VIIa tail by using the yeast two-hybrid system. Here, we report on shroom2, a submembranous PDZ domain-containing protein that is associated with the tight junctions in multiple embryonic and adult epithelia. Shroom2 directly interacts with the C-terminal MyTH4-FERM domain of Myosin VIIa and with F-actin. In addition, a shroom2 fragment containing the region of interaction with F-actin was able to protect actin filaments from cytochalasin-D-induced disruption in MDCK cells. Transfection experiments in MDCK and LE (L fibroblasts that express E-cadherin) cells led us to conclude that shroom2 is targeted to the cell-cell junctions in the presence of tight junctions only. In Ca(2+)-switch experiments on MDCK cells, ZO-1 (also known as TJP1) preceded GFP-tagged shroom2 at the differentiating tight junctions. ZO-1 directly interacts with the serine- and proline-rich region of shroom2 in vitro. Moreover, the two proteins colocalize in vivo at mature tight junctions, and could be coimmunoprecipitated from brain and cochlear extracts. We suggest that shroom2 and ZO-1 form a tight-junction-associated scaffolding complex, possibly linked to Myosin VIIa, that bridges the junctional membrane to the underlying cytoskeleton, thereby contributing to the stabilization of these junctions.
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PHR1, an integral membrane protein of the inner ear sensory cells, directly interacts with Myosin 1c and Myosin VIIa.
Journal of Cell Science, 2005Co-Authors: Raphael Etournay, Stephane Blanchard, Aziz El-amraoui, Amel Bahloul, Isabelle Roux, Guillaume Pézeron, Nicolas Michalski, Laurent Daviet, Jean-pierre Hardelin, Pierre LegrainAbstract:By using the yeast two-hybrid technique, we identified a candidate protein ligand of the Myosin 1c tail, PHR1, and found that this protein can also bind to the Myosin VIIa tail. PHR1 is an integral membrane protein that contains a pleckstrin homology (PH) domain. Myosin 1c and Myosin VIIa are two unconventional Myosins present in the inner ear sensory cells. We showed that PHR1 immunoprecipitates with either Myosin tail by using protein extracts from cotransfected HEK293 cells. In vitro binding assays confirmed that PHR1 directly interacts with these two Myosins. In both cases the binding involves the PH domain. In vitro interactions between PHR1 and the Myosin tails were not affected by the presence or absence of Ca2+ and calmodulin. Finally, we found that PHR1 is able to dimerise. As PHR1 is expressed in the vestibular and cochlear sensory cells, its direct interactions with the Myosin 1c and VIIa tails are likely to play a role in anchoring the actin cytoskeleton to the plasma membrane of these cells. Moreover, as both Myosins have been implicated in the mechanotransduction slow adaptation process that takes place in the hair bundles, we propose that PHR1 is also involved in this process.
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Unconventional Myosin VIIa and vezatin, two proteins crucial for Listeria entry into epithelial cells.
Journal of cell science, 2004Co-Authors: Sandra Sousa, Aziz El-amraoui, Christine Petit, Marc Lecuit, Didier Cabanes, Pascale CossartAbstract:Listeria monocytogenes is a bacterial pathogen with the capacity to invade non-phagocytic cells. This dynamic process involves coordinated membrane remodelling and actin cytoskeleton rearrangements. Although some of the molecular factors promoting these events have been identified, the driving force allowing internalization is unknown. One of the receptors for L. monocytogenes on epithelial cells is E-cadherin, a transmembrane protein normally involved in homophilic interactions that allow cell-cell contacts at the adherens junctions. E-cadherin has to be connected to the actin cytoskeleton to mediate strong cell-cell adhesion and to trigger Listeria entry; alpha- and beta-catenins play key roles in these processes. We have recently identified an unconventional Myosin, Myosin VIIa and its ligand vezatin, at the adherens junctions of polarized epithelial cells. Here, we demonstrate by pharmacological and genetic approaches that both Myosin VIIa and vezatin are crucial for Listeria internalization. These results provide the first evidence for the role of an unconventional Myosin in bacterial internalization and a novel example of the exploitation of mammalian proteins, by a pathogen, to establish a successful infection.
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Myosin VIIa, harmonin and cadherin 23, three Usher I gene products that cooperate to shape the sensory hair cell bundle
The EMBO journal, 2002Co-Authors: Batiste Boëda, Stephane Blanchard, Aziz El-amraoui, Amel Bahloul, Laurent Daviet, Isabelle Perfettini, Richard J. Goodyear, Karl R. Fath, Spencer L. Shorte, Jan ReinersAbstract: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.
