Overlapping and distinct functions for cofilin, coronin and Aip1 in actin dynamics in vivo

Abstract

SummaryActin-filament disassembly is crucial for actin-based motility, to control filament network architecture and to regenerate subunits for assembly. Here, we examined the roles of three actin cytoskeletal proteins, coronin, cofilin and Aip1, which have been suggested to combine in various ways to control actin dynamics by promoting or regulating disassembly. We studied their functions during the endocytosis process in budding yeast, where actin-filament dynamics at the cortical actin 'patch' contribute to the formation and movement of endocytic vesicles. We found that all three proteins were recruited during the late phase of the life of the actin patch. They all arrived at the same time, when actin and other actin-associated proteins were leaving the patch. Cofilin point mutations influenced the localization of coronin and Aip1, but the complete loss of coronin had no effect on localization of cofilin or Aip1. Using quantitative patch motion analysis and comparing mutant alleles, the phenotypes for mutations of the three genes showed some commonalities, but also some striking differences. Cofilin was clearly the most important; it displayed the most severe mutant phenotypes affecting actin-patch assembly and movement. Together, the results suggest that all three proteins work together to promote actin disassembly, but not in a simple way, and not with equal importance.

A High Precision Survey of the Molecular Dynamics of Mammalian Clathrin-Mediated Endocytosis

“…Here, we showed that the onset of actin polymerization did indeed precede the final burst of dynamin recruitment by ,20 s, consistent with a role for actin polymerization early in the invagination stage of CME and the later recruitment of dynamin to the deeply invaginated CCP, where it executed scission [9] (Figure 8). We also discovered that coronin1B and cofilin, proteins involved in the down-regulation of actin polymerization and F-actin severing, respectively, were recruited at later time points, again similar to yeast endocytosis [24,72,73]. Third, it was proposed that scission of endocytic invaginations in yeast is triggered by a PI-phosphatase that dephosphorylates PiP2 and thus induces a line tension in the membrane neck [74].…”

Mechanism of a Concentration-dependent Switch between Activation and Inhibition of Arp2/3 Complex by Coronin

“…Activation of Arp2/3 Is Required for Normal Actin Patch Dynamics in Budding Yeast-In wild-type S. cerevisiae, actin and actin-binding proteins assemble to their peak concentrations in the patches in about 8 s, after which the patches separate from the cortex and move into the cytoplasm (15,19,21). In the crn1⌬ strain, the duration of the assembly phase of an average actin patch is increased by 2 s (13,19).…”

“…In the crn1⌬ strain, the duration of the assembly phase of an average actin patch is increased by 2 s (13,19). To determine whether activation of Arp2/3 complex by Crn1 influences the dynamics of patch assembly, we deleted CRN1 from a haploid strain and reintegrated mutant or wild-type CRN1 genes under control of their native promoters into the leu2-3,112 locus (see supplemental Table S1 for all strains used in this study).…”

“…In endocytic patches, Crn1 reaches an average peak concentration of 93 molecules in budding yeast and 400 -500 molecules in fission yeast when compared with 5000 -7000 molecules of actin (19,33). Crn1 recruitment is delayed when compared with actin, and its concentration peaks about 4 s later than the core actin module (19,33).…”

“…Five of these proteins have been shown to activate the complex in vitro, and in vivo experiments have demonstrated that despite some functional overlap, all five NPFs contribute to actin patch assembly (13)(14)(15)(16). Coronin, 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 then disassemble as the vesicle moves into the cytoplasm to fuse with endosomes (20). Actin polymerization is thought to provide force for invagination of endocytic vesicles and may propel the vesicle into the cytoplasm (21).…”