Talin depletion reveals independence of initial cell spreading from integrin activation and traction

Abstract

Cell spreading, adhesion and remodelling of the extracellular matrix (eCM) involve bi-directional signalling and physical linkages between the eCM, integrins and the cell cytoskeleton [1][2][3] . the actinbinding proteins talin1 and 2 link ligand-bound integrins to the actin cytoskeleton and increase the affinity of integrin for the eCM 4-6 . Here we report that depletion of talin2 in talin1-null (talin1 −/− ) cells did not affect the initiation of matrix-activated spreading or src family kinase (sFK) activation, but abolished the eCM-integrin-cytoskeleton linkage and sustained cell spreading and adhesion. specifically, focal adhesion assembly, focal adhesion kinase (FAK) signalling and traction force generation on substrates were severely affected. the talin1 head domain restored β1 integrin activation but only full-length talin1 restored the eCM-cytoskeleton linkage and normal cytoskeleton organization. Our results demonstrate three biochemically distinct steps in fibronectin-activated cell spreading and adhesion: 1) fibronectin-integrin binding and initiation of spreading, 2) fast cell spreading and 3) focal adhesion formation and substrate traction. We suggest that talin is not required for initial cell spreading. However, talin provides the important mechanical linkage between ligandbound integrins and the actin cytoskeleton required to catalyse focal adhesion-dependent pathways.Disruption of the talin1 gene in undifferentiated embryonic stem (ES) cells severely disrupts cell adhesion and cytoskeleton organization 7 . However, fibroblasts derived from talin1 −/− ES cells spread and adhere normally, presumably due to increased expression of the closely related talin2 (refs 7,8 ). To study the role of talin in initial cell responses to ECM signals, we constructed a mouse talin2 short interfering RNA (siRNA) expression plasmid and transfected it into the talin1 −/− fibroblasts. We observed that talin2 siRNA-expressing talin1 −/− cells began to round up in culture after 48 h. After 3.5 days, 77 ± 11% of the cells became rounded (mean ± s.d., n > 450, 3 repetitions), whereas most of the control siRNA-expressing cells showed an elongated morphology in culture. Co-transfection of a GFP-talin1 plasmid rescued the morphology change induced by talin2 siRNA (11 ± 3% and 15 ± 8% rounded up in control siRNA transfected and GFP-talin1 and talin2 siRNA co-transfected cells, respectively; mean 3Correspondence should be addressed to M.P.S. (e-mail: ms2001@columbia.edu). Note: Supplementary Information is available on the Nature Cell Biology website. AUTHOR CONTRIBUTIONS X.Z. performed all the experiments in M.P.S. lab with significant support from G.J. and Y.C.; S.J.M. provided the cell line and important information; D.R.C. provided important direction on the project. Supplementary Information, Fig. S1a, b). Immunoblot analyses with a pantalin antibody 8d4 and a talin2-specific antibody showed a significant reduction in talin2 expression 3.5 days after talin2 siRNA transfection, whereas the levels of vinculin and...

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.…”

“…Nevertheless, both isoforms are expressed in most cell types (24) (Fig. 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.…”

Targeting Cell Spreading: A Method of Sensitizing Metastatic Tumor Cells to TRAIL-Induced Apoptosis

“…Integrin-linked kinase (ILK) forms a signaling hub at the b-integrin tail, signaling downstream through parvin proteins, PINCH, and NCK-2 (19,20). Another adaptor is talin, which binds both to the tail of b-integrins and to actin, relaying signals which aid actin polymerization, leading to cell spreading (21). The nonreceptor tyrosine kinase FAK is activated by integrin-dependent cell attachment, and binds to and activates another nonreceptor tyrosine kinase, Src.…”

Contribution of Bone Tissue Modulus to Breast Cancer Metastasis to Bone

“…Specific inhibitors to Src tyrosine kinases, which are required to regulate integrin-cytoskeleton interactions, have shown promise in preclinical studies, reducing both tumor burden and incidence in bone [97,98] as well as metastasis to bone [99]. After activation, SFKs promote coupling of the cytoskeleton to integrins through talin [100][101][102], which enables mechanical forces on the integrins to engage the integrin/matrix catch bond [50]. While the detailed mechanisms governing translation of mechanical forces sensed within cell-matrix adhesions to biochemical changes are not completely known, changes in the conformation of integrins in response to a rigid matrix are thought to result in tension-dependent changes in conformation of integrinassociated proteins, such as SFK, p130Cas and vinculin [96].…”