Myosin VI and branched actin filaments mediate membrane constriction and fission of melanosomal tubule carriers

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.

[1]  K. Rottner,et al.  Cortactin: Cell Functions of A Multifaceted Actin-Binding Protein. , 2017, Trends in cell biology.

[2]  G. Voth,et al.  Friction Mediates Scission of Tubular Membranes Scaffolded by BAR Proteins , 2017, Cell.

[3]  Sabine Bardin,et al.  Routing of the RAB6 secretory pathway towards the lysosome related organelle of melanocytes , 2017, Nature Communications.

[4]  P. Thomason,et al.  Mroh1, a lysosomal regulator localized by WASH-generated actin , 2017, Journal of Cell Science.

[5]  F. Buss,et al.  MYO6 is targeted by Salmonella virulence effectors to trigger PI3-kinase signaling and pathogen invasion into host cells , 2017, Proceedings of the National Academy of Sciences.

[6]  Jean Salamero,et al.  eC-CLEM: Flexible Multidimensional Registration Software for Correlative Microscopies with Refined Accuracy Mapping , 2017, Microscopy and Microanalysis.

[7]  R. Heintzmann,et al.  Optical photon reassignment with increased axial resolution by structured illumination , 2016, Methods and applications in fluorescence.

[8]  R. Insall,et al.  WASH drives early recycling from macropinosomes and phagosomes to maintain surface phagocytic receptors , 2016, Proceedings of the National Academy of Sciences.

[9]  G. van Loo,et al.  Cellular Functions of Optineurin in Health and Disease. , 2016, Trends in immunology.

[10]  R. Insall,et al.  Loss of strumpellin in the melanocytic lineage impairs the WASH Complex but does not affect coat colour , 2016, Pigment cell & melanoma research.

[11]  Anand R. Patwardhan,et al.  Ions switch off darkness: role of TPC2 in melanosomes , 2016, Pigment Cell & Melanoma Research.

[12]  G. van Niel,et al.  PMEL Amyloid Fibril Formation: The Bright Steps of Pigmentation , 2016, International journal of molecular sciences.

[13]  T. Galli,et al.  BLOC-1 and BLOC-3 regulate VAMP7 cycling to and from melanosomes via distinct tubular transport carriers , 2016, The Journal of cell biology.

[14]  E. Oancea,et al.  A melanosomal two-pore sodium channel regulates pigmentation , 2016, Scientific Reports.

[15]  G. Raposo,et al.  Illuminating the dark side of recycling endosomes , 2016, Cell cycle.

[16]  K. Christian,et al.  TPC2 controls pigmentation by regulating melanosome pH and size , 2016, Proceedings of the National Academy of Sciences.

[17]  G. Raposo,et al.  BLOC-1 Brings Together the Actin and Microtubule Cytoskeletons to Generate Recycling Endosomes , 2016, Current Biology.

[18]  R. Weigert,et al.  Arp2/3-mediated F-actin formation controls regulated exocytosis in vivo , 2015, Nature Communications.

[19]  Riddhi Atul Jani,et al.  STX13 regulates cargo delivery from recycling endosomes during melanosome biogenesis , 2015, Journal of Cell Science.

[20]  G. Guillemin,et al.  Defects in optineurin- and myosin VI-mediated cellular trafficking in amyotrophic lateral sclerosis. , 2015, Human molecular genetics.

[21]  G. Raposo,et al.  Exosomes released by keratinocytes modulate melanocyte pigmentation , 2015, Nature Communications.

[22]  M. Marks,et al.  An intracellular anion channel critical for pigmentation , 2014, eLife.

[23]  E. C. Dell'Angelica,et al.  Myosin Vc Interacts with Rab32 and Rab38 Proteins and Works in the Biogenesis and Secretion of Melanosomes* , 2014, The Journal of Biological Chemistry.

[24]  J. Hammer,et al.  Melanosome transfer: it is best to give and receive. , 2014, Current opinion in cell biology.

[25]  C. Delevoye.  Melanin transfer: the keratinocytes are more than gluttons. , 2014, The Journal of investigative dermatology.

[26]  Jean Salamero,et al.  The CryoCapsule: Simplifying Correlative Light to Electron Microscopy , 2014, Traffic.

[27]  A. Gautreau,et al.  Function and regulation of the endosomal fusion and fission machineries. , 2014, Cold Spring Harbor perspectives in biology.

[28]  A. Gautreau,et al.  Retromer-mediated endosomal protein sorting: all WASHed up! , 2013, Trends in cell biology.

[29]  R. Insall,et al.  WASH is required for lysosomal recycling and efficient autophagic and phagocytic digestion , 2013, Molecular biology of the cell.

[30]  G. Raposo,et al.  Lysosome-related organelles: unusual compartments become mainstream. , 2013, Current opinion in cell biology.

