The dynamic F-actin crosslinker alpha-actinin is tailored for contractile ring assembly during cytokinesis in Schizosaccharomyces pombe

Actin builds the basic units of many cellular processes in eukaryotes. Various actin structures are built and maintained by distinct sets of actin-binding proteins. An important cell process that highly relies on the actin network is cytokinesis, the last step in cell division. During cytokinesis, the assembly and constriction of the contractile ring are mediated by the coordinated action of diverse actin-binding proteins, which nucleate, bundle, sever, and travel along actin filaments. My two projects investigated the functions and regulations of actin crosslinkers and nucleators, using two model organisms.;In the first project, I examined the crosslinker alpha-actinin SpAin1, the primary actin bundling protein for cytokinesis in fission yeast Schizosaccharomyces pombe. SpAin1 has a similar domain organization as animal isoforms, with an actin-binding domain followed by spectrin repeats that facilitate dimerization. My goal was to elucidate the actin binding and bundling properties of SpAin1, and determine whether the specific properties are physiologically important for contractile ring function. Using bulk sedimentation assays and single filament TIRF microscopy observation, I determined that SpAin1 binds and bundles actin filaments of mixed polarity. However, compared to human alpha-actinins, SpAin1 bundles actin filaments with a significantly lower efficiency. The basis for a lower bundling efficiency is that SpAin1 is significantly more dynamic than human alpha-actinin HsACTN 4 on filaments, with shorter resident times on both single and bundled filaments. I predict that SpAin1's inefficient, versatile, and dynamic filament bundling properties are suitable for contractile ring dynamics in fission yeast, because other studies have shown that excessive actin filament bundling might reduce the overall actin dynamics during ring formation, resulting in defects in the assembly of the contractile ring. I tested this prediction by determining the effect on contractile ring assembly and constriction with a mutant version of SpAin1(R216E) that bundles F-actin more efficiently than the wild type SpAin1. I found that increasing the overall bundling efficiency by this mutant causes severe cytokinesis defects, during both ring assembly and ring constriction.;Cytokinesis in animal cells also requires contractile rings, but it remains unclear how they are assembled and maintained. The actin nucleator/elongator formin CYK-1 is essential for contractile ring assembly in the nematode worm Caenorhabditis elegans early embryo, and is hypothesized to be regulated by the small GTPase RhoA. CYK-1 contains actin assembly formin-homology domains 1 and 2 FH1FH2, which are flanked by putative N-terminal Diaphanous inhibitory domain DID and C-terminal Diaphanous autoregulatory domain DAD. Previously the Kovar laboratory found that CYK-1 stimulates the rapid assembly of actin monomers bound to profilin (Neidt et al., 2008). Here I report experiments that investigate whether CYK-1 is processive, and whether CYK-1 is auto-inhibited and activated by RhoA. First, by utilizing TIRF microscopy to directly observe the elongation of individual actin filaments associated with CYK-1 immobilized on the coverslip, I found that CYK-1 is processive in both the absence and presence of profilin. I observed buckling filaments as they processively elongate from immobilized CYK-1. Next, I tested whether CYK-1 is auto-inhibited by mixing its N- and C-terminal halves in actin assembly assays in vitro. Utilizing TIRF-microscopy and seeded assembly assays we found that CYK-1-mediated actin nucleation, but not CYK-1 mediated filament elongation, is auto-inhibited through direct association of its N- and C-terminal regulatory domains. Point mutations within the DID and DAD domains completely abolish auto-inhibition. Furthermore, CYK-1 can be partially activated by non-hydrolyzable GTP analog GMP-PNP bound RhoA, but not GDP RhoA or GMP-PNP Cdc42 and Rac. In summary, we determined that CYK-1 is a processive formin whose nucleation activity is regulated by autoinhibition, and is partially activated by RhoA. Other mechanisms must be in place to turn off CYK-1-mediated actin filament elongation.