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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

The talin head is essential for integrin function in Drosophila.(a–c) Maternal-zygotic talin  embryos (shown in a) rescued with either full-length WT talinGFP transgene (b) or headless talinGFP transgene (c) and stained for F-actin (green) and integrin (magenta). (d–f) Integrin-dependent phenotypes germband retraction (d), dorsal closure (e) and muscle attachment (f) were assayed in talin- embryos, WT-talin-rescued embryos, and headless-talin-rescued embryos. The talin head was required for all three processess assayed. Scale bar = 100 µm. (g–h) Recruitment of ubi-promoter driven, GFP-tagged full-length WT talin and headless talin to sites of adhesion was assayed in wild-type embryos (g) and in a talin  background (h). In a WT background, headless-talinGFP competed less well with endogenous talin and was only weakly recruited to sites of adhesion compared to WT (***p<0.001); in the absence of endogenous talin, headless-talin was well recruited to sites of adhesion. (i) FRAP experiments on talinGFP-WT and headless-talinGFP reveal that headless talin is much less stable at sites of adhesion than talinGFP-WT. (j–m) Confocal z-stacks of stage 17 maternal/zygotic-mutant embryos rescued with either full length WT talin (j,l) or headless-talin (k,m). (j–k) adhesions stained for talin (green in j–k; grey in j′–k′) and integrin (red in j–k). In the absence of the talin head, talin was still well recruited. (l–m) Muscles stained for talin (magenta in l–m) and paxillin (green in l–m; grey in l′–m′). Paxillin was not well recruited to adhesions. Scale bars: a–e = 100 µm; j–m = 20 µm.
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pgen-1004756-g002: The talin head is essential for integrin function in Drosophila.(a–c) Maternal-zygotic talin embryos (shown in a) rescued with either full-length WT talinGFP transgene (b) or headless talinGFP transgene (c) and stained for F-actin (green) and integrin (magenta). (d–f) Integrin-dependent phenotypes germband retraction (d), dorsal closure (e) and muscle attachment (f) were assayed in talin- embryos, WT-talin-rescued embryos, and headless-talin-rescued embryos. The talin head was required for all three processess assayed. Scale bar = 100 µm. (g–h) Recruitment of ubi-promoter driven, GFP-tagged full-length WT talin and headless talin to sites of adhesion was assayed in wild-type embryos (g) and in a talin background (h). In a WT background, headless-talinGFP competed less well with endogenous talin and was only weakly recruited to sites of adhesion compared to WT (***p<0.001); in the absence of endogenous talin, headless-talin was well recruited to sites of adhesion. (i) FRAP experiments on talinGFP-WT and headless-talinGFP reveal that headless talin is much less stable at sites of adhesion than talinGFP-WT. (j–m) Confocal z-stacks of stage 17 maternal/zygotic-mutant embryos rescued with either full length WT talin (j,l) or headless-talin (k,m). (j–k) adhesions stained for talin (green in j–k; grey in j′–k′) and integrin (red in j–k). In the absence of the talin head, talin was still well recruited. (l–m) Muscles stained for talin (magenta in l–m) and paxillin (green in l–m; grey in l′–m′). Paxillin was not well recruited to adhesions. Scale bars: a–e = 100 µm; j–m = 20 µm.

Mentions: We sought to identify roles of the talin head beyond integrin binding and activation. To this end, using the same approach described above, we replaced endogenous talin with a ubiquitously expressed construct that deletes the talin head (residues 1–448) but leaves the rest of talin intact: headless-talinGFP. Importantly,head deletion resulted in severe phenotypes resembling complete loss of talin (Fig. 2a–c). GBR and DC were severely disrupted (Fig. 2d–e), as was stable muscle attachment to the ECM (Fig. 2f). While the headless-talinGFP localized poorly in the presence of endogenous talin it exhibited robust localization in talin embryos (Fig. 2g, h) even though its overall expression appeared somewhat lower compared to wild type (Supplemental Fig. S2). Therefore headless-talinGFP was able to retain functional interactions that supported recruitment to sites of adhesion. Nonetheless, FRAP analysis showed that headless-talinGFP was substantially less stable at sites of adhesion (Fig. 2i). In addition, the adhesion complex that is normally recruited to sites of adhesion by talin ([25], [32]; Fig. 2j,l), was absent or severely reduced in headless-talinGFP rescue embryos (Fig. 2k,m). These results show that deletion of the head results in severe defects in recruitment and stabilization of the adhesion complex; consequently, loss of talin head function blocks talin-dependent morphogenetic events.


