Abstract
The Arf GTPase-activating protein (Arf GAP) with SH3 domain, ankyrin repeat and PH domain 1 (ASAP1) establishes a connection between the cell membrane and the cortical actin cytoskeleton. The formation, maintenance, and turnover of actin filaments and bundles in the actin cortex are important for cell adhesion, invasion, and migration. Here, using actin cosedimentation, polymerization, and depolymerization assays, along with total internal reflection fluorescence (TIRF), confocal, and EM analyses, we show that the N-terminal N-BAR domain of ASAP1 directly binds to F-actin. We found that ASAP1 homodimerization aligns F-actin in predominantly unipolar bundles and stabilizes them against depolymerization. Furthermore, the ASAP1 N-BAR domain moderately reduced the spontaneous polymerization of G-actin. The overexpression of the ASAP1 BAR-PH tandem domain in fibroblasts induced the formation of actin-filled projections more effectively than did full-length ASAP1. An ASAP1 construct that lacked the N-BAR domain failed to induce cellular projections. Our results suggest that ASAP1 regulates the dynamics and the formation of higher-order actin structures, possibly through direct binding to F-actin via its N-BAR domain. We propose that ASAP1 is a hub protein for dynamic protein-protein interactions in mechanosensitive structures, such as focal adhesions, invadopodia, and podosomes, that are directly implicated in oncogenic events. The effect of ASAP1 on actin dynamics puts a spotlight on its function as a central signaling molecule that regulates the dynamics of the actin cytoskeleton by transmitting signals from the plasma membrane.
Highlights
The spatiotemporal reorganization of the actin cytoskeleton is often coupled with the deformation of membranes in cellular processes including migration, adhesion, and division [1,2,3]
Considering the pivotal role of ASAP1 in filamentous actin (F-actin)-rich structures, such as circular dorsal ruffles, focal adhesions, invadopodia, and podosomes, we investigated whether ASAP1 could directly interact with F-actin
After we examined the effect of ASAP1BARPH on actin dynamics in vitro, we tested whether F-actin regulates the ASAP1 GTPase-activating protein (GAP) activity, which is important for the hydrolysis of GTP bound to the small GTPase Arf1 during cytoskeletal reorganization [27]
Summary
The spatiotemporal reorganization of the actin cytoskeleton is often coupled with the deformation of membranes in cellular processes including migration, adhesion, and division [1,2,3]. In agreement with the bulk kinetic assays, increasing concentrations of ASAP1BARPH decreased actin polymerization rates in TIRF microscopy assays in which the elongation of actin filaments was observed in real time (Fig. 4G). Some actin-binding proteins and BAR domain-containing proteins were previously shown to induce the formation of cellular projections [29,30,31,32].
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