All metazoan cells carry transmembrane receptors of the integrin family, which couple the contractile force of the actomyosin cytoskeleton to the extracellular environment. In agreement with this principle, rapidly migrating leukocytes use integrin-mediated adhesion when moving over two-dimensional surfaces. As migration on two-dimensional substrates naturally overemphasizes the role of adhesion, the contribution of integrins during three-dimensional movement of leukocytes within tissues has remained controversial. We studied the interplay between adhesive, contractile and protrusive forces during interstitial leukocyte chemotaxis in vivo and in vitro. We ablated all integrin heterodimers from murine leukocytes, and show here that functional integrins do not contribute to migration in three-dimensional environments. Instead, these cells migrate by the sole force of actin-network expansion, which promotes protrusive flowing of the leading edge. Myosin II-dependent contraction is only required on passage through narrow gaps, where a squeezing contraction of the trailing edge propels the rigid nucleus.

Wnt genes comprise a large family of secreted polypeptides that are expressed in spatially and tissue-restricted patterns during vertebrate embryonic development. Mutational analysis in mice has shown the importance of Wnts in controlling diverse developmental processes such as patterning of the body axis, central nervous system and limbs, and the regulation of inductive events during organogenesis. Although many components of the Wnt signalling pathway have been identified, little is known about how Wnts and their cognate Frizzled receptors signal to downstream effector molecules. Here we present evidence that a new member of the low-density lipoprotein (LDL)-receptor-related protein family, LRP6 (ref. 3), is critical for Wnt signalling in mice. Embryos homozygous for an insertion mutation in the LRP6 gene exhibit developmental defects that are a striking composite of those caused by mutations in individual Wnt genes. Furthermore, we show a genetic enhancement of a Wnt mutant phenotype in mice lacking one functional copy of LRP6. Together, our results support a broad role for LRP6 in the transduction of several Wnt signals in mammals.

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...

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Platelet adhesion and aggregation at sites of vascular injury are essential for normal hemostasis but may also lead to pathological thrombus formation, causing diseases such as myocardial infarction or stroke. Heterodimeric receptors of the integrin family play a central role in the adhesion and aggregation of platelets. In resting platelets, integrins exhibit a low affinity state for their ligands, and they shift to a high affinity state at sites of vascular injury. It has been proposed that direct binding of the cytoskeletal protein talin1 to the cytoplasmic domain of the integrin β subunits is necessary and sufficient to trigger the activation of integrins to this high affinity state, but direct in vivo evidence in support of this hypothesis is still lacking. Here, we show that platelets from mice lacking talin1 are unable to activate integrins in response to all known major platelet agonists while other cellular functions are still preserved. As a consequence, mice with talin-deficient platelets display a severe hemostatic defect and are completely resistant to arterial thrombosis. Collectively, these experiments demonstrate that talin is required for inside-out activation of platelet integrins in hemostasis and thrombosis.

Integrins are critical for hemostasis and thrombosis because they mediate both platelet adhesion and aggregation. Talin is an integrin-binding cytoplasmic adaptor that is a central organizer of focal adhesions, and loss of talin phenocopies integrin deletion in Drosophila. Here, we have examined the role of talin in mammalian integrin function in vivo by selectively disrupting the talin1 gene in mouse platelet precursor megakaryocytes. Talin null megakaryocytes produced circulating platelets that exhibited normal morphology yet manifested profoundly impaired hemostatic function. Specifically, platelet-specific deletion of talin1 led to spontaneous hemorrhage and pathological bleeding. Ex vivo and in vitro studies revealed that loss of talin1 resulted in dramatically impaired integrin αIIbβ3-mediated platelet aggregation and β1 integrin–mediated platelet adhesion. Furthermore, loss of talin1 strongly inhibited the activation of platelet β1 and β3 integrins in response to platelet agonists. These data establish that platelet talin plays a crucial role in hemostasis and provide the first proof that talin is required for the activation and function of mammalian α2β1 and αIIbβ3 integrins in vivo.

Podocytes are specialized actin-rich epithelial cells that line the kidney glomerular filtration barrier. The interface between the podocyte and the glomerular basement membrane requires integrins, and defects in either α 3 or β 1 integrin, or the α 3 β 1 ligand laminin result in nephrotic syndrome in murine models. The large cytoskeletal protein talin1 is not only pivotal for integrin activation, but also directly links integrins to the actin cytoskeleton. Here, we found that mice lacking talin1 specifically in podocytes display severe proteinuria, foot process effacement, and kidney failure. Loss of talin1 in podocytes caused only a modest reduction in β 1 integrin activation, podocyte cell adhesion, and cell spreading; however, the actin cytoskeleton of podocytes was profoundly altered by the loss of talin1. Evaluation of murine models of glomerular injury and patients with nephrotic syndrome revealed that calpain-induced talin1 cleavage in podocytes might promote pathogenesis of nephrotic syndrome. Furthermore, pharmacologic inhibition of calpain activity following glomerular injury substantially reduced talin1 cleavage, albuminuria, and foot process effacement. Collectively, these findings indicate that podocyte talin1 is critical for maintaining the integrity of the glomerular filtration barrier and provide insight into the pathogenesis of nephrotic syndrome.

We have used gene disruption to isolate two talin (−/−) ES cell mutants that contain no intact talin. The undifferentiated cells (a) were unable to spread on gelatin or laminin and grew as rounded colonies, although they were able to spread on fibronectin (b) showed reduced adhesion to laminin, but not fibronectin (c) expressed much reduced levels of β1 integrin, although levels of α5 and αV were wild-type (d) were less polarized with increased membrane protrusions compared with a vinculin (−/−) ES cell mutant (e) were unable to assemble vinculin or paxillin-containing focal adhesions or actin stress fibers on fibronectin, whereas vinculin (−/−) ES cells were able to assemble talin-containing focal adhesions. Both talin (−/−) ES cell mutants formed embryoid bodies, but differentiation was restricted to two morphologically distinct cell types. Interestingly, these differentiated talin (−/−) ES cells were able to spread and form focal adhesion-like structures containing vinculin and paxillin on fibronectin. Moreover, the levels of the β1 integrin subunit were comparable to those in wild-type ES cells. We conclude that talin is essential for β1 integrin expression and focal adhesion assembly in undifferentiated ES cells, but that a subset of differentiated cells are talin independent for both characteristics.