Studies on the morphology and spreading of human endothelial cells define key inter- and intramolecular interactions for talin1

Abstract

Talin binds to and activates integrins and is thought to couple them to cytoskeletal actin. However, functional studies on talin have been restricted by the fact that most cells express two talin isoforms. Here we show that human umbilical vein endothelial cells (HUVEC) express only talin1, and that talin1 knockdown inhibited focal adhesion (FA) assembly preventing the cells from maintaining a spread morphology, a phenotype that was rescued by GFP-mouse talin1. Thus HUVEC offer an ideal model system in which to conduct talin structure/function studies. Talin contains an N-terminal FERM domain (comprised of F1, F2 and F3 domains) and a C-terminal flexible rod. The F3 FERM domain binds β-integrin tails, and mutations in F3 that inhibited integrin binding (W359A) or activation (L325R) severely compromised the ability of GFP-talin1 to rescue the talin1 knockdown phenotype despite the presence of a second integrin-binding site in the talin rod. 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.

Talin determines the nanoscale architecture of focal adhesions

“…siRNA-mediated depletion of endogenous human talin1 results in impaired cell spreading and migration (Fig. S1 D-J), as well as FA assembly defects, which can be rescued by coexpression of a mouse talin1 GFP fusion as described previously (25,26). The HUVEC platform thus allows substitution of endogenous talin1 with engineered talin constructs.…”

“…S1 A-C), and talin2 is able to support FA assembly and cell spreading in talin1-null mouse fibroblasts (25). To study the structural role of talin in FAs, we therefore chose primary human umbilical vein endothelial cells (HUVECs), which express only talin1 (26) (Fig. S1 A-C).…”

“…HUVECs (passage number <15) were cultured in a 5% CO 2 , 37°C humidified atmosphere in Medium 200 (M-200-500; Life Technologies) supplemented with large vessel endothelial factors (A14608-01; Life Technologies). siRNA-mediated knockdown of human Talin1 in HUVECs was performed as previously described (26), using custom stealth RNAi siRNA duplexes (oligo sequence: 5′-CCAAGAACGGAAACCUGCCAGA-GUU-3′; Life Technologies). To transfect cells with expression vectors and/or siRNA, cells were trypsinized and electroporated using the Neon platform (Life Technologies).…”

Subcellular Localization of Talin Is Regulated by Inter-domain Interactions

“…This idea agrees with the presence of lipid-binding sites in other subdomains of the talin head that are also critical for integrin activation. In addition to the F3 domain (26), a series of basic residues in the F1 domain (21) and a series of basic residues in the F2 domain, called the membrane orientation patch (MOP) (31), are all involved in talin-membrane association, integrin activation, and for the ability of talin to support focal adhesion assembly (36). The structure of the THD reveals an open, extended conformation in which all three lipid-binding sites on the surface of THD are orientated toward the membrane (22).…”

“…These lipid-binding sites, which are aligned along one surface of the extended FERM domain, make extensive contacts with the plasma membrane (22) and play a critical role in integrin activation (21,26,31). The rod domain of talin consists of 62 amphipathic ␣-helices that are assembled into a series of ␣-helical bundles (33,34), and autoinhibitory interactions between THD and the rod domain regulate the functions of talin including its interactions with integrins (17,(35)(36)(37)(38)(39)(40).…”