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The talin head domain reinforces integrin-mediated adhesion by promoting adhesion complex stability and clustering.

Ellis SJ, Lostchuck E, Goult BT, Bouaouina M, Fairchild MJ, López-Ceballos P, Calderwood DA, Tanentzapf G - PLoS Genet. (2014)

Bottom Line: Intriguingly, subsequent studies showed that canonical inside-out activation of integrin might not take place in flies.Consistent with this, a mutation in talin that specifically blocks its ability to activate mammalian integrins does not significantly impinge on talin function during fly development.Importantly, we provide evidence that this mutation blocks integrin clustering in vivo.

View Article: PubMed Central - PubMed

Affiliation: Department of Cellular and Physiological Sciences, University of British Columbia, Vancouver, Canada.

ABSTRACT
Talin serves an essential function during integrin-mediated adhesion in linking integrins to actin via the intracellular adhesion complex. In addition, the N-terminal head domain of talin regulates the affinity of integrins for their ECM-ligands, a process known as inside-out activation. We previously showed that in Drosophila, mutating the integrin binding site in the talin head domain resulted in weakened adhesion to the ECM. Intriguingly, subsequent studies showed that canonical inside-out activation of integrin might not take place in flies. Consistent with this, a mutation in talin that specifically blocks its ability to activate mammalian integrins does not significantly impinge on talin function during fly development. Here, we describe results suggesting that the talin head domain reinforces and stabilizes the integrin adhesion complex by promoting integrin clustering distinct from its ability to support inside-out activation. Specifically, we show that an allele of talin containing a mutation that disrupts intramolecular interactions within the talin head attenuates the assembly and reinforcement of the integrin adhesion complex. Importantly, we provide evidence that this mutation blocks integrin clustering in vivo. We propose that the talin head domain is essential for regulating integrin avidity in Drosophila and that this is crucial for integrin-mediated adhesion during animal development.

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Related in: MedlinePlus

rhea17 encodes a hypomorphic talin protein which disrupts talin head function.(a–b) The rhea17 allele is characterized by a missense mutation in a conserved glycine residue in the F3 lobe of the talin head FERM domain, G340E. (c–g) Whole mount stage 17 embryos stained for F-actin (green) and integrin (magenta) reveal that rhea17 mutant embryos (g) harbour severe morphogenetic phenotypes in GBR (c) and DC (d), as well as muscle detachment defects (e) compared to WT heterozygous embryos (f). Phenotypic analysis of rhea17 over the rhea79 deficiency increased the penetrance of all phenotypes. (h–j) αPS2-integrin recruitment was measured in WT (h,i) and rhea17 (h,j) stage 16 embryonic muscles stained for integrin. Integrin was recruited at WT levels in rhea17 embryos. (k–m) Talin recruitment was measured in WT (k,i) and rhea17 (k,m) stage 16 embryonic muscles stained for talin. Talin was well recruited in rhea17 embryos. (n) Activation of human integrins by fly talin head constructs was measured in CHO cells. The G340E mutation was sufficient to abrogate integrin activation compared to WT. (o) Quantitative Western blot analyses of relative levels of talin normalized to beta-actin levels in flies heterozygous for either the rhea79 talin  mutation (left lane) or the rhea17 mutant allele (right lane). Scale bars: f–g = 100 µm; i–j; l-m = 20 µm.
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pgen-1004756-g003: rhea17 encodes a hypomorphic talin protein which disrupts talin head function.(a–b) The rhea17 allele is characterized by a missense mutation in a conserved glycine residue in the F3 lobe of the talin head FERM domain, G340E. (c–g) Whole mount stage 17 embryos stained for F-actin (green) and integrin (magenta) reveal that rhea17 mutant embryos (g) harbour severe morphogenetic phenotypes in GBR (c) and DC (d), as well as muscle detachment defects (e) compared to WT heterozygous embryos (f). Phenotypic analysis of rhea17 over the rhea79 deficiency increased the penetrance of all phenotypes. (h–j) αPS2-integrin recruitment was measured in WT (h,i) and rhea17 (h,j) stage 16 embryonic muscles stained for integrin. Integrin was recruited at WT levels in rhea17 embryos. (k–m) Talin recruitment was measured in WT (k,i) and rhea17 (k,m) stage 16 embryonic muscles stained for talin. Talin was well recruited in rhea17 embryos. (n) Activation of human integrins by fly talin head constructs was measured in CHO cells. The G340E mutation was sufficient to abrogate integrin activation compared to WT. (o) Quantitative Western blot analyses of relative levels of talin normalized to beta-actin levels in flies heterozygous for either the rhea79 talin mutation (left lane) or the rhea17 mutant allele (right lane). Scale bars: f–g = 100 µm; i–j; l-m = 20 µm.

