Effects of the myosin inhibitor 2,3-butanedione monoxime on the physiology of fission yeast

Abstract

F-actin and associated myosins are thought to take part in a wide range of cellular processes, like motility and contraction, polarized growth, and secretion. The reagent 2,3-butanedione monoxime (BDM) is a well characterized inhibitor of the contraction of vertebrate muscle that reversibly affects myosin function and influences the intracellular concentration of Ca2+. Here we describe the influence of BDM on growth and division of the fission yeast Schizosaccharomyces pombe. At concentrations from 1-30 mM, BDM gradually inhibited formation and growth of S. pombe colonies on agar plates, with a lethal effect at > or = 15 mM. In strains of S. pombe that were blocked by elevated temperature from entry into mitosis, drug treatment reversibly decreased microtubule-independent tip growth and septation, with an IC50 value around 12 mM; nuclear division, on the other hand, was essentially unaffected by up to 15 mM BDM. At 30 mM BDM the secretion of invertase, which required both F-actin and microtubules, was decreased to the same extent as that seen when cytochalasin D was used to disrupt F-actin. However, the actin cytoskeleton was insensitive to up to 10 mM BDM, while the actin patches lost their polar distribution at 20-30 mM BDM. Cells treated with 5-20 mM BDM for 3 hours and then high pressure frozen did not show an accumulation of secretory vesicles. However, 10 mM BDM treatment disorganized the fungal cell wall, resulting in some unusually thick parts lying next to regions were the wall was almost absent. These defects could be rescued by incubating the cells in inhibitors of glucanases. Osmolytic stabilization with sorbitol rescued the effect of 15 mM BDM on colony survival, indicating that the secretion of wall components and/or wall-modifying enzymes may be the principal reason for cell death caused by BDM. Our results are consistent with the hypothesis that BDM influences actin-dependent processes in fission yeast and that actomyosin-dependent motility contributes to the secretory process of tip growth.