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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

G340 maintains an intermolecular interaction between F2 and F3 that couples their activity.(a) The conserved role of G340 (G331 in mammalian talin2, shown here) is to stabilize the domain orientation of F2 and F3 (a), which work together to induce integrin activation and stabilize that talin head at the plasma membrane. Modelling based on known structures (see [15]) of mouse talin2 and the integrin cytoplasmic tail suggests that the G340E mutation would disrupt the tight apposition of F2 and F3, thus allowing them to behave as independent modules. (b) In vitro expression of WT (left lane), L334R (middle lane), and G340E (right lane) constructs reveals proteolytic sensitivity of G340E compared to WT and L334R. (c) MALDI-TOF mass spectrometry and peptide mass fingerprinting were used to identify that the sequence of the truncated fragment of the talin head observed in (b) corresponded to the F0-F2 domains indicating F3 was often cleaved in the G340E mutant. (d–e) The recruitment of talin was measured in neomycin treated embryos (d) and in rhea17 embryos (e). Talin recruitment was significantly reduced in neomycin-treated compared to controls (*p<0.05). In contrast there was no such reduction in the rhea17 embryos suggesting the G340E mutant talin protein interacts with the membrane as well as the WT protein.
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pgen-1004756-g006: G340 maintains an intermolecular interaction between F2 and F3 that couples their activity.(a) The conserved role of G340 (G331 in mammalian talin2, shown here) is to stabilize the domain orientation of F2 and F3 (a), which work together to induce integrin activation and stabilize that talin head at the plasma membrane. Modelling based on known structures (see [15]) of mouse talin2 and the integrin cytoplasmic tail suggests that the G340E mutation would disrupt the tight apposition of F2 and F3, thus allowing them to behave as independent modules. (b) In vitro expression of WT (left lane), L334R (middle lane), and G340E (right lane) constructs reveals proteolytic sensitivity of G340E compared to WT and L334R. (c) MALDI-TOF mass spectrometry and peptide mass fingerprinting were used to identify that the sequence of the truncated fragment of the talin head observed in (b) corresponded to the F0-F2 domains indicating F3 was often cleaved in the G340E mutant. (d–e) The recruitment of talin was measured in neomycin treated embryos (d) and in rhea17 embryos (e). Talin recruitment was significantly reduced in neomycin-treated compared to controls (*p<0.05). In contrast there was no such reduction in the rhea17 embryos suggesting the G340E mutant talin protein interacts with the membrane as well as the WT protein.

Mentions: We have shown that talin protein in rhea17 mutants fails to cluster integrins and reinforce integrin-mediated adhesions. However, the data presented so far does not explain the mechanism by which this effect is mediated. When put into the context of the large body of knowledge that exists about the structure of the talin head, the nature of molecular lesion in rhea17 provides some intriguing hints about this mechanism. Specifically, the G340E mutation is expected to disrupt the coordinated movement of the F2 and F3 domains which is essential for activation and clustering. Structural modeling of the talin head, using the solved crystal structure of the mouse talin head in complex with β1-integrin ([15]; PDB number 3G9W), predicted that residue equivalent to G340 (G331) is located on the surface of F3 at its interface with F2 (Fig. 6a, inset). In the WT talin head this glycine allows the close packing of these two sub-domains. However, substitution for a glutamate inserts a charged carboxyl group into this close gap disrupting the fixed orientation of the F2 and F3 domains, which should allow them to move independently of one another. Since it has been proposed that a tri-partite interaction between integrin, talin head, and the phospholipid bilayer is required to facilitate stable adhesion and to promote inside-out signaling, it is quite possible that disrupting the coordination of F2 and F3 would destabilize these interactions. Consistent with such an effect the G340E mutation rendered the talin head domain proteolytically sensitive to cleavage of the F3 subdomain from the F0-F2 subdomains (Fig. 6b). Furthermore, we used MALDI-TOF mass spectrometry and peptide mass fingerprinting to confirm that the cleaved fragment we observed was indeed the F0-F2 domain, indicating that the F3 had been lost (Fig. 6c). This in vitro evidence is in line with the assertion that F2 and F3 become structurally uncoupled from one another when G340 is mutated.