David S Williams - One of the best experts on this subject based on the ideXlab platform.
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Cell Motility and the Cytoskeleton 40:261–271 (1998) Myosin VIIa as a Common Component of Cilia and Microvilli
2013Co-Authors: Uwe Wolfrum, Igor P Udovichenko, Xinran Liu, Angelika Schmitt, David S WilliamsAbstract:The distribution of Myosin VIIa, which is defective or absent in Usher syndrome 1B, was studied in a variety of tissues by immunomicroscopy. The primary aim was to determine whether this putative actin-based mechanoenzyme is a common component of cilia. Previously, it has been proposed that defective ciliary function might be the basis of some forms of Usher syndrome. Myosin VIIa was detected in cilia from cochlear hair cells, olfactory neurons, kidney distal tubules, and lung bronchi. It was also found to cofractionate with the axonemal fraction of retinal photoreceptor cells. Immunolabeling appeared most concentrated in the periphery of the transition zone of the cilia. This general presence of a Myosin in cilia is surprising, given that cilia are dominated by microtubules, and not actin filaments. In addition to cilia, Myosin VIIa was also found in actin-rich microvilli of different types of cell. We conclude that Myosin VIIa is a common component of cilia an
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A novel allele of Myosin VIIa reveals a critical function for the C-terminal FERM domain for melanosome transport in retinal pigment epithelial cells.
The Journal of neuroscience : the official journal of the Society for Neuroscience, 2009Co-Authors: Martin Schwander, David S Williams, Concepción Lillo, Daniel Gibbs, Vanda S. Lopes, Anna Sczaniecka, David L. Delano, Lisa M. Tarantino, Tim Wiltshire, Ulrich MüllerAbstract:Mutations in the head and tail domains of the motor protein Myosin VIIa (MYO7A) cause deaf-blindness (Usher syndrome type 1B, USH1B) and nonsyndromic deafness (DFNB2, DFNA11). The head domain binds to F-actin and serves as the MYO7A motor domain, but little is known about the function of the tail domain. In a genetic screen, we have identified polka mice, which carry a mutation (c.5742 + 5G > A) that affects splicing of the MYO7A transcript and truncates the MYO7A tail domain at the C-terminal FERM domain. In the inner ear, expression of the truncated MYO7A protein is severely reduced, leading to defects in hair cell development. In retinal pigment epithelial (RPE) cells, the truncated MYO7A protein is expressed at comparative levels to wild-type protein but fails to associate with and transport melanosomes. We conclude that the C-terminal FERM domain of MYO7A is critical for melanosome transport in RPE cells. Our findings also suggest that MYO7A mutations can lead to tissue-specific effects on protein levels, which may explain why some mutations in MYO7A lead to deafness without retinal impairment.
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Physical and Functional Interaction between Protocadherin 15 and Myosin VIIa in Mechanosensory Hair Cells
The Journal of neuroscience : the official journal of the Society for Neuroscience, 2006Co-Authors: Mathias Senften, Piotr Kazmierczak, Tama Hasson, David S Williams, Martin Schwander, Concepción Lillo, Jung-bum Shin, Gwenaëlle S. G. Géléoc, Peter G. Gillespie, Jeffrey R. HoltAbstract:Hair cells of the mammalian inner ear are the mechanoreceptors that convert sound-induced vibrations into electrical signals. The molecular mechanisms that regulate the development and function of the mechanically sensitive organelle of hair cells, the hair bundle, are poorly defined. We link here two gene products that have been associated with deafness and hair bundle defects, protocadherin 15 (PCDH15) and Myosin VIIa (MYO7A), into a common pathway. We show that PCDH15 binds to MYO7A and that both proteins are expressed in an overlapping pattern in hair bundles. PCDH15 localization is perturbed in MYO7A-deficient mice, whereas MYO7A localization is perturbed in PCDH15-deficient mice. Like MYO7A, PCDH15 is critical for the development of hair bundles in cochlear and vestibular hair cells, controlling hair bundle morphogenesis and polarity. Cochlear and vestibular hair cells from PCDH15-deficient mice also show defects in mechanotransduction. Together, our findings suggest that PCDH15 and MYO7A cooperate to regulate the development and function of the mechanically sensitive hair bundle.
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Role of Myosin VIIa and Rab27a in the motility and localization of RPE melanosomes.
