The Experts below are selected from a list of 2112 Experts worldwide ranked by ideXlab platform
Folma Buss - One of the best experts on this subject based on the ideXlab platform.
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Myosin VI facilitates connexin 43 gap junction accretion
Journal of Cell Science, 2017Co-Authors: Prabuddha Sengupta, Bennett J Waxse, Geoffrey G Hesketh, Jennifer Lippincottschwartz, Folma BussAbstract:In this study, we demonstrate Myosin VI enrichment at Cx43 (also known as GJA1)-containing gap junctions (GJs) in heart tissue, primary cardiomyocytes and cell culture models. In primary cardiac tissue and in fibroblasts from the Myosin VI-null mouse as well as in tissue culture cells transfected with siRNA against Myosin VI, we observe reduced GJ plaque size with a concomitant reduction in intercellular communication, as shown by fluorescence recovery after photobleaching (FRAP) and a new method of selective calcein administration. Analysis of the molecular role of Myosin VI in Cx43 trafficking indicates that Myosin VI is dispensable for the delivery of Cx43 to the cell surface and connexon movement in the plasma membrane. Furthermore, we cannot corroborate clathrin or Dab2 localization at gap junctions and we do not observe a function for the Myosin-VI-Dab2 complex in clathrin-dependent endocytosis of annular gap junctions. Instead, we found that Myosin VI was localized at the edge of Cx43 plaques by using total internal reflection fluorescence (TIRF) microscopy and use FRAP to identify a plaque accretion defect as the primary manifestation of Myosin VI loss in Cx43 homeostasis. A fuller understanding of this derangement may explain the cardiomyopathy or gliosis associated with the loss of Myosin VI.
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loss of cargo binding in the human Myosin VI deafness mutant r1166x leads to increased actin filament binding
Biochemical Journal, 2016Co-Authors: Susan D. Arden, David A Tumbarello, John Kendrickjones, Tariq Butt, Folma BussAbstract:Mutations in Myosin VI have been associated with autosomal-recessive (DFNB37) and autosomal-dominant (DFNA22) deafness in humans. Here, we characterise an Myosin VI nonsense mutation (R1166X) that was identified in a family with hereditary hearing loss in Pakistan. This mutation leads to the deletion of the C-terminal 120 amino acids of the Myosin VI cargo-binding domain, which includes the WWY-binding motif for the adaptor proteins LMTK2, Tom1 as well as Dab2. Interestingly, compromising Myosin VI vesicle-binding ability by expressing Myosin VI with the R1166X mutation or with single point mutations in the adaptor-binding sites leads to increased F-actin binding of this Myosin in VItro and in VIvo As our results highlight the importance of cargo attachment for regulating actin binding to the motor domain, we perform a detailed characterisation of adaptor protein binding and identify single amino acids within Myosin VI required for binding to cargo adaptors. We not only show that the adaptor proteins can directly interact with the cargo-binding tail of Myosin VI, but our in VItro studies also suggest that multiple adaptor proteins can bind simultaneously to non-overlapping sites in the Myosin VI tail. In conclusion, our characterisation of the human Myosin VI deafness mutant (R1166X) suggests that defects in cargo binding may leave Myosin VI in a primed/activated state with an increased actin-binding ability.
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the autophagy receptor tax1bp1 and the molecular motor Myosin VI are required for clearance of salmonella typhimurium by autophagy
PLOS Pathogens, 2015Co-Authors: David A Tumbarello, Susan D. Arden, John Kendrickjones, Paul T Manna, Mark D Allen, Mark Bycroft, Folma BussAbstract:Autophagy plays a key role during Salmonella infection, by eliminating these pathogens following escape into the cytosol. In this process, selective autophagy receptors, including the Myosin VI adaptor proteins optineurin and NDP52, have been shown to recognize cytosolic pathogens. Here, we demonstrate that Myosin VI and TAX1BP1 are recruited to ubiquitylated Salmonella and play a key role in xenophagy. The absence of TAX1BP1 causes an accumulation of ubiquitin-positive Salmonella, whereas loss of Myosin VI leads to an increase in ubiquitylated and LC3-positive bacteria. Our structural studies demonstrate that the ubiquitin-binding site of TAX1BP1 overlaps with the Myosin VI binding site and point mutations in the TAX1BP1 zinc finger domains that affect ubiquitin binding also ablate binding to Myosin VI. This mutually exclusive binding and the association of TAX1BP1 with LC3 on the outer limiting membrane of autophagosomes may suggest a molecular mechanism for recruitment of this motor to autophagosomes. The predominant role of TAX1BP1, a paralogue of NDP52, in xenophagy is supported by our evolutionary analysis, which demonstrates that functionally intact NDP52 is missing in Xenopus and mice, whereas TAX1BP1 is expressed in all vertebrates analysed. In summary, this work highlights the importance of TAX1BP1 as a novel autophagy receptor in Myosin VI-mediated xenophagy. Our study identifies essential new machinery for the autophagy-dependent clearance of Salmonella typhimurium and suggests modulation of Myosin VI motor actiVIty as a potential therapeutic target in cellular immunity.
