Mechanisms for activating the actin-related protein 2/3 (Arp2/3) complex have been the focus of many recent studies. Here, we identify a novel mode of Arp2/3 complex regulation mediated by the highly conserved actin binding protein coronin. Yeast coronin (Crn1) physically associates with the Arp2/3 complex and inhibits WA- and Abp1-activated actin nucleation in vitro. The inhibition occurs specifically in the absence of preformed actin filaments, suggesting that Crn1 may restrict Arp2/3 complex activity to the sides of filaments. The inhibitory activity of Crn1 resides in its coiled coil domain. Localization of Crn1 to actin patches in vivo and association of Crn1 with the Arp2/3 complex also require its coiled coil domain. Genetic studies provide in vivo evidence for these interactions and activities. Overexpression of CRN1 causes growth arrest and redistribution of Arp2 and Crn1p into aberrant actin loops. These defects are suppressed by deletion of the Crn1 coiled coil domain and by arc35-26, an allele of the p35 subunit of the Arp2/3 complex. Further in vivo evidence that coronin regulates the Arp2/3 complex comes from the observation that crn1 and arp2 mutants display an allele-specific synthetic interaction. This work identifies a new form of regulation of the Arp2/3 complex and an important cellular function for coronin.

Background Actin polymerization by Arp2/3 complex must be tightly regulated to promote endocytosis. While many Arp2/3 complex activators have been identified, mechanisms for its negative regulation have remained more elusive. We analyzed the yeast arp2-7 allele, which is biochemically unique in causing unregulated actin assembly in vitro in the absence of Arp2/3 activators. Results We examined endocytosis in arp2-7 mutants by live cell imaging of Sla1-GFP, a coat marker, and Abp1-RFP, which marks the later actin phase of endocytosis. Sla1-GFP and Abp1-RFP lifetimes were accelerated in arp2-7 mutants, which is opposite to actin nucleation-impaired arp2 alleles or deletions of Arp2/3 activators. We performed a screen for multi-copy suppressors of arp2-7 and identified SYP1, a FCHO1 homologue, which contains F-BAR and AP-2μ homology domains. Over-expression of SYP1 in arp2-7 cells slowed Sla1-GFP lifetimes closer to wild type cells. Further, purified Syp1 directly inhibited Las17/WASp stimulation of Arp2/3 complex-mediated actin assembly. This activity was mapped to a fragment of Syp1 located between its F-BAR and AP-2μ homology domains, and depends on non-VCA sequences in Las17/WASp. Conclusions Together, these data identify Syp1 as a novel negative regulator of WASp-Arp2/3 complex that helps choreograph the precise timing of actin assembly during endocytosis.

Actin nucleation by the Arp2/3 complex is under tight control, remaining inactive until stimulation by nucleation-promoting factors (NPFs). Although multiple NPFs are expressed in most cell types, little is known about how they are coordinated and whether they perform similar or distinct functions. We examined genetic relationships among the four S. cerevisiae NPFs. Combining las17D with pan1-101 or myo3Dmyo5D was lethal at all temperatures, whereas combining pan1-101 with myo3Dmyo5D showed no genetic interaction and abp1D partially suppressed las17D. These data suggest that NPFs have distinct and overlapping functions in vivo. We also tested genetic interactions between each NPF mutant and seven different temperature-sensitive arp2 alleles and purified mutant Arp2/3 complexes to compare their activities. Two arp2 alleles with mutations at the barbed end were severely impaired in nucleation, providing the first experimental evidence that Arp2 nucleates actin at its barbed end in vitro and in vivo.