[31]  Zhen-Ge Luo,et al.  Myosin Vb controls biogenesis of post-Golgi Rab10 carriers during axon development , 2013, Nature Communications.

[32]  F. Buss,et al.  Myosin VI and its cargo adaptors – linking endocytosis and autophagy , 2013, Journal of Cell Science.

[33]  B. Nolen,et al.  Small molecules CK-666 and CK-869 inhibit actin-related protein 2/3 complex by blocking an activating conformational change. , 2013, Chemistry & biology.

[34]  M. Welch,et al.  Regulation of integrin trafficking, cell adhesion, and cell migration by WASH and the Arp2/3 complex , 2012, Cytoskeleton.

[35]  F. Barr,et al.  BLOC-3 Mutated in Hermansky-Pudlak Syndrome Is a Rab32/38 Guanine Nucleotide Exchange Factor , 2012, Current Biology.

[36]  D. Manstein,et al.  Kinetic properties and small-molecule inhibition of human myosin-6 , 2012, FEBS letters.

[37]  F. Buss,et al.  Autophagy-receptors link myosin VI to autophagosomes to mediate Tom1-dependent autophagosome maturation and fusion with the lysosome , 2012, Nature Cell Biology.

[38]  Anna Espinal,et al.  Ultrastructural dynamics of proteins involved in endocytic budding , 2012, Proceedings of the National Academy of Sciences.

[39]  D. Billadeau,et al.  Trafficking defects in WASH-knockout fibroblasts originate from collapsed endosomal and lysosomal networks , 2012, Molecular biology of the cell.

[40]  D. Drubin,et al.  Myosin 1E coordinates actin assembly and cargo trafficking during clathrin-mediated endocytosis , 2012, Molecular biology of the cell.

[41]  E. Derivery,et al.  Actin Polymerization Controls the Organization of WASH Domains at the Surface of Endosomes , 2012, PloS one.

[42]  Hari Shroff,et al.  Resolution Doubling in Live, Multicellular Organisms via Multifocal Structured Illumination Microscopy , 2012, Nature Methods.

[43]  G. Raposo,et al.  Myosin VI Regulates Actin Dynamics and Melanosome Biogenesis , 2012, Traffic.

[44]  Marcus J. Taylor,et al.  A Feedback Loop between Dynamin and Actin Recruitment during Clathrin-Mediated Endocytosis , 2012, PLoS biology.

[45]  M. Seaman,et al.  Recruitment of the endosomal WASH complex is mediated by the extended 'tail' of Fam21 binding to the retromer protein Vps35. , 2012, The Biochemical journal.

[46]  Bernard Hoflack,et al.  Bridging membrane and cytoskeleton dynamics in the secretory and endocytic pathways , 2011, Nature Cell Biology.

[47]  M. Prevost,et al.  Clathrin phosphorylation is required for actin recruitment at sites of bacterial adhesion and internalization , 2011, The Journal of cell biology.

[48]  Comert Kural,et al.  Actin dynamics counteract membrane tension during clathrin-mediated endocytosis , 2011, Nature Cell Biology.

[49]  R. Insall,et al.  Actin polymerization driven by WASH causes V-ATPase retrieval and vesicle neutralization before exocytosis , 2011, The Journal of cell biology.

[50]  John A.G. Briggs,et al.  Correlated fluorescence and 3D electron microscopy with high sensitivity and spatial precision , 2011, The Journal of cell biology.

[51]  J. Sellers,et al.  Myosin VI and its binding partner optineurin are involved in secretory vesicle fusion at the plasma membrane , 2011, Molecular biology of the cell.

[52]  G. Chalancon,et al.  Rab and actomyosin-dependent fission of transport vesicles at the Golgi complex , 2010, Nature Cell Biology.

[53]  K. Gaus,et al.  Actin Dynamics Drive Membrane Reorganization and Scission in Clathrin-Independent Endocytosis , 2010, Cell.

[54]  P. De Camilli,et al.  Coordinated actions of actin and BAR proteins upstream of dynamin at endocytic clathrin-coated pits. , 2009, Developmental cell.

[55]  M. Gardel,et al.  Identification and characterization of a small molecule inhibitor of formin-mediated actin assembly. , 2009, Chemistry & biology.

[56]  D. Billadeau,et al.  A FAM21-containing WASH complex regulates retromer-dependent sorting. , 2009, Developmental cell.

[57]  E. Derivery,et al.  The Arp2/3 activator WASH controls the fission of endosomes through a large multiprotein complex. , 2009, Developmental cell.

[58]  T. Pollard,et al.  Characterization of two classes of small molecule inhibitors of Arp2/3 complex , 2009, Nature.

[59]  K. Ayscough,et al.  Under Pressure: the Differential Requirements for Actin during Yeast and Mammalian Endocytosis , 2009, Nature Cell Biology.