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Myosin VI small insert isoform maintains exocytosis by tethering secretory granules to the cortical actin
Journal of Cell Biology, 2013Co-Authors: Vanesa M Tomatis, Folma Buss, John Kendrickjones, Andreas Papadopulos, Nancy T Malintan, Sally Martin, Tristan Wallis, Rachel S Gormal, Frederic A MeunierAbstract:Before undergoing neuroexocytosis, secretory granules (SGs) are mobilized and tethered to the cortical actin network by an unknown mechanism. Using an SG pull-down assay and mass spectrometry, we found that Myosin VI was recruited to SGs in a Ca2+-dependent manner. Interfering with Myosin VI function in PC12 cells reduced the density of SGs near the plasma membrane without affecting their biogenesis. Myosin VI knockdown selectively impaired a late phase of exocytosis, consistent with a replenishment defect. This exocytic defect was selectively rescued by expression of the Myosin VI small insert (SI) isoform, which efficiently tethered SGs to the cortical actin network. These Myosin VI SI–specific effects were prevented by deletion of a c-Src kinase phosphorylation DYD motif, identified in silico. Myosin VI SI thus recruits SGs to the cortical actin network, potentially VIa c-Src phosphorylation, thereby maintaining an active pool of SGs near the plasma membrane.
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dynamic exchange of Myosin VI on endocytic structures
Journal of Biological Chemistry, 2012Co-Authors: Susan D. Arden, Folma Buss, John Kendrickjones, Lisa M Bond, James R SellersAbstract:The actin-based molecular motor Myosin VI functions in the endocytic uptake pathway, both during the early stages of clathrin-mediated uptake and in later transport to/from early endosomes. This study uses fluorescence recovery after photobleaching (FRAP) to examine the turnover rate of Myosin VI during endocytosis. The results demonstrate that Myosin VI turns over dynamically on endocytic structures with a characteristic half-life common to both the large insert isoform of Myosin VI on clathrin-coated structures and the no-insert isoform on early endosomes. This half-life is shared by the Myosin VI-binding partner Dab2 and is identical for full-length Myosin VI and the cargo-binding tail region. The 4-fold slower half-life of an artificially dimerized construct of Myosin VI on clathrin-coated structures suggests that wild type Myosin VI does not function as a stable dimer, but either as a monomer or in a monomer/dimer equilibrium. Taken together, these FRAP results offer insight into both the basic turnover dynamics and the monomer/dimer nature of Myosin VI.
John Kendrickjones - One of the best experts on this subject based on the ideXlab platform.
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loss of cargo binding in the human Myosin VI deafness mutant r1166x leads to increased actin filament binding
Biochemical Journal, 2016Co-Authors: Susan D. Arden, David A Tumbarello, John Kendrickjones, Tariq Butt, Folma BussAbstract:Mutations in Myosin VI have been associated with autosomal-recessive (DFNB37) and autosomal-dominant (DFNA22) deafness in humans. Here, we characterise an Myosin VI nonsense mutation (R1166X) that was identified in a family with hereditary hearing loss in Pakistan. This mutation leads to the deletion of the C-terminal 120 amino acids of the Myosin VI cargo-binding domain, which includes the WWY-binding motif for the adaptor proteins LMTK2, Tom1 as well as Dab2. Interestingly, compromising Myosin VI vesicle-binding ability by expressing Myosin VI with the R1166X mutation or with single point mutations in the adaptor-binding sites leads to increased F-actin binding of this Myosin in VItro and in VIvo As our results highlight the importance of cargo attachment for regulating actin binding to the motor domain, we perform a detailed characterisation of adaptor protein binding and identify single amino acids within Myosin VI required for binding to cargo adaptors. We not only show that the adaptor proteins can directly interact with the cargo-binding tail of Myosin VI, but our in VItro studies also suggest that multiple adaptor proteins can bind simultaneously to non-overlapping sites in the Myosin VI tail. In conclusion, our characterisation of the human Myosin VI deafness mutant (R1166X) suggests that defects in cargo binding may leave Myosin VI in a primed/activated state with an increased actin-binding ability.
