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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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rhea17 disrupts integrin clustering.(a–a′) Clones of cells lacking talin, marked by the absence of GFP (a), failed to cluster integrins into adhesions (a′). (b,b′) Clones of cells expressing the rhea17 mutant allele of talin (marked by absence of GFP in b) also failed to cluster integrins (b′). (c–d) Expression of a full length talin point mutant that specifically disrupts IBS-1 binding (c, talinGFP*R367A, LI>AA, see Ellis et al, 2011) or that specifically disrupts integrin activation (talinGFP*L334R) restored integrin adhesions (c′, d′) within the clones of cells (arrow) lacking endogenous talin and the GFP marker (c,d). The red outline demarcates the position of the clones. Note that in d, cell outlines are also marked with GFP due to localization of the talinGFP*L334R protein to basolateral membranes. (e–j) Recruitment of integrins to MTJs was measured in stage 16 and stage 17 for both control (e,g,i) and rhea17 mutant embryos (f,h,j). In contrast to control embryos (***p<0.001), rhea17 mutant embryos did not exhibit an increase in integrin recruitment to MTJs during this developmental transition. (k–l) FRAP analysis revealed the mobile fraction of integrin-YFP was higher than respective controls in embryos treated with neomycin (k; ***p<0.001) or in rhea17 zygotic mutant embryos (l; ***p<0.001). Since these two FRAP experiments employed different genetic backgrounds and protocols in preparation for FRAP (ie. embryos in k were subjected to a drug delivery protocol), they necessitated two separate controls. In k, the control was established from vehicle-treated wild type embryos expressing integrin-YFP. In l, the controls were taken from heterozygous talin mutant embryos. Scale bars: a–d = 10 µm; g–i; h–j = 20 µm.
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pgen-1004756-g004: rhea17 disrupts integrin clustering.(a–a′) Clones of cells lacking talin, marked by the absence of GFP (a), failed to cluster integrins into adhesions (a′). (b,b′) Clones of cells expressing the rhea17 mutant allele of talin (marked by absence of GFP in b) also failed to cluster integrins (b′). (c–d) Expression of a full length talin point mutant that specifically disrupts IBS-1 binding (c, talinGFP*R367A, LI>AA, see Ellis et al, 2011) or that specifically disrupts integrin activation (talinGFP*L334R) restored integrin adhesions (c′, d′) within the clones of cells (arrow) lacking endogenous talin and the GFP marker (c,d). The red outline demarcates the position of the clones. Note that in d, cell outlines are also marked with GFP due to localization of the talinGFP*L334R protein to basolateral membranes. (e–j) Recruitment of integrins to MTJs was measured in stage 16 and stage 17 for both control (e,g,i) and rhea17 mutant embryos (f,h,j). In contrast to control embryos (***p<0.001), rhea17 mutant embryos did not exhibit an increase in integrin recruitment to MTJs during this developmental transition. (k–l) FRAP analysis revealed the mobile fraction of integrin-YFP was higher than respective controls in embryos treated with neomycin (k; ***p<0.001) or in rhea17 zygotic mutant embryos (l; ***p<0.001). Since these two FRAP experiments employed different genetic backgrounds and protocols in preparation for FRAP (ie. embryos in k were subjected to a drug delivery protocol), they necessitated two separate controls. In k, the control was established from vehicle-treated wild type embryos expressing integrin-YFP. In l, the controls were taken from heterozygous talin mutant embryos. Scale bars: a–d = 10 µm; g–i; h–j = 20 µm.

Mentions: The phenotype observed in rhea17 embryos cannot be explained by a defect in integrin activation alone since our data demonstrates that blocking activation by itself does not give rise to a severe phenotype (Fig. 1). Therefore, we hypothesized that the underlying cause of the rhea17 phenotype is due to a defect in another function associated with talin: integrin clustering [19], [21]. Integrin clustering in the fly can be assessed using a well-established in vivo assay in the context of the fly imaginal wing disc epithelium [25], [32], [36]. In the imaginal wing disc integrins mediate adhesion between the epithelial layers and form discrete puncta that colocalize with other adhesion complex components including talin, on the basal surface of the epithelium [32]. In the absence of talin these clusters fail to form, indicating a role for talin in integrin clustering (Fig. 4a; [32]). Interestingly, clonal patches of homozygous rhea17 mutant cells also failed to form integrin clusters (Fig. 4b). In comparison, neither the R367A mutation, nor the L334R mutation, disrupted integrin clustering (Fig. 4c–d; [25]). These results are in line with the hypothesis that the G340E mutation in rhea17 directly impinges on the ability of talin to cluster integrins.


