The process by which clathrin-coated vesicles are produced involves interactions of multifunctional adaptor proteins with the plasma membrane, as well as with clathrin and several accessory proteins and phosphoinositides. Here we review recent findings highlighting new insights into mechanisms underlying clathrin-dependent endocytosis.

No abstract

Since human immunodeficiency virus (HIV)-

Actin is implicated in membrane fusion, but the precise mechanisms remain unclear. We showed earlier that membrane organelles catalyze the de novo assembly of F-actin that then facilitates the fusion between latex bead phagosomes and a mixture of early and late endocytic organelles. Here, we correlated the polymerization and organization of F-actin with phagosome and endocytic organelle fusion processes in vitro by using biochemistry and light and electron microscopy. When membrane organelles and cytosol were incubated at 37°C with ATP, cytosolic actin polymerized rapidly and became organized into bundles and networks adjacent to membrane organelles. By 30-min incubation, a gel-like state was formed with little further polymerization of actin thereafter. Also during this time, the bulk of in vitro fusion events occurred between phagosomes/endocytic organelles. The fusion between latex bead phagosomes and late endocytic organelles, or between late endocytic organelles themselves was facilitated by actin, but we failed to detect any effect of perturbing F-actin polymerization on early endosome fusion. Consistent with this, late endosomes, like phagosomes, could nucleate F-actin, whereas early endosomes could not. We propose that actin assembled by phagosomes or late endocytic organelles can provide tracks for fusion-partner organelles to move vectorially toward them, via membranebound myosins, to facilitate fusion. INTRODUCTIONActin is essential for many cellular processes and dynamic polymerization/depolymerization of actin filaments is a critical property of all eukaryotic cells (Mitchison and Cramer, 1996;Carlier, 1998;Machesky and Insall, 1999;Small et al., 1999;Amann and Pollard, 2000). Much of the F-actin assembled in cells is intimately associated with membranes, especially the plasma membrane (Tilney, 1976;Hoglund et al., 1980;Lindberg et al., 1981;Carraway and Carraway, 1989;Dickinson and Purich, 2002). The first links between F-actin and membranes were reported by Tilney and Cardell (1970) who demonstrated the role of plasma membranes in actin nucleation, and Orci et al. (1972) who showed that cytochalasin can either stimulate or inhibit exocytic fusion, depending on the conditions. In addition to exocytosis (Bernstein et al., 1998;Lang et al., 2000;Bader et al., 2002), actin is now known to be involved in many other trafficking events, including phagocytosis (Swanson et al., 1999; Desjardin and Griffiths, 2003) and transcytosis (Durrbach et al., 2000). In the endocytic pathway, actin and myosins are essential for two different transport steps: clathrin-dependent internalization from the plasma membrane and transport to lysosomes (Durrbach et al., 1996;Riezman et al., 1996;Buss et al., 2001). More recently, actin has also been shown to be directly involved in homotypic fusion between yeast vacuoles (see DISCUSSION).Our interest in actin emerged from our analysis of an in vitro phagosome-endocytic organelle fusion assay that used latex bead phagosomes (LBP) and early endosomes (EE) and late endocytic org...

The mechanisms by which in vivo electroporation (EP) improves the potency of i.m. DNA vaccination were characterized by using the hepatitis C virus nonstructural (NS) 3/4A gene. Following a standard i.m. injection of DNA with or without in vivo EP, plasmid levels peaked immediately at the site of injection and decreased by 4 logs the first week. In vivo EP did not promote plasmid persistence and, depending on the dose, the plasmid was cleared or almost cleared after 60 days. In vivo imaging and immunohistochemistry revealed that protein expression was restricted to the injection site despite the detection of significant levels of plasmid in adjacent muscle groups. In vivo EP increased and prolonged NS3/4A protein expression levels as well as an increased infiltration of CD3+ T cells at the injection site. These factors most likely additively contributed to the enhanced and broadened priming of NS3/4A-specific Abs, CD4+ T cells, CD8+ T cells, and γ-IFN production. The primed CD8+ responses were functional in vivo, resulting in elimination of hepatitis C virus NS3/4A-expressing liver cells in transiently transgenic mice. Collectively, the enhanced protein expression and inflammation at the injection site following in vivo EP contributed to the priming of in vivo functional immune responses. These localized effects most likely help to insure that the strength and duration of the responses are maintained when the vaccine is tested in larger animals, including rabbits and humans. Thus, the combined effects mediated by in vivo EP serves as a potent adjuvant for the NS3/4A-based DNA vaccine.