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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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Differentiation capacity of talin (−/−) A28 ES cells. (A) Wild-type ES cells (HM1) and talin (−/−) A28 ES cells were used to  generate embryoid bodies that were cultured in the absence of LIF for 8 d, and then were plated onto gelatin-coated tissue culture  dishes. Embryoid bodies after 8 d in culture in the absence of LIF (d8), d8 embryoid bodies 1 d after plating onto gelatin-coated dishes  (d8+1d), d8 embryoid bodies 2 d after plating onto gelatin-coated (d8+2d). (B) Examples of some of the morphologically distinct cell  types (including giant cells; arrowheads) and structures (arrows) which were observed when d8 wild-type HM1 embryoid bodies were  cultured on gelatin-coated plates for 7 d (d8+7d). The two right hand panels show the only two morphologically distinct cell types observed when the talin (−/−) A28 d8 embryoid bodies were cultured under identical conditions. Bars, 200 μm.
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Figure 8: Differentiation capacity of talin (−/−) A28 ES cells. (A) Wild-type ES cells (HM1) and talin (−/−) A28 ES cells were used to generate embryoid bodies that were cultured in the absence of LIF for 8 d, and then were plated onto gelatin-coated tissue culture dishes. Embryoid bodies after 8 d in culture in the absence of LIF (d8), d8 embryoid bodies 1 d after plating onto gelatin-coated dishes (d8+1d), d8 embryoid bodies 2 d after plating onto gelatin-coated (d8+2d). (B) Examples of some of the morphologically distinct cell types (including giant cells; arrowheads) and structures (arrows) which were observed when d8 wild-type HM1 embryoid bodies were cultured on gelatin-coated plates for 7 d (d8+7d). The two right hand panels show the only two morphologically distinct cell types observed when the talin (−/−) A28 d8 embryoid bodies were cultured under identical conditions. Bars, 200 μm.

Mentions: Both wild-type ES cells and the talin (−/−) A28 ES cell mutant form embryoid bodies of a similar size and appearance when grown in the absence of LIF for 8 d (Fig. 8). This is not unexpected as the process is dependent on cadherin-mediated cell–cell interactions (Larue et al., 1996), and talin is specifically localized to integrin-containing cell–matrix junctions (Geiger et al., 1985). When wild-type embryoid bodies were plated on gelatin-coated tissue culture dishes, cells spread out from the central cell mass as a continuous sheet over a period of 24–48 h (Fig. 8 A), and formed an extensive cell monolayer within 5 d. In contrast, only a few cells had begun to spread out from the margins of the embryoid bodies formed by the talin (−/−) A28 ES cell mutants after 24 h, and those cells that did migrate over a 48-h period migrated as individual cells rather than as a continuous cell sheet (Fig. 8 A). The leading edge of the migrating cells from the wild-type embryoid bodies always contained giant cells (Fig. 8 A, arrowheads). These were never seen in the cells emerging from the talin (−/−) A28 embryoid bodies. After 7 d on gelatin, wild-type embryoid bodies gave rise to a variety of cell types with distinct morphologies, including beating cardiomyocytes and giant cells (Fig. 8 B, arrowheads), as well as organized structures (Fig. 8 B, arrows). In contrast, the talin (−/−) A28 embryoid bodies typically gave rise to only two morphologically distinct cell types, and no organized structures were observed (Fig. 8 B). Essentially identical results were obtained with the talin (−/−) J26 ES cell mutant (data not shown). Deletion of β1-integrins in mouse F9 teratocarcinoma cells has also been reported to block morphological differentiation (Stephens et al., 1993).


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)

Differentiation capacity of talin (−/−) A28 ES cells. (A) Wild-type ES cells (HM1) and talin (−/−) A28 ES cells were used to  generate embryoid bodies that were cultured in the absence of LIF for 8 d, and then were plated onto gelatin-coated tissue culture  dishes. Embryoid bodies after 8 d in culture in the absence of LIF (d8), d8 embryoid bodies 1 d after plating onto gelatin-coated dishes  (d8+1d), d8 embryoid bodies 2 d after plating onto gelatin-coated (d8+2d). (B) Examples of some of the morphologically distinct cell  types (including giant cells; arrowheads) and structures (arrows) which were observed when d8 wild-type HM1 embryoid bodies were  cultured on gelatin-coated plates for 7 d (d8+7d). The two right hand panels show the only two morphologically distinct cell types observed when the talin (−/−) A28 d8 embryoid bodies were cultured under identical conditions. Bars, 200 μm.
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Figure 8: Differentiation capacity of talin (−/−) A28 ES cells. (A) Wild-type ES cells (HM1) and talin (−/−) A28 ES cells were used to generate embryoid bodies that were cultured in the absence of LIF for 8 d, and then were plated onto gelatin-coated tissue culture dishes. Embryoid bodies after 8 d in culture in the absence of LIF (d8), d8 embryoid bodies 1 d after plating onto gelatin-coated dishes (d8+1d), d8 embryoid bodies 2 d after plating onto gelatin-coated (d8+2d). (B) Examples of some of the morphologically distinct cell types (including giant cells; arrowheads) and structures (arrows) which were observed when d8 wild-type HM1 embryoid bodies were cultured on gelatin-coated plates for 7 d (d8+7d). The two right hand panels show the only two morphologically distinct cell types observed when the talin (−/−) A28 d8 embryoid bodies were cultured under identical conditions. Bars, 200 μm.
Mentions: Both wild-type ES cells and the talin (−/−) A28 ES cell mutant form embryoid bodies of a similar size and appearance when grown in the absence of LIF for 8 d (Fig. 8). This is not unexpected as the process is dependent on cadherin-mediated cell–cell interactions (Larue et al., 1996), and talin is specifically localized to integrin-containing cell–matrix junctions (Geiger et al., 1985). When wild-type embryoid bodies were plated on gelatin-coated tissue culture dishes, cells spread out from the central cell mass as a continuous sheet over a period of 24–48 h (Fig. 8 A), and formed an extensive cell monolayer within 5 d. In contrast, only a few cells had begun to spread out from the margins of the embryoid bodies formed by the talin (−/−) A28 ES cell mutants after 24 h, and those cells that did migrate over a 48-h period migrated as individual cells rather than as a continuous cell sheet (Fig. 8 A). The leading edge of the migrating cells from the wild-type embryoid bodies always contained giant cells (Fig. 8 A, arrowheads). These were never seen in the cells emerging from the talin (−/−) A28 embryoid bodies. After 7 d on gelatin, wild-type embryoid bodies gave rise to a variety of cell types with distinct morphologies, including beating cardiomyocytes and giant cells (Fig. 8 B, arrowheads), as well as organized structures (Fig. 8 B, arrows). In contrast, the talin (−/−) A28 embryoid bodies typically gave rise to only two morphologically distinct cell types, and no organized structures were observed (Fig. 8 B). Essentially identical results were obtained with the talin (−/−) J26 ES cell mutant (data not shown). Deletion of β1-integrins in mouse F9 teratocarcinoma cells has also been reported to block morphological differentiation (Stephens et al., 1993).

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