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...

Let's go bananas: revisiting the endocytic BAR code

“…The observation that members of these F-BAR and N-BAR subfamily members with their different, distinct shapes nevertheless appear together and also show a synchronous disappearance after scission when the highly curved membrane neck collapses (Taylor et al, 2011) thus was unexpected. Interestingly, their dynamic appearance and disappearance parallels the temporal signatures of two of the main binding partners of syndapins, amphiphysins and endophilins, dynamin and N-WASP (David et al, 1996;Ringstad et al, 1997;Qualmann and Kelly, 2000;Kessels and Qualmann, 2002;Otsuki et al, 2003;Yamada et al, 2009; Figure 4A-C).…”

Membrane shaping by the Bin/amphiphysin/Rvs (BAR) domain protein superfamily

“…Many of these factors, in addition to their BAR/F-BAR domains, also contain a Src homology 3 domain (SH3) domain that allows them to associate with other endocytic and cytoskeletal proteins, such as the GTPase dynamin (mediating vesicle fission), or N-WASP (an initiator for actin polymerization) (Fig. 3a) [6,15,[55][56][57][58][59]. During clathrin-mediated endocytosis, F-BAR domain proteins typically arrive early to the site of endocytosis, and may be involved in the nucleation of CCPs [60][61][62][63].…”

The Ca2+ channel subunit CaVβ2a‐subunit down‐regulates voltage‐activated ion current densities by disrupting actin‐dependent traffic in chromaffin cells

“…2S). On the other hand, amphiphysin-SH3 by itself does not influence actin polymerization (Yamada et al, 2009), but increases Ca 2+ current densities (Fig. 2S).…”

“…As SH3 domains regulate cellular functions by forming homodimers (Harkiolaki et al, 2006;Kristensen et al, 2006;Padrick et al, 2008;Yamada et al, 2009) and the effects of b2a-SH3 on Ca 2+ current densities in Xenopus oocytes depend on its dimerization (Miranda-Laferte et al, 2011), we evaluated the effect of substituting cysteine 113 in rat b2a-SH3 (Swiss-Prot entry:Q8VGC3) by alanine. The resulting recombinant protein (b2a-SH3 C113) does not form dimers, as demonstrated by size-exclusion chromatography and blue native polyacrylamide gel electrophoresis; further, its migration pattern was not modified by treatment with sodium dodecyl sulfate or DL-dithiothreitol, (Miranda-Laferte et al, 2011).…”