Abstract
Arp2/3 complex is a key actin filament nucleator that assembles branched actin networks in response to cellular signals. The activity of Arp2/3 complex is regulated by both activating and inhibitory proteins. Coronins make up a large class of actin-binding proteins previously shown to inhibit Arp2/3 complex. Although coronins are known to play a role in controlling actin dynamics in diverse processes, including endocytosis and cell motility, the precise mechanism by which they regulate Arp2/3 complex is unclear. We conducted a detailed biochemical analysis of budding yeast coronin, Crn1, and found that it not only inhibits Arp2/3 complex but also activates it. We mapped regions required for activation and found that Crn1 contains a sequence called CA, which is conserved in WASp/Scar proteins, the prototypical activators of Arp2/3 complex. Point mutations in CA abolished activation of Arp2/3 complex by Crn1 in vitro. Confocal microscopy and quantitative actin patch tracking showed that these mutants had defective endocytic actin patch dynamics in Saccharomyces cerevisiae, indicating that activation of Arp2/3 complex by coronin is required for normal actin dynamics in vivo. The switch between the dual modes of regulation by Crn1 is controlled by concentration, and low concentrations of Crn1 enhance filament binding by Arp2/3 complex, whereas high concentrations block binding. Our data support a direct tethering recruitment model for activation of Arp2/3 complex by Crn1 and suggest that Crn1 indirectly inhibits Arp2/3 complex by blocking it from binding actin filaments.
Highlights
An Arp2/3 complex inhibitor present in yeast actin patches, plays a role in regulating the dynamics of the patches [13, 17,18,19], which assemble at the cortex as the endocytic vesicle forms and disassemble as the vesicle moves into the cytoplasm to fuse with endosomes [20]
Crn1 Is an Arp2/3 Complex Activator with Similarities to Both Cortactin and WASp/Scar Proteins—We show here that Crn1 activates Arp2/3 complex both on its own and synergistically with Las17-VCA
We found that Crn1 failed to bind actin monomers, so we concluded that Crn1 does not activate the complex by recruiting actin monomers to the branch junction, a mechanism proposed for the VCA region of WASp/Scar [29, 34]
Summary
Budding yeast Arp2/3 complex, Crn, Las17-VCA, and N-WASp-VCA proteins were purified as described in the supplemental material. Actin was purified and labeled with pyrene iodoacetamide, and the increase in fluorescence was measured to monitor polymerization as described previously [22]. Sedimentation velocity experiments were carried out in a Beckman XL-I analytical ultracentrifuge as described in the supplemental material. Fluorescence anisotropy competition experiments were carried out as described previously [22] and analyzed as described in the supplemental material. Detailed procedures for all other methods can be found in the supplemental material
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