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the autophagy receptor tax1bp1 and the molecular motor Myosin VI are required for clearance of salmonella typhimurium by autophagy
PLOS Pathogens, 2015Co-Authors: David A Tumbarello, Susan D. Arden, John Kendrickjones, Paul T Manna, Mark D Allen, Mark Bycroft, Folma BussAbstract:Autophagy plays a key role during Salmonella infection, by eliminating these pathogens following escape into the cytosol. In this process, selective autophagy receptors, including the Myosin VI adaptor proteins optineurin and NDP52, have been shown to recognize cytosolic pathogens. Here, we demonstrate that Myosin VI and TAX1BP1 are recruited to ubiquitylated Salmonella and play a key role in xenophagy. The absence of TAX1BP1 causes an accumulation of ubiquitin-positive Salmonella, whereas loss of Myosin VI leads to an increase in ubiquitylated and LC3-positive bacteria. Our structural studies demonstrate that the ubiquitin-binding site of TAX1BP1 overlaps with the Myosin VI binding site and point mutations in the TAX1BP1 zinc finger domains that affect ubiquitin binding also ablate binding to Myosin VI. This mutually exclusive binding and the association of TAX1BP1 with LC3 on the outer limiting membrane of autophagosomes may suggest a molecular mechanism for recruitment of this motor to autophagosomes. The predominant role of TAX1BP1, a paralogue of NDP52, in xenophagy is supported by our evolutionary analysis, which demonstrates that functionally intact NDP52 is missing in Xenopus and mice, whereas TAX1BP1 is expressed in all vertebrates analysed. In summary, this work highlights the importance of TAX1BP1 as a novel autophagy receptor in Myosin VI-mediated xenophagy. Our study identifies essential new machinery for the autophagy-dependent clearance of Salmonella typhimurium and suggests modulation of Myosin VI motor actiVIty as a potential therapeutic target in cellular immunity.
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Myosin VI small insert isoform maintains exocytosis by tethering secretory granules to the cortical actin
Journal of Cell Biology, 2013Co-Authors: Vanesa M Tomatis, Folma Buss, John Kendrickjones, Andreas Papadopulos, Nancy T Malintan, Sally Martin, Tristan Wallis, Rachel S Gormal, Frederic A MeunierAbstract:Before undergoing neuroexocytosis, secretory granules (SGs) are mobilized and tethered to the cortical actin network by an unknown mechanism. Using an SG pull-down assay and mass spectrometry, we found that Myosin VI was recruited to SGs in a Ca2+-dependent manner. Interfering with Myosin VI function in PC12 cells reduced the density of SGs near the plasma membrane without affecting their biogenesis. Myosin VI knockdown selectively impaired a late phase of exocytosis, consistent with a replenishment defect. This exocytic defect was selectively rescued by expression of the Myosin VI small insert (SI) isoform, which efficiently tethered SGs to the cortical actin network. These Myosin VI SI–specific effects were prevented by deletion of a c-Src kinase phosphorylation DYD motif, identified in silico. Myosin VI SI thus recruits SGs to the cortical actin network, potentially VIa c-Src phosphorylation, thereby maintaining an active pool of SGs near the plasma membrane.
