title: Role of lipids and actin in the formation of clathrin-coated pits.
abstract: Assembly of clathrin-coated pits and their maturation into coated vesicles requires coordinated interactions between specific lipids and several structural and regulatory proteins. In the presence of primary alcohols, phospholipase D generates phosphatidylalcohols instead of PA, reducing stimulation of phosphatidyl inositol 5-kinase (PI5K) and hence decreasing formation of phosphoinositide-4,5-biphosphate (PIP(2)). Using live-cell imaging, we have shown that acute treatment of cells with 1-butanol or other small primary alcohols induces rapid disassembly of coated pits at the plasma membrane and blocks appearance of new ones. Addition of exogenous PIP(2) reverses this effect. Coated pits and vesicles reappear synchronously upon removal of 1-butanol; we have used this synchrony to assess the role of actin in coated vesicle assembly. Prolonged inhibition of actin polymerization by latrunculin A or cytochalasin D reduced by approximately 50% the frequency of coated pit formation without affecting maturation into coated vesicles. As in control cells, removal of 1-butanol in the continued presence of an actin depolymerizer led to synchronous appearance of new pits, which matured normally. Thus, remodeling of the actin cytoskeleton is not essential for clathrin-coated vesicle assembly but may indirectly affect the nucleation of clathrin-coated pits.
authors.affiliation: Department of Cell Biology and the CBR Institute for Biomedical Research, Harvard Medical School, Boston, 200 Longwood Ave, MA 02115, USA.
authors.forename: Emmanuel
authors.initials: E
authors.lastname: Boucrot
authors.forename: Saveez
authors.initials: S
authors.lastname: Saffarian
authors.forename: Ramiro
authors.initials: R
authors.lastname: Massol
authors.forename: Tomas
authors.initials: T
authors.lastname: Kirchhausen
authors.forename: Marcelo
authors.initials: M
authors.lastname: Ehrlich
mesh_terms: D020001:1-Butanol / Q000494:pharmacology*; D000199:Actins / Q000378:metabolism / Q000502:physiology*; D000818:Animals; D019556:COS Cells; D002460:Cell Line; D002522:Chlorocebus aethiops; D022163:Clathrin-Coated Vesicles / Q000187:drug effects / Q000378:metabolism*; D003034:Coated Pits, Cell-Membrane / Q000187:drug effects / Q000378:metabolism; D003599:Cytoskeleton / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D008055:Lipids / Q000737:chemistry*; D008081:Liposomes / Q000378:metabolism; D010716:Phosphatidylinositols / Q000737:chemistry; D014162:Transfection; D014168:Transferrin / Q000493:pharmacokinetics
chemical_list: D000199:Actins; D008055:Lipids; D008081:Liposomes; D010716:Phosphatidylinositols; D014168:Transferrin; D020001:1-Butanol