Mentions: Thus far, we have shown that talin-head mediated integrin activation plays only a minor role in talin function during fly develeopment and that despite this, the head domain has other essential functions in mediating integrin-based Cell-ECM adhesion. To uncover the mechanism underlying additional roles for talin we turned to a previously isolated allele of talin, rhea17. This allele encodes a talin protein containing a missense mutation in the talin head and importantly, produces a phenotype that is similar to that observed when the talin head is deleted in full (Fig. 2b, Fig. 3f). The rhea17 allele was originally uncovered in a screen for dominant enhancers of a hypomorphic integrin allele [32]. We sequenced the rhea17 allele (see materials and methods and Supplemental Fig. S3) and found that it contains a mutation that replaces a conserved glycine, G340 (G331 in mammalian talin1, G334 in mammalian talin2), to a glutamate (G340E; Fig. 3a–b). Phenotypic analysis of embryos homozygous for this mutation (Fig. 3f–g) showed that integrin-dependent morphogenetic processes GBR and DC as well as stable muscle attachment to the ECM were severely disrupted compared to heterozygous controls (Fig. 3f–g, c–e). Interestingly, embryos that had a GBR phenotype were more likely to have a DC phenotype. For example, while 38% of the total population of rhea17 mutant embryos analyzed displayed DC defects, amongst those that also have GBR defects, the proportion of embryos with DC defects increased to about 63%. This connection seems likely, because the morphology of the amnioserosa, an extra-embryonic tissue required for both GBR and DC [33], [34], is defective as a result of GBR failure. To ensure that the phenotypes we observed in embryos homozygous for the rhea17 mutation were not due to the accumulation of background mutations, we analyzed the phenotype of embryos trans-heterozygous for the rhea17 allele and a talin allele (rhea17/Df). This revealed an even stronger phenotype, suggesting that the phenotype observed in the rhea17 homozygous mutants is not due to background mutations. Furthermore, this implied that the rhea17 allele is a hypomorphic mutation that retains some functionality in comparison to complete loss of talin (Fig. 3c–e). Of note, the GBR phenotype of rhea17/Df embryos was stronger than that of rhea79 talin mutant embryos (Fig. 3c). It is thus possible that the rhea17 might be acting in a dominant negative fashion in this process. This is consistent with what we have previously observed for some mutations in integrin that give rise to stronger GBR phenotypes than the loss of function mutants [35]. Another possibility is that the rhea79 allele may have accumulated background mutations that slightly suppress the talin phenotype.


The talin head domain reinforces integrin-mediated adhesion by promoting adhesion complex stability and clustering.

Ellis SJ, Lostchuck E, Goult BT, Bouaouina M, Fairchild MJ, López-Ceballos P, Calderwood DA, Tanentzapf G - PLoS Genet. (2014)

rhea17 encodes a hypomorphic talin protein which disrupts talin head function.(a–b) The rhea17 allele is characterized by a missense mutation in a conserved glycine residue in the F3 lobe of the talin head FERM domain, G340E. (c–g) Whole mount stage 17 embryos stained for F-actin (green) and integrin (magenta) reveal that rhea17 mutant embryos (g) harbour severe morphogenetic phenotypes in GBR (c) and DC (d), as well as muscle detachment defects (e) compared to WT heterozygous embryos (f). Phenotypic analysis of rhea17 over the rhea79 deficiency increased the penetrance of all phenotypes. (h–j) αPS2-integrin recruitment was measured in WT (h,i) and rhea17 (h,j) stage 16 embryonic muscles stained for integrin. Integrin was recruited at WT levels in rhea17 embryos. (k–m) Talin recruitment was measured in WT (k,i) and rhea17 (k,m) stage 16 embryonic muscles stained for talin. Talin was well recruited in rhea17 embryos. (n) Activation of human integrins by fly talin head constructs was measured in CHO cells. The G340E mutation was sufficient to abrogate integrin activation compared to WT. (o) Quantitative Western blot analyses of relative levels of talin normalized to beta-actin levels in flies heterozygous for either the rhea79 talin  mutation (left lane) or the rhea17 mutant allele (right lane). Scale bars: f–g = 100 µm; i–j; l-m = 20 µm.
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Related In: Results  -  Collection

