title: Cholesterol-sensitive Cdc42 activation regulates actin polymerization for endocytosis via the GEEC pathway.
abstract: Glycosyl-phosphatidylinositol (GPI)-anchored proteins (GPI-APs) are present at the surface of living cells in cholesterol dependent nanoscale clusters. These clusters appear to act as sorting signals for the selective endocytosis of GPI-APs via a Cdc42-regulated, dynamin and clathrin-independent pinocytic pathway called the GPI-AP-enriched early endosomal compartments (GEECs) pathway. Here we show that endocytosis via the GEECs pathway is inhibited by mild depletion of cholesterol, perturbation of actin polymerization or overexpression of the Cdc42/Rac-interactive-binding (CRIB) motif of neural Wiskott-Aldrich syndrome protein (N-WASP). Consistent with the involvement of Cdc42-based actin nanomachinery, nascent endocytic vesicles containing cargo for the GEEC pathway co-localize with fluorescent protein-tagged isoforms of Cdc42, CRIB domain, N-WASP and actin; high-resolution electron microscopy on plasma membrane sheets reveals Cdc42-labelled regions rich in green fluorescent protein-GPI. Using total internal reflection fluorescence microscopy at the single-molecule scale, we find that mild cholesterol depletion alters the dynamics of actin polymerization at the cell surface by inhibiting Cdc42 activation and consequently its stabilization at the cell surface. These results suggest that endocytosis into GEECs occurs through a cholesterol-sensitive, Cdc42-based recruitment of the actin polymerization machinery.
authors.affiliation: National Centre for Biological Sciences, UAS-GKVK Campus, Bellary Road, Bangalore 560065, India.
authors.forename: Rahul
authors.initials: R
authors.lastname: Chadda
authors.forename: Mark T
authors.initials: MT
authors.lastname: Howes
authors.forename: Sarah J
authors.initials: SJ
authors.lastname: Plowman
authors.forename: John F
authors.initials: JF
authors.lastname: Hancock
authors.forename: Robert G
authors.initials: RG
authors.lastname: Parton
authors.forename: Satyajit
authors.initials: S
authors.lastname: Mayor
mesh_terms: D000199:Actins / Q000378:metabolism* / Q000648:ultrastructure; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D016466:CHO Cells; D002460:Cell Line; D002784:Cholesterol / Q000378:metabolism*; D002966:Clathrin / Q000378:metabolism / Q000648:ultrastructure; D006224:Cricetinae; D003412:Cricetulus; D034281:Dynamins / Q000378:metabolism / Q000648:ultrastructure; D004705:Endocytosis; D017261:Glycosylphosphatidylinositols / Q000378:metabolism*; D049452:Green Fluorescent Proteins / Q000378:metabolism; D051316:Wiskott-Aldrich Syndrome Protein / Q000737:chemistry / Q000378:metabolism* / Q000648:ultrastructure; D020764:cdc42 GTP-Binding Protein / Q000378:metabolism* / Q000648:ultrastructure
chemical_list: D000199:Actins; D002966:Clathrin; D017261:Glycosylphosphatidylinositols; D051316:Wiskott-Aldrich Syndrome Protein; D049452:Green Fluorescent Proteins; D002784:Cholesterol; D020764:cdc42 GTP-Binding Protein; D034281:Dynamins