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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 talin ES cell mutants. (A) 100  μg of total cellular protein from wild-type ES cells (HM1) and  talin ES cell mutants, C39 (+/−) and A28 (−/−) was Western  blotted onto Hybond C and probed for talin, vinculin, α-actinin,  paxillin, pp125 focal adhesion kinase (FAK), β1 integrin, α5 integrin, and αV integrin. The panel labeled Talin* is a long exposure  of a blot probed for talin. The β1, αV, and α5 integrin subunits  were detected in nonreducing gels. (B) A second Western blot  with similar amounts of total cellular protein from the above cell  lines plus a second talin (−/−) ES line (J26), confirms that the reduction in β1 integrin levels is characteristic of talin (−/−) ES  cell lines.
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Figure 2: Western blot analysis of talin ES cell mutants. (A) 100 μg of total cellular protein from wild-type ES cells (HM1) and talin ES cell mutants, C39 (+/−) and A28 (−/−) was Western blotted onto Hybond C and probed for talin, vinculin, α-actinin, paxillin, pp125 focal adhesion kinase (FAK), β1 integrin, α5 integrin, and αV integrin. The panel labeled Talin* is a long exposure of a blot probed for talin. The β1, αV, and α5 integrin subunits were detected in nonreducing gels. (B) A second Western blot with similar amounts of total cellular protein from the above cell lines plus a second talin (−/−) ES line (J26), confirms that the reduction in β1 integrin levels is characteristic of talin (−/−) ES cell lines.

Mentions: The expression of talin in these various ES cell clones, grown in the presence of LIF to suppress differentiation, was analyzed by Western blotting using a monoclonal antibody (TD77) raised against human platelet talin. This antibody, which recognizes an epitope at the extreme COOH-terminal region (residues 2464–2541) of talin (Bolton et al., 1997), detected a single talin polypeptide in the wild-type ES cells. The talin (+/−) ES cell clone C39 showed a significant reduction in talin immunoreactivity. However, these cells expressed two talin polypeptides, one which comigrated with the wild-type protein, the other migrating with slightly increased mobility (Fig. 2, A and B). The talin (−/−) ES cell mutants A28 and J26 showed almost complete loss of talin immunoreactivity (Fig. 2, A and B), although prolonged exposure of the blots showed that clone A28 (Fig. 2 A) and J26 (data not shown) expressed very low levels of a truncated talin polypeptide that comigrated with the truncated talin polypeptide expressed in the (+/−) C39 ES cell line. The results clearly establish that both the talin (−/−) A28 and J26 ES cell mutants express no intact talin. The levels of two other cytoskeletal proteins associated with the cytoplasmic face of focal adhesions, vinculin and α-actinin, were also somewhat reduced in the talin (−/−) A28 mutant, although the levels of paxillin and pp125FAK were similar to those in wild-type cells (Fig. 2 A). There was also a dramatic reduction in the levels of the β1 integrin subunit in both the talin (−/−) ES cell mutants (Fig. 2, A and B) whereas the levels of both α5 and αV integrin subunits were comparable to those in wild-type ES cells. The results show that loss of talin is associated with a reduction in the levels of a number of other focal adhesion proteins, most notably the β1 integrin subunit.


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 talin ES cell mutants. (A) 100  μg of total cellular protein from wild-type ES cells (HM1) and  talin ES cell mutants, C39 (+/−) and A28 (−/−) was Western  blotted onto Hybond C and probed for talin, vinculin, α-actinin,  paxillin, pp125 focal adhesion kinase (FAK), β1 integrin, α5 integrin, and αV integrin. The panel labeled Talin* is a long exposure  of a blot probed for talin. The β1, αV, and α5 integrin subunits  were detected in nonreducing gels. (B) A second Western blot  with similar amounts of total cellular protein from the above cell  lines plus a second talin (−/−) ES line (J26), confirms that the reduction in β1 integrin levels is characteristic of talin (−/−) ES  cell lines.
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Related In: Results  -  Collection

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Figure 2: Western blot analysis of talin ES cell mutants. (A) 100 μg of total cellular protein from wild-type ES cells (HM1) and talin ES cell mutants, C39 (+/−) and A28 (−/−) was Western blotted onto Hybond C and probed for talin, vinculin, α-actinin, paxillin, pp125 focal adhesion kinase (FAK), β1 integrin, α5 integrin, and αV integrin. The panel labeled Talin* is a long exposure of a blot probed for talin. The β1, αV, and α5 integrin subunits were detected in nonreducing gels. (B) A second Western blot with similar amounts of total cellular protein from the above cell lines plus a second talin (−/−) ES line (J26), confirms that the reduction in β1 integrin levels is characteristic of talin (−/−) ES cell lines.
Mentions: The expression of talin in these various ES cell clones, grown in the presence of LIF to suppress differentiation, was analyzed by Western blotting using a monoclonal antibody (TD77) raised against human platelet talin. This antibody, which recognizes an epitope at the extreme COOH-terminal region (residues 2464–2541) of talin (Bolton et al., 1997), detected a single talin polypeptide in the wild-type ES cells. The talin (+/−) ES cell clone C39 showed a significant reduction in talin immunoreactivity. However, these cells expressed two talin polypeptides, one which comigrated with the wild-type protein, the other migrating with slightly increased mobility (Fig. 2, A and B). The talin (−/−) ES cell mutants A28 and J26 showed almost complete loss of talin immunoreactivity (Fig. 2, A and B), although prolonged exposure of the blots showed that clone A28 (Fig. 2 A) and J26 (data not shown) expressed very low levels of a truncated talin polypeptide that comigrated with the truncated talin polypeptide expressed in the (+/−) C39 ES cell line. The results clearly establish that both the talin (−/−) A28 and J26 ES cell mutants express no intact talin. The levels of two other cytoskeletal proteins associated with the cytoplasmic face of focal adhesions, vinculin and α-actinin, were also somewhat reduced in the talin (−/−) A28 mutant, although the levels of paxillin and pp125FAK were similar to those in wild-type cells (Fig. 2 A). There was also a dramatic reduction in the levels of the β1 integrin subunit in both the talin (−/−) ES cell mutants (Fig. 2, A and B) whereas the levels of both α5 and αV integrin subunits were comparable to those in wild-type ES cells. The results show that loss of talin is associated with a reduction in the levels of a number of other focal adhesion proteins, most notably the β1 integrin subunit.

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