Studies on the morphology and spreading of human endothelial cells define key inter- and intramolecular interactions for talin1.
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 beta-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.
Kopp
Petra M
PM
Department of Biochemistry, University of Leicester, Leicester, UK.
Bate
Neil
N
Hansen
Tania M
TM
Brindle
Nicholas P J
NP
Praekelt
Uta
U
Debrand
Emmanuel
E
Coleman
Stacey
S
Mazzeo
Daniela
D
Goult
Benjamin T
BT
Gingras
Alexandre R
AR
Pritchard
Catrin A
CA
Critchley
David R
DR
Monkley
Susan J
SJ
D000199:Actins / Q000235:genetics / Q000378:metabolism; D000818:Animals; D002448:Cell Adhesion / Q000502:physiology; D002465:Cell Movement / Q000502:physiology; D042783:Endothelial Cells / Q000166:cytology / Q000378:metabolism* / Q000502:physiology; D022001:Focal Adhesions / Q000502:physiology; D055785:Gene Knockdown Techniques; D006801:Humans; D016023:Integrins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D010641:Phenotype; D016608:Talin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014162:Transfection; D014471:Umbilical Veins / Q000166:cytology; D015854:Up-Regulation
D000199:Actins; D016023:Integrins; C518832:TLN1 protein, human; D016608:Talin