Studies on the morphology and spreading of human endothelial cells define key inter- and intramolecular interactions for talin1.
Bottom Line: The talin rod contains several actin-binding sites (ABS), and mutations in the C-terminal ABS that reduced actin-binding impaired talin1 function, whereas those that increased binding resulted in more stable FAs.The results show that both the N-terminal integrin and C-terminal actin-binding functions of talin are essential to cell spreading and FA assembly.Finally, mutations that relieve talin auto-inhibition resulted in the rapid and excessive production of FA, highlighting the importance of talin regulation within the cell.
Affiliation: Department of Biochemistry, University of Leicester, Leicester, UK.Show MeSH
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Mentions: Talin can adopt an auto-inhibited conformation due to an interaction between the N-terminal F3 FERM domain and a C-terminal domain in the talin rod (residues 1655–1822) that masks the integrin-binding site in F3 (Goksoy et al., 2008; Goult et al., 2009a) (Fig. 6B). We have identified several mutations in the talin rod that abolish this interaction (Goult et al., 2009a), and we therefore studied the effects of two of these mutations on cell spreading and FA formation. HUVEC transfected with the talin1 siRNA and expressing either GFP-talin1 T1676E or GFP-talin1 E1770A were well spread (Fig. 6A) although the T1676E mutation provoked a transient increase in the number of arborized cells at 5 h (Fig. 6C). However, the most striking effect of these mutations was the increased rate of FA formation, with the first FA appearing as early as 30 min after replating, while in cells transfected with wild-type GFP-talin1, the first FA were only observed after 1 h (Fig. 6E and F). The number of FA continued to increase, and after 24 h there were 3 times more FA in cells expressing either the T1676E or the E1770A mutant than in controls (Fig. 6E and F), although FA size was not affected (Fig. S4B). Both these GFP-talin1 mutants localised to peripheral FA as well as FB-like adhesions (Fig. 6A arrows) whereas wild-type GFP-talin1 was predominantly localised in FA. The results indicate that pathways that regulate the talin head/rod interaction must play a key role in controlling the rate and extent of FA assembly.
Affiliation: Department of Biochemistry, University of Leicester, Leicester, UK.