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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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Generation and analysis of ES cell  lines with disrupted vinculin alleles. (A) Targeting of the vinculin gene. On the top line is  a schematic of the gene structure of vinculin  with the introns as lines and exons as numbered boxes. The second line represents the  generic vinculin positive/negative targeting  vector where a Neo or Hyg selectable marker  has been inserted into the unique MscI (M)  site in exon 5. The bottom part represents the  resulting targeted alleles with either Neo or  Hyg inserted into the MscI site of exon 5. ES  cell clones were screened by Southern blotting using EcoRI digests that were probed  with the 5′ probe indicated. The wild-type  band detected is 7.0-kb whereas targeted alleles containing either Neo or Hyg give rise to  bands of 6.5 kb. E, EcoRI; B, BamHI; M, MscI;  X, XbaI. An example of such a Southern blot  is shown in B. (C) Western blot analysis of  wild-type ES cells (HM1), and vinculin ES cell  mutants ES2 (+/−) and D7 (−/−). (100 μg  protein loaded per well). The blots were  probed with antibodies to talin, vinculin, α-actinin and VASP. (D) Morphology of the vinculin (−/−) D7 ES cell mutant on gelatin and fibronectin coated dishes 48 h after plating. Note  the more rounded morphology of the D7 colonies on gelatin. (E) Localization of talin and  F-actin in wild-type ES cells (clone HM1) and  the vinculin (−/−) D7 ES cell mutant by immunofluoresence. Bars: (D) 50 μm; (E) 5 μm.
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Figure 6: Generation and analysis of ES cell lines with disrupted vinculin alleles. (A) Targeting of the vinculin gene. On the top line is a schematic of the gene structure of vinculin with the introns as lines and exons as numbered boxes. The second line represents the generic vinculin positive/negative targeting vector where a Neo or Hyg selectable marker has been inserted into the unique MscI (M) site in exon 5. The bottom part represents the resulting targeted alleles with either Neo or Hyg inserted into the MscI site of exon 5. ES cell clones were screened by Southern blotting using EcoRI digests that were probed with the 5′ probe indicated. The wild-type band detected is 7.0-kb whereas targeted alleles containing either Neo or Hyg give rise to bands of 6.5 kb. E, EcoRI; B, BamHI; M, MscI; X, XbaI. An example of such a Southern blot is shown in B. (C) Western blot analysis of wild-type ES cells (HM1), and vinculin ES cell mutants ES2 (+/−) and D7 (−/−). (100 μg protein loaded per well). The blots were probed with antibodies to talin, vinculin, α-actinin and VASP. (D) Morphology of the vinculin (−/−) D7 ES cell mutant on gelatin and fibronectin coated dishes 48 h after plating. Note the more rounded morphology of the D7 colonies on gelatin. (E) Localization of talin and F-actin in wild-type ES cells (clone HM1) and the vinculin (−/−) D7 ES cell mutant by immunofluoresence. Bars: (D) 50 μm; (E) 5 μm.

Mentions: We have used a similar strategy to generate ES cells in which both copies of the vinculin gene have been disrupted (Fig. 6, A and B). The vinculin (−/−) D7 ES cell line expressed no intact vinculin (Fig. 6 C), although low levels of a truncated vinculin polypeptide could be detected on prolonged exposure of the Western blots. However, the levels of three vinculin-binding proteins, talin, α-actinin, and VASP (Fig. 6 C) as well as β1-integrin (data not shown) were comparable to those in wild-type ES cells. The vinculin (−/−) D7 ES cell line showed a tendency to form rounded colonies when grown under standard culture conditions on gelatin-coated tissue culture dishes, although on fibronectin, their morphology was similar to that of wild-type ES cells. These results are similar to those with the talin (−/−) ES cell mutants that also formed rounded colonies on gelatin, but were able to spread on fibronectin.


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)

Generation and analysis of ES cell  lines with disrupted vinculin alleles. (A) Targeting of the vinculin gene. On the top line is  a schematic of the gene structure of vinculin  with the introns as lines and exons as numbered boxes. The second line represents the  generic vinculin positive/negative targeting  vector where a Neo or Hyg selectable marker  has been inserted into the unique MscI (M)  site in exon 5. The bottom part represents the  resulting targeted alleles with either Neo or  Hyg inserted into the MscI site of exon 5. ES  cell clones were screened by Southern blotting using EcoRI digests that were probed  with the 5′ probe indicated. The wild-type  band detected is 7.0-kb whereas targeted alleles containing either Neo or Hyg give rise to  bands of 6.5 kb. E, EcoRI; B, BamHI; M, MscI;  X, XbaI. An example of such a Southern blot  is shown in B. (C) Western blot analysis of  wild-type ES cells (HM1), and vinculin ES cell  mutants ES2 (+/−) and D7 (−/−). (100 μg  protein loaded per well). The blots were  probed with antibodies to talin, vinculin, α-actinin and VASP. (D) Morphology of the vinculin (−/−) D7 ES cell mutant on gelatin and fibronectin coated dishes 48 h after plating. Note  the more rounded morphology of the D7 colonies on gelatin. (E) Localization of talin and  F-actin in wild-type ES cells (clone HM1) and  the vinculin (−/−) D7 ES cell mutant by immunofluoresence. Bars: (D) 50 μm; (E) 5 μm.
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Figure 6: Generation and analysis of ES cell lines with disrupted vinculin alleles. (A) Targeting of the vinculin gene. On the top line is a schematic of the gene structure of vinculin with the introns as lines and exons as numbered boxes. The second line represents the generic vinculin positive/negative targeting vector where a Neo or Hyg selectable marker has been inserted into the unique MscI (M) site in exon 5. The bottom part represents the resulting targeted alleles with either Neo or Hyg inserted into the MscI site of exon 5. ES cell clones were screened by Southern blotting using EcoRI digests that were probed with the 5′ probe indicated. The wild-type band detected is 7.0-kb whereas targeted alleles containing either Neo or Hyg give rise to bands of 6.5 kb. E, EcoRI; B, BamHI; M, MscI; X, XbaI. An example of such a Southern blot is shown in B. (C) Western blot analysis of wild-type ES cells (HM1), and vinculin ES cell mutants ES2 (+/−) and D7 (−/−). (100 μg protein loaded per well). The blots were probed with antibodies to talin, vinculin, α-actinin and VASP. (D) Morphology of the vinculin (−/−) D7 ES cell mutant on gelatin and fibronectin coated dishes 48 h after plating. Note the more rounded morphology of the D7 colonies on gelatin. (E) Localization of talin and F-actin in wild-type ES cells (clone HM1) and the vinculin (−/−) D7 ES cell mutant by immunofluoresence. Bars: (D) 50 μm; (E) 5 μm.
Mentions: We have used a similar strategy to generate ES cells in which both copies of the vinculin gene have been disrupted (Fig. 6, A and B). The vinculin (−/−) D7 ES cell line expressed no intact vinculin (Fig. 6 C), although low levels of a truncated vinculin polypeptide could be detected on prolonged exposure of the Western blots. However, the levels of three vinculin-binding proteins, talin, α-actinin, and VASP (Fig. 6 C) as well as β1-integrin (data not shown) were comparable to those in wild-type ES cells. The vinculin (−/−) D7 ES cell line showed a tendency to form rounded colonies when grown under standard culture conditions on gelatin-coated tissue culture dishes, although on fibronectin, their morphology was similar to that of wild-type ES cells. These results are similar to those with the talin (−/−) ES cell mutants that also formed rounded colonies on gelatin, but were able to spread on fibronectin.

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