Journal of cell science, 2004Co-Authors: Daniel Gibbs, Karen P Steel, Concepción Lillo, Sassan M Azarian, Junko Kitamoto, Adriana E Klomp, Richard T Libby, David S WilliamsAbstract:Myosin VIIa functions in the outer retina, and loss of this function causes human blindness in Usher syndrome type 1B (USH1B). In mice with mutant Myo7a, melanosomes in the retinal pigmented epithelium (RPE) are distributed abnormally. In this investigation we detected many proteins in RPE cells that could potentially participate in melanosome transport, but of those tested, only Myosin VIIa and Rab27a were found to be required for normal distribution. Two other expressed proteins, melanophilin and Myosin Va, both of which are required for normal melanosome distribution in melanocytes, were not required in RPE, despite the association of Myosin Va with the RPE melanosome fraction. Both Myosin VIIa and Myosin Va were immunodetected broadly in sections of the RPE, overlapping with a region of apical filamentous actin. Some 70-80% of the Myosin VIIa in RPE cells was detected on melanosome membranes by both subcellular fractionation of RPE cells and quantitative immunoelectron microscopy, consistent with a role for Myosin VIIa in melanosome motility. Time-lapse microscopy of melanosomes in primary cultures of mouse RPE cells demonstrated that the melanosomes move in a saltatory manner, interrupting slow movements with short bursts of rapid movement (>1 RR01183m/second). In RPE cells from Myo7a-null mice, both the slow and rapid movements still occurred, except that more melanosomes underwent rapid movements, and each movement extended approximately five times longer (and further). Hence, our studies demonstrate the presence of many potential effectors of melanosome motility and localization in the RPE, with a specific requirement for Rab27a and Myosin VIIa, which function by transporting and constraining melanosomes within a region of filamentous actin. The presence of two distinct melanosome velocities in both control and Myo7a-null RPE cells suggests the involvement of at least two motors other than Myosin VIIa in melanosome motility, most probably, a microtubule motor and Myosin Va.
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Abnormal phagocytosis by retinal pigmented epithelium that lacks Myosin VIIa, the Usher syndrome 1B protein
Proceedings of the National Academy of Sciences of the United States of America, 2003Co-Authors: Daniel Gibbs, Junko Kitamoto, David S WilliamsAbstract:Abstract Mutations in the Myosin VIIa gene (MYO7A) cause Usher syndrome type 1B (USH1B), a major type of the deaf-blind disorder, Usher syndrome. We have studied mutant phenotypes in the retinas of Myo7a mutant mice (shaker1), with the aim of elucidating the role(s) of Myosin VIIa in the retina and what might underlie photoreceptor degeneration in USH1B patients. A photoreceptor defect has been described. Here, we report that the phagocytosis of photoreceptor outer segment disks by the retinal pigment epithelium (RPE) is abnormal in Myo7a null mice. Both in vivo and in primary cultures of RPE cells, the transport of ingested disks out of the apical region is inhibited in the absence of Myo7a. The results with the cultured RPE cells were the same, irrespective of whether the disks came from wild-type or mutant mice, thus demonstrating that the RPE is the source of this defect. The inhibited transport seems to delay phagosome–lysosomal fusion, as the degradation of ingested disks was slower in mutant RPE. Moreover, fewer packets of disk membranes were ingested in vivo, possibly because retarded removal of phagosomes from the apical processes inhibited the ingestion of additional disk membranes. We conclude that Myo7a is required for the normal processing of ingested disk membranes in the RPE, primarily in the basal transport of phagosomes into the cell body where they then fuse with lysosomes. Because the phagocytosis of photoreceptor disks by the RPE has been shown to be critical for photoreceptor cell viability, this defect likely contributes to the progressive blindness in USH1B.
Karen P Steel - One of the best experts on this subject based on the ideXlab platform.
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Role of Myosin VIIa and Rab27a in the motility and localization of RPE melanosomes.
