Tropomyosin and actin isoforms modulate the localization of tropomyosin strands on actin filaments

Tropomyosin is present in virtually all eucaryotic cells, where it function s to modulate actin-myosin interaction and to stabilize actin filament stru cture. In striated muscle, tropomyosin regulates contractility by stericall y blocking myosin-binding sites on actin in the relaxed state. On activatio n, tropomyosin moves away from these sites in two steps. one induced by Ca2 + binding to troponin and a second by the binding of myosin to actin. In sm ooth muscle and non-muscle cells, where troponin is absent, the precise rol e and structural dynamics of tropomyosin on actin are poorly understood. He re, the location of tropomyosin on F-actin filaments free of troponin and o ther actin-binding proteins was determined to better understand the structu ral basis of its functioning in muscle and non-muscle cells. Using electron microscopy and three-dimensional image reconstruction, the association of a diverse set of wild-type and mutant actin and tropomyosin isoforms, from both muscle and nonmuscle sources, was investigated. Tropomyosin position o n actin appeared to be defined by two sets of binding interactions and trop omyosin localized on either the inner or the outer domain of actin, dependi ng on the specific actin or tropomyosin isoform examined. Since these equil ibrium positions depended on minor amino acid sequence differences among is oforms, we conclude that the energy barrier between thin filament states is small. Our results imply that, in striated muscles, troponin and myosin se rve to stabilize tropomyosin in inhibitory and activating states, respectiv ely. In addition, they are consistent with tropomyosin-dependent cooperativ e switching on and off of actomyosin-based motility. Finally, the locations of tropomyosin that we have determined suggest the possibility of signific ant competition between tropomyosin and other cellular actin-binding protei ns. Based on these results, we present a general framework for tropomyosin modulation of motility and cytoskeletal modelling. (C) 2000 Academic Press.