Limits...
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.

Show MeSH

Related in: MedlinePlus

rhea17 disrupts adhesion complex reinforcement and adhesion consolidation.WT and rhea17 embryonic muscles stained for talin (red in a–d; grey in a′–d′) and integrin (green in a–d) at stage 16 (a–b) and stage 17 (c–d). (e) The recruitment of talin to adhesions (normalized to integrin levels; see materials and methods) was comparable between WT and rhea17 in stage 16 embryos. However, although talin was maintained at sites of adhesion, its recruitment was not reinforced in rhea17 embryos in stage 17 embryos (e). (f–j). WT and rhea17 embryos stained for integrin (green in f–i) and PINCH (red in f–i, grey in f′–i′) at stage 16 (f–g) and stage 17 (h–i). PINCH recruitment was not reinforced in stage 17 rhea17 embryos as determined by measuring the ratio of anti-PINCH fluorescence intensity relative to integrin intensity at MTJs. (j; see Materials and Methods). (k–o) WT and rhea17 embryos stained for integrin (green in k–n) and pFAK (red in k–n; grey in k′–n′). pFAK recruitment was not reinforced in stage 17 rhea17 embryos as determined by measuring the ratio of anti-pFAK fluorescence intensity relative to integrin intensity at MTJs (o; see Materials and Methods). (p–r) MTJ length was measured in control heterozygous (p) and rhea17 mutant (q) embryos (see materials and methods). MTJs were significantly longer in rhea17 mutants compared to control embryos (****p<0.0001). Scale bars: a–n = 50 µm; p–q = 10 µm.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4230843&req=5

pgen-1004756-g005: rhea17 disrupts adhesion complex reinforcement and adhesion consolidation.WT and rhea17 embryonic muscles stained for talin (red in a–d; grey in a′–d′) and integrin (green in a–d) at stage 16 (a–b) and stage 17 (c–d). (e) The recruitment of talin to adhesions (normalized to integrin levels; see materials and methods) was comparable between WT and rhea17 in stage 16 embryos. However, although talin was maintained at sites of adhesion, its recruitment was not reinforced in rhea17 embryos in stage 17 embryos (e). (f–j). WT and rhea17 embryos stained for integrin (green in f–i) and PINCH (red in f–i, grey in f′–i′) at stage 16 (f–g) and stage 17 (h–i). PINCH recruitment was not reinforced in stage 17 rhea17 embryos as determined by measuring the ratio of anti-PINCH fluorescence intensity relative to integrin intensity at MTJs. (j; see Materials and Methods). (k–o) WT and rhea17 embryos stained for integrin (green in k–n) and pFAK (red in k–n; grey in k′–n′). pFAK recruitment was not reinforced in stage 17 rhea17 embryos as determined by measuring the ratio of anti-pFAK fluorescence intensity relative to integrin intensity at MTJs (o; see Materials and Methods). (p–r) MTJ length was measured in control heterozygous (p) and rhea17 mutant (q) embryos (see materials and methods). MTJs were significantly longer in rhea17 mutants compared to control embryos (****p<0.0001). Scale bars: a–n = 50 µm; p–q = 10 µm.

Mentions: Both the fact that the talin head was required for adhesion complex assembly (Fig. 2), and that the G340E mutation interfered with integrin clustering (Fig. 4), led us to hypothesize that the rhea17 mutation might give rise to defective adhesion complex assembly and maintenance. Since MTJs grow and mature over several hours of embryonic development (stages 16–17), they serve as a useful system to study the maturation of integrin-mediated adhesions. In WT embryos talin is localized at MTJs as they form during stage 15 and then undergoes substantial enrichment between stages 16 and 17 as Cell-ECM adhesions are consolidated and reinforced (Fig. 5a,c,e). Recruitment of other adhesion complex components including PINCH (Fig. 5f,h,j) and pFAK (Fig. 5k,m,o) also occurred at stage 16 and was maintained through stage 17. In rhea17 embryos, although talin is well recruited to MTJs by stage 16, its recruitment is not reinforced in stage 17 (Fig. 5b,d,e). Strikingly, other adhesion complex components such as PINCH (Fig. 5g,i,j) and pFAK (Fig. 5l,n,o) were also initially recruited to MTJs at stage 16 at levels comparable to WT, but by stage 17, the levels were drastically reduced. Intriguingly, we found that MTJs are longer in rhea17 embryos compared to WT controls, further suggesting a failure in adhesion maturation and consolidation (Fig. 5p–r). Altogether, these data demonstrate that the G340E mutation in the talin head impinges on the ability to reinforce integrin-mediated adhesions, consistent with a defect in adhesion maturation, leading to the breakdown of the Cell-ECM adhesion.