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 disrupts integrin clustering.(a–a′) Clones of cells lacking talin, marked by the absence of GFP (a), failed to cluster integrins into adhesions (a′). (b,b′) Clones of cells expressing the rhea17 mutant allele of talin (marked by absence of GFP in b) also failed to cluster integrins (b′). (c–d) Expression of a full length talin point mutant that specifically disrupts IBS-1 binding (c, talinGFP*R367A, LI>AA, see Ellis et al, 2011) or that specifically disrupts integrin activation (talinGFP*L334R) restored integrin adhesions (c′, d′) within the clones of cells (arrow) lacking endogenous talin and the GFP marker (c,d). The red outline demarcates the position of the clones. Note that in d, cell outlines are also marked with GFP due to localization of the talinGFP*L334R protein to basolateral membranes. (e–j) Recruitment of integrins to MTJs was measured in stage 16 and stage 17 for both control (e,g,i) and rhea17 mutant embryos (f,h,j). In contrast to control embryos (***p<0.001), rhea17 mutant embryos did not exhibit an increase in integrin recruitment to MTJs during this developmental transition. (k–l) FRAP analysis revealed the mobile fraction of integrin-YFP was higher than respective controls in embryos treated with neomycin (k; ***p<0.001) or in rhea17 zygotic mutant embryos (l; ***p<0.001). Since these two FRAP experiments employed different genetic backgrounds and protocols in preparation for FRAP (ie. embryos in k were subjected to a drug delivery protocol), they necessitated two separate controls. In k, the control was established from vehicle-treated wild type embryos expressing integrin-YFP. In l, the controls were taken from heterozygous talin mutant embryos. Scale bars: a–d = 10 µm; g–i; h–j = 20 µm.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4230843&req=5

pgen-1004756-g004: rhea17 disrupts integrin clustering.(a–a′) Clones of cells lacking talin, marked by the absence of GFP (a), failed to cluster integrins into adhesions (a′). (b,b′) Clones of cells expressing the rhea17 mutant allele of talin (marked by absence of GFP in b) also failed to cluster integrins (b′). (c–d) Expression of a full length talin point mutant that specifically disrupts IBS-1 binding (c, talinGFP*R367A, LI>AA, see Ellis et al, 2011) or that specifically disrupts integrin activation (talinGFP*L334R) restored integrin adhesions (c′, d′) within the clones of cells (arrow) lacking endogenous talin and the GFP marker (c,d). The red outline demarcates the position of the clones. Note that in d, cell outlines are also marked with GFP due to localization of the talinGFP*L334R protein to basolateral membranes. (e–j) Recruitment of integrins to MTJs was measured in stage 16 and stage 17 for both control (e,g,i) and rhea17 mutant embryos (f,h,j). In contrast to control embryos (***p<0.001), rhea17 mutant embryos did not exhibit an increase in integrin recruitment to MTJs during this developmental transition. (k–l) FRAP analysis revealed the mobile fraction of integrin-YFP was higher than respective controls in embryos treated with neomycin (k; ***p<0.001) or in rhea17 zygotic mutant embryos (l; ***p<0.001). Since these two FRAP experiments employed different genetic backgrounds and protocols in preparation for FRAP (ie. embryos in k were subjected to a drug delivery protocol), they necessitated two separate controls. In k, the control was established from vehicle-treated wild type embryos expressing integrin-YFP. In l, the controls were taken from heterozygous talin mutant embryos. Scale bars: a–d = 10 µm; g–i; h–j = 20 µm.
Mentions: The phenotype observed in rhea17 embryos cannot be explained by a defect in integrin activation alone since our data demonstrates that blocking activation by itself does not give rise to a severe phenotype (Fig. 1). Therefore, we hypothesized that the underlying cause of the rhea17 phenotype is due to a defect in another function associated with talin: integrin clustering [19], [21]. Integrin clustering in the fly can be assessed using a well-established in vivo assay in the context of the fly imaginal wing disc epithelium [25], [32], [36]. In the imaginal wing disc integrins mediate adhesion between the epithelial layers and form discrete puncta that colocalize with other adhesion complex components including talin, on the basal surface of the epithelium [32]. In the absence of talin these clusters fail to form, indicating a role for talin in integrin clustering (Fig. 4a; [32]). Interestingly, clonal patches of homozygous rhea17 mutant cells also failed to form integrin clusters (Fig. 4b). In comparison, neither the R367A mutation, nor the L334R mutation, disrupted integrin clustering (Fig. 4c–d; [25]). These results are in line with the hypothesis that the G340E mutation in rhea17 directly impinges on the ability of talin to cluster integrins.

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