Dynamic Interaction of Amphiphysin with N-WASP Regulates Actin Assembly

Abstract

Amphiphysin 1, an endocytic adaptor concentrated at synapses that couples clathrin-mediated endocytosis to dynamin-dependent fission, was also shown to have a regulatory role in actin dynamics. Here, we report that amphiphysin 1 interacts with N-WASP and stimulates N-WASP-and Arp2/3-dependent actin polymerization. Both the Src homology 3 and the N-BAR domains are requiredforthisstimulation.Acidicliposome-triggered,N-WASPdependent actin polymerization is strongly impaired in brain cytosol of amphiphysin 1 knock-out mice. FRET-FLIM analysis of Sertoli cells, where endogenously expressed amphiphysin 1 colocalizes with N-WASP in peripheral ruffles, confirmed the association between the two proteins in vivo. This association undergoes regulation and is enhanced by stimulating phosphatidylserine receptors on the cell surface with phosphatidylserine-containing liposomes that trigger ruffle formation. These results indicate that actin regulation is a key function of amphiphysin 1 and that such function cooperates with the endocytic adaptor role and membrane shaping/curvature sensing properties of the protein during the endocytic reaction.The dynamic nature of the actin cytoskeleton is crucial for a variety of cellular events, including cell morphogenesis, cell migration, and intracellular membrane traffic (1). Actin polymerization is stimulated by a variety of actin regulatory proteins, prominent among which are WASP family proteins that function by triggering Arp2/3-mediated actin nucleation (2). Activation of WASP family proteins, in turn, is controlled by factors that bind these proteins and release an autoinhibitory intramolecular interaction that prevents their VCA domain from interacting with the Arp2/3 complex. 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). In addition to a C-terminal SH3 domain, known to bind the GTPase dynamin and the phosphoinositide phosphatase synaptojanin (6), amphiphysin 1 contains an N-terminal BAR domain, a curved protein module that binds lipid bilayers and generates and senses curvature (7,8). It also contains binding motifs for clathrin and for the clathrin adaptor AP-2 (9). Hence, amphiphysin 1 was primarily studied as an endocytic protein capable of assembling at the neck of endocytic pits and of coupling clathrin-mediated budding to dynamin-mediated fission (10, 11). However, regulatory roles of amphiphysin 1 in actin cytoskeleton have also been suggested by studies of neuronal growth cones (12) and by the function of the amphiphysin homologue in yeast, . 81-86-235-7126; E-mail: kohji@md.okayama-u.ac.jp. 4 The abbrevi...

A role for the dynamin-like protein Vps1 during endocytosis in yeast

“…2B) was reminiscent of the behaviour noted for rvs167⌬ cells (Kaksonen et al, 2005). These data suggest that the function of Vps1 could be linked to, or overlap with, that of amphiphysins, as has been suggested from studies in mammalian cells (Ferguson et al, 2009;Itoh et al, 2005;Yamada et al, 2009). To investigate possible interplay between Vps1 and Rvs167, single deletion strains were crossed to determine any genetic interaction.…”

Spermiation

“…25 Amphiphysin I, present in TBCs, 99 has binding sites for clathrin and dynamin and was recently shown to be an N-WASPinteracting protein in Sertoli cells. 97 This interaction promoted actin polymerization in vitro, 97 revealing a mechanism whereby amphiphysin I can modulate actin dynamics. 97 Amphiphysin I null mice are reported to have reduced numbers of TBCs and an increase in retained spermatids in stage VIII ( Table 2).…”

Regulation of Hip1r by epsin controls the temporal and spatial coupling of actin filaments to clathrin-coated pits

“…Once activated by phosphorylation, Hip1r could directly regulate and orient actin filament formation, or act as a scaffold to recruit other proteins that regulate actin. Whether direct or not, this pathway probably involves the protein Arp2/3, a key regulator of polymerization of actin filaments that interacts with many proteins involved in clathrin-mediated endocytosis via N-WASP and localizes to internalizing clathrin pits in Dictyostelium and a wide range of other eukaryotes (Schafer, 2002;Merrifield et al, 2004;Heinrich et al, 2008;Yamada et al, 2009). Understanding how this regulatory pathway functions will be key to understanding the complexity of how clathrin-coated pits are integrated with the dynamic actin cytoskeleton.…”