Tropomyosins are rodlike coiled coil dimers that form continuous polymers along the major groove of most actin filaments. In striated muscle, tropomyosin regulates the actin-myosin interaction and, hence, contraction of muscle. Tropomyosin also contributes to most, if not all, functions of the actin cytoskeleton, and its role is essential for the viability of a wide range of organisms. The ability of tropomyosin to contribute to the many functions of the actin cytoskeleton is related to the temporal and spatial regulation of expression of tropomyosin isoforms. Qualitative and quantitative changes in tropomyosin isoform expression accompany morphogenesis in a range of cell types. The isoforms are segregated to different intracellular pools of actin filaments and confer different properties to these filaments. Mutations in tropomyosins are directly involved in cardiac and skeletal muscle diseases. Alterations in tropomyosin expression directly contribute to the growth and spread of cancer. The functional specificity of tropomyosins is related to the collaborative interactions of the isoforms with different actin binding proteins such as cofilin, gelsolin, Arp 2/3, myosin, caldesmon, and tropomodulin. It is proposed that local changes in signaling activity may be sufficient to drive the assembly of isoform-specific complexes at different intracellular sites.

We have constructed a mammalian expression vector consisting of 3 kilobases of the human fi-actin gene 5' flanking sequence plus 5' untranslated region and intervening sequence I linked at the 3' splice site to a short DNA polylinker sequence containing unique Sal I, HindIII, and BamHI restriction endonuclease sites followed by a simian virus 40 (SV40) polyadenylylation signal. Two derivatives, containing the selection markers obtained from pSV2gpt or pSV2neo, were also generated. We find that the promoter activity of this vector is as great or greater than that of the SV40 early promoter in a variety of human and rodent cells. The vector was used to generate y-actin and 8-tubulin antisense transcripts in human fibroblast cell lines. The antisense transcripts accumulate to levels comparable with that of the highly abundant y-actin and .-tubulin mRNAs.

We report the complete nucleotide sequence of a human ,B actin cDNA. Both the 5' and 3' untranslated regions of the sequence are similar (>809%) to the analogous regions of the rat 8-actin gene reported by Nudel et al (1983). When a segment of the 3' untranslated region is used as a radiolabelled probe, strong hybridization to chick I8 actin mRNA is seen. This conservation of sequences suggests that strong selective pressures operate on non-translated segments of,B actin mRNA.

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.

cDNA clones encoding three classes of human actins have been isolated and characterized. The first two classes (y and ,B, cytoplasmic actins) were obtained from a cDNA library constructed from simian virus 40-transformed human fibroblast mRNA, and the third class (a, muscle actin) was obtained from a cDNA library constructed from adult human muscle mRNA. A new approach was developed to enrich for full-length cDNAs. The human fibroblast cDNA plasmid library was linearized with restriction enzymes that did not cut the inserts of interest; it was then size-fractionated on gels, and the chimeric molecules of optimal length were selected for retransformation of bacteria. When the resulting clones were screened for actin-coding sequences it was found that some fulllength cDNAs were enriched as much as 50-to 100-fold relative to the original frequency of full-length clones in the total library. Two types of clones were distinguished. One of these clones encodes y actin and contains 100 base pairs of 5' untranslated region, the entire protein coding region, and the 3' untranslated region. The second class encodes 1 actin, and the longest such clone contains 45 base pairs of 5' untranslated region plus the remainder of the mRNA extending to the polyadenylic acid tail. A third class, obtained from the human muscle cDNA library, encodes a actin and contains 100 base pairs of 5' untranslated region, the entire coding region, and the 3' untranslated region. Analysis of the DNA sequences of the 5' end of the clones demonstrated that although 13-and y-actin

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...

More than a billion humans worldwide are predicted to be completely deficient in the fast skeletal muscle fiber protein alpha-actinin-3 owing to homozygosity for a premature stop codon polymorphism, R577X, in the ACTN3 gene. The R577X polymorphism is associated with elite athlete status and human muscle performance, suggesting that alpha-actinin-3 deficiency influences the function of fast muscle fibers. Here we show that loss of alpha-actinin-3 expression in a knockout mouse model results in a shift in muscle metabolism toward the more efficient aerobic pathway and an increase in intrinsic endurance performance. In addition, we demonstrate that the genomic region surrounding the 577X null allele shows low levels of genetic variation and recombination in individuals of European and East Asian descent, consistent with strong, recent positive selection. We propose that the 577X allele has been positively selected in some human populations owing to its effect on skeletal muscle metabolism.

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