We observed live fission yeast expressing pairs of functional fluorescent fusion proteins to test the popular model that the cytokinetic contractile ring assembles from a single myosin II progenitor or a Cdc12p-Cdc15p spot. Under our conditions, the anillin-like protein Mid1p establishes a broad band of small dots or nodes in the cortex near the nucleus. These nodes mature by the addition of conventional myosin II (Myo2p, Cdc4p, and Rlc1p), IQGAP (Rng2p), pombe Cdc15 homology protein (Cdc15p), and formin (Cdc12p). The nodes coalesce laterally into a compact ring when Cdc12p and profilin Cdc3p stimulate actin polymerization. We did not observe assembly of contractile rings by extension of a leading cable from a single spot or progenitor. Arp2/3 complex and its activators accumulate in patches near the contractile ring early in anaphase B, but are not concentrated in the contractile ring and are not required for assembly of the contractile ring. Their absence delays late steps in cytokinesis, including septum formation and cell separation.

We report time courses of the accumulation and loss of 16 fluorescent fusion proteins at sites of clathrin-mediated endocytosis in fission yeast. Mathematical modeling shows that dendritic nucleation hypothesis can account for the kinetics of actin assembly in vivo and disassembly requires actin filament severing along with depolymerization.

SUMMARY Fission yeast cells utilize Arp2/3 complex and formin to assemble diverse filamentous actin (F-actin) networks within a common cytoplasm for endocytosis, division and polarization. Although these homeostatic F-actin networks are usually investigated separately, competition for a limited pool of actin monomers (G-actin) helps regulate their size and density. However, the mechanism by which G-actin is correctly distributed between rival F-actin networks is not clear. Using a combination of cell biological approaches and in vitro reconstitution of competition between actin assembly factors, we discovered that the small G-actin binding protein profilin directly inhibits Arp2/3 complex-mediated actin assembly. Profilin is therefore required for formin to compete effectively with excess Arp2/3 complex for limited G-actin, and to assemble F-actin for contractile ring formation in dividing cells.

Yeast actin patches are dynamic structures that form at the sites of cell growth and are thought to play a role in endocytosis. We used biochemical analysis and live cell imaging to investigate actin patch assembly in fission yeast Schizosaccharomyces pombe. Patch assembly proceeds via two parallel pathways: one dependent on WASp Wsp1p and verprolin Vrp1p converges with another dependent on class 1 myosin Myo1p to activate the actin-related protein 2/3 (Arp2/3) complex. Wsp1p activates Arp2/3 complex via a conventional mechanism, resulting in branched filaments. Myo1p is a weaker Arp2/3 complex activator that makes unstable branches and is enhanced by verprolin. During patch assembly in vivo, Wsp1p and Vrp1p arrive first independent of Myo1p. Arp2/3 complex associates with nascent activator patches over 6–9 s while remaining stationary. After reaching a maximum concentration, Arp2/3 complex patches move centripetally as activator proteins dissociate. Genetic dependencies of patch formation suggest that patch formation involves cross talk between Myo1p and Wsp1p/Vrp1p pathways.

Summary Controlling the quantity and size of organelles through competition for a limited supply of components is quickly emerging as an important cellular regulatory mechanism [1]. Cells assemble diverse actin filament (F-actin) networks for fundamental processes including division, motility, and polarization [2–4]. F-actin polymerization is tightly regulated by activation of assembly factors such as the Arp2/3 complex and formins at specific times and places. We directly tested an additional hypothesis that diverse F-actin networks are in homeostasis, whereby competition for actin monomers (G-actin) is critical for regulating F-actin network size. Here we show that inhibition of Arp2/3 complex in the fission yeast Schizosaccharomyces pombe not only eliminates Arp2/3 complex-mediated endocytic actin patches, but also induces a dramatic excess of formin-assembled F-actin. Conversely, disruption of formin increases the density of Arp2/3 complex-mediated patches. Furthermore, modifying actin levels significantly perturbs the fission yeast actin cytoskeleton. Increasing actin favors Arp2/3 complex-mediated actin assembly, whereas decreasing actin favors formin-mediated contractile rings. Therefore, the specific actin concentration in a cell is critical, and competition for G-actin helps regulate the proper amount of F-actin assembly for diverse processes.