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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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Adhesive properties of the talin (−/−) ES cell  mutants. Wild-type ES cells  (HM1, +/+) and the talin ES  cell mutants C39.S2 (+/−)  A28 (−/−) and J26 (−/−)  were trypsinized to single cell  suspension and then plated  onto fibronectin (A) or laminin (B) coated wells for 1 h  either in the presence (black  bars) or absence (gray bars)  of 10 mM EDTA. Cells that  did not adhere in this time were washed off, and the number of adherent cells quantitated by staining with toluidine blue followed by the  extraction of bound dye. Absorbance was read at 630 nm. The results are the mean of three separate experiments conducted in triplicate. Bars represent standard errors.
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Figure 4: Adhesive properties of the talin (−/−) ES cell mutants. Wild-type ES cells (HM1, +/+) and the talin ES cell mutants C39.S2 (+/−) A28 (−/−) and J26 (−/−) were trypsinized to single cell suspension and then plated onto fibronectin (A) or laminin (B) coated wells for 1 h either in the presence (black bars) or absence (gray bars) of 10 mM EDTA. Cells that did not adhere in this time were washed off, and the number of adherent cells quantitated by staining with toluidine blue followed by the extraction of bound dye. Absorbance was read at 630 nm. The results are the mean of three separate experiments conducted in triplicate. Bars represent standard errors.

Mentions: The ability of wild-type ES cells, and the various talin ES cell mutants to adhere to fibronectin and laminin is shown in Fig. 4. Wild-type ES cells adhered to both fibronectin and laminin to about the same degree over a 1-h period, whereas adhesion to gelatin or collagen was very low (OD630 < 0.1). Adhesion was abolished in the presence of 10 mM EDTA demonstrating a requirement for divalent cations, a characteristic of integrin-mediated adhesion. Although both talin (−/−) ES cell mutants expressed much reduced levels of β1 integrin (Fig. 2), their adhesion to fibronectin was identical to that of wild-type ES cells. In contrast, they showed a significant reduction in adhesion to laminin. The reduced adhesion of the talin (−/−) ES cell mutants to laminin is consistent with the fact that they spread less well on laminin than 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)

Adhesive properties of the talin (−/−) ES cell  mutants. Wild-type ES cells  (HM1, +/+) and the talin ES  cell mutants C39.S2 (+/−)  A28 (−/−) and J26 (−/−)  were trypsinized to single cell  suspension and then plated  onto fibronectin (A) or laminin (B) coated wells for 1 h  either in the presence (black  bars) or absence (gray bars)  of 10 mM EDTA. Cells that  did not adhere in this time were washed off, and the number of adherent cells quantitated by staining with toluidine blue followed by the  extraction of bound dye. Absorbance was read at 630 nm. The results are the mean of three separate experiments conducted in triplicate. Bars represent standard errors.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 4: Adhesive properties of the talin (−/−) ES cell mutants. Wild-type ES cells (HM1, +/+) and the talin ES cell mutants C39.S2 (+/−) A28 (−/−) and J26 (−/−) were trypsinized to single cell suspension and then plated onto fibronectin (A) or laminin (B) coated wells for 1 h either in the presence (black bars) or absence (gray bars) of 10 mM EDTA. Cells that did not adhere in this time were washed off, and the number of adherent cells quantitated by staining with toluidine blue followed by the extraction of bound dye. Absorbance was read at 630 nm. The results are the mean of three separate experiments conducted in triplicate. Bars represent standard errors.
Mentions: The ability of wild-type ES cells, and the various talin ES cell mutants to adhere to fibronectin and laminin is shown in Fig. 4. Wild-type ES cells adhered to both fibronectin and laminin to about the same degree over a 1-h period, whereas adhesion to gelatin or collagen was very low (OD630 < 0.1). Adhesion was abolished in the presence of 10 mM EDTA demonstrating a requirement for divalent cations, a characteristic of integrin-mediated adhesion. Although both talin (−/−) ES cell mutants expressed much reduced levels of β1 integrin (Fig. 2), their adhesion to fibronectin was identical to that of wild-type ES cells. In contrast, they showed a significant reduction in adhesion to laminin. The reduced adhesion of the talin (−/−) ES cell mutants to laminin is consistent with the fact that they spread less well on laminin than 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