Journal of cell science, 2004Co-Authors: Daniel Gibbs, Karen P Steel, Concepción Lillo, Sassan M Azarian, Junko Kitamoto, Adriana E Klomp, Richard T Libby, David S WilliamsAbstract:Myosin VIIa functions in the outer retina, and loss of this function causes human blindness in Usher syndrome type 1B (USH1B). In mice with mutant Myo7a, melanosomes in the retinal pigmented epithelium (RPE) are distributed abnormally. In this investigation we detected many proteins in RPE cells that could potentially participate in melanosome transport, but of those tested, only Myosin VIIa and Rab27a were found to be required for normal distribution. Two other expressed proteins, melanophilin and Myosin Va, both of which are required for normal melanosome distribution in melanocytes, were not required in RPE, despite the association of Myosin Va with the RPE melanosome fraction. Both Myosin VIIa and Myosin Va were immunodetected broadly in sections of the RPE, overlapping with a region of apical filamentous actin. Some 70-80% of the Myosin VIIa in RPE cells was detected on melanosome membranes by both subcellular fractionation of RPE cells and quantitative immunoelectron microscopy, consistent with a role for Myosin VIIa in melanosome motility. Time-lapse microscopy of melanosomes in primary cultures of mouse RPE cells demonstrated that the melanosomes move in a saltatory manner, interrupting slow movements with short bursts of rapid movement (>1 RR01183m/second). In RPE cells from Myo7a-null mice, both the slow and rapid movements still occurred, except that more melanosomes underwent rapid movements, and each movement extended approximately five times longer (and further). Hence, our studies demonstrate the presence of many potential effectors of melanosome motility and localization in the RPE, with a specific requirement for Rab27a and Myosin VIIa, which function by transporting and constraining melanosomes within a region of filamentous actin. The presence of two distinct melanosome velocities in both control and Myo7a-null RPE cells suggests the involvement of at least two motors other than Myosin VIIa in melanosome motility, most probably, a microtubule motor and Myosin Va.
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A missense mutation in Myosin VIIa prevents aminoglycoside accumulation in cochlear hair cells
1999Co-Authors: Guy P. Richardson, Steve D M Brown, David S Williams, Andrew Forge, Corné J. Kros, J. Fleming, Walter Marcotti, D Becker, Julian R. Thorpe, Karen P SteelAbstract:Myosin VIIa is expressed by sensory hair cells in the inner ear and proximal tubule cells in the kidney, the two primary targets of aminoglycoside antibiotics. Using cochlear cultures prepared from early postnatal Myo7a6J mice with a missense mutation in the head region of the Myosin VIIa molecule we show that this Myosin is required for aminoglycoside accumulation in cochlear hair cells. Hair cells in homozygous mutant Myo7a6J cochlear cultures have disorganized hair bundles, but are otherwise morphologically normal and transduce. However, and in contrast to hair cells from heterozygous Myo7a6J cultures, the homozygous Myo7a6J hair cells do not accumulate [3H]gentamicin and do not exhibit an ototoxic response on exposure to aminoglycoside. Possible roles for Myosin VIIa in the process of aminoglycoside accumulation are discussed.
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A missense mutation in Myosin VIIa prevents aminoglycoside accumulation in early postnatal cochlear hair cells.
Annals of the New York Academy of Sciences, 1999Co-Authors: Guy P. Richardson, Steve D M Brown, David S Williams, Andrew Forge, Corné J. Kros, J. Fleming, Walter Marcotti, Julian R. Thorpe, David L. Becker, Karen P SteelAbstract:Myosin VIIa is expressed by sensory hair cells in the inner ear and proximal tubule cells in the kidney, the two primary targets of aminoglycoside antibiotics. Using cochlear cultures prepared from early postnatal Myo7a6J mice with a missense mutation in the head region of the Myosin VIIa molecule we show that this Myosin is required for aminoglycoside accumulation in cochlear hair cells. Hair cells in homozygous mutant Myo7a6J cochlear cultures have disorganized hair bundles, but are otherwise morphologically normal and transduce. However, and in contrast to hair cells from heterozygous Myo7a6J cultures, the homozygous Myo7a6J hair cells do not accumulate [3H]gentamicin and do not exhibit an ototoxic response on exposure to aminoglycoside. Possible roles for Myosin VIIa in the process of aminoglycoside accumulation are discussed.
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Myosin VIIa Participates in Opsin Transport through The Photoreceptor Cilium
The Journal of neuroscience : the official journal of the Society for Neuroscience, 1999Co-Authors: Xinran Liu, Karen P Steel, Igor P Udovichenko, Stephen D.m. Brown, David S WilliamsAbstract:Two types of Usher syndrome, a blindness-deafness disorder, result from mutations in the Myosin VIIa gene. As for most other unconventional Myosins, little is known about the function or functions of Myosin VIIa. Here, we studied the photoreceptor cells of mice with mutant Myosin VIIa by electron immunomicroscopy and microscopic autoradiography. We found evidence that Myosin VIIa functions in the connecting cilium of each photoreceptor cell and participates in the transport of opsin through this structure. These findings provide the first direct evidence that opsin travels along the connecting cilium en route to the outer segment. They demonstrate that a Myosin may function in a cilium and suggest that abnormal opsin transport might contribute to blindness in Usher syndrome.
