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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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Morphology of the talin ES cell mutants plated on gelatin and fibronectin. Wild-type ES cells (clone HM1) and the two talin  (−/−) ES cell lines A28 and J26, were trypsinized to obtain a single cell suspension and plated onto gelatin-coated (A) or fibronectin-coated (B) tissue culture plastic. These cells were then photographed after 4 and 48 h. Both talin (−/−) ES cell mutants spread more  slowly than wild-type ES cells on gelatin and fibronectin (4 h time point), and both showed evidence of extensive membrane blebbing on  gelatin (see arrows, and inset for high power view) and on fibronectin. After 48 h on gelatin, both talin (−/−) ES cell mutants formed  colonies with a rounded, refractile appearance which was quite distinct from the well spread morphology of the colonies formed by wild-type ES cells. On fibronectin, the talin (−/−) ES cell mutants behaved much more like the wild-type ES cells, although the colonies  formed were still less well spread than those formed by wild-type cells. Note the macropinocytic vesicles that occur at high frequency in  the talin (−/−) ES cell mutants on both gelatin and fibronectin. Bars: (A and B) 50 μm; (inset) 20 μm.
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Figure 3: Morphology of the talin ES cell mutants plated on gelatin and fibronectin. Wild-type ES cells (clone HM1) and the two talin (−/−) ES cell lines A28 and J26, were trypsinized to obtain a single cell suspension and plated onto gelatin-coated (A) or fibronectin-coated (B) tissue culture plastic. These cells were then photographed after 4 and 48 h. Both talin (−/−) ES cell mutants spread more slowly than wild-type ES cells on gelatin and fibronectin (4 h time point), and both showed evidence of extensive membrane blebbing on gelatin (see arrows, and inset for high power view) and on fibronectin. After 48 h on gelatin, both talin (−/−) ES cell mutants formed colonies with a rounded, refractile appearance which was quite distinct from the well spread morphology of the colonies formed by wild-type ES cells. On fibronectin, the talin (−/−) ES cell mutants behaved much more like the wild-type ES cells, although the colonies formed were still less well spread than those formed by wild-type cells. Note the macropinocytic vesicles that occur at high frequency in the talin (−/−) ES cell mutants on both gelatin and fibronectin. Bars: (A and B) 50 μm; (inset) 20 μm.

Mentions: The construct was linearized with NotI and electroporated into 1 × 108 ES cells (clone HM1). G418 was used to select for cells containing the plasmid, and gancyclovir was used to select against cells in which the construct had integrated randomly. Resistant colonies were transferred to four 96-well microtiter plates. The cells were then grown to confluence, replica plated, and genomic DNA from one set was analyzed by Southern blotting. The wild-type talin gene gives rise to a 13.8-kb EcoRI fragment that is detected by the probe indicated, whereas the Neo targeted allele gives rise to a novel 7.6-kb fragment due the presence of an EcoRI site within the Neo gene (Fig. 1, C and D). The result for one clone (C39) which is heterozygous at the talin locus is shown in Fig. 1 E. Gancylcovir lead to a twofold enrichment in homologous recombinants, and the talin gene was targeted in ∼1 in 3 of the G418/gancyclovir-resistant clones. To inactivate the second talin allele, an equivalent targeting vector was constructed in which the Neo gene was replaced by a Hyg selection marker. This was electroporated into the talin (+/−) ES cell clone C39, and EcoRI digests of genomic DNA from 384 hygromycin resistant clones screened for targeting of the second allele. The targeted allele should give rise to a novel 6.8-kb fragment, with corresponding loss of the remaining wild-type 13.8-kb fragment (Fig. 1, C and D). Only 1 clone (A28) was obtained which displayed this genotype (Fig. 1 E). This may be because the talin cells adhere only weakly to gelatin-coated plates (see Fig. 3), and are lost during the selection procedures. The talin (+/−) ES cell clone C39 was used in a second experiment with the Hyg targeting vector, and an additional clone (J26) in which both talin alleles had been disrupted was isolated (Fig. 1 E).