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dynamic exchange of Myosin VI on endocytic structures
Journal of Biological Chemistry, 2012Co-Authors: Susan D. Arden, Folma Buss, John Kendrickjones, Lisa M Bond, James R SellersAbstract:The actin-based molecular motor Myosin VI functions in the endocytic uptake pathway, both during the early stages of clathrin-mediated uptake and in later transport to/from early endosomes. This study uses fluorescence recovery after photobleaching (FRAP) to examine the turnover rate of Myosin VI during endocytosis. The results demonstrate that Myosin VI turns over dynamically on endocytic structures with a characteristic half-life common to both the large insert isoform of Myosin VI on clathrin-coated structures and the no-insert isoform on early endosomes. This half-life is shared by the Myosin VI-binding partner Dab2 and is identical for full-length Myosin VI and the cargo-binding tail region. The 4-fold slower half-life of an artificially dimerized construct of Myosin VI on clathrin-coated structures suggests that wild type Myosin VI does not function as a stable dimer, but either as a monomer or in a monomer/dimer equilibrium. Taken together, these FRAP results offer insight into both the basic turnover dynamics and the monomer/dimer nature of Myosin VI.
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autophagy receptors link Myosin VI to autophagosomes to mediate tom1 dependent autophagosome maturation and fusion with the lysosome
Nature Cell Biology, 2012Co-Authors: David A Tumbarello, Susan D. Arden, Bennett J Waxse, Nicholas A Bright, John Kendrickjones, Folma BussAbstract:Autophagy targets pathogens, damaged organelles and protein aggregates for lysosomal degradation. These ubiquitylated cargoes are recognized by specific autophagy receptors, which recruit LC3-positive membranes to form autophagosomes. Subsequently, autophagosomes fuse with endosomes and lysosomes, thus facilitating degradation of their content; however, the machinery that targets and mediates fusion of these organelles with autophagosomes remains to be established. Here we demonstrate that Myosin VI, in concert with its adaptor proteins NDP52, optineurin, T6BP and Tom1, plays a crucial role in autophagy. We identify Tom1 as a Myosin VI binding partner on endosomes, and demonstrate that loss of Myosin VI and Tom1 reduces autophagosomal delivery of endocytic cargo and causes a block in autophagosome-lysosome fusion. We propose that Myosin VI delivers endosomal membranes containing Tom1 to autophagosomes by docking to NDP52, T6BP and optineurin, thereby promoting autophagosome maturation and thus driVIng fusion with lysosomes.
Lee H Sweeney - One of the best experts on this subject based on the ideXlab platform.
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Myosin VI deafness mutation prevents the initiation of processive runs on actin
Proceedings of the National Academy of Sciences of the United States of America, 2015Co-Authors: Olena Pylypenko, Anne Houdusse, Zhaohui Yang, Lin Song, Ai Shima, Lee H SweeneyAbstract:Mutations in the reverse-direction Myosin, Myosin VI, are associated with deafness in humans and mice. A Myosin VI deafness mutation, D179Y, which is in the transducer of the motor, uncoupled the release of the ATP hydrolysis product, inorganic phosphate (Pi), from dependency on actin binding and destroyed the ability of single dimeric molecules to move processively on actin filaments. We observed that processive movement is rescued if ATP is added to the mutant dimer following binding of both heads to actin in the absence of ATP, demonstrating that the mutation selectively destroys the initiation of processive runs at physiological ATP levels. A drug (omecamtiv) that accelerates the actin-activated actiVIty of cardiac Myosin was able to rescue processiVIty of the D179Y mutant dimers at physiological ATP concentrations by slowing the actin-independent release of Pi. Thus, it may be possible to create Myosin VI-specific drugs that rescue the function of deafness-causing mutations.
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role of insert 1 of Myosin VI in modulating nucleotide affinity
Journal of Biological Chemistry, 2011Co-Authors: Olena Pylypenko, Alan B Zong, Anne Houdusse, Xiaoyan Liu, Lin Song, Gaelle Squires, Lee H SweeneyAbstract:Myosin VI is unique in its directionality among Myosin superfamily members and also displays a slow and strain-dependent rate of ATP binding that allows for gating between its heads. In this study we demonstrate that leucine 310 is positioned by a class VI-specific insert, insert-1, so as to account for the selective hindrance of ATP versus ADP binding. Mutation of leucine 310 to glycine removes all influence of insert-1 on ATP binding. Furthermore, by analyzing Myosin VI structures with either leucine 310 substituted to a glycine or complete removal of insert-1, we conclude that nucleotides may initially bind to Myosin by their purine rings before docking their phosphate moieties. Otherwise, insert-1 could not exert a differential influence on ATP versus ADP binding.
