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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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Analysis of wild-type ES cells and the talin and vinculin  mutants by time lapse video interference microscopy. (a) Wild-type ES cells (b) the talin (−/−) A28 ES cell mutant and (c) the  vinculin (−/−) D7 ES cell mutant taken from DRIMAPS recordings processed to reveal the regions of protrusion (green) and retraction (red) of the cell margin during a 2-min interval. The grey  levels inside the cells represent the dry mass distribution on an  arbitrary scale. (d). Median values of cell polarity measured as  the distance in μm between the centroids of the protrusion and  retraction areas over a 5-min period. The rectangles and bars indicate the distributions of data values. (e). Power spectra of the  variations in protrusion of the wild-type ES cells (black), talin  (−/−) A28 ES cell mutant (red) and the vinculin (−/−) D7 ES  cell mutant cells (green). The power (vertical axis) in arbitrary  units reveals how much of the variance in the data is attributable  to each frequency. Bars: (A–C) 20 μm.
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Figure 7: Analysis of wild-type ES cells and the talin and vinculin mutants by time lapse video interference microscopy. (a) Wild-type ES cells (b) the talin (−/−) A28 ES cell mutant and (c) the vinculin (−/−) D7 ES cell mutant taken from DRIMAPS recordings processed to reveal the regions of protrusion (green) and retraction (red) of the cell margin during a 2-min interval. The grey levels inside the cells represent the dry mass distribution on an arbitrary scale. (d). Median values of cell polarity measured as the distance in μm between the centroids of the protrusion and retraction areas over a 5-min period. The rectangles and bars indicate the distributions of data values. (e). Power spectra of the variations in protrusion of the wild-type ES cells (black), talin (−/−) A28 ES cell mutant (red) and the vinculin (−/−) D7 ES cell mutant cells (green). The power (vertical axis) in arbitrary units reveals how much of the variance in the data is attributable to each frequency. Bars: (A–C) 20 μm.

Mentions: Wild-type ES cells and the talin (−/−) A28 and vinculin (−/−) D7 ES cell mutants were plated on fibronectin-coated coverslips and their behavior recorded over a period of 20 h using interference microscopy. The rate of increase in dry mass of individual cells served as a check on the viability of the cultures. All cultures grew at a mean rate of just over 5% h−1 with no significant differences due to genotype in an ANOVA test. Cell spreading and cell shape are all determined by the patterns of protrusion and retraction of the cell margin (Dunn et al., 1997) and, therefore, we examined these patterns directly. Fig. 7, a–c shows a sample of protrusions (green) and retractions (red) for cells of the three genotypes during a 2-min interval. Compared with the wild-type ES cells (Fig. 7 a), the talin (−/−) A28 ES cell mutants (Fig. 7 b) showed large bleb-like protrusions evenly interspersed with retractions around the cell periphery, whereas the vinculin (−/−) D7 ES cell mutant (Fig. 7 c) showed greater polarity, with the central cell displaying a large lamellar protrusion opposite an extensive retracting margin. Fig. 7 d is a summary of measurements of the mean polarity which we have previously defined as the distance in μm separating the centroids of protrusion and retraction over a 5-min period (Dunn et al., 1997). In this plot, the solid discs represent the median values and the distributions of the data are indicated by the rectangles that span 50% of data values, and the “bars” that span 80% of values. In ANOVA tests, the polarity of the talin (−/−) A28 ES cell mutant was significantly suppressed compared with wild-type ES cells (P < 0.05) whereas the polarity of the vinculin (−/−) D7 ES cell mutant was significantly increased (P < 0.01).


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)

Analysis of wild-type ES cells and the talin and vinculin  mutants by time lapse video interference microscopy. (a) Wild-type ES cells (b) the talin (−/−) A28 ES cell mutant and (c) the  vinculin (−/−) D7 ES cell mutant taken from DRIMAPS recordings processed to reveal the regions of protrusion (green) and retraction (red) of the cell margin during a 2-min interval. The grey  levels inside the cells represent the dry mass distribution on an  arbitrary scale. (d). Median values of cell polarity measured as  the distance in μm between the centroids of the protrusion and  retraction areas over a 5-min period. The rectangles and bars indicate the distributions of data values. (e). Power spectra of the  variations in protrusion of the wild-type ES cells (black), talin  (−/−) A28 ES cell mutant (red) and the vinculin (−/−) D7 ES  cell mutant cells (green). The power (vertical axis) in arbitrary  units reveals how much of the variance in the data is attributable  to each frequency. Bars: (A–C) 20 μm.
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Figure 7: Analysis of wild-type ES cells and the talin and vinculin mutants by time lapse video interference microscopy. (a) Wild-type ES cells (b) the talin (−/−) A28 ES cell mutant and (c) the vinculin (−/−) D7 ES cell mutant taken from DRIMAPS recordings processed to reveal the regions of protrusion (green) and retraction (red) of the cell margin during a 2-min interval. The grey levels inside the cells represent the dry mass distribution on an arbitrary scale. (d). Median values of cell polarity measured as the distance in μm between the centroids of the protrusion and retraction areas over a 5-min period. The rectangles and bars indicate the distributions of data values. (e). Power spectra of the variations in protrusion of the wild-type ES cells (black), talin (−/−) A28 ES cell mutant (red) and the vinculin (−/−) D7 ES cell mutant cells (green). The power (vertical axis) in arbitrary units reveals how much of the variance in the data is attributable to each frequency. Bars: (A–C) 20 μm.
Mentions: Wild-type ES cells and the talin (−/−) A28 and vinculin (−/−) D7 ES cell mutants were plated on fibronectin-coated coverslips and their behavior recorded over a period of 20 h using interference microscopy. The rate of increase in dry mass of individual cells served as a check on the viability of the cultures. All cultures grew at a mean rate of just over 5% h−1 with no significant differences due to genotype in an ANOVA test. Cell spreading and cell shape are all determined by the patterns of protrusion and retraction of the cell margin (Dunn et al., 1997) and, therefore, we examined these patterns directly. Fig. 7, a–c shows a sample of protrusions (green) and retractions (red) for cells of the three genotypes during a 2-min interval. Compared with the wild-type ES cells (Fig. 7 a), the talin (−/−) A28 ES cell mutants (Fig. 7 b) showed large bleb-like protrusions evenly interspersed with retractions around the cell periphery, whereas the vinculin (−/−) D7 ES cell mutant (Fig. 7 c) showed greater polarity, with the central cell displaying a large lamellar protrusion opposite an extensive retracting margin. Fig. 7 d is a summary of measurements of the mean polarity which we have previously defined as the distance in μm separating the centroids of protrusion and retraction over a 5-min period (Dunn et al., 1997). In this plot, the solid discs represent the median values and the distributions of the data are indicated by the rectangles that span 50% of data values, and the “bars” that span 80% of values. In ANOVA tests, the polarity of the talin (−/−) A28 ES cell mutant was significantly suppressed compared with wild-type ES cells (P < 0.05) whereas the polarity of the vinculin (−/−) D7 ES cell mutant was significantly increased (P < 0.01).

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