Amphiphysin 1 Is Important for Actin Polymerization during Phagocytosis

Abstract

Amphiphysin 1 is involved in clathrin-mediated endocytosis. In this study, we demonstrate that amphiphysin 1 is essential for cellular phagocytosis and that it is critical for actin polymerization. Phagocytosis in Sertoli cells was induced by stimulating phosphatidylserine receptors. This stimulation led to the formation of actin-rich structures, including ruffles, phagocytic cups, and phagosomes, all of which showed an accumulation of amphiphysin 1. Knocking out amphiphysin 1 by RNA interference in the cells resulted in the reduction of ruffle formation, actin polymerization, and phagocytosis. Phagocytosis was also drastically decreased in amph 1 (؊/؊) Sertoli cells. In addition, phosphatidylinositol-4,5-bisphosphate-induced actin polymerization was decreased in the knockout testis cytosol. The addition of recombinant amphiphysin 1 to the cytosol restored the polymerization process. Ruffle formation in small interfering RNA-treated cells was recovered by the expression of constitutively active Rac1, suggesting that amphiphysin 1 functions upstream of the protein. These findings support that amphiphysin 1 is important in the regulation of actin dynamics and that it is required for phagocytosis. INTRODUCTIONPhagocytosis is a form of endocytosis in which large molecules are engulfed by the plasma membrane and internalized for digestion (Greenberg and Grinstein, 2002;Niedergang and Chavrier, 2004). In vertebrates, macrophages and testicular Sertoli cells are the predominant phagocytes. Phagocytosis contributes to removal of foreign bodies, bacteria, and microbe-infected and apoptotic cells. Binding of these objects to the receptors, such as Fc␥ receptors or phosphatidylserine receptors, on the surface of the macrophage initiates membrane remodeling and reorganization of the actin cytoskeleton. Cytoskeletal changes cause the cells to extend pseudopods that then engulf the objects (Anderem and Underhill, 1999).Sertoli cells participate in the maturation of germ cells and the release of sperm (Russell et al., 1990). These cells are highly phagocytic and are responsible for eliminating the residual cytoplasm of spermatids (Chemes, 1986;Clermont et al., 1987;Morales and Clermont, 1991). The phagocytosis is also used to remove apoptotic germ cells (Shiratsuchi et al., 1997(Shiratsuchi et al., , 1999Kawasaki et al., 2002). Phagocytosis in Sertoli cells is initiated by the recognition of phosphatidylserine (PS) found on the germ cell surface by PS receptors (Shiratsuchi et al., 1997(Shiratsuchi et al., , 1999Blanco-Rodriguez and Martinez-Garcia, 1999;Kawasaki et al., 2002). The class B scavenger family receptors SR-BI and CD36 are found on the surface of Sertoli cells where they function in PS recognition (Shiratsuchi et al., 1997(Shiratsuchi et al., , 1999Kawasaki et al., 2002;Gillot et al., 2005). In the testis, Sertoli cells express high levels of amphiphysin 1 (Watanabe et al., 2001;Kamitani et al., 2002). The expression of amphiphysin, along with that of dynamin 2, increases with sexual development (Watanabe et al., 2...

Dynamic interaction of amphiphysin with N-WASP regulates actin assembly

“…Rabbit anti-Myc antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Monoclonal anti-amphiphysin 1 antibodies (monoclonal antibody 3) were as described previously (3). Rabbit anti-PAN-WAVE antibody was a kind gift from Dr. Scita (IFOM Istituto Fondazione Italiana Ricerca sul Cancro di Oncologia Molecolare Via Adamello 16, Italy).…”

“…They were allowed to adapt to their environment for more than 1 week before initiating the experiments. Ser-W3 cells, a rat Sertoli cell line, were cultured with Dulbecco's modified Eagle's medium containing 10% fetal bovine serum at 37°C under 5% CO 2 (3).…”

“…Preparation of Liposomes-For stimulation of Ser-W3 cells, small unilamellar liposomes containing 70% PC and 30% PS were prepared by sonication as described previously (3). For actin polymerization assay, small liposomes of uniform size, ϳ100 nm in diameter, composed of 50% PS/50% PC, 50% phosphatidylethanolamine/50% PC, 50% PI/50% PC, 50% phosphatidic acid/50% PC, or 10% PI(4,5)P 2 /90% PC were used.…”

“…As extensively shown for N-WASP, many such factors are proteins that bind to the N-WASP proline-rich region via the SH3 4 domain. Recently, we have found that the SH3 domain containing protein amphiphysin 1 stimulates actin polymerization during phagocytosis in testicular Sertoli cells, and this effect requires interactions of its C-terminal SH3 domain (3). Amphiphysin 1 is an endocytic adaptor present at high levels in brain at neuronal synapses but is also expressed at significant levels in Sertoli cells (4,5).…”

Chemotactic activation ofDictyosteliumAGC-family kinases AKT and PKBR1 requires separate but coordinated functions of PDK1 and TORC2

“…SHAPS, however, is part of the BIN/amphiphysin family. Consistent with the targeted phosphorylation of SHAPS following chemotactic stimulation, BIN/amphiphysin proteins are implicated in a wide array of membrane processes, including actin function, interaction with small GTPases and other signaling pathways, and clathrin-mediated endocytosis (Frost et al, 2009;Peter et al, 2004;Yamada et al, 2007). PHAPS and SHAPS may function as adapters to recruit components to membrane compartments and are potentially regulated by differential states of phosphorylation.…”