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Myosin VI dimerization triggers an unfolding of a three helix bundle in order to extend its reach
Molecular Cell, 2009Co-Authors: Monalisa Mukherjea, Mirko Travaglia, Daniel Safer, Anne Houdusse, Paul R. Selvin, Julie Ménétrey, Clara Franziniarmstrong, P Llinas, Lee H SweeneyAbstract:Summary Myosin VI challenges the prevailing theory of how Myosin motors move on actin: the lever arm hypothesis. While the reverse directionality and large powerstroke of Myosin VI can be attributed to unusual properties of a subdomain of the motor (converter with a unique insert), these adaptations cannot account for the large step size on actin. Either the lever arm hypothesis needs modification, or Myosin VI has some unique form of extension of its lever arm. We determined the structure of the region immediately distal to the lever arm of the motor and show that it is a three-helix bundle. Based on C-terminal truncations that display the normal range of step sizes on actin, CD, fluorescence studies, and a partial deletion of the bundle, we demonstrate that this bundle unfolds upon dimerization of two Myosin VI monomers. This unconventional mechanism generates an extension of the lever arm of Myosin VI.
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the structural basis for the large powerstroke of Myosin VI
Cell, 2007Co-Authors: Julie Ménétrey, Lee H Sweeney, Monalisa Mukherjea, Paola Llinas, Anne HoudusseAbstract:Due to a unique addition to the lever arm-positioning region (converter), class VI Myosins move in the opposite direction (toward the minus-end of actin filaments) compared to other characterized Myosin classes. However, the large size of the Myosin VI lever arm swing (powerstroke) cannot be explained by our current VIew of the structural transitions that occur within the Myosin motor. We have solved the crystal structure of a fragment of the Myosin VI motor in the structural state that represents the starting point for movement on actin; the pre-powerstroke state. Unexpectedly, the converter itself rearranges to achieve a conformation that has not been seen for other Myosins. This results in a much larger powerstroke than is achievable without the converter rearrangement. Moreover, it proVIdes a new mechanism that could be exploited to increase the powerstroke of yet to be characterized plus-end-directed Myosin classes.
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how Myosin VI coordinates its heads during processive movement
The EMBO Journal, 2007Co-Authors: Lee H Sweeney, Alan B Zong, Paul R. Selvin, Zhaohui Yang, Hyokeun Park, Steven S RosenfeldAbstract:A processive molecular motor must coordinate the enzymatic state of its two catalytic domains in order to prevent premature detachment from its track. For Myosin V, internal strain produced when both heads of are attached to an actin track prevents completion of the lever arm swing of the lead head and blocks ADP release. However, this mechanism cannot work for Myosin VI, since its lever arm positions are reversed. Here, we demonstrate that Myosin VI gating is achieved instead by blocking ATP binding to the lead head once it has released its ADP. The structural basis for this unique gating mechanism involves an insert near the nucleotide binding pocket that is found only in class VI Myosin. Reverse strain greatly favors binding of ADP to the lead head, which makes it possible for Myosin VI to function as a processive transporter as well as an actin-based anchor. While this mechanism is unlike that of any other Myosin superfamily member, it bears remarkable similarities to that of another processive motor from a different superfamily—kinesin I.
Susan D. Arden - One of the best experts on this subject based on the ideXlab platform.
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loss of cargo binding in the human Myosin VI deafness mutant r1166x leads to increased actin filament binding
Biochemical Journal, 2016Co-Authors: Susan D. Arden, David A Tumbarello, John Kendrickjones, Tariq Butt, Folma BussAbstract:Mutations in Myosin VI have been associated with autosomal-recessive (DFNB37) and autosomal-dominant (DFNA22) deafness in humans. Here, we characterise an Myosin VI nonsense mutation (R1166X) that was identified in a family with hereditary hearing loss in Pakistan. This mutation leads to the deletion of the C-terminal 120 amino acids of the Myosin VI cargo-binding domain, which includes the WWY-binding motif for the adaptor proteins LMTK2, Tom1 as well as Dab2. Interestingly, compromising Myosin VI vesicle-binding ability by expressing Myosin VI with the R1166X mutation or with single point mutations in the adaptor-binding sites leads to increased F-actin binding of this Myosin in VItro and in VIvo As our results highlight the importance of cargo attachment for regulating actin binding to the motor domain, we perform a detailed characterisation of adaptor protein binding and identify single amino acids within Myosin VI required for binding to cargo adaptors. We not only show that the adaptor proteins can directly interact with the cargo-binding tail of Myosin VI, but our in VItro studies also suggest that multiple adaptor proteins can bind simultaneously to non-overlapping sites in the Myosin VI tail. In conclusion, our characterisation of the human Myosin VI deafness mutant (R1166X) suggests that defects in cargo binding may leave Myosin VI in a primed/activated state with an increased actin-binding ability.
