These experiments identified a pathway, involving Dia2- and Arp2/3-promoted actin filament nucleation and several functionally distinct tropomyosins, that is required for generation of contractile actomyosin arrays in cells.

The specific functions of greater than 40 vertebrate nonmuscle tropomyosins (Tms) are poorly understood. In this article we have tested the ability of two Tm isoforms, TmBr3 and the human homologue of Tm5 (hTM5 NM1 ), to regulate actin filament function. We found that these Tms can differentially alter actin filament organization, cell size, and shape. hTm5 NM1 was able to recruit myosin II into stress fibers, which resulted in decreased lamellipodia and cellular migration. In contrast, TmBr3 transfection induced lamellipodial formation, increased cellular migration, and reduced stress fibers. Based on coimmunoprecipitation and colocalization studies, TmBr3 appeared to be associated with actin-depolymerizing factor/cofilin (ADF)-bound actin filaments. Additionally, the Tms can specifically regulate the incorporation of other Tms into actin filaments, suggesting that selective dimerization may also be involved in the control of actin filament organization. We conclude that Tm isoforms can be used to specify the functional properties and molecular composition of actin filaments and that spatial segregation of isoforms may lead to localized specialization of actin filament function. INTRODUCTIONThe actin microfilament network is a primary cytoskeletal system involved in the development and maintenance of morphology within cells. The dynamic nature of the actinbased system and its organization is thought to regulate specific structural changes within different cellular regions (Gunning et al., 1998b). The function and form of the actin cytoskeleton is largely determined by actin-binding proteins that are associated with the polymeric structure. Tropomyosins (Tms), along with actin, are integral components of the microfilament cytoskeleton, although not all actin filaments have Tms bound to them (Bamburg, 1999). Tms bind largely by electrostatic charge to the helical groove of the actin filament and the Ͼ40 isoforms are obtained by alternative splicing from four genes, of which almost all are nonmuscle variants (Lees-Miller et al., 1990;Goodwin et al., 1991;Beisel and Kennedy, 1994;Dufour et al., 1998;Cooley and Bergtrom, 2001). Although a considerable amount of information exists as to the biochemical regulation of microfilament dynamics, little is known about the function of this large family of proteins in vertebrate nonmuscle cells.In vitro studies have shown that nonmuscle Tms are able to differentially protect actin from the severing action of gelsolin (Ishikawa et al., 1989) and can regulate the MgATPase activity of myosins to varying degrees (Fanning et al., 1994). The different binding strengths to actin are thought to impart a range of stability to the filaments (Matsumura and Yamashiro-Matsumura, 1985;Hitchcock-DeGregori et al., 1988;Pittenger et al., 1995). The impact of Tms on vertebrate cell morphology is poorly understood even though studies suggest the importance of Tm isoforms in regulating Article published online ahead of print. Mol. Biol. Cell 10.1091/ mbc.E02-04 -0244. Article and publication dat...

No abstract