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)

G340 maintains an intermolecular interaction between F2 and F3 that couples their activity.(a) The conserved role of G340 (G331 in mammalian talin2, shown here) is to stabilize the domain orientation of F2 and F3 (a), which work together to induce integrin activation and stabilize that talin head at the plasma membrane. Modelling based on known structures (see [15]) of mouse talin2 and the integrin cytoplasmic tail suggests that the G340E mutation would disrupt the tight apposition of F2 and F3, thus allowing them to behave as independent modules. (b) In vitro expression of WT (left lane), L334R (middle lane), and G340E (right lane) constructs reveals proteolytic sensitivity of G340E compared to WT and L334R. (c) MALDI-TOF mass spectrometry and peptide mass fingerprinting were used to identify that the sequence of the truncated fragment of the talin head observed in (b) corresponded to the F0-F2 domains indicating F3 was often cleaved in the G340E mutant. (d–e) The recruitment of talin was measured in neomycin treated embryos (d) and in rhea17 embryos (e). Talin recruitment was significantly reduced in neomycin-treated compared to controls (*p<0.05). In contrast there was no such reduction in the rhea17 embryos suggesting the G340E mutant talin protein interacts with the membrane as well as the WT protein.
© Copyright Policy
Related In: Results  -  Collection

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

pgen-1004756-g006: G340 maintains an intermolecular interaction between F2 and F3 that couples their activity.(a) The conserved role of G340 (G331 in mammalian talin2, shown here) is to stabilize the domain orientation of F2 and F3 (a), which work together to induce integrin activation and stabilize that talin head at the plasma membrane. Modelling based on known structures (see [15]) of mouse talin2 and the integrin cytoplasmic tail suggests that the G340E mutation would disrupt the tight apposition of F2 and F3, thus allowing them to behave as independent modules. (b) In vitro expression of WT (left lane), L334R (middle lane), and G340E (right lane) constructs reveals proteolytic sensitivity of G340E compared to WT and L334R. (c) MALDI-TOF mass spectrometry and peptide mass fingerprinting were used to identify that the sequence of the truncated fragment of the talin head observed in (b) corresponded to the F0-F2 domains indicating F3 was often cleaved in the G340E mutant. (d–e) The recruitment of talin was measured in neomycin treated embryos (d) and in rhea17 embryos (e). Talin recruitment was significantly reduced in neomycin-treated compared to controls (*p<0.05). In contrast there was no such reduction in the rhea17 embryos suggesting the G340E mutant talin protein interacts with the membrane as well as the WT protein.
Mentions: We have shown that talin protein in rhea17 mutants fails to cluster integrins and reinforce integrin-mediated adhesions. However, the data presented so far does not explain the mechanism by which this effect is mediated. When put into the context of the large body of knowledge that exists about the structure of the talin head, the nature of molecular lesion in rhea17 provides some intriguing hints about this mechanism. Specifically, the G340E mutation is expected to disrupt the coordinated movement of the F2 and F3 domains which is essential for activation and clustering. Structural modeling of the talin head, using the solved crystal structure of the mouse talin head in complex with β1-integrin ([15]; PDB number 3G9W), predicted that residue equivalent to G340 (G331) is located on the surface of F3 at its interface with F2 (Fig. 6a, inset). In the WT talin head this glycine allows the close packing of these two sub-domains. However, substitution for a glutamate inserts a charged carboxyl group into this close gap disrupting the fixed orientation of the F2 and F3 domains, which should allow them to move independently of one another. Since it has been proposed that a tri-partite interaction between integrin, talin head, and the phospholipid bilayer is required to facilitate stable adhesion and to promote inside-out signaling, it is quite possible that disrupting the coordination of F2 and F3 would destabilize these interactions. Consistent with such an effect the G340E mutation rendered the talin head domain proteolytically sensitive to cleavage of the F3 subdomain from the F0-F2 subdomains (Fig. 6b). Furthermore, we used MALDI-TOF mass spectrometry and peptide mass fingerprinting to confirm that the cleaved fragment we observed was indeed the F0-F2 domain, indicating that the F3 had been lost (Fig. 6c). This in vitro evidence is in line with the assertion that F2 and F3 become structurally uncoupled from one another when G340 is mutated.

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