[60]  S. Whelan,et al.  Vesicular Stomatitis Virus Enters Cells through Vesicles Incompletely Coated with Clathrin That Depend upon Actin for Internalization , 2009, PLoS pathogens.

[61]  A. Verkleij,et al.  Electron tomography of early melanosomes: Implications for melanogenesis and the generation of fibrillar amyloid sheets , 2008, Proceedings of the National Academy of Sciences.

[62]  V. Hearing,et al.  Modifying skin pigmentation - approaches through intrinsic biochemistry and exogenous agents. , 2008, Drug discovery today. Disease mechanisms.

[63]  D. Tobin,et al.  The silver locus product (Silv/gp100/Pmel17) as a new tool for the analysis of melanosome transfer in human melanocyte–keratinocyte co‐culture , 2008, Experimental dermatology.

[64]  Y. Wada,et al.  Vacuolar-type H+-ATPase with the a3 isoform is the proton pump on premature melanosomes , 2008, Cell and Tissue Research.

[65]  F. Buss,et al.  Myosin VI and its interacting protein LMTK2 regulate tubule formation and transport to the endocytic recycling compartment , 2007, Journal of Cell Science.

[66]  F. Buss,et al.  Myosin VI is required for sorting of AP-1B–dependent cargo to the basolateral domain in polarized MDCK cells , 2007, The Journal of cell biology.

[67]  M. Mooseker,et al.  Myosin 1E interacts with synaptojanin‐1 and dynamin and is involved in endocytosis , 2007, FEBS letters.

[68]  G. Spudich,et al.  Myosin VI targeting to clathrin-coated structures and dimerization is mediated by binding to Disabled-2 and PtdIns(4,5)P2 , 2007, Nature Cell Biology.

[69]  T. Hasson,et al.  Binding of internalized receptors to the PDZ domain of GIPC/synectin recruits myosin VI to endocytic vesicles , 2006, Proceedings of the National Academy of Sciences.

[70]  G. Oster,et al.  Endocytic vesicle scission by lipid phase boundary forces , 2006, Proceedings of the National Academy of Sciences.

[71]  T. Pollard,et al.  Interactions of WASp, myosin-I, and verprolin with Arp2/3 complex during actin patch assembly in fission yeast , 2005, The Journal of cell biology.

[72]  G. Spudich,et al.  Optineurin links myosin VI to the Golgi complex and is involved in Golgi organization and exocytosis , 2005, The Journal of cell biology.

[73]  M. Sheetz,et al.  Mitochondrial Function and Actin Regulate Dynamin-Related Protein 1-Dependent Mitochondrial Fission , 2005, Current Biology.

[74]  T. Hasson,et al.  Regulation of Myosin‐VI Targeting to Endocytic Compartments , 2004, Traffic.

[75]  Rong Li,et al.  Dynamics of Yeast Myosin I Evidence for a Possible Role in Scission of Endocytic Vesicles , 2004, Current Biology.

[76]  James A. Spudich,et al.  The Mechanism of Myosin VI Translocation and Its Load-Induced Anchoring , 2004, Cell.

[77]  S. Chasserot-Golaz,et al.  Regulated exocytosis in neuroendocrine cells: a role for subplasmalemmal Cdc42/N-WASP-induced actin filaments. , 2003, Molecular biology of the cell.

[78]  B. Humbel,et al.  Influence of aldehyde fixation on the morphology of endosomes and lysosomes: quantitative analysis and electron tomography , 2003, Journal of microscopy.

[79]  T. Hasson,et al.  Myo6 facilitates the translocation of endocytic vesicles from cell peripheries. , 2003, Molecular biology of the cell.

[80]  Timothy J Mitchison,et al.  Dissecting Temporal and Spatial Control of Cytokinesis with a Myosin II Inhibitor , 2003, Science.

[81]  K. Steel,et al.  Loss of myosin VI reduces secretion and the size of the Golgi in fibroblasts from Snell's waltzer mice , 2003, The EMBO journal.

[82]  J. Luzio,et al.  Myosin VI isoform localized to clathrin‐coated vesicles with a role in clathrin‐mediated endocytosis , 2001, The EMBO journal.

[83]  G. Raposo,et al.  Distinct Protein Sorting and Localization to Premelanosomes, Melanosomes, and Lysosomes in Pigmented Melanocytic Cells✪ , 2001, The Journal of cell biology.

[84]  Daniel Safer,et al.  Myosin VI is an actin-based motor that moves backwards , 1999, Nature.

[85]  M. Brown,et al.  Visualization of acidic organelles in intact cells by electron microscopy. , 1984, Proceedings of the National Academy of Sciences of the United States of America.

[86]  G. Raposo,et al.  Analyzing Lysosome-Related Organelles by Electron Microscopy. , 2017, Methods in molecular biology.

[87]  A. Straight,et al.  Specificity of blebbistatin, an inhibitor of myosin II , 2004, Journal of Muscle Research & Cell Motility.