Cell motility proceeds by cycles of edge protrusion, adhesion, and retraction. Whether these functions are coordinated by biochemical or biomechanical processes is unknown. We find that myosin II pulls the rear of the lamellipodial actin network, causing upward bending, edge retraction, and initiation of new adhesion sites. The network then separates from the edge and condenses over the myosin. Protrusion resumes as lamellipodial actin regenerates from the front and extends rearward until it reaches newly assembled myosin, initiating the next cycle. Upward bending, observed by evanescence and electron microscopy, results in ruffle formation when adhesion strength is low. Correlative fluorescence and electron microscopy shows that the regenerating lamellipodium forms a cohesive, separable layer of actin above the lamellum. Thus, actin polymerization periodically builds a mechanical link, the lamellipodium, connecting myosin motors with the initiation of adhesion sites, suggesting that the major functions driving motility are coordinated by a biomechanical process.

Autosomal dominant polycystic kidney disease (ADPKD) describes a group of at least three genetically distinct disorders with almost identical clinical features that collectively affects 1:1,000 of the population. Affected individuals typically develop large cystic kidneys and approximately one half develop end-stage renal disease by their seventh decade. It has been suggested that the diseases result from defects in interactive factors involved in a common pathway. The recent discovery of the genes for the two most common forms of ADPKD has provided an opportunity to test this hypothesis. We describe a previously unrecognized coiled-coil domain within the C terminus of the PKD1 gene product, polycystin, and demonstrate that it binds specifically to the C terminus of PKD2. Homotypic interactions involving the C terminus of each are also demonstrated. We show that naturally occurring pathogenic mutations of PKD1 and PKD2 disrupt their associations. We have characterized the structural basis of their heterotypic interactions by deletional and site-specific mutagenesis. Our data suggest that PKD1 and PKD2 associate physically in vivo and may be partners of a common signalling cascade involved in tubular morphogenesis.

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...

Nonmuscle myosin IIA (NMM-IIA) is involved in the formation of focal adhesions and neurite retraction. However, the role of NMM-IIA in these functions remains largely unknown. Using RNA interference as a tool to decrease NMM-IIA expression, we have found that NMM-IIA is the major myosin involved in traction force generation and retrograde F-actin flow in mouse embryonic fibroblast cells. Quantitative analyses revealed that approximately 60% of traction force on fibronectin-coated surfaces is contributed by NMM-IIA and approximately 30% by NMM-IIB. The retrograde F-actin flow decreased dramatically in NMM-IIA-depleted cells, but seemed unaffected by NMM-IIB deletion. In addition, we found that depletion of NMM-IIA caused cells to spread at a higher rate and to a greater area on fibronectin substrates during the early spreading period, whereas deletion of NMM-IIB appeared to have no effect on spreading. The distribution of NMM-IIA was concentrated on the dorsal surface and approached the ventral surface in the periphery, whereas NMM-IIB was primarily concentrated around the nucleus and to a lesser extent at the ventral surface in cell periphery. Our results suggest that NMM-IIA is involved in generating a coherent cytoplasmic contractile force from one side of the cell to the other through the cross-linking and the contraction of dorsal actin filaments.

SummaryMaintaining a physical connection across cytoplasm is crucial for many biological processes such as matrix force generation, cell motility, cell shape and tissue development. However, in the absence of stress fibers, the coherent structure that transmits force across the cytoplasm is not understood. We find that nonmuscle myosin-II (NMII) contraction of cytoplasmic actin filaments establishes a coherent cytoskeletal network irrespective of the nature of adhesive contacts. When NMII activity is inhibited during cell spreading by Rho kinase inhibition, blebbistatin, caldesmon overexpression or NMIIA RNAi, the symmetric traction forces are lost and cell spreading persists, causing cytoplasm fragmentation by membrane tension that results in 'C' or dendritic shapes. Moreover, local inactivation of NMII by chromophore-assisted laser inactivation causes local loss of coherence. Actin filament polymerization is also required for cytoplasmic coherence, but microtubules and intermediate filaments are dispensable. Loss of cytoplasmic coherence is accompanied by loss of circumferential actin bundles. We suggest that NMIIA creates a coherent actin network through the formation of circumferential actin bundles that mechanically link elements of the peripheral actin cytoskeleton where much of the force is generated during spreading. Adelstein, 2008;Vicente-Manzanares et al., 2009;Wylie and Chantler, 2008). NMIIA and NMIIB are the primary force generators in fibroblasts (Cai et al., 2006;Lo et al., 2004). Phosphorylation of myosin light chains (MLC), primarily by Rho kinases (ROCK) and MLC kinase (Totsukawa et al., 2004), regulates the NMII activity. ROCK has multiple protein targets including MLC, MLC phosphatase, adducin and moesin (Totsukawa et al., 2004). ROCK activates NMII by phosphorylating MLC and also by inactivating MLC phosphatase to inhibit MLC dephosphorylation (Totsukawa et al., 2004). Specific inhibitors have been developed for studying the functions of NMII, i.e. Y27632 inhibits ROCK and blebbistatin inhibits the ATPase activity of myosin-II. In addition to MLC phosphatase, some other proteins also negatively regulate NMII activity. For example, caldesmon interacts with actin, myosin-II and tropomyosin, and inhibits the ATPase activity of myosin-II (Marston et al., 1998). Caldesmon overexpression causes suppression of traction forces and focal adhesions (Helfman et al., 1999). Thus, there are a variety of ways to inhibit force generation on substrates.The plasma membrane limits the spreading of cells on substrates, and tension in the plasma membrane inhibits the ability of actin to polymerize at the periphery (Raucher and Sheetz, 2000). Although the tension in the membrane is typically very low (Sheetz, 2001), it can influence the behavior of cells (Keren et al., 2008) and the final shape of cells is heavily influenced by the final membrane area.We here demonstrate that NMIIA is crucial for the mechanical coherence of cytoplasm. Inhibition of NMII contractility or depletion of NMIIA causes cytoplasm to ...