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Shaker-1 mutations reveal roles for Myosin VIIa in both development and function of cochlear hair cells
Development (Cambridge England), 1998Co-Authors: Tim Self, James Walsh, Steve D M Brown, J. Fleming, Mary Mahony, Karen P SteelAbstract:The mouse shaker-1 locus, Myo7a, encodes Myosin VIIa and mutations in the orthologous gene in humans cause Usher syndrome type 1B or non-syndromic deafness. Myo7a is expressed very early in sensory hair cell development in the inner ear. We describe the effects of three mutations on cochlear hair cell development and function. In the Myo7a816SB and Myo7a6J mutants, stereocilia grow and form rows of graded heights as normal, but the bundles become progressively more disorganised. Most of these mutants show no gross electrophysiological responses, but some did show evidence of hair cell depolarisation despite the disorganisation of their bundles. In contrast, the original shaker-1 mutants, Myo7ash1, had normal early development of stereocilia bundles, but still showed abnormal cochlear responses. These findings suggest that Myosin VIIa is required for normal stereocilia bundle organisation and has a role in the function of cochlear hair cells.
Mitsuo Ikebe - One of the best experts on this subject based on the ideXlab platform.
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Human Myosin VIIa Is a Very Slow Processive Motor Protein on Various Cellular Actin Structures
The Journal of biological chemistry, 2017Co-Authors: Osamu Sato, Tsuyoshi Sakai, Ryosuke Tanaka, Takeomi Mizutani, Tomonobu M. Watanabe, Reiko Ikebe, Satoshi Komatsu, Yoshikazu Tsukasaki, Mitsuo IkebeAbstract:Human Myosin VIIa (MYO7A) is an actin-linked motor protein associated with human Usher syndrome (USH) type 1B, which causes human congenital hearing and visual loss. Although it has been thought that the role of human Myosin VIIa is critical for USH1 protein tethering with actin and transportation along actin bundles in inner-ear hair cells, Myosin VIIa's motor function remains unclear. Here, we studied the motor function of the tail-truncated human Myosin VIIa dimer (HM7AΔTail/LZ) at the single-molecule level. We found that the HM7AΔTail/LZ moves processively on single actin filaments with a step size of 35 nm. Dwell-time distribution analysis indicated an average waiting time of 3.4 s, yielding ∼0.3 s−1 for the mechanical turnover rate; hence, the velocity of HM7AΔTail/LZ was extremely slow, at 11 nm·s−1. We also examined HM7AΔTail/LZ movement on various actin structures in demembranated cells. HM7AΔTail/LZ showed unidirectional movement on actin structures at cell edges, such as lamellipodia and filopodia. However, HM7AΔTail/LZ frequently missed steps on actin tracks and exhibited bidirectional movement at stress fibers, which was not observed with tail-truncated Myosin Va. These results suggest that the movement of the human Myosin VIIa motor protein is more efficient on lamellipodial and filopodial actin tracks than on stress fibers, which are composed of actin filaments with different polarity, and that the actin structures influence the characteristics of cargo transportation by human Myosin VIIa. In conclusion, Myosin VIIa movement appears to be suitable for translocating USH1 proteins on stereocilia actin bundles in inner-ear hair cells.
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Structure and Regulation of the Movement of Human Myosin VIIa
The Journal of biological chemistry, 2015Co-Authors: Tsuyoshi Sakai, Osamu Sato, Reiko Ikebe, Hyun Suk Jung, Masafumi D. Yamada, Dong Ju You, Mitsuo IkebeAbstract: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 Ca(2+). 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 Ca(2+), 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.
