Abstract
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 beta1 integrin, although levels of alpha5 and alphaV 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 beta1 integrin subunit were comparable to those in wild-type ES cells. We conclude that talin is essential for beta1 integrin expression and focal adhesion assembly in undifferentiated ES cells, but that a subset of differentiated cells are talin independent for both characteristics.
Highlights
The interaction between animal cells and the adhesive glycoproteins of the extracellular matrix is mediated by the integrin family of cell adhesion molecules, the cytoplasmic domains of which are thought to be linked in many, but not all cases, to the actin cytoskeleton (Burridge et al, 1996)
Because the differentiated talin (Ϫ/Ϫ) A28 and J26 ES cells that migrate out of embryoid bodies are able to adopt a spread morphology on gelatin-coated dishes, we investigated whether these cells could form focal adhesions and actin stress fibers when plated on fibronectin
The most notable features of the undifferentiated talin (Ϫ/Ϫ) ES cell mutants are extensive membrane blebbing, an accumulation of macropinocytic vesicles and an inability to spread on gelatin or laminin on which the cells grow as loosely attached colonies
Summary
The interaction between animal cells and the adhesive glycoproteins of the extracellular matrix is mediated by the integrin family of cell adhesion molecules, the cytoplasmic domains of which are thought to be linked in many, but not all cases, to the actin cytoskeleton (Burridge et al, 1996). Proteins that have been suggested to participate in this link include talin, vinculin and ␣-actinin (Jockusch et al, 1995) which colocalize with integrins in the specialized cell–matrix junctions or focal adhesions formed when cultured cells are grown on rigid supports. ␣-Actinin has been reported to bind directly to 1-integrin cytoplasmic domain peptides (Otey et al 1990) Such data have lead to a model of the focal adhesion in which integrins are linked to F-actin either directly via talin or ␣-actinin, or indirectly via interac-. Studies with a chimeric molecule containing the extracellular domain of N-cadherin fused to the transmembrane and cytoplasmic domains of 1-integrin support the view that the 1-integrin cytoplasmic domain plays a key role in localizing talin to cell– matrix rather than cell–cell junctions (Geiger et al, 1992). The phenotypic properties of these cells are consistent with the hypothesis that talin plays a key role in cell–matrix interactions
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