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pgen-1004756-g003: rhea17 encodes a hypomorphic talin protein which disrupts talin head function.(a–b) The rhea17 allele is characterized by a missense mutation in a conserved glycine residue in the F3 lobe of the talin head FERM domain, G340E. (c–g) Whole mount stage 17 embryos stained for F-actin (green) and integrin (magenta) reveal that rhea17 mutant embryos (g) harbour severe morphogenetic phenotypes in GBR (c) and DC (d), as well as muscle detachment defects (e) compared to WT heterozygous embryos (f). Phenotypic analysis of rhea17 over the rhea79 deficiency increased the penetrance of all phenotypes. (h–j) αPS2-integrin recruitment was measured in WT (h,i) and rhea17 (h,j) stage 16 embryonic muscles stained for integrin. Integrin was recruited at WT levels in rhea17 embryos. (k–m) Talin recruitment was measured in WT (k,i) and rhea17 (k,m) stage 16 embryonic muscles stained for talin. Talin was well recruited in rhea17 embryos. (n) Activation of human integrins by fly talin head constructs was measured in CHO cells. The G340E mutation was sufficient to abrogate integrin activation compared to WT. (o) Quantitative Western blot analyses of relative levels of talin normalized to beta-actin levels in flies heterozygous for either the rhea79 talin mutation (left lane) or the rhea17 mutant allele (right lane). Scale bars: f–g = 100 µm; i–j; l-m = 20 µm.
Mentions: Thus far, we have shown that talin-head mediated integrin activation plays only a minor role in talin function during fly develeopment and that despite this, the head domain has other essential functions in mediating integrin-based Cell-ECM adhesion. To uncover the mechanism underlying additional roles for talin we turned to a previously isolated allele of talin, rhea17. This allele encodes a talin protein containing a missense mutation in the talin head and importantly, produces a phenotype that is similar to that observed when the talin head is deleted in full (Fig. 2b, Fig. 3f). The rhea17 allele was originally uncovered in a screen for dominant enhancers of a hypomorphic integrin allele [32]. We sequenced the rhea17 allele (see materials and methods and Supplemental Fig. S3) and found that it contains a mutation that replaces a conserved glycine, G340 (G331 in mammalian talin1, G334 in mammalian talin2), to a glutamate (G340E; Fig. 3a–b). Phenotypic analysis of embryos homozygous for this mutation (Fig. 3f–g) showed that integrin-dependent morphogenetic processes GBR and DC as well as stable muscle attachment to the ECM were severely disrupted compared to heterozygous controls (Fig. 3f–g, c–e). Interestingly, embryos that had a GBR phenotype were more likely to have a DC phenotype. For example, while 38% of the total population of rhea17 mutant embryos analyzed displayed DC defects, amongst those that also have GBR defects, the proportion of embryos with DC defects increased to about 63%. This connection seems likely, because the morphology of the amnioserosa, an extra-embryonic tissue required for both GBR and DC [33], [34], is defective as a result of GBR failure. To ensure that the phenotypes we observed in embryos homozygous for the rhea17 mutation were not due to the accumulation of background mutations, we analyzed the phenotype of embryos trans-heterozygous for the rhea17 allele and a talin allele (rhea17/Df). This revealed an even stronger phenotype, suggesting that the phenotype observed in the rhea17 homozygous mutants is not due to background mutations. Furthermore, this implied that the rhea17 allele is a hypomorphic mutation that retains some functionality in comparison to complete loss of talin (Fig. 3c–e). Of note, the GBR phenotype of rhea17/Df embryos was stronger than that of rhea79 talin mutant embryos (Fig. 3c). It is thus possible that the rhea17 might be acting in a dominant negative fashion in this process. This is consistent with what we have previously observed for some mutations in integrin that give rise to stronger GBR phenotypes than the loss of function mutants [35]. Another possibility is that the rhea79 allele may have accumulated background mutations that slightly suppress the talin phenotype.

Bottom Line: Intriguingly, subsequent studies showed that canonical inside-out activation of integrin might not take place in flies.Consistent with this, a mutation in talin that specifically blocks its ability to activate mammalian integrins does not significantly impinge on talin function during fly development.Importantly, we provide evidence that this mutation blocks integrin clustering in vivo.

View Article: PubMed Central - PubMed

Affiliation: Department of Cellular and Physiological Sciences, University of British Columbia, Vancouver, Canada.

ABSTRACT
Talin serves an essential function during integrin-mediated adhesion in linking integrins to actin via the intracellular adhesion complex. In addition, the N-terminal head domain of talin regulates the affinity of integrins for their ECM-ligands, a process known as inside-out activation. We previously showed that in Drosophila, mutating the integrin binding site in the talin head domain resulted in weakened adhesion to the ECM. Intriguingly, subsequent studies showed that canonical inside-out activation of integrin might not take place in flies. Consistent with this, a mutation in talin that specifically blocks its ability to activate mammalian integrins does not significantly impinge on talin function during fly development. Here, we describe results suggesting that the talin head domain reinforces and stabilizes the integrin adhesion complex by promoting integrin clustering distinct from its ability to support inside-out activation. Specifically, we show that an allele of talin containing a mutation that disrupts intramolecular interactions within the talin head attenuates the assembly and reinforcement of the integrin adhesion complex. Importantly, we provide evidence that this mutation blocks integrin clustering in vivo. We propose that the talin head domain is essential for regulating integrin avidity in Drosophila and that this is crucial for integrin-mediated adhesion during animal development.

Show MeSH
Related in: MedlinePlus