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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

Integrin-binding to the talin head, but not integrin activation, is required for muscle attachment.(a) Schematic of key domains in talin for this study. The talin head is contains an N-terminal atypical FERM domain [10] and a C-terminal rod domain comprised of 13 helical bundles [59]. (b) Alignment of residues 325–375 of fly talin F3 domain with human talin isoforms. Dark blue indicates identical residues between homologues, lighter blue indicates similar residues. The mutations utilized to study integrin activation are indicated with an arrowhead. (c–e) Integrin-dependent phenotypes germband retraction (c), dorsal closure (d) and muscle attachment (e) were assayed in talin- embryos, WT talinGFP-rescued embryos, and talinGFP*L334R-rescued embryos. Apart from mild muscle detachment in about 20% of embryos, the talinGFP*L334R transgene was able to rescue all phenotypes such that the embryos hatched to the larval stages. (f–g) Maternal zygotic talin  embryos rescued with either full-length WT talinGFP transgene (f) or talinGFP*L334R mutant transgene (g) and stained for F-actin (green) and βPS-integrin (magenta). (h–j) MTJs of talin  embryos rescued with either talinGFP-WT (h), talinGFP*R367A (i), talinGFP*L334R (j). Embryos were stained for anti- αPS2-integrin (green in h–j; grey in h′–j′) and tiggrin, a Drosophila ECM molecule (red in h–j). (h″–j″) Average intensity profiles for integrin and tiggrin across the widths of the boxed areas in h–j. Tiggrin and integrin completely overlapped at MTJs in WT talin rescue embryos (h″), but were separated from one another in talin- embryos rescued with talinGFP*R367A (i–i″). Overlap between tiggrin and integrin was maintained in talin- embryos rescued with talinGFP*L334R (j–j″). The pink arrowheads mark the sites of separated integrin and ECM signal. (k) Activation of human integrins by fly talin head constructs was measured in CHO cells. The L334R mutation was sufficient to abrogate integrin activation. (l–m) Recruitment of ubi-promoter driven full-length WT talinGFP and talinGFP*L334R to sites of adhesion was assayed in talin  (l) and in wild-type embryos (m). Compared to WT TalinGFP, TalinGFP*L334R was well recruited in a background devoid of any endogenous talin (l–l″; **p<0.01), but competed less well in the presence of endogenous talin and was only weakly recruited to sites of adhesion compared to WT, which was robustly recruited (m–m″; ***p<0.001). (n) FRAP experiments on WT talinGFP and talinGFP*L334R reveal that talinGFP*L334R is much less stable at sites of adhesion than WT talinGFP. Scale bars: f–g  = 100 µm; h–j;l–m = 20 µm.
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pgen-1004756-g001: Integrin-binding to the talin head, but not integrin activation, is required for muscle attachment.(a) Schematic of key domains in talin for this study. The talin head is contains an N-terminal atypical FERM domain [10] and a C-terminal rod domain comprised of 13 helical bundles [59]. (b) Alignment of residues 325–375 of fly talin F3 domain with human talin isoforms. Dark blue indicates identical residues between homologues, lighter blue indicates similar residues. The mutations utilized to study integrin activation are indicated with an arrowhead. (c–e) Integrin-dependent phenotypes germband retraction (c), dorsal closure (d) and muscle attachment (e) were assayed in talin- embryos, WT talinGFP-rescued embryos, and talinGFP*L334R-rescued embryos. Apart from mild muscle detachment in about 20% of embryos, the talinGFP*L334R transgene was able to rescue all phenotypes such that the embryos hatched to the larval stages. (f–g) Maternal zygotic talin embryos rescued with either full-length WT talinGFP transgene (f) or talinGFP*L334R mutant transgene (g) and stained for F-actin (green) and βPS-integrin (magenta). (h–j) MTJs of talin embryos rescued with either talinGFP-WT (h), talinGFP*R367A (i), talinGFP*L334R (j). Embryos were stained for anti- αPS2-integrin (green in h–j; grey in h′–j′) and tiggrin, a Drosophila ECM molecule (red in h–j). (h″–j″) Average intensity profiles for integrin and tiggrin across the widths of the boxed areas in h–j. Tiggrin and integrin completely overlapped at MTJs in WT talin rescue embryos (h″), but were separated from one another in talin- embryos rescued with talinGFP*R367A (i–i″). Overlap between tiggrin and integrin was maintained in talin- embryos rescued with talinGFP*L334R (j–j″). The pink arrowheads mark the sites of separated integrin and ECM signal. (k) Activation of human integrins by fly talin head constructs was measured in CHO cells. The L334R mutation was sufficient to abrogate integrin activation. (l–m) Recruitment of ubi-promoter driven full-length WT talinGFP and talinGFP*L334R to sites of adhesion was assayed in talin (l) and in wild-type embryos (m). Compared to WT TalinGFP, TalinGFP*L334R was well recruited in a background devoid of any endogenous talin (l–l″; **p<0.01), but competed less well in the presence of endogenous talin and was only weakly recruited to sites of adhesion compared to WT, which was robustly recruited (m–m″; ***p<0.001). (n) FRAP experiments on WT talinGFP and talinGFP*L334R reveal that talinGFP*L334R is much less stable at sites of adhesion than WT talinGFP. Scale bars: f–g  = 100 µm; h–j;l–m = 20 µm.