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)

The talin head is essential for integrin function in Drosophila.(a–c) Maternal-zygotic talin  embryos (shown in a) rescued with either full-length WT talinGFP transgene (b) or headless talinGFP transgene (c) and stained for F-actin (green) and integrin (magenta). (d–f) Integrin-dependent phenotypes germband retraction (d), dorsal closure (e) and muscle attachment (f) were assayed in talin- embryos, WT-talin-rescued embryos, and headless-talin-rescued embryos. The talin head was required for all three processess assayed. Scale bar = 100 µm. (g–h) Recruitment of ubi-promoter driven, GFP-tagged full-length WT talin and headless talin to sites of adhesion was assayed in wild-type embryos (g) and in a talin  background (h). In a WT background, headless-talinGFP competed less well with endogenous talin and was only weakly recruited to sites of adhesion compared to WT (***p<0.001); in the absence of endogenous talin, headless-talin was well recruited to sites of adhesion. (i) FRAP experiments on talinGFP-WT and headless-talinGFP reveal that headless talin is much less stable at sites of adhesion than talinGFP-WT. (j–m) Confocal z-stacks of stage 17 maternal/zygotic-mutant embryos rescued with either full length WT talin (j,l) or headless-talin (k,m). (j–k) adhesions stained for talin (green in j–k; grey in j′–k′) and integrin (red in j–k). In the absence of the talin head, talin was still well recruited. (l–m) Muscles stained for talin (magenta in l–m) and paxillin (green in l–m; grey in l′–m′). Paxillin was not well recruited to adhesions. Scale bars: a–e = 100 µm; j–m = 20 µm.
© Copyright Policy
Related In: Results  -  Collection

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pgen-1004756-g002: The talin head is essential for integrin function in Drosophila.(a–c) Maternal-zygotic talin embryos (shown in a) rescued with either full-length WT talinGFP transgene (b) or headless talinGFP transgene (c) and stained for F-actin (green) and integrin (magenta). (d–f) Integrin-dependent phenotypes germband retraction (d), dorsal closure (e) and muscle attachment (f) were assayed in talin- embryos, WT-talin-rescued embryos, and headless-talin-rescued embryos. The talin head was required for all three processess assayed. Scale bar = 100 µm. (g–h) Recruitment of ubi-promoter driven, GFP-tagged full-length WT talin and headless talin to sites of adhesion was assayed in wild-type embryos (g) and in a talin background (h). In a WT background, headless-talinGFP competed less well with endogenous talin and was only weakly recruited to sites of adhesion compared to WT (***p<0.001); in the absence of endogenous talin, headless-talin was well recruited to sites of adhesion. (i) FRAP experiments on talinGFP-WT and headless-talinGFP reveal that headless talin is much less stable at sites of adhesion than talinGFP-WT. (j–m) Confocal z-stacks of stage 17 maternal/zygotic-mutant embryos rescued with either full length WT talin (j,l) or headless-talin (k,m). (j–k) adhesions stained for talin (green in j–k; grey in j′–k′) and integrin (red in j–k). In the absence of the talin head, talin was still well recruited. (l–m) Muscles stained for talin (magenta in l–m) and paxillin (green in l–m; grey in l′–m′). Paxillin was not well recruited to adhesions. Scale bars: a–e = 100 µm; j–m = 20 µm.
Mentions: We sought to identify roles of the talin head beyond integrin binding and activation. To this end, using the same approach described above, we replaced endogenous talin with a ubiquitously expressed construct that deletes the talin head (residues 1–448) but leaves the rest of talin intact: headless-talinGFP. Importantly,head deletion resulted in severe phenotypes resembling complete loss of talin (Fig. 2a–c). GBR and DC were severely disrupted (Fig. 2d–e), as was stable muscle attachment to the ECM (Fig. 2f). While the headless-talinGFP localized poorly in the presence of endogenous talin it exhibited robust localization in talin embryos (Fig. 2g, h) even though its overall expression appeared somewhat lower compared to wild type (Supplemental Fig. S2). Therefore headless-talinGFP was able to retain functional interactions that supported recruitment to sites of adhesion. Nonetheless, FRAP analysis showed that headless-talinGFP was substantially less stable at sites of adhesion (Fig. 2i). In addition, the adhesion complex that is normally recruited to sites of adhesion by talin ([25], [32]; Fig. 2j,l), was absent or severely reduced in headless-talinGFP rescue embryos (Fig. 2k,m). These results show that deletion of the head results in severe defects in recruitment and stabilization of the adhesion complex; consequently, loss of talin head function blocks talin-dependent morphogenetic events.

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