Limits...
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.

Show MeSH

Related in: MedlinePlus

Localization of talin, vinculin, paxillin, and F-actin in  undifferentiated talin (−/−) ES cell mutants by immunofluoresence. Wild-type (+/+) HM1 ES cells 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. Wild-type ES cells form focal  adhesions containing talin, vinculin and associated actin filaments. The talin (−/−) A28 mutant failed to assemble vinculin or  paxillin-containing focal adhesions and lacked actin stress fibers.  Bar, 20 μm.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2132864&req=5

Figure 5: Localization of talin, vinculin, paxillin, and F-actin in undifferentiated talin (−/−) ES cell mutants by immunofluoresence. Wild-type (+/+) HM1 ES cells 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. Wild-type ES cells form focal adhesions containing talin, vinculin and associated actin filaments. The talin (−/−) A28 mutant failed to assemble vinculin or paxillin-containing focal adhesions and lacked actin stress fibers. Bar, 20 μm.

Mentions: Undifferentiated wild-type ES cells (HM1) and the talin (−/−) A28 ES cell mutant grown in the presence of LIF were plated on coverslips coated with fibronectin and analyzed for their ability to form vinculin-containing focal adhesions and associated actin stress fibers after 48 h (Fig. 5). Wild-type ES cells adopted a well-spread morphology and assembled numerous actin stress fibers that terminated in focal adhesions that stained for talin, vinculin, and paxillin. In contrast, the talin (−/−) A28 ES cell mutant formed large rounded colonies with few if any spread cells. They failed to assemble any actin stress fibers, and actin staining was limited to the cell margins with diffuse cytoplasmic staining. There was no evidence of talin localized in focal adhesions, although the cells showed some diffuse staining for talin. Similarly, vinculin and paxillin staining was diffuse with only occasional punctate staining for vinculin at the margins of the cell aggregates. The results for the J26 talin (−/−) ES cell mutant were identical (data not shown). These findings establish that loss of talin compromises the ability of undifferentiated mouse ES cells to form focal adhesions and associated actin stress fibers on fibronectin, although talin is not required to support integrin-mediated adhesion to 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)

Localization of talin, vinculin, paxillin, and F-actin in  undifferentiated talin (−/−) ES cell mutants by immunofluoresence. Wild-type (+/+) HM1 ES cells 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. Wild-type ES cells form focal  adhesions containing talin, vinculin and associated actin filaments. The talin (−/−) A28 mutant failed to assemble vinculin or  paxillin-containing focal adhesions and lacked actin stress fibers.  Bar, 20 μm.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 5: Localization of talin, vinculin, paxillin, and F-actin in undifferentiated talin (−/−) ES cell mutants by immunofluoresence. Wild-type (+/+) HM1 ES cells 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. Wild-type ES cells form focal adhesions containing talin, vinculin and associated actin filaments. The talin (−/−) A28 mutant failed to assemble vinculin or paxillin-containing focal adhesions and lacked actin stress fibers. Bar, 20 μm.
Mentions: Undifferentiated wild-type ES cells (HM1) and the talin (−/−) A28 ES cell mutant grown in the presence of LIF were plated on coverslips coated with fibronectin and analyzed for their ability to form vinculin-containing focal adhesions and associated actin stress fibers after 48 h (Fig. 5). Wild-type ES cells adopted a well-spread morphology and assembled numerous actin stress fibers that terminated in focal adhesions that stained for talin, vinculin, and paxillin. In contrast, the talin (−/−) A28 ES cell mutant formed large rounded colonies with few if any spread cells. They failed to assemble any actin stress fibers, and actin staining was limited to the cell margins with diffuse cytoplasmic staining. There was no evidence of talin localized in focal adhesions, although the cells showed some diffuse staining for talin. Similarly, vinculin and paxillin staining was diffuse with only occasional punctate staining for vinculin at the margins of the cell aggregates. The results for the J26 talin (−/−) ES cell mutant were identical (data not shown). These findings establish that loss of talin compromises the ability of undifferentiated mouse ES cells to form focal adhesions and associated actin stress fibers on fibronectin, although talin is not required to support integrin-mediated adhesion to 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