Disruption of the Talin Gene Compromises Focal Adhesion Assembly in Undifferentiated but Not Differentiated Embryonic Stem Cells

Priddle, Helen; Hemmings, Lance; Monkley, Susan J.; Woods, Alison J.; Patel, Bipin; Sutton, Deborah H.; Dunn, Graham; Zicha, Daniel; Critchley, David R.

doi:10.1083/jcb.142.4.1121

Cited by 168 publications

(156 citation statements)

References 66 publications

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“…The latter possibility is suggested by biochemical studies of ␤ integrin cytoplasmic tails demonstrating that the Y-A mutation disrupts interaction with the cytoskeletal protein talin (Calderwood et al 2002) and the observation that talin-deficient ES cells lose surface expression of ␤1 integrins (Priddle et al 1998). To address this question, mutant ␤1 integrin expression and function were next studied in homozy- Figure 1.…”

Section: Resultsmentioning

confidence: 99%

In vivo β1 integrin function requires phosphorylation-independent regulation by cytoplasmic tyrosines

Chen

Zou²,

Sarratt³

et al. 2006

Genes Dev.

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Integrins are heterodimeric adhesion receptors associated with bidirectional signaling. In vitro studies support a role for the binding of evolutionarily conserved tyrosine motifs (NPxY) in the ␤ integrin cytoplasmic tail to phosphotyrosine-binding (PTB) domain-containing proteins, an interaction proposed to be dynamically regulated by tyrosine phosphorylation. Here we show that replacement of both ␤1 integrin cytoplasmic tyrosines with alanines, resulting in the loss of all PTB domain interaction, causes complete loss of ␤1 integrin function in vivo. In contrast, replacement of ␤1 integrin cytoplasmic tyrosines with phenylalanines, a mutation that prevents tyrosine phosphorylation, conserves in vivo integrin function. These results have important implications for the molecular mechanism and regulation of integrin function.Supplemental material is available at http://www.genesdev.org.Received January 9, 2006; revised version accepted February 17, 2006. Integrin receptors bridge extracellular matrix proteins and intracellular cytoskeletal and signaling proteins and are essential regulators of cellular behavior and function (for review, see Hynes 2002). Since the cytoplasmic tails of integrin receptors are relatively short and lack intrinsic enzymatic activity, integrin signals are believed to be mediated by protein-protein interactions. Biochemical studies have identified a large number of intracellular cytoskeletal and signaling proteins that are capable of binding integrin cytoplasmic tails, but a clear picture of how these many potential protein-protein interactions result in bidirectional integrin signals has not yet emerged.A highly recognizable motif in the integrin cytoplasmic tail is NPxY, two of which are found in the cytoplasmic tails of ␤ integrin subunits. Biochemical and structural studies have demonstrated that NPxY motifs bind phosphotyrosine-binding (PTB) domains and that the specificity of NPxY-PTB domain interactions is conferred by both the NPxY motif and the PTB domain (for review, see Uhlik et al. 2005). Some PTB domains (e.g., the Shc [Src homology 2 domain-containing] transforming protein 1 PTB domain) require phosphotyrosines for high-affinity interaction with NPxY motifs, while others (e.g., the talin PTB domain) bind through hydrophobic interaction with nonphosphorylated tyrosine or phenylalanine residues. The specificity and diversity of NPxY-PTB domain interactions have recently been proposed as a molecular mechanism by which integrin cytoplasmic tails transmit a variety of bidirectional signals . Biochemical studies performed in vitro suggest that distinct ␤ integrin cytoplasmic tails bind distinct PTB domain-containing proteins, and that tyrosine phosphorylation can alter these interactions through addition of a charged phosphate group to the hydrophobic tyrosine aromatic ring Garcia-Alvarez et al. 2003). Dynamic regulation of NPxY-PTB domain binding by tyrosine phosphorylation is therefore a potentially elegant means of explaining how short integrin cytoplasmic tails transmit di...

