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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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Western blot analysis of  differentiated ES cells. 100 μg of total cellular protein from d8 embryoid bodies made from wild-type  (HM1) ES cells or talin (−/−) mutant ES cells (A28 and J26) was resolved by SDS-PAGE, blotted onto  Hybond C and probed for talin, vinculin, and β1 integrin.
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Figure 10: Western blot analysis of differentiated ES cells. 100 μg of total cellular protein from d8 embryoid bodies made from wild-type (HM1) ES cells or talin (−/−) mutant ES cells (A28 and J26) was resolved by SDS-PAGE, blotted onto Hybond C and probed for talin, vinculin, and β1 integrin.

Mentions: Western blot analysis of both the differentiated talin (−/−) ES cell mutants showed that although these cells still expressed no intact talin, the levels of β1 integrin subunit as well as vinculin were comparable with those in differentiated wild-type ES cells (Fig. 10). Therefore, it is apparent that differentiation of the talin (−/−) ES cell mutants in vitro generates cells in which neither β1 integrin subunit levels or the assembly of focal adhesions and associated actin filaments is dependent on talin.


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)

Western blot analysis of  differentiated ES cells. 100 μg of total cellular protein from d8 embryoid bodies made from wild-type  (HM1) ES cells or talin (−/−) mutant ES cells (A28 and J26) was resolved by SDS-PAGE, blotted onto  Hybond C and probed for talin, vinculin, and β1 integrin.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2132864&req=5

Figure 10: Western blot analysis of differentiated ES cells. 100 μg of total cellular protein from d8 embryoid bodies made from wild-type (HM1) ES cells or talin (−/−) mutant ES cells (A28 and J26) was resolved by SDS-PAGE, blotted onto Hybond C and probed for talin, vinculin, and β1 integrin.
Mentions: Western blot analysis of both the differentiated talin (−/−) ES cell mutants showed that although these cells still expressed no intact talin, the levels of β1 integrin subunit as well as vinculin were comparable with those in differentiated wild-type ES cells (Fig. 10). Therefore, it is apparent that differentiation of the talin (−/−) ES cell mutants in vitro generates cells in which neither β1 integrin subunit levels or the assembly of focal adhesions and associated actin filaments is dependent on talin.

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