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)

Morphology of the talin ES cell mutants plated on gelatin and fibronectin. Wild-type ES cells (clone HM1) and the two talin  (−/−) ES cell lines A28 and J26, were trypsinized to obtain a single cell suspension and plated onto gelatin-coated (A) or fibronectin-coated (B) tissue culture plastic. These cells were then photographed after 4 and 48 h. Both talin (−/−) ES cell mutants spread more  slowly than wild-type ES cells on gelatin and fibronectin (4 h time point), and both showed evidence of extensive membrane blebbing on  gelatin (see arrows, and inset for high power view) and on fibronectin. After 48 h on gelatin, both talin (−/−) ES cell mutants formed  colonies with a rounded, refractile appearance which was quite distinct from the well spread morphology of the colonies formed by wild-type ES cells. On fibronectin, the talin (−/−) ES cell mutants behaved much more like the wild-type ES cells, although the colonies  formed were still less well spread than those formed by wild-type cells. Note the macropinocytic vesicles that occur at high frequency in  the talin (−/−) ES cell mutants on both gelatin and fibronectin. Bars: (A and B) 50 μm; (inset) 20 μm.
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Figure 3: Morphology of the talin ES cell mutants plated on gelatin and fibronectin. Wild-type ES cells (clone HM1) and the two talin (−/−) ES cell lines A28 and J26, were trypsinized to obtain a single cell suspension and plated onto gelatin-coated (A) or fibronectin-coated (B) tissue culture plastic. These cells were then photographed after 4 and 48 h. Both talin (−/−) ES cell mutants spread more slowly than wild-type ES cells on gelatin and fibronectin (4 h time point), and both showed evidence of extensive membrane blebbing on gelatin (see arrows, and inset for high power view) and on fibronectin. After 48 h on gelatin, both talin (−/−) ES cell mutants formed colonies with a rounded, refractile appearance which was quite distinct from the well spread morphology of the colonies formed by wild-type ES cells. On fibronectin, the talin (−/−) ES cell mutants behaved much more like the wild-type ES cells, although the colonies formed were still less well spread than those formed by wild-type cells. Note the macropinocytic vesicles that occur at high frequency in the talin (−/−) ES cell mutants on both gelatin and fibronectin. Bars: (A and B) 50 μm; (inset) 20 μm.
Mentions: The construct was linearized with NotI and electroporated into 1 × 108 ES cells (clone HM1). G418 was used to select for cells containing the plasmid, and gancyclovir was used to select against cells in which the construct had integrated randomly. Resistant colonies were transferred to four 96-well microtiter plates. The cells were then grown to confluence, replica plated, and genomic DNA from one set was analyzed by Southern blotting. The wild-type talin gene gives rise to a 13.8-kb EcoRI fragment that is detected by the probe indicated, whereas the Neo targeted allele gives rise to a novel 7.6-kb fragment due the presence of an EcoRI site within the Neo gene (Fig. 1, C and D). The result for one clone (C39) which is heterozygous at the talin locus is shown in Fig. 1 E. Gancylcovir lead to a twofold enrichment in homologous recombinants, and the talin gene was targeted in ∼1 in 3 of the G418/gancyclovir-resistant clones. To inactivate the second talin allele, an equivalent targeting vector was constructed in which the Neo gene was replaced by a Hyg selection marker. This was electroporated into the talin (+/−) ES cell clone C39, and EcoRI digests of genomic DNA from 384 hygromycin resistant clones screened for targeting of the second allele. The targeted allele should give rise to a novel 6.8-kb fragment, with corresponding loss of the remaining wild-type 13.8-kb fragment (Fig. 1, C and D). Only 1 clone (A28) was obtained which displayed this genotype (Fig. 1 E). This may be because the talin cells adhere only weakly to gelatin-coated plates (see Fig. 3), and are lost during the selection procedures. The talin (+/−) ES cell clone C39 was used in a second experiment with the Hyg targeting vector, and an additional clone (J26) in which both talin alleles had been disrupted was isolated (Fig. 1 E).

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