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the autophagy receptor tax1bp1 and the molecular motor Myosin VI are required for clearance of salmonella typhimurium by autophagy
PLOS Pathogens, 2015Co-Authors: David A Tumbarello, Susan D. Arden, John Kendrickjones, Paul T Manna, Mark D Allen, Mark Bycroft, Folma BussAbstract:Autophagy plays a key role during Salmonella infection, by eliminating these pathogens following escape into the cytosol. In this process, selective autophagy receptors, including the Myosin VI adaptor proteins optineurin and NDP52, have been shown to recognize cytosolic pathogens. Here, we demonstrate that Myosin VI and TAX1BP1 are recruited to ubiquitylated Salmonella and play a key role in xenophagy. The absence of TAX1BP1 causes an accumulation of ubiquitin-positive Salmonella, whereas loss of Myosin VI leads to an increase in ubiquitylated and LC3-positive bacteria. Our structural studies demonstrate that the ubiquitin-binding site of TAX1BP1 overlaps with the Myosin VI binding site and point mutations in the TAX1BP1 zinc finger domains that affect ubiquitin binding also ablate binding to Myosin VI. This mutually exclusive binding and the association of TAX1BP1 with LC3 on the outer limiting membrane of autophagosomes may suggest a molecular mechanism for recruitment of this motor to autophagosomes. The predominant role of TAX1BP1, a paralogue of NDP52, in xenophagy is supported by our evolutionary analysis, which demonstrates that functionally intact NDP52 is missing in Xenopus and mice, whereas TAX1BP1 is expressed in all vertebrates analysed. In summary, this work highlights the importance of TAX1BP1 as a novel autophagy receptor in Myosin VI-mediated xenophagy. Our study identifies essential new machinery for the autophagy-dependent clearance of Salmonella typhimurium and suggests modulation of Myosin VI motor actiVIty as a potential therapeutic target in cellular immunity.
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dynamic exchange of Myosin VI on endocytic structures
Journal of Biological Chemistry, 2012Co-Authors: Susan D. Arden, Folma Buss, John Kendrickjones, Lisa M Bond, James R SellersAbstract:The actin-based molecular motor Myosin VI functions in the endocytic uptake pathway, both during the early stages of clathrin-mediated uptake and in later transport to/from early endosomes. This study uses fluorescence recovery after photobleaching (FRAP) to examine the turnover rate of Myosin VI during endocytosis. The results demonstrate that Myosin VI turns over dynamically on endocytic structures with a characteristic half-life common to both the large insert isoform of Myosin VI on clathrin-coated structures and the no-insert isoform on early endosomes. This half-life is shared by the Myosin VI-binding partner Dab2 and is identical for full-length Myosin VI and the cargo-binding tail region. The 4-fold slower half-life of an artificially dimerized construct of Myosin VI on clathrin-coated structures suggests that wild type Myosin VI does not function as a stable dimer, but either as a monomer or in a monomer/dimer equilibrium. Taken together, these FRAP results offer insight into both the basic turnover dynamics and the monomer/dimer nature of Myosin VI.
