Genetic interaction between profilin and myosin II reveals a potential role for myosin II in actin filament disassemblyin vivo

Abstract

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 fission yeast, we found that, myo2-S1 (myo2-G515D), a myosin II mutant allele was capable of rescuing lethality caused by compromise of mechanisms involved in actin cable / ring assembly and stability. 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 severely compromised in contraction upon ATP addition, suggesting that Myo2-S1p was defective in actin binding and / or motor activity. These studies strongly suggest a role for Myo2p in actin cytoskeletal disassembly and turnover, and that compromise of this activity leads to genetic suppression of mutants defective in actin cable assembly / stability. Introduction:Actin is a highly conserved cytoskeletal polymer forming protein that is key to a vast array of physiological processes in the three domains of life. In eukaryotes, the actin cytoskeleton plays essential roles in cell polarity, morphogenesis, migration, and cytokinesis (Pollard and Wu 2010, Cheffings, Burroughs et al. 2016, Misu, Takebayashi et al. 2017, Rottner, Faix et al. 2017, Skruber, Read et al. 2018. A balance of factors that control filament nucleation and stability and those that promote its disassembly exquisitely regulates the functions of the actin cytoskeleton (Lee and Dominguez 2010). Over the past three decades, the fission yeast Schizosaccharomyces pombe has emerged as an attractive organism for the study of the actin cytoskeleton and its role in cell polarity and division (Pollard and Wu 2010, Cheffings, Burroughs et al. 2016, Chiou, Balasubramanian et al. 2017. This is particularly due to the fact that many of the actin cytoskeletal proteins can be characterized through easily identifiable lethal mutant phenotypes (Wertman, Drubin et al. 1992, Holtzman, Wertman et al. 1994, Balasubramanian, McCollum et al. 1998). In fission yeast, theArp2/3 complex nucleates actin patches (Pelham and Chang 2001, Sirotkin, Berro et al. 2010), whereas linear actin cables and cytokinetic actomyosin rings are assembled by formins For3 (actin cables) (Feierbach and Chang 2001) and Cdc12p (cytokinetic actomyosin rings) (Chang, Drubin et al. 1997) and the actin-binding protein Cdc3-profilin (Balasubramanian, Hirani et al. 1994). The coiled-coil actin binding protein Cdc8 tropomyosin ensures stability of actin cables and actomyosin rings (Liu and Bretscher 1989, Balasubramanian, Helfman et al. 1992, Gunning, Hardeman et al. 2015). The fission yeast actin cytoskeleton undergoes dramatic disassembly and turnover (Kovar, Sirotkin et al. 2011). Treatment of cells with the actin polymerization inhibitor latrunculin A causes complete loss of actin cables...