SummaryCytokinesis in many eukaryotes requires an actomyosin-based contractile ring [1]. In fission yeast, cytokinesis involves the type II myosins Myo2p and Myp2p and the type V myosin Myo51p [2]. A recent study by Laplante et al.[3], using deletion mutants of myp2 and myo51 and the mis-sense mutant myo2-E1 [4], concluded that each myosin has distinct functions and proposed that Myp2p plays the dominant role in actomyosin ring contraction. Here we present evidence that Myo2p, not Myp2p, is likely to be the major motor driving actomyosin ring contractility. Since the previous work [3] was performed at 25°C, the permissive temperature for myo2-E1, we compared cytokinesis timings in myo2-E1 and myo2Δ at 25°C and found that myo2-E1 is only partially compromised at 25°C. Furthermore, we find that myp2Δ and myp2Δ myo51Δ double mutants contract actomyosin rings at ∼90% of the rate of wild-type cells at 30°C and 36°C, suggesting that Myp2p plays a minimal role in ring contraction at these temperatures. Finally, ring contraction in our myo2-E1 strain took longer at 25°C than previously reported [3]. Although faster-acting alleles of myo2 will be required to evaluate its contribution at 25°C, our work establishes that Myo2p is the major motor involved in ring contraction, under most, if not all, conditions.

This work investigates the mitotic stage-dependent mobility of fission yeast actomyosin ring proteins in the cytokinetic ring using fluorescence recovery after photobleaching. It reveals a cell cycle–dependent mobility change in the F-BAR protein Cdc15.

Cytokinesis in many eukaryotes requires a contractile actomyosin ring that is placed at the division site. In fission yeast, which is an attractive organism for the study of cytokinesis, actomyosin ring assembly and contraction requires the myosin II heavy chain Myo2p. Although myo2-E1, a temperature-sensitive mutant defective in the upper 50 kDa domain of Myo2p, has been studied extensively, the molecular basis of the cytokinesis defect is not understood. Here, we isolate myo2-E1-Sup2, an intragenic suppressor that contains the original mutation in myo2-E1 (G345R) and a second mutation in the upper 50 kDa domain (Y297C). Unlike myo2-E1-Sup1, a previously characterized myo2-E1 suppressor, myo2-E1-Sup2 reverses actomyosin ring contraction defects in vitro and in vivo. Structural analysis of available myosin motor domain conformations suggests that a steric clash in myo2-E1, which is caused by the replacement of a glycine with a bulky arginine, is relieved in myo2-E1-Sup2 by mutation of a tyrosine to a smaller cysteine. Our work provides insight into the function of the upper 50 kDa domain of Myo2p, informs a molecular basis for the cytokinesis defect in myo2-E1, and may be relevant to the understanding of certain cardiomyopathies.

The actin cytoskeleton plays a variety of roles in eukaryotic cell physiology, ranging from cell polarity and migration to cytokinesis. Key to the function of the actin cytoskeleton is the mechanisms that control its assembly, stability, and turnover. Through genetic analyses in S. pombe, we found that, myo2-S1 ( myo2-G515D), a myosin II mutant allele, was capable of rescuing lethality caused by partial defects in actin nucleation / stability caused, for example, through compromised function of the actin-binding protein Cdc3-profilin. The mutation in myo2-S1 affects the activation loop of Myosin II, which is involved in physical interaction with subdomain 1 of actin and in stimulating the ATPase activity of Myosin. Consistently, actomyosin rings in myo2-S1 cell ghosts were unstable and severely compromised in contraction upon ATP addition. These studies strongly suggest a role for Myo2 in actin cytoskeletal disassembly and turnover in vivo, and that compromise of this activity leads to genetic suppression of mutants defective in actin filament assembly / stability at the division site. [Media: see text]

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