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autophagy receptors link Myosin VI to autophagosomes to mediate tom1 dependent autophagosome maturation and fusion with the lysosome
Nature Cell Biology, 2012Co-Authors: David A Tumbarello, Susan D. Arden, Bennett J Waxse, Nicholas A Bright, John Kendrickjones, Folma BussAbstract:Autophagy targets pathogens, damaged organelles and protein aggregates for lysosomal degradation. These ubiquitylated cargoes are recognized by specific autophagy receptors, which recruit LC3-positive membranes to form autophagosomes. Subsequently, autophagosomes fuse with endosomes and lysosomes, thus facilitating degradation of their content; however, the machinery that targets and mediates fusion of these organelles with autophagosomes remains to be established. Here we demonstrate that Myosin VI, in concert with its adaptor proteins NDP52, optineurin, T6BP and Tom1, plays a crucial role in autophagy. We identify Tom1 as a Myosin VI binding partner on endosomes, and demonstrate that loss of Myosin VI and Tom1 reduces autophagosomal delivery of endocytic cargo and causes a block in autophagosome-lysosome fusion. We propose that Myosin VI delivers endosomal membranes containing Tom1 to autophagosomes by docking to NDP52, T6BP and optineurin, thereby promoting autophagosome maturation and thus driVIng fusion with lysosomes.
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overexpression of Myosin VI in prostate cancer cells enhances psa and vegf secretion but has no effect on endocytosis
Oncogene, 2010Co-Authors: Claudia Puri, Susan D. Arden, John Kendrickjones, Margarita V Chibalina, Antonina J Kruppa, Folma BussAbstract:Tissue expression microarrays, employed to determine the players and mechanisms leading to prostate cancer development, have consistently shown that Myosin VI, a unique actin-based motor, is upregulated in medium-grade human prostate cancers. Thus, to understand the role of Myosin VI in prostate cancer development, we have characterized its intracellular localization and function in the prostate cancer cell line LNCaP. Using light and electron microscopy, we identified Myosin VI on Rab5-positive early endosomes, as well as on recycling endosomes and the trans-Golgi network. Intracellular targeting seems to involve two Myosin VI-interacting proteins, GIPC and LMTK2, both of which can be co-immunoprecipitated with Myosin VI from LNCaP cells. The absence of Disabled-2 (Dab2), a tumour suppressor and Myosin VI-binding partner, inhibits recruitment of Myosin VI to endocytic structures at the plasma membrane in LNCaP cells, but interestingly has no effect on endocytosis. Small interfering RNA-mediated downregulation of Myosin VI expression results in a significant reduction in prostate-specific antigen (PSA) and vascular endothelial growth factor (VEGF) secretion in LNCaP cells. Our results suggest that in prostate cancer cells, Myosin VI regulates protein secretion, but the overexpression of Myosin VI has no major impact on clathrin-mediated endocytosis.
Anne Houdusse - One of the best experts on this subject based on the ideXlab platform.
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Myosin VI and branched actin filaments mediate membrane constriction and fission of melanosomal tubule carriers
Journal of Cell Biology, 2018Co-Authors: Lea Ripoll, Xavier Heiligenstein, Ilse Hurbain, Lia Domingues, Florent Figon, Karl J Petersen, Megan K Dennis, Anne HoudusseAbstract:Vesicular and tubular transport intermediates regulate organellar cargo dynamics. Transport carrier release involves local and profound membrane remodeling before fission. Pinching the neck of a budding tubule or vesicle requires mechanical forces, likely exerted by the action of molecular motors on the cytoskeleton. Here, we show that Myosin VI, together with branched actin filaments, constricts the membrane of tubular carriers that are then released from melanosomes, the pigment containing lysosome-related organelles of melanocytes. By combining superresolution fluorescence microscopy, correlative light and electron microscopy, and biochemical analyses, we find that Myosin VI motor actiVIty mediates severing by constricting the neck of the tubule at specific melanosomal subdomains. Pinching of the tubules involves the cooperation of the Myosin adaptor optineurin and the actiVIty of actin nucleation machineries, including the WASH and Arp2/3 complexes. The fission and release of these tubules allows for the export of components from melanosomes, such as the SNARE VAMP7, and promotes melanosome maturation and transfer to keratinocytes. Our data reveal a new Myosin VI– and actin-dependent membrane fission mechanism required for organelle function.