Mentions: We sought to introduce a mutation into the talin head that disrupted its ability to activate integrins but not other aspects of its function. We relied on the extensive knowledge of talin structure generated by previous NMR and crystallographic analysis of the talin-head interaction with integrin in order to do this. Previous studies identified a mutation (Fig. 1a–b; L325R in talin1, L331R in talin2) in mammalian talin that specifically abrogates the integrin-activating function of talin, but does not substantially affect the ability of the talin head to bind to integrin [28]. When this mutation is introduced into the talin head, it blocks the conformational change in integrin that drives activation. The residue identified specifically attenuates the interaction between the talin head and integrin at the membrane proximal region of the β-integrin cytoplasmic tail, while maintaining the interaction between the talin head and the distal regions of the β-integrin cytoplasmic tail [28]. We introduced this mutation into fly talin (L334R) to study its effects.


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)

Integrin-binding to the talin head, but not integrin activation, is required for muscle attachment.(a) Schematic of key domains in talin for this study. The talin head is contains an N-terminal atypical FERM domain [10] and a C-terminal rod domain comprised of 13 helical bundles [59]. (b) Alignment of residues 325–375 of fly talin F3 domain with human talin isoforms. Dark blue indicates identical residues between homologues, lighter blue indicates similar residues. The mutations utilized to study integrin activation are indicated with an arrowhead. (c–e) Integrin-dependent phenotypes germband retraction (c), dorsal closure (d) and muscle attachment (e) were assayed in talin- embryos, WT talinGFP-rescued embryos, and talinGFP*L334R-rescued embryos. Apart from mild muscle detachment in about 20% of embryos, the talinGFP*L334R transgene was able to rescue all phenotypes such that the embryos hatched to the larval stages. (f–g) Maternal zygotic talin  embryos rescued with either full-length WT talinGFP transgene (f) or talinGFP*L334R mutant transgene (g) and stained for F-actin (green) and βPS-integrin (magenta). (h–j) MTJs of talin  embryos rescued with either talinGFP-WT (h), talinGFP*R367A (i), talinGFP*L334R (j). Embryos were stained for anti- αPS2-integrin (green in h–j; grey in h′–j′) and tiggrin, a Drosophila ECM molecule (red in h–j). (h″–j″) Average intensity profiles for integrin and tiggrin across the widths of the boxed areas in h–j. Tiggrin and integrin completely overlapped at MTJs in WT talin rescue embryos (h″), but were separated from one another in talin- embryos rescued with talinGFP*R367A (i–i″). Overlap between tiggrin and integrin was maintained in talin- embryos rescued with talinGFP*L334R (j–j″). The pink arrowheads mark the sites of separated integrin and ECM signal. (k) Activation of human integrins by fly talin head constructs was measured in CHO cells. The L334R mutation was sufficient to abrogate integrin activation. (l–m) Recruitment of ubi-promoter driven full-length WT talinGFP and talinGFP*L334R to sites of adhesion was assayed in talin  (l) and in wild-type embryos (m). Compared to WT TalinGFP, TalinGFP*L334R was well recruited in a background devoid of any endogenous talin (l–l″; **p<0.01), but competed less well in the presence of endogenous talin and was only weakly recruited to sites of adhesion compared to WT, which was robustly recruited (m–m″; ***p<0.001). (n) FRAP experiments on WT talinGFP and talinGFP*L334R reveal that talinGFP*L334R is much less stable at sites of adhesion than WT talinGFP. Scale bars: f–g  = 100 µm; h–j;l–m = 20 µm.
© Copyright Policy
Related In: Results  -  Collection

