We have used gene disruption to isolate two talin (−/−) ES cell mutants that contain no intact talin. The undifferentiated cells (a) were unable to spread on gelatin or laminin and grew as rounded colonies, although they were able to spread on fibronectin (b) showed reduced adhesion to laminin, but not fibronectin (c) expressed much reduced levels of β1 integrin, although levels of α5 and αV were wild-type (d) were less polarized with increased membrane protrusions compared with a vinculin (−/−) ES cell mutant (e) were unable to assemble vinculin or paxillin-containing focal adhesions or actin stress fibers on fibronectin, whereas vinculin (−/−) ES cells were able to assemble talin-containing focal adhesions. Both talin (−/−) ES cell mutants formed embryoid bodies, but differentiation was restricted to two morphologically distinct cell types. Interestingly, these differentiated talin (−/−) ES cells were able to spread and form focal adhesion-like structures containing vinculin and paxillin on fibronectin. Moreover, the levels of the β1 integrin subunit were comparable to those in wild-type ES cells. We conclude that talin is essential for β1 integrin expression and focal adhesion assembly in undifferentiated ES cells, but that a subset of differentiated cells are talin independent for both characteristics.

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Abstract. To define the actin-binding site within the N11 2 -terminal domain (residues 1-245) of chick smooth muscle a-actinin, we expressed a series of a-actinin deletion mutants in monkey Cos cells . Mutant a-actinins in which residues 2-19, 217-242, and 196-242 were deleted still retained the ability to target to actin filaments and filament ends, suggesting that the actinbinding site is located within residues 20-195. When a truncated a-actinin (residues 1-290) was expressed in Cos cells, the protein localized exclusively to filament ends. This activity was retained by a deletion mutant lacking residues 196-242, confirming that these are not essential for actin binding . The actinbinding site in a-actinin was further defined by expressing both wild-type and mutant actin-binding domains as fusion proteins in E. coli. Analysis of the ability of a-ACTININ is a rod-shaped F-actin cross-linking protein found in both muscle and nonmuscle cells at sites where actin is attached to a variety of intracellular structures. It is a homodimer with subunits of molecular mass 94-103 kD arranged in an antiparallel orientation . Smooth, skeletal, and nonmuscle isoforms of the protein have been identified, the only clear functional difference being that binding of the nonmuscle isoform to actin is inhibited by calcium, whereas the muscle isoforms are calcium insensitive in this respect (reviewed in Blanchard et al ., 1989) . Analysis of the deduced sequence of the chick smooth muscle isoform of the protein shows that a-actinin can be divided into three distinct domains, an NH2-terminal actin-binding domain spanning Address reprint requests to David R. Critchley, Department of Biochemistry, University of Leicester, University Road, Leicester, UK LEI 7RH .Since this work was submitted for publication, Bresnick et al . (Bresnick, A . R., P. A . Janmey, and J . Condeelis . 1991 . J. Mol. Chem. 268 :12989-12993) have shown that antibodies to the 27 amino acids implicated in binding of ABP120 to actin can immune precipitate ABP120 from cell lysates, suggesting that these residues are on the surface of the protein as expected for an actin-binding site. Fab' fragments of the antibody inhibited ABP120 binding to actin, and the synthetic 27 mer inhibited actin cross-linking by ABP120. Evidence was also presented that the synthetic 27 mer cosediments with actin .

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We have shown previously that the N-terminal actin-binding domain of cc-actinin retains activity when expressed in E. coil a5 a fusion protein with glutathione-S-transferase. In the present study we have made a series of N-and C-terminal deletions within this domain and show that an actin-binding site is contained within residues 120--134. Amino acid substitutions within this region indicate that several highly ¢on~.ed hydrophobic residues are involved in binding to F-aetin. The hypotlaesis that the interaction between :r-aetinin and F-actin is predominantly hydrophobic in nature is supported by the observation that binding is relatively independent of salt concentration.a-Actinin; Actin-binding site; Dystrophin;/~-Sp~etri~t; ABP-120; Cytoskeleton