Abstract
The Arp2/3 complex is essential for actin assembly and motility in many cell processes, and a large number of proteins have been found to bind and regulate it in vitro. A critical challenge is to understand the actions of these proteins in cells, especially in settings where multiple regulators are present. In a systematic study of the sequential multicomponent actin assembly processes that accompany endocytosis in yeast, we examined and compared the roles of WASp, two type-I myosins, and two other Arp2/3 activators, along with that of coronin, which is a proposed inhibitor of Arp2/3. Quantitative analysis of high-speed fluorescence imaging revealed individual functions for the regulators, manifested in part by novel phenotypes. We conclude that Arp2/3 regulators have distinct and overlapping roles in the processes of actin assembly that drive endocytosis in yeast. The formation of the endocytic actin patch, the creation of the endocytic vesicle, and the movement of the vesicle into the cytoplasm display distinct dependencies on different Arp2/3 regulators. Knowledge of these roles provides insight into the in vivo relevance of the dendritic nucleation model for actin assembly.
Highlights
Dynamic networks of branched actin filaments are frequently found adjacent to membranes and appear to play a role in many cellular process
An important challenge for the field is to understand how the activities of multiple Arp2/3-activating proteins are coordinated in vivo. Do these activators have overlapping functions or does each of these proteins have a unique role in the formation of a proper network? We addressed this question by investigating the roles of all of the proposed Arp2/3 regulatory proteins in the assembly and movement of cortical actin patches in Saccharomyces cerevisiae
Endocytosis occurs at the plasma membrane in association with the assembly and movement of cortical actin patches, which contain six Arp2/3 regulators
Summary
Dynamic networks of branched actin filaments are frequently found adjacent to membranes and appear to play a role in many cellular process (reviewed in [1]). The dendritic nucleation model provides a framework to understand how networks of branched actin filaments assemble and generate a pushing force [2,3,4]. A key step in the formation of a branched actin filament network is the activation of the Arp2/3 complex, which nucleates a new actin filament from the side of an existing filament. This new growing filament pushes against the membrane. Arp2/3 normally exists in an inactive state and requires an activator protein to induce a large conformational change, which allows for nucleation of a new actin filament (reviewed in [1,5,6]). An Arp2/3 inhibitor, stabilizes Arp2/3 in the inactive conformation [7]
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