title: Impairing actin filament or syndapin functions promotes accumulation of clathrin-coated vesicles at the apical plasma membrane of acinar epithelial cells.
abstract: In this article, we investigate the contributions of actin filaments and accessory proteins to apical clathrin-mediated endocytosis in primary rabbit lacrimal acini. Confocal fluorescence and electron microscopy revealed that cytochalasin D promoted apical accumulation of clathrin, alpha-adaptin, dynamin, and F-actin and increased the amounts of coated pits and vesicles at the apical plasma membrane. Sorbitol density gradient analysis of membrane compartments showed that cytochalasin D increased [14C]dextran association with apical membranes from stimulated acini, consistent with functional inhibition of apical endocytosis. Recombinant syndapin SH3 domains interacted with lacrimal acinar dynamin, neuronal Wiskott-Aldrich Syndrome protein (N-WASP), and synaptojanin; their introduction by electroporation elicited remarkable accumulation of clathrin, accessory proteins, and coated pits at the apical plasma membrane. These SH3 domains also significantly (p </= 0.05) increased F-actin, with substantial colocalization of dynamin and N-WASP with the additional filaments. Coelectroporation with the VCA domain of N-WASP blocked the increase in F-actin and reversed the morphological changes indicative of impaired apical endocytosis. We suggest that transient modulation of actin polymerization by syndapins through activation of the Arp2/3 complex via N-WASP coordinates dynamin-mediated vesicle fission at the apical plasma membrane of acinar epithelia. Trapping of assembled F-actin intermediates during this process by cytochalasin D or syndapin SH3 domains impairs endocytosis.
authors.affiliation: Department of Pharmaceutical Sciences, University of Southern California, Los Angeles, California 90033, USA.
authors.forename: Silvia R
authors.initials: SR
authors.lastname: Da Costa
authors.forename: Eunbyul
authors.initials: E
authors.lastname: Sou
authors.forename: Jiansong
authors.initials: J
authors.lastname: Xie
authors.forename: Francie A
authors.initials: FA
authors.lastname: Yarber
authors.forename: Curtis T
authors.initials: CT
authors.lastname: Okamoto
authors.forename: Michael
authors.initials: M
authors.lastname: Pidgeon
authors.forename: Michael M
authors.initials: MM
authors.lastname: Kessels
authors.forename: Austin K
authors.initials: AK
authors.lastname: Mircheff
authors.forename: Joel E
authors.initials: JE
authors.lastname: Schechter
authors.forename: Britta
authors.initials: B
authors.lastname: Qualmann
authors.forename: Sarah F
authors.initials: SF
authors.lastname: Hamm-Alvarez
mesh_terms: D008841:Actin Cytoskeleton / Q000187:drug effects; D051377:Actin-Related Protein 2; D051378:Actin-Related Protein 3; D000199:Actins / Q000378:metabolism* / Q000502:physiology; D000818:Animals; D022181:COP-Coated Vesicles / Q000378:metabolism* / Q000502:physiology; D002352:Carrier Proteins / Q000378:metabolism* / Q000502:physiology; D002458:Cell Fractionation; D002462:Cell Membrane / Q000378:metabolism* / Q000502:physiology; D002478:Cells, Cultured; D003001:Cloning, Molecular; D015638:Cytochalasin D / Q000494:pharmacology; D003598:Cytoskeletal Proteins / Q000378:metabolism; D034281:Dynamins / Q000378:metabolism; D004705:Endocytosis; D004847:Epithelial Cells / Q000378:metabolism* / Q000648:ultrastructure; D005260:Female; D007765:Lacrimal Apparatus / Q000378:metabolism; D018613:Microscopy, Confocal; D008854:Microscopy, Electron; D009419:Nerve Tissue Proteins / Q000378:metabolism; D010744:Phosphoric Monoester Hydrolases / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011817:Rabbits; D051317:Wiskott-Aldrich Syndrome Protein, Neuronal
chemical_list: D051377:Actin-Related Protein 2; D051378:Actin-Related Protein 3; D000199:Actins; D002352:Carrier Proteins; D003598:Cytoskeletal Proteins; D009419:Nerve Tissue Proteins; C117738:Pacsin1 protein, rat; D051317:Wiskott-Aldrich Syndrome Protein, Neuronal; D015638:Cytochalasin D; C097702:synaptojanin; D010744:Phosphoric Monoester Hydrolases; D034281:Dynamins