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Single Molecule Characterization of Human Myosin VIIa
Biophysical Journal, 2014Co-Authors: Osamu Sato, Tsuyoshi Sakai, Ryosuke Tanaka, Takeomi Mizutani, Tomonobu M. Watanabe, Reiko Ikebe, Mitsuo IkebeAbstract:Human Myosin VIIa is responsible for Usher Syndrome (USH) type 1B. Although human Myosin VIIa is thought to be involved in transportation of USH1 proteins and melanosomes, it is obscure that human Myosin VIIa is a suitable motor as a cargo transporter.In this study, we analyzed the motor characteristics of tail truncated human Myosin VIIa (HM7DTail/LZ) at single molecule level. We found that HM7DTail/LZ moves processively with large ∼35 nm forward and small ∼23 nm backward steps at physiological ATP concentration. The forward step of HM7DTail/LZ was slightly larger than that of Drosophila Myosin VIIa [∼30 nm, Yang et al. (2006) PNAS, 103, 5746-5751]. The average run-length of HM7DTail/LZ was ∼0.7 µm on single actin filaments. This means that HM7DTail/LZ can move ∼20 steps on single actin filaments on average.Dwell time distribution gave the average waiting time of ∼3.4 s, yielding 0.3 s−1 for the mechanical turnover rate. This rate is consistent with the Vmax value of actin activated ATPase activity (∼0.3 s−1) of tailless human Myosin VIIa. On the other hands, the velocity of HM7DTail/LZ was extremely slow, 11 nm/sec. This value is ∼7 times slower than that of Drosophila Myosin VIIa [72 nm/s, Yang et al. (2006)]. Recent studies have suggested that mammalian Myosin VIIa may function in tethering melanosomes on actin filaments. Thus, the slow velocity may imply that human Myosin VIIa is more suitable for cargo-actin tethering motor than Drosophila Myosin VIIa, and may link transportation processes such as USH protein transportation. We recently found that human Myosin VIIa localizes at the filopodia tip in cells when it dimerizes [Sakai et al. (2011) PNAS,108, 7028-7033]. Currently, we are examining the movement of HM7DTail/LZ on actin bundles in filopodia using de-membraned cell system. The result is underway.
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Cargo binding activates Myosin VIIa motor function in cells
Proceedings of the National Academy of Sciences of the United States of America, 2011Co-Authors: Tsuyoshi Sakai, Reiko Ikebe, Nobuhisa Umeki, Mitsuo IkebeAbstract:Myosin VIIa, thought to be involved in human auditory function, is a gene responsible for human Usher syndrome type 1B, which causes hearing and visual loss. Recent studies have suggested that it can move processively if it forms a dimer. Nevertheless, it exists as a monomer in vitro, unlike the well-known two-headed processive Myosin Va. Here we studied the molecular mechanism, which is currently unknown, of activating Myosin VIIa as a cargo-transporting motor. Human Myosin VIIa was present throughout cytosol, but it moved to the tip of filopodia upon the formation of dimer induced by dimer-inducing reagent. The forced dimer of Myosin VIIa translocated its cargo molecule, MyRip, to the tip of filopodia, whereas Myosin VIIa without the forced dimer-forming module does not translocate to the filopodial tips. These results suggest that dimer formation of Myosin VIIa is important for its cargo-transporting activity. On the other hand, Myosin VIIa without the forced dimerization module became translocated to the filopodial tips in the presence of cargo complex, i.e., MyRip/Rab27a, and transported its cargo complex to the tip. Coexpression of MyRip promoted the association of Myosin VIIa to vesicles and the dimer formation. These results suggest that association of Myosin VIIa monomers with membrane via the MyRip/Rab27a complex facilitates the cargo-transporting activity of Myosin VIIa, which is achieved by cluster formation on the membrane, where it possibly forms a dimer. Present findings support that MyRip, a cargo molecule, functions as an activator of Myosin VIIa transporter function.
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The tail binds to the head-neck domain, inhibiting ATPase activity of Myosin VIIa.
Proceedings of the National Academy of Sciences of the United States of America, 2009Co-Authors: Nobuhisa Umeki, Tsuyoshi Sakai, Reiko Ikebe, Shinya Watanabe, Hyun Suk Jung, Roger Craig, Mitsuo IkebeAbstract:Myosin VIIa is an unconventional Myosin, responsible for human Usher syndrome type 1B, which causes hearing and visual loss. Here, we studied the molecular mechanism of regulation of Myosin VIIa, which is currently unknown. Although it was originally thought that Myosin VIIa is a dimeric Myosin, our electron microscopic (EM) observations revealed that full-length Drosophila Myosin VIIa (DM7A) is a monomer. Interestingly, the tail domain markedly inhibits the actin-activated ATPase activity of tailless DM7A at low Ca(2+) but not high Ca(2+). By examining various deletion constructs, we found that deletion of the distal IQ domain, the C-terminal region of the tail, and the N-terminal region of the tail abolishes the tail-induced inhibition of ATPase activity. Single-particle EM analysis of full-length DM7A at low Ca(2+) suggests that the tail folds back on to the head, where it contacts both the motor core domain and the neck domain, forming an inhibited conformation. We concluded that unconventional Myosin that may be present a monomer in the cell can be regulated by intramolecular interaction of the tail with the head.
