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Disruption of the talin gene compromises focal adhesion assembly in undifferentiated but not differentiated embryonic stem cells.

Priddle H, Hemmings L, Monkley S, Woods A, Patel B, Sutton D, Dunn GA, Zicha D, Critchley DR - J. Cell Biol. (1998)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, University of Leicester, Leicester LE1 7RH, United Kingdom.

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.

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Localization of talin, vinculin, paxillin, and F-actin in  differentiated talin (−/−) ES cell mutants by immunofluoresence. Cells derived from the embryoid bodies formed by wild-type (+/+) ES cells (HM1) and the talin (−/−) A28 ES cell mutant were seeded onto fibronectin-coated glass coverslips. After  48 h, the cells were stained for talin and F-actin, vinculin and  F-actin or paxillin and F-actin. Differentiated HM1 cells displayed a variety of morphologies, but all showed strong staining  for talin, vinculin and paxillin in focal adhesions at the ends of actin filaments. The differentiated talin (−/−) ES cell mutants  adopted a spread morphology with clear evidence of vinculin and  paxillin-containing focal adhesion-like structures at the ends of  actin filaments. However, the structures formed were less extensive than those seen in wild-type ES cells. Bar, 20 μm.
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Figure 9: Localization of talin, vinculin, paxillin, and F-actin in differentiated talin (−/−) ES cell mutants by immunofluoresence. Cells derived from the embryoid bodies formed by wild-type (+/+) ES cells (HM1) and the talin (−/−) A28 ES cell mutant were seeded onto fibronectin-coated glass coverslips. After 48 h, the cells were stained for talin and F-actin, vinculin and F-actin or paxillin and F-actin. Differentiated HM1 cells displayed a variety of morphologies, but all showed strong staining for talin, vinculin and paxillin in focal adhesions at the ends of actin filaments. The differentiated talin (−/−) ES cell mutants adopted a spread morphology with clear evidence of vinculin and paxillin-containing focal adhesion-like structures at the ends of actin filaments. However, the structures formed were less extensive than those seen in wild-type ES cells. Bar, 20 μm.

Mentions: Because the differentiated talin (−/−) 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 differentiated cells derived from wild-type embryoid bodies showed a variety of morphologies, and the organization of the actin cytoarchitecture within these cells took on a variety of forms. However, in all cases there was prominent staining for talin, vinculin and paxillin at the ends of actin filaments (Fig. 9). Interestingly, the differentiated talin (−/−) A28 cells also contained actin filaments, and in some cells, the ends of these filaments showed staining for vinculin and paxillin, but not talin. Identical results were obtained with the talin (−/−) J26 ES cell mutant (data not shown).


Disruption of the talin gene compromises focal adhesion assembly in undifferentiated but not differentiated embryonic stem cells.

Priddle H, Hemmings L, Monkley S, Woods A, Patel B, Sutton D, Dunn GA, Zicha D, Critchley DR - J. Cell Biol. (1998)

Localization of talin, vinculin, paxillin, and F-actin in  differentiated talin (−/−) ES cell mutants by immunofluoresence. Cells derived from the embryoid bodies formed by wild-type (+/+) ES cells (HM1) and the talin (−/−) A28 ES cell mutant were seeded onto fibronectin-coated glass coverslips. After  48 h, the cells were stained for talin and F-actin, vinculin and  F-actin or paxillin and F-actin. Differentiated HM1 cells displayed a variety of morphologies, but all showed strong staining  for talin, vinculin and paxillin in focal adhesions at the ends of actin filaments. The differentiated talin (−/−) ES cell mutants  adopted a spread morphology with clear evidence of vinculin and  paxillin-containing focal adhesion-like structures at the ends of  actin filaments. However, the structures formed were less extensive than those seen in wild-type ES cells. Bar, 20 μm.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2132864&req=5

Figure 9: Localization of talin, vinculin, paxillin, and F-actin in differentiated talin (−/−) ES cell mutants by immunofluoresence. Cells derived from the embryoid bodies formed by wild-type (+/+) ES cells (HM1) and the talin (−/−) A28 ES cell mutant were seeded onto fibronectin-coated glass coverslips. After 48 h, the cells were stained for talin and F-actin, vinculin and F-actin or paxillin and F-actin. Differentiated HM1 cells displayed a variety of morphologies, but all showed strong staining for talin, vinculin and paxillin in focal adhesions at the ends of actin filaments. The differentiated talin (−/−) ES cell mutants adopted a spread morphology with clear evidence of vinculin and paxillin-containing focal adhesion-like structures at the ends of actin filaments. However, the structures formed were less extensive than those seen in wild-type ES cells. Bar, 20 μm.
Mentions: Because the differentiated talin (−/−) 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 differentiated cells derived from wild-type embryoid bodies showed a variety of morphologies, and the organization of the actin cytoarchitecture within these cells took on a variety of forms. However, in all cases there was prominent staining for talin, vinculin and paxillin at the ends of actin filaments (Fig. 9). Interestingly, the differentiated talin (−/−) A28 cells also contained actin filaments, and in some cells, the ends of these filaments showed staining for vinculin and paxillin, but not talin. Identical results were obtained with the talin (−/−) J26 ES cell mutant (data not shown).

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, University of Leicester, Leicester LE1 7RH, United Kingdom.

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.

Show MeSH
Related in: MedlinePlus