Abstract
ABSTRACTTropomyosin is a coiled-coil protein that binds and regulates actin filaments. The tropomyosin gene in Schizosaccharomyces pombe, cdc8, is required for formation of actin cables, contractile rings, and polar localization of actin patches. The roles of conserved residues were investigated in gene replacement mutants. The work validates an evolution-based approach to identify tropomyosin functions in living cells and sites of potential interactions with other proteins. A cdc8 mutant with near-normal actin affinity affects patch polarization and vacuole fusion, possibly by affecting Myo52p, a class V myosin, function. The presence of labile residual cell attachments suggests a delay in completion of cell division and redistribution of cell patches following cytokinesis. Another mutant with a mild phenotype is synthetic negative with GFP-fimbrin, inferring involvement of the mutated tropomyosin sites in interaction between the two proteins. Proteins that assemble in the contractile ring region before actin do so in a mutant cdc8 strain that cannot assemble condensed actin rings, yet some cells can divide. Of general significance, LifeAct-GFP negatively affects the actin cytoskeleton, indicating caution in its use as a biomarker for actin filaments.
Highlights
Actin-based cellular mechanisms in cytokinesis, intracellular transport, and establishment of cellular polarity in eukaryotes are universal
Since cdc8R121A can divide without formation of a discrete actincontaining contractile ring, we investigated the localization of proteins that are found in the area of the ring and known to be regulated by tropomyosin in fission yeast and/or mammalian cells: myosin II, fimbrin, and formin (Cdc12p) (Kovar et al, 2011)
Analysis of the effects of site-specific mutations at conserved surface residues in fission yeast tropomyosin indicates the involvement of conserved residues in specific functions
Summary
Actin-based cellular mechanisms in cytokinesis, intracellular transport, and establishment of cellular polarity in eukaryotes are universal. Tropomyosin is a core actin regulatory protein, known for its role in regulating muscle contraction (Lehman and Craig, 2008), and is found in most eukaryotes It is a two-chained α-helical coiled-coil protein that binds end-to-end along the length of both sides of the actin filament. In this position, tropomyosin regulates actin cytoskeleton dynamics. It stabilizes the actin filament and protects it against the actions of DNase I (Hitchcock et al, 1976), cofilin
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