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 adhesion complex reinforcement and adhesion consolidation.WT and rhea17 embryonic muscles stained for talin (red in a–d; grey in a′–d′) and integrin (green in a–d) at stage 16 (a–b) and stage 17 (c–d). (e) The recruitment of talin to adhesions (normalized to integrin levels; see materials and methods) was comparable between WT and rhea17 in stage 16 embryos. However, although talin was maintained at sites of adhesion, its recruitment was not reinforced in rhea17 embryos in stage 17 embryos (e). (f–j). WT and rhea17 embryos stained for integrin (green in f–i) and PINCH (red in f–i, grey in f′–i′) at stage 16 (f–g) and stage 17 (h–i). PINCH recruitment was not reinforced in stage 17 rhea17 embryos as determined by measuring the ratio of anti-PINCH fluorescence intensity relative to integrin intensity at MTJs. (j; see Materials and Methods). (k–o) WT and rhea17 embryos stained for integrin (green in k–n) and pFAK (red in k–n; grey in k′–n′). pFAK recruitment was not reinforced in stage 17 rhea17 embryos as determined by measuring the ratio of anti-pFAK fluorescence intensity relative to integrin intensity at MTJs (o; see Materials and Methods). (p–r) MTJ length was measured in control heterozygous (p) and rhea17 mutant (q) embryos (see materials and methods). MTJs were significantly longer in rhea17 mutants compared to control embryos (****p<0.0001). Scale bars: a–n = 50 µm; p–q = 10 µm.
© Copyright Policy
Related In: Results  -  Collection

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

pgen-1004756-g005: rhea17 disrupts adhesion complex reinforcement and adhesion consolidation.WT and rhea17 embryonic muscles stained for talin (red in a–d; grey in a′–d′) and integrin (green in a–d) at stage 16 (a–b) and stage 17 (c–d). (e) The recruitment of talin to adhesions (normalized to integrin levels; see materials and methods) was comparable between WT and rhea17 in stage 16 embryos. However, although talin was maintained at sites of adhesion, its recruitment was not reinforced in rhea17 embryos in stage 17 embryos (e). (f–j). WT and rhea17 embryos stained for integrin (green in f–i) and PINCH (red in f–i, grey in f′–i′) at stage 16 (f–g) and stage 17 (h–i). PINCH recruitment was not reinforced in stage 17 rhea17 embryos as determined by measuring the ratio of anti-PINCH fluorescence intensity relative to integrin intensity at MTJs. (j; see Materials and Methods). (k–o) WT and rhea17 embryos stained for integrin (green in k–n) and pFAK (red in k–n; grey in k′–n′). pFAK recruitment was not reinforced in stage 17 rhea17 embryos as determined by measuring the ratio of anti-pFAK fluorescence intensity relative to integrin intensity at MTJs (o; see Materials and Methods). (p–r) MTJ length was measured in control heterozygous (p) and rhea17 mutant (q) embryos (see materials and methods). MTJs were significantly longer in rhea17 mutants compared to control embryos (****p<0.0001). Scale bars: a–n = 50 µm; p–q = 10 µm.
Mentions: Both the fact that the talin head was required for adhesion complex assembly (Fig. 2), and that the G340E mutation interfered with integrin clustering (Fig. 4), led us to hypothesize that the rhea17 mutation might give rise to defective adhesion complex assembly and maintenance. Since MTJs grow and mature over several hours of embryonic development (stages 16–17), they serve as a useful system to study the maturation of integrin-mediated adhesions. In WT embryos talin is localized at MTJs as they form during stage 15 and then undergoes substantial enrichment between stages 16 and 17 as Cell-ECM adhesions are consolidated and reinforced (Fig. 5a,c,e). Recruitment of other adhesion complex components including PINCH (Fig. 5f,h,j) and pFAK (Fig. 5k,m,o) also occurred at stage 16 and was maintained through stage 17. In rhea17 embryos, although talin is well recruited to MTJs by stage 16, its recruitment is not reinforced in stage 17 (Fig. 5b,d,e). Strikingly, other adhesion complex components such as PINCH (Fig. 5g,i,j) and pFAK (Fig. 5l,n,o) were also initially recruited to MTJs at stage 16 at levels comparable to WT, but by stage 17, the levels were drastically reduced. Intriguingly, we found that MTJs are longer in rhea17 embryos compared to WT controls, further suggesting a failure in adhesion maturation and consolidation (Fig. 5p–r). Altogether, these data demonstrate that the G340E mutation in the talin head impinges on the ability to reinforce integrin-mediated adhesions, consistent with a defect in adhesion maturation, leading to the breakdown of the Cell-ECM adhesion.

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