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Section: Resultsmentioning

confidence: 99%

In vivo β1 integrin function requires phosphorylation-independent regulation by cytoplasmic tyrosines

Chen

Zou²,

Sarratt³

et al. 2006

Genes Dev.

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“…A small population (<5%) of FlnA-containing platelets could be detected in some animals, suggesting mosaic GATA1-Cre expression, rendering incomplete FlnA loxP excision. Whether these platelets contain full-length FlnA or a truncated form, as shown for talin (Priddle et al, 1998), remains to be investigated. The high efficiency of gene inactivation is similar to that described for the widely used megakaryocytespecific Pf4-Cre transgenic mouse and posits the GATA1-Cre mouse as a strong alternative for studies in megakaryocytes and platelets (Léon et al, 2007;Petrich et al, 2007;Tiedt et al, 2007;Hitchcock et al, 2008;Wen et al, 2009).…”

Section: Discussionmentioning

confidence: 99%

A novel interaction between FlnA and Syk regulates platelet ITAM-mediated receptor signaling and function

Falet¹,

Pollitt²,

Begonja³

et al. 2010

J Cell Biol

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“…Moreover, the downregulation of vinculin in Balb\c 3T3 cells with the use of antisense technology resulted in cells that were less well spread, assembled smaller focal adhesions and were more motile [53], whereas over-expression of vinculin in the same cell line led to the assembly of larger focal adhesions and a lower motility of cells [54]. However, gene disruption studies have shown that, whereas talin is essential to the assembly of focal adhesions in mouse embryonic stem cells, vinculin is not [14]. Neither is vinculin essential for focal adhesion assembly in mouse F9 embryonic carcinoma cells, although the F9 cells were less well spread and showed decreased adhesion to laminin and fibronectin [55].…”

Section: Figure 7 Domain Structure Of Talinmentioning

confidence: 99%

“…Down-regulation of talin expression in HeLa cells slowed down the rate of cell spreading and caused a decrease in the size of focal adhesions [11] ; microinjection of antibodies against talin into fibroblasts led to the disruption of focal adhesions and actin stress fibres [9,12], as did certain talin polypeptides [13]. Finally, mouse embryonic stem cells in which both copies of the talin gene were disrupted showed extensive membrane blebbing, a decrease in cell polarity and an inability to form focal adhesions [14].…”

Section: Introductionmentioning

confidence: 99%

Talin contains three similar vinculin-binding sites predicted to form an amphipathic helix

et al. 1999

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Using recombinant talin polypeptides and an SDS\PAGE-blot overlay assay, we have previously identified three regions of talin that are involved in binding to vinculin [Gilmore, Wood, Ohanian, Jackson, Patel, Rees, Hynes and Critchley (1993) J. Cell Biol. 122, 337-347]. We have confirmed these observations by using a yeast two-hybrid assay and shown that talin residues 498-656, 852-950 and 1929-2029 are each capable of binding to vinculin residues 1-258. We have further defined the three vinculin-binding sites in talin to residues 607-636, 852-876 and 1944-1969 ; alignment of these sequences shows 59 % similarity, although there are only two identical residues. Predictions of secondary structure indicate that this vinculin-binding motif forms an amphipathic α-helix. The hydrophobic face of helix 607-636 contains three aligned leucines (residues 608, 615 and 622), which show conservative substitutions in the other two

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Disruption of the Talin Gene Compromises Focal Adhesion Assembly in Undifferentiated but Not Differentiated Embryonic Stem Cells

Cited by 168 publications

References 66 publications

In vivo β1 integrin function requires phosphorylation-independent regulation by cytoplasmic tyrosines

In vivo β1 integrin function requires phosphorylation-independent regulation by cytoplasmic tyrosines

A novel interaction between FlnA and Syk regulates platelet ITAM-mediated receptor signaling and function

Talin contains three similar vinculin-binding sites predicted to form an amphipathic helix

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