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Myosin VI deafness mutation prevents the initiation of processive runs on actin
Proceedings of the National Academy of Sciences of the United States of America, 2015Co-Authors: Olena Pylypenko, Anne Houdusse, Zhaohui Yang, Lin Song, Ai Shima, Lee H SweeneyAbstract:Mutations in the reverse-direction Myosin, Myosin VI, are associated with deafness in humans and mice. A Myosin VI deafness mutation, D179Y, which is in the transducer of the motor, uncoupled the release of the ATP hydrolysis product, inorganic phosphate (Pi), from dependency on actin binding and destroyed the ability of single dimeric molecules to move processively on actin filaments. We observed that processive movement is rescued if ATP is added to the mutant dimer following binding of both heads to actin in the absence of ATP, demonstrating that the mutation selectively destroys the initiation of processive runs at physiological ATP levels. A drug (omecamtiv) that accelerates the actin-activated actiVIty of cardiac Myosin was able to rescue processiVIty of the D179Y mutant dimers at physiological ATP concentrations by slowing the actin-independent release of Pi. Thus, it may be possible to create Myosin VI-specific drugs that rescue the function of deafness-causing mutations.
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Myosin VI Must Dimerize and Deploy Its Unusual Lever Arm in Order to Perform Its Cellular Roles
Cell reports, 2014Co-Authors: Monalisa Mukherjea, Daniel Safer, Anne Houdusse, Zhaohui Yang, Virginie Ropars, M. Yusuf Ali, Carlos Kikuti, Helena Sirkia, David M. Warshaw, H. Lee SweeneyAbstract:Summary It is unclear whether the reverse-direction Myosin (Myosin VI) functions as a monomer or dimer in cells and how it generates large movements on actin. We deleted a stable, single-α-helix (SAH) domain that has been proposed to function as part of a lever arm to amplify movements without impact on in VItro movement or in VIvo functions. A Myosin VI construct that used this SAH domain as part of its lever arm was able to take large steps in VItro but did not rescue in VIvo functions. It was necessary for Myosin VI to internally dimerize, triggering unfolding of a three-helix bundle and calmodulin binding in order to step normally in VItro and rescue endocytosis and Golgi morphology in Myosin VI-null fibroblasts. A model for Myosin VI emerges in which cargo binding triggers dimerization and unfolds the three-helix bundle to create a lever arm essential for in VIvo functions.
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Processive Steps in the Reverse Direction Require Uncoupling of the Lead Head Lever Arm of Myosin VI.
Molecular cell, 2012Co-Authors: Julie Ménétrey, H. Lee Sweeney, Xiaoyan Liu, T. Isabet, Virginie Ropars, Monalisa Mukherjea, Olena Pylypenko, Javier Pérez, Patrice Vachette, Anne HoudusseAbstract:Summary Myosin VI is the only known reverse-direction Myosin motor. It has an unprecedented means of amplifying movements within the motor involVIng rearrangements of the converter subdomain at the C terminus of the motor and an unusual lever arm projecting from the converter. While the average step size of a Myosin VI dimer is 30–36 nm, the step size is highly variable, presenting a challenge to the lever arm mechanism by which all Myosins are thought to move. Herein, we present structures of Myosin VI that reveal regions of compliance that allow an uncoupling of the lead head when movement is modeled on actin. The location of the compliance restricts the possible actin binding sites and predicts the observed stepping behaVIor. The model reveals that Myosin VI, unlike plus-end directed Myosins, does not use a pure lever arm mechanism, but instead steps with a mechanism analogous to the kinesin neck-linker uncoupling model.
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role of insert 1 of Myosin VI in modulating nucleotide affinity
Journal of Biological Chemistry, 2011Co-Authors: Olena Pylypenko, Alan B Zong, Anne Houdusse, Xiaoyan Liu, Lin Song, Gaelle Squires, Lee H SweeneyAbstract:Myosin VI is unique in its directionality among Myosin superfamily members and also displays a slow and strain-dependent rate of ATP binding that allows for gating between its heads. In this study we demonstrate that leucine 310 is positioned by a class VI-specific insert, insert-1, so as to account for the selective hindrance of ATP versus ADP binding. Mutation of leucine 310 to glycine removes all influence of insert-1 on ATP binding. Furthermore, by analyzing Myosin VI structures with either leucine 310 substituted to a glycine or complete removal of insert-1, we conclude that nucleotides may initially bind to Myosin by their purine rings before docking their phosphate moieties. Otherwise, insert-1 could not exert a differential influence on ATP versus ADP binding.