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

pgen-1004756-g001: Integrin-binding to the talin head, but not integrin activation, is required for muscle attachment.(a) Schematic of key domains in talin for this study. The talin head is contains an N-terminal atypical FERM domain [10] and a C-terminal rod domain comprised of 13 helical bundles [59]. (b) Alignment of residues 325–375 of fly talin F3 domain with human talin isoforms. Dark blue indicates identical residues between homologues, lighter blue indicates similar residues. The mutations utilized to study integrin activation are indicated with an arrowhead. (c–e) Integrin-dependent phenotypes germband retraction (c), dorsal closure (d) and muscle attachment (e) were assayed in talin- embryos, WT talinGFP-rescued embryos, and talinGFP*L334R-rescued embryos. Apart from mild muscle detachment in about 20% of embryos, the talinGFP*L334R transgene was able to rescue all phenotypes such that the embryos hatched to the larval stages. (f–g) Maternal zygotic talin embryos rescued with either full-length WT talinGFP transgene (f) or talinGFP*L334R mutant transgene (g) and stained for F-actin (green) and βPS-integrin (magenta). (h–j) MTJs of talin embryos rescued with either talinGFP-WT (h), talinGFP*R367A (i), talinGFP*L334R (j). Embryos were stained for anti- αPS2-integrin (green in h–j; grey in h′–j′) and tiggrin, a Drosophila ECM molecule (red in h–j). (h″–j″) Average intensity profiles for integrin and tiggrin across the widths of the boxed areas in h–j. Tiggrin and integrin completely overlapped at MTJs in WT talin rescue embryos (h″), but were separated from one another in talin- embryos rescued with talinGFP*R367A (i–i″). Overlap between tiggrin and integrin was maintained in talin- embryos rescued with talinGFP*L334R (j–j″). The pink arrowheads mark the sites of separated integrin and ECM signal. (k) Activation of human integrins by fly talin head constructs was measured in CHO cells. The L334R mutation was sufficient to abrogate integrin activation. (l–m) Recruitment of ubi-promoter driven full-length WT talinGFP and talinGFP*L334R to sites of adhesion was assayed in talin (l) and in wild-type embryos (m). Compared to WT TalinGFP, TalinGFP*L334R was well recruited in a background devoid of any endogenous talin (l–l″; **p<0.01), but competed less well in the presence of endogenous talin and was only weakly recruited to sites of adhesion compared to WT, which was robustly recruited (m–m″; ***p<0.001). (n) FRAP experiments on WT talinGFP and talinGFP*L334R reveal that talinGFP*L334R is much less stable at sites of adhesion than WT talinGFP. Scale bars: f–g  = 100 µm; h–j;l–m = 20 µm.
Mentions: We sought to introduce a mutation into the talin head that disrupted its ability to activate integrins but not other aspects of its function. We relied on the extensive knowledge of talin structure generated by previous NMR and crystallographic analysis of the talin-head interaction with integrin in order to do this. Previous studies identified a mutation (Fig. 1a–b; L325R in talin1, L331R in talin2) in mammalian talin that specifically abrogates the integrin-activating function of talin, but does not substantially affect the ability of the talin head to bind to integrin [28]. When this mutation is introduced into the talin head, it blocks the conformational change in integrin that drives activation. The residue identified specifically attenuates the interaction between the talin head and integrin at the membrane proximal region of the β-integrin cytoplasmic tail, while maintaining the interaction between the talin head and the distal regions of the β-integrin cytoplasmic tail [28]. We introduced this mutation into fly talin (L334R) to study its effects.

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