Fimbrin and Tropomyosin Competition Regulates Endocytosis and Cytokinesis Kinetics in Fission Yeast

SummaryBackgroundTropomyosin is an important actin filament-stabilizing protein that controls the access of other essential proteins to filaments, including myosin motors, Arp2/3 complex, formin, and cofilin. It is therefore critical to establish mechanisms for regulating the actin filament binding of tropomyosin. We examined how the actin filament crosslinking protein fimbrin Fim1p and tropomyosin Cdc8p affect each other's ability to bind filaments, localize to particular cellular structures, and regulate filament severing by cofilin Adf1p in fission yeast Schizosaccharomyces pombe.ResultsWe discovered a novel mechanism for regulating actin filament dynamics in fission yeast. Fim1p inhibits Cdc8p binding to actin filaments in vitro, which permits Adf1p-mediated severing in the presence of Cdc8p. In cells, the balance between Fim1p and Cdc8p is important for both endocytic actin patch kinetics and contractile ring assembly during cytokinesis. High Fim1p concentrations prevent Cdc8p from associating with actin patches, allowing rapid patch turnover and motility. In the absence of Fim1p, ectopic localization of Cdc8p to actin patches increases patch lifetime while decreasing patch motility. Fim1p and Cdc8p also play antagonistic roles during cytokinesis, in which the deletion of Fim1p rescues the contractile ring assembly defects caused by mutation of Cdc8p.ConclusionFimbrin Fim1p dissociates tropomyosin Cdc8p from actin filaments, permitting cofilin Adf1p-mediated severing. Therefore, we propose that in addition to actin filament crosslinking, Fim1p has a novel role as a positive actin-binding “selector” protein that promotes the access of other proteins to actin filaments by inhibiting Cdc8p

The Cytokinesis Formins from the Nematode Worm and Fission Yeast Differentially Mediate Actin Filament Assembly*

Formins drive actin filament assembly for diverse cellular processes including motility, establishing polarity, and cell division. To investigate the mechanism of contractile ring assembly in animal cells, we directly compared the actin assembly properties of formins required for cytokinesis in the nematode worm early embryo (CYK-1) and fission yeast (Cdc12p). Like Cdc12p and most other formins, CYK-1 nucleates actin filament assembly and remains processively associated with the elongating barbed end while facilitating the addition of profilin-actin above the theoretical diffusion-limited rate. However, specific properties differ significantly between Cdc12p and CYK-1. Cdc12p efficiently nucleates filaments that in the presence of profilin elongate at approximately the same rate as control filaments without formin (∼10.0 subunits/s). CYK-1 is an inefficient nucleator but allows filaments to elongate profilin-actin 6-fold faster than Cdc12p (∼60 subunits/s). Both Cdc12p and CYK-1 bind to pre-assembled actin filaments with low nanomolar affinity, but CYK-1 dissociates 2 orders of magnitude more quickly. However, CYK-1 rapidly re-associates with free barbed ends. Cdc12p allows barbed ends to elongate in the presence of excess capping protein, whereas capping protein inhibits CYK-1-mediated actin assembly. Therefore, these evolutionarily diverse formins can drive contractile ring assembly by a generally similar mechanism, but cells with unique dimensions and physical parameters might require proteins with carefully tuned actin assembly properties

Role of Tropomyosin in Formin-mediated Contractile Ring Assembly in Fission Yeast

Like animal cells, fission yeast divides by assembling actin filaments into a contractile ring. In addition to formin Cdc12p and profilin, the single tropomyosin isoform SpTm is required for contractile ring assembly. Cdc12p nucleates actin filaments and remains processively associated with the elongating barbed end while driving the addition of profilin-actin. SpTm is thought to stabilize mature filaments, but it is not known how SpTm localizes to the contractile ring and whether SpTm plays a direct role in Cdc12p-mediated actin polymerization. Using “bulk” and single actin filament assays, we discovered that Cdc12p can recruit SpTm to actin filaments and that SpTm has diverse effects on Cdc12p-mediated actin assembly. On its own, SpTm inhibits actin filament elongation and depolymerization. However, Cdc12p completely overcomes the combined inhibition of actin nucleation and barbed end elongation by profilin and SpTm. Furthermore, SpTm increases the length of Cdc12p-nucleated actin filaments by enhancing the elongation rate twofold and by allowing them to anneal end to end. In contrast, SpTm ultimately turns off Cdc12p-mediated elongation by “trapping” Cdc12p within annealed filaments or by dissociating Cdc12p from the barbed end. Therefore, SpTm makes multiple contributions to contractile ring assembly during and after actin polymerization