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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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Targeting of the  talin locus in ES cells. (A)  Gene structure of talin locus  around the targeting site.  The position of the first two  coding exons of talin is  shown in relation to a restriction map of the mouse ES  cell talin genomic DNA in  that region. Exons are represented by rectangles (black  being untranslated and grey  being coding) and introns as solid lines. The arrow indicates the  translation start. (B) Talin targeting construct with Neo replacing  the sequence between the BamHI and HindIII sites found in exons 1 and 2, respectively. (C) Targeted talin alleles showing the  replacement of BamHI–HindIII fragment with either Neo or Hyg  and the location of the newly introduced EcoRI (E) sites. (D) Expected fragment sizes and positions when genomic DNA from either wild-type, Neo targeted, or Hyg targeted ES cells is digested  with EcoRI and probed with the 5′ external probe shown. (E)  Southern blots of targeted ES cell clones. EcoRI digested genomic DNA from wild-type ES cells (HM1), talin (+/−) ES cells  with one allele targeted by the Neo vector (C39), and talin (−/−)  ES cells with one allele targeted by the Neo vector, the other by  the Hyg vector (A28 and J26). The clone C39.S2 which is (+/−)  at the talin locus was isolated during attempts to inactivate the  second allele with the Hyg targeting vector, and is a useful control for any phenotypic changes which might arise during Hyg selection. E, EcoRI; H, HindIII; B, BamHI; S, SacI.
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Figure 1: Targeting of the talin locus in ES cells. (A) Gene structure of talin locus around the targeting site. The position of the first two coding exons of talin is shown in relation to a restriction map of the mouse ES cell talin genomic DNA in that region. Exons are represented by rectangles (black being untranslated and grey being coding) and introns as solid lines. The arrow indicates the translation start. (B) Talin targeting construct with Neo replacing the sequence between the BamHI and HindIII sites found in exons 1 and 2, respectively. (C) Targeted talin alleles showing the replacement of BamHI–HindIII fragment with either Neo or Hyg and the location of the newly introduced EcoRI (E) sites. (D) Expected fragment sizes and positions when genomic DNA from either wild-type, Neo targeted, or Hyg targeted ES cells is digested with EcoRI and probed with the 5′ external probe shown. (E) Southern blots of targeted ES cell clones. EcoRI digested genomic DNA from wild-type ES cells (HM1), talin (+/−) ES cells with one allele targeted by the Neo vector (C39), and talin (−/−) ES cells with one allele targeted by the Neo vector, the other by the Hyg vector (A28 and J26). The clone C39.S2 which is (+/−) at the talin locus was isolated during attempts to inactivate the second allele with the Hyg targeting vector, and is a useful control for any phenotypic changes which might arise during Hyg selection. E, EcoRI; H, HindIII; B, BamHI; S, SacI.

Mentions: A mouse talin genomic clone (5Tλ2) was analyzed by restriction enzyme digestion and Southern blotting using oligonucleotide probes from the 5′ end of the talin cDNA. Sequencing of genomic fragments hybridizing to these probes allowed the boundaries of the first two coding exons to be identified (Fig. 1 A). The first coding exon is 163 bp long and contains 127 bp of coding sequence. Codon 44 is split by an intron between coding exons 1 and 2. The second coding exon contains 97 bp ending at codon 76. A 2.4-kb BamHI fragment containing the 5′ end of the first coding exon and a 6.2-kb HindIII fragment containing the 3′ end of the second coding exon were cloned either side of the Neo gene in the vector pX53, which also contains a TK negative selection marker (Fig. 1 B). Homologous recombination of this construct with the talin gene should lead to deletion of codons 37–66 and the fusion of the Neo gene with the residual parts of coding exons 1 and 2.


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)

Targeting of the  talin locus in ES cells. (A)  Gene structure of talin locus  around the targeting site.  The position of the first two  coding exons of talin is  shown in relation to a restriction map of the mouse ES  cell talin genomic DNA in  that region. Exons are represented by rectangles (black  being untranslated and grey  being coding) and introns as solid lines. The arrow indicates the  translation start. (B) Talin targeting construct with Neo replacing  the sequence between the BamHI and HindIII sites found in exons 1 and 2, respectively. (C) Targeted talin alleles showing the  replacement of BamHI–HindIII fragment with either Neo or Hyg  and the location of the newly introduced EcoRI (E) sites. (D) Expected fragment sizes and positions when genomic DNA from either wild-type, Neo targeted, or Hyg targeted ES cells is digested  with EcoRI and probed with the 5′ external probe shown. (E)  Southern blots of targeted ES cell clones. EcoRI digested genomic DNA from wild-type ES cells (HM1), talin (+/−) ES cells  with one allele targeted by the Neo vector (C39), and talin (−/−)  ES cells with one allele targeted by the Neo vector, the other by  the Hyg vector (A28 and J26). The clone C39.S2 which is (+/−)  at the talin locus was isolated during attempts to inactivate the  second allele with the Hyg targeting vector, and is a useful control for any phenotypic changes which might arise during Hyg selection. E, EcoRI; H, HindIII; B, BamHI; S, SacI.
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Related In: Results  -  Collection

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Figure 1: Targeting of the talin locus in ES cells. (A) Gene structure of talin locus around the targeting site. The position of the first two coding exons of talin is shown in relation to a restriction map of the mouse ES cell talin genomic DNA in that region. Exons are represented by rectangles (black being untranslated and grey being coding) and introns as solid lines. The arrow indicates the translation start. (B) Talin targeting construct with Neo replacing the sequence between the BamHI and HindIII sites found in exons 1 and 2, respectively. (C) Targeted talin alleles showing the replacement of BamHI–HindIII fragment with either Neo or Hyg and the location of the newly introduced EcoRI (E) sites. (D) Expected fragment sizes and positions when genomic DNA from either wild-type, Neo targeted, or Hyg targeted ES cells is digested with EcoRI and probed with the 5′ external probe shown. (E) Southern blots of targeted ES cell clones. EcoRI digested genomic DNA from wild-type ES cells (HM1), talin (+/−) ES cells with one allele targeted by the Neo vector (C39), and talin (−/−) ES cells with one allele targeted by the Neo vector, the other by the Hyg vector (A28 and J26). The clone C39.S2 which is (+/−) at the talin locus was isolated during attempts to inactivate the second allele with the Hyg targeting vector, and is a useful control for any phenotypic changes which might arise during Hyg selection. E, EcoRI; H, HindIII; B, BamHI; S, SacI.
Mentions: A mouse talin genomic clone (5Tλ2) was analyzed by restriction enzyme digestion and Southern blotting using oligonucleotide probes from the 5′ end of the talin cDNA. Sequencing of genomic fragments hybridizing to these probes allowed the boundaries of the first two coding exons to be identified (Fig. 1 A). The first coding exon is 163 bp long and contains 127 bp of coding sequence. Codon 44 is split by an intron between coding exons 1 and 2. The second coding exon contains 97 bp ending at codon 76. A 2.4-kb BamHI fragment containing the 5′ end of the first coding exon and a 6.2-kb HindIII fragment containing the 3′ end of the second coding exon were cloned either side of the Neo gene in the vector pX53, which also contains a TK negative selection marker (Fig. 1 B). Homologous recombination of this construct with the talin gene should lead to deletion of codons 37–66 and the fusion of the Neo gene with the residual parts of coding exons 1 and 2.

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