Actin Polymerization Does Not Provide Direct Mechanical Forces for Vesicle Fission during Clathrin-Mediated Endocytosis

Actin polymerization is important for vesicle fission during clathrin-mediated endocytosis (CME), and it has been proposed that actin polymerization may promote vesicle fission during CME by providing direct mechanical forces. However, there is no direct evidence in support of this hypothesis. In the present study, the role of actin polymerization in vesicle fission was tested by analyzing the kinetics of the endocytic tubular membrane neck (the fission-pore) with cell-attached capacitance measurements to detect CME of single vesicles in a millisecond time resolution in mouse chromaffin cells. Inhibition in dynamin GTPase activity increased the fission-pore conductance (Gp), supporting the mechanical role of dynamin GTPase in vesicle fission. However, disruptions in actin polymerization did not alter the fission-pore conductance Gp, thus arguing against the force-generating role of actin polymerization in vesicle fission during CME. Similar to disruptions of actin polymerization, cholesterol depletion results in an increase in the fission-pore duration, indicating a role for cholesterol-dependent membrane reorganization in vesicle fission. Further experiments suggested that actin polymerization and cholesterol might function in vesicle fission during CME in the same pathway. Our results thus support a model in which actin polymerization promotes vesicle fission during CME by inducing cholesterol-dependent membrane reorganization.

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

[2]  M. Lindau,et al.  Synaptotagmin 1 Is Necessary for the Ca2+ Dependence of Clathrin-Mediated Endocytosis , 2012, Journal of Neuroscience.

[3]  Kenneth A. Taylor,et al.  Structural Organization of the Actin Cytoskeleton at Sites of Clathrin-Mediated Endocytosis , 2011, Current Biology.

[4]  R. Chan,et al.  Synaptojanin 1-mediated PI(4,5)P2 hydrolysis is modulated by membrane curvature and facilitates membrane fission. , 2011, Developmental cell.

[5]  J. Cooper,et al.  Actin dynamics and endocytosis in yeast and mammals. , 2010, Current opinion in biotechnology.

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

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

[8]  David G. Drubin,et al.  The Mechanochemistry of Endocytosis , 2009, PLoS biology.

[9]  Emanuele Cocucci,et al.  Distinct Dynamics of Endocytic Clathrin-Coated Pits and Coated Plaques , 2009, PLoS biology.

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

[11]  S. Schmid,et al.  Real-Time Visualization of Dynamin-Catalyzed Membrane Fission and Vesicle Release , 2008, Cell.

[12]  Wei Wu,et al.  Rapid bulk endocytosis and its kinetics of fission pore closure at a central synapse , 2007, Proceedings of the National Academy of Sciences.

[13]  D. Fletcher,et al.  Actin polymerization serves as a membrane domain switch in model lipid bilayers. , 2006, Biophysical journal.

[14]  Leon Lagnado,et al.  Clathrin-Mediated Endocytosis Is the Dominant Mechanism of Vesicle Retrieval at Hippocampal Synapses , 2006, Neuron.

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

[16]  T. Kirchhausen,et al.  Dynasore, a cell-permeable inhibitor of dynamin. , 2006, Developmental cell.

[17]  M. Kaksonen,et al.  Harnessing actin dynamics for clathrin-mediated endocytosis , 2006, Nature Reviews Molecular Cell Biology.

[18]  P. Camilli,et al.  GTP-dependent twisting of dynamin implicates constriction and tension in membrane fission , 2006, Nature.

[19]  David Zenisek,et al.  Coupling between Clathrin-Coated-Pit Invagination, Cortactin Recruitment, and Membrane Scission Observed in Live Cells , 2005, Cell.

[20]  P. Bassereau,et al.  Role of curvature and phase transition in lipid sorting and fission of membrane tubules , 2005, The EMBO journal.

[21]  Sandra L Schmid,et al.  A dynamic actin cytoskeleton functions at multiple stages of clathrin-mediated endocytosis. , 2004, Molecular biology of the cell.

[22]  K. Chandran,et al.  Endocytosis by Random Initiation and Stabilization of Clathrin-Coated Pits , 2004, Cell.

[23]  Watt W. Webb,et al.  Imaging coexisting fluid domains in biomembrane models coupling curvature and line tension , 2003, Nature.

[24]  M. McNiven,et al.  Dynamin at the actin-membrane interface. , 2003, Current opinion in cell biology.

[25]  S. Schmid,et al.  Dynamin GTPase domain mutants block endocytic vesicle formation at morphologically distinct stages. , 2001, Molecular biology of the cell.

[26]  M. Lindau,et al.  Resolution of patch capacitance recordings and of fusion pore conductances in small vesicles. , 2000, Biophysical journal.

[27]  M. Stowell,et al.  Nucleotide-dependent conformational changes in dynamin: evidence for a mechanochemical molecular spring , 1999, Nature Cell Biology.

[28]  W. Almers,et al.  Endocytic vesicles move at the tips of actin tails in cultured mast cells , 1999, Nature Cell Biology.

[29]  B. Deurs,et al.  Extraction of cholesterol with methyl-beta-cyclodextrin perturbs formation of clathrin-coated endocytic vesicles. , 1999, Molecular biology of the cell.

[30]  S. Schmid,et al.  Induction of mutant dynamin specifically blocks endocytic coated vesicle formation , 1994, The Journal of cell biology.

[31]  M. Lindau,et al.  Exo-endocytosis and closing of the fission pore during endocytosis in single pituitary nerve terminals internally perfused with high calcium concentrations. , 1994, Proceedings of the National Academy of Sciences of the United States of America.