We created two new mutants of fission yeast cofilin to investigate why cytokinesis in many organisms depends on this small actin-binding protein. These mutant cofilins bound actin monomers normally, but bound and severed ADP-actin filaments much slower than wild-type cofilin. Cells depending on mutant cofilins condensed nodes, precursors of the contractile ring, into clumps rather than rings. Starting from clumped nodes, mutant cells slowly assembled rings from diverse intermediate structures including spiral strands containing actin filaments and other contractile ring proteins. This process in mutant cells depended on α-actinin. These slowly assembled contractile rings constricted at a normal rate but with more variability, indicating ring constriction is not very sensitive to defects in severing by cofilin. Computer simulations of the search-capture-pull and release model of contractile ring formation predicted that nodes clump when the release step is slow, so cofilin severing of actin filament connections between nodes likely contributes to the release step.

Aip1 (actin interacting protein 1) is ubiquitous in eukaryotic organisms, where it cooperates with cofilin to disassemble actin filaments, but neither its mechanism of action nor its biological functions have been clear. We purified both fission yeast and human Aip1 and investigated their biochemical activities with or without cofilin. Both types of Aip1 bind actin filaments with micromolar affinities and weakly nucleate actin polymerization. Aip1 increases up to 12-fold the rate that high concentrations of yeast or human cofilin sever actin filaments, most likely by competing with cofilin for binding to the side of actin filaments, reducing the occupancy of the filaments by cofilin to a range favorable for severing. Aip1 does not cap the barbed ends of filaments severed by cofilin. Fission yeast lacking Aip1 are viable and assemble cytokinetic contractile rings normally, but rings in these Δaip1 cells accumulate 30% less myosin II. Further, these mutant cells initiate the ingression of cleavage furrows earlier than normal, shortening the stage of cytokinetic ring maturation by 50%. The Δaip1 mutation has negative genetic interactions with deletion mutations of both capping protein subunits and cofilin mutations with severing defects, but no genetic interaction with deletion of coronin.Aip1 cooperates with cofilin to disassemble actin filaments. Results Aip1 increases the rate of filament severing by cofilin by binding the sides of actin filaments, and Δaip1 mutants have cytokinesis defects. Conclusion Aip1 promotes actin filament severing by the high concentrations of cofilin in cells. Significance We provide the first evidence that Aip1 promotes filament severing and illustrate its importance to cytokinesis.

Aurora kinases are highly conserved proteins with important roles in mitosis. Metazoans contain two kinases, Aurora A and B, which contribute distinct functions at the spindle poles and the equatorial region respectively. It is not currently known whether the specialized functions of the two kinases arose after their duplication in animal cells or were already present in their ancestral kinase. We show thatDictyostelium discoideumcontains a single Aurora kinase, DdAurora, that displays characteristics of both Aurora A and B. Like Aurora A, DdAurora has an extended N-terminal domain with an A-box sequence and localizes at the spindle poles during early mitosis. Like Aurora B, DdAurora binds to its partner DdINCENP and localizes on centromeres at metaphase, the central spindle during anaphase, and the cleavage furrow at the end of cytokinesis. DdAurora also has several unusual properties. DdAurora remains associated with centromeres in anaphase, and this association does not require an interaction with DdINCENP. DdAurora then localizes at the cleavage furrow, but only at the end of cytokinesis. This localization is dependent on DdINCENP and the motor proteins Kif12 and myosin II. Thus, DdAurora may represent the ancestral kinase that gave rise to the different Aurora kinases in animals and also those in other organisms.