How physical force is sensed by cells and transduced into cellular signaling pathways is poorly understood. Previously, we showed that tyrosine phosphorylation of p130Cas (Cas) in a cytoskeletal complex is involved in force-dependent activation of the small GTPase Rap1. Here, we mechanically extended bacterially expressed Cas substrate domain protein (CasSD) in vitro and found a remarkable enhancement of phosphorylation by Src family kinases with no apparent change in kinase activity. Using an antibody that recognized extended CasSD in vitro, we observed Cas extension in intact cells in the peripheral regions of spreading cells, where higher traction forces are expected and where phosphorylated Cas was detected, suggesting that the in vitro extension and phosphorylation of CasSD are relevant to physiological force transduction. Thus, we propose that Cas acts as a primary force sensor, transducing force into mechanical extension and thereby priming phosphorylation and activation of downstream signaling.

Summary Cell rearrangements shape the Drosophila embryo through spatially regulated changes in cell shape and adhesion. We show that Bazooka/Par-3 (Baz) is required for the planar polarized distribution of myosin II and adherens junction proteins and polarized intercalary behavior is disrupted in baz mutants. The myosin II activator Rho-kinase is asymmetrically enriched at anterior and posterior borders of intercalating cells in a pattern complementary to Baz. Loss of Rho-kinase results in expansion of the Baz domain and activated Rho-kinase is sufficient to exclude Baz from the cortex. The planar polarized distribution of Baz requires its C-terminal domain. Rho-kinase can phosphorylate this domain and inhibit its interaction with phosphoinositide membrane lipids, suggesting a mechanism by which Rho-kinase could regulate Baz association with the cell cortex. These results demonstrate that Rho-kinase plays an instructive role in planar polarity by targeting Baz/Par-3 and myosin II to complementary cortical domains.

Cells sense and respond to mechanical force. However, the mechanisms of transduction of extracellular matrix (ECM) forces to biochemical signals are not known. After removing the cell membrane and soluble proteins by Triton X-100 extraction, we found that the remaining complex (Triton cytoskeletons) activated Rap1 upon stretch. Rap1 guanine nucleotide exchange factor, C3G, was required for this activation; C3G as well as the adaptor protein, CrkII, in cell extract bound to Triton cytoskeletons in a stretch-dependent manner. CrkII binding, which was Cas dependent, correlated with stretch-dependent tyrosine phosphorylation of proteins in Triton cytoskeletons including Cas at the contacts with ECM. These in vitro findings were compatible with in vivo observations of stretch-enhanced phosphotyrosine signals, accumulation of CrkII at cell-ECM contacts, and CrkII-Cas colocalization. We suggest that mechanical force on Triton cytoskeletons activates local tyrosine phosphorylation, which provides docking sites for cytosolic proteins, and initiates signaling to activate Rap1.

The suprachiasmatic nucleus (SCN) is the neuroanatomical locus of the mammalian circadian pacemaker. Here we demonstrate that an abrupt shift in the light/dark (LD) cycle disrupts the synchronous oscillation of circadian components in the rat SCN. The phases of the RNA cycles of the period genes Per1 and Per2 and the cryptochrome gene Cry1 shifted rapidly in the ventrolateral, photoreceptive region of the SCN, but were relatively slow to shift in the dorsomedial region. During the period of desynchrony, the animals displayed increased nighttime rest, the timing of which was inversely correlated with the expression of Per1 mRNA in the dorsomedial SCN. Molecular resynchrony required approximately 6 d after a 10 hr delay and 9 approximately 13 d after a 6 hr advance of the LD cycle and was accompanied by the reemergence of normal rest-activity patterns. This dissociation and slow resynchronization of endogenous oscillators within the SCN after an LD cycle shift suggests a mechanism for the physiological symptoms that constitute jet lag.

The interaction between "switch I/effector domain" of Ha-Ras and the Ras-binding domain (RBD, amino acid 51-131) of Raf-1 is essential for signal transduction. However, the importance of the "activator domain" (approximately corresponding to amino acids 26 -28 and 40 -49) of Ha-Ras and of the "cysteine-rich region" (CRR, amino acids 152-184) of Raf-1 have also been proposed. Here, we found that Raf-1 CRR interacts directly with Ha-Ras independently of RBD and that participation of CRR is necessary for efficient Ras-Raf binding. Furthermore, Ha-Ras carrying mutations (N26G and V45E) in the activator domain failed to bind CRR, whereas they bound RBD normally. On the contrary, Ha-Ras carrying mutations in the switch I/effector domain exhibited severely reduced ability to bind RBD, whereas their ability to bind CRR was unaffected. Mutants that bound to either RBD or CRR alone failed to activate Raf-1. Ha-Ras without post-translational modifications, which lacks the ability to activate Raf-1, selectively lost the ability to bind CRR. These results suggest that the activator domain of Ha-Ras participates in activation of Raf-1 through interaction with CRR and that post-translational modifications of Ha-Ras are required for this interaction.Ras belongs to a family of small GTP-binding proteins and plays essential roles in the regulation of cell proliferation and differentiation (for a review, see Ref. 1). Like other GTP-binding proteins, the GTP-bound form of Ras is active and able to interact with its effectors whereas the GDP-bound form is not. X-ray crystallographic studies showed that, on the protein surface of mammalian Ha-Ras, the conformation of two regions named "switch I" (Asp ) flanking the switch I have also been shown to be critical for effector activation (6 -9). These residues have been proposed to constitute the "activator domain" (Fig. 1A) (11). The activator domain is also exposed on the surface of Ha-Ras but its conformation is not much altered by GDP/GTP exchange (10, 11). In addition, post-translational modifications of Ha-Ras have also been shown to be crucial for its biological function (for a review, see Ref. 12). However, it is presently unclear how these individual structural features are involved in effector activation.Raf-1, a 74-kDa cytoplasmic serine/threonine protein kinase regulating the mitogen-activated protein kinase cascade, is one of the major effectors of Ha-Ras (for a review, see Ref. 13). Raf-1 shares three regions of conservation, termed CR1, 1 CR2, and CR3, with other Raf isoforms and homologs (Fig. 1B) (13). CR1 and CR2 are located in the N-terminal half of Raf-1, and CR3 corresponds to the C-terminal kinase domain. Activation of Raf-1 by N-terminal truncations indicates that the N-terminal portion plays an important regulatory role (14). The minimal region of Raf-1 responsible for the interaction with Ha-Ras has been mapped into 81 amino acids in CR1, RBD (amino acids 51-131) (15), and mutational analyses have suggested that RBD interacts directly with the switch I of . Howeve...

Actomyosin contraction powers the sealing of epithelial sheets during embryogenesis and wound closure; however, the mechanisms are poorly understood. After laser ablation wounding of Madin–Darby canine kidney cell monolayers, we observed distinct steps in wound closure from time-lapse images of myosin distribution during resealing. Immediately upon wounding, actin and myosin II regulatory light chain accumulated at two locations: (1) in a ring adjacent to the tight junction that circumscribed the wound and (2) in fibers at the base of the cell in membranes extending over the wound site. Rho-kinase activity was required for assembly of the myosin ring, and myosin II activity was required for contraction but not for basal membrane extension. As it contracted, the myosin ring moved toward the