Abstract
The fission yeast Schizosaccharomyces pombe serves as a model system for studying role of actin cytoskeleton, since it has simple actin cytoskeletons and is genetically tractable. In contrast, biochemical approaches using this organism are still developing; fission yeast actin has so far not been isolated in its native form and characterized, and therefore, biochemical assays of fission yeast actin-binding proteins (ABPs) or myosin have been performed using rabbit skeletal muscle actin that may interact with the fission yeast ABPs in a manner different from fission yeast actin. Here, we report a novel method for isolating functionally active actin from fission yeast cells. The highly purified fission yeast actin polymerized with kinetics somewhat different from those of muscle actin and forms filaments that are structurally indistinguishable from skeletal muscle actin filaments. The fission yeast actin was a significantly weaker activator of Mg(2+)-ATPase of HMM of skeletal muscle myosin than muscle actin. The fission yeast profilin Cdc3 suppressed polymerization of fission yeast actin more effectively than that of muscle actin and showed an affinity for fission yeast actin higher than for muscle actin. The establishment of purification of fission yeast actin will enable reconstruction of physiologically relevant interactions between the actin and fission yeast ABPs or myosins and contribute to clarification of function of actin cytoskeleton in various cellular activities.
Highlights
The actin cytoskeleton is involved in many cellular motile processes, such as muscle contraction, migration of the cell, cytoplasmic streaming, organelle positioning, cell morphogenesis, and cytokinesis
Fission yeast actin was purified to 96 –99% homogeneity, and typically 0.3– 0.5 mg of actin was obtained from 25 ml of packed cells (Table S1)
We purified the fission yeast actin, which was competent to polymerize for the first time and investigated its biochemical properties
Summary
The actin cytoskeleton is involved in many cellular motile processes, such as muscle contraction, migration of the cell, cytoplasmic streaming, organelle positioning, cell morphogenesis, and cytokinesis. The others are regulation of the assembly feature of actin by actinmodulating proteins and formation of three-dimensional structures of actin filaments by various actin-cross-linking proteins, activities of both of which may be further controlled by upstream signaling pathways Analyses of both assembly properties of actin and actions of various actin-binding proteins are requisite for understanding the dynamic nature of each actin cytoskeleton. The fission yeast Schizosaccharomyces pombe serves as a good model system for studying the actin cytoskeletal organization, since the cells have only three distinct F-actin structures: cortical patches located mainly at the growing cell tip; cables running longitudinally along the cell; and the contractile ring, which forms during mitosis [1, 2] These structures are constructed and destroyed during the cell cycle. Some nonmuscle ABPs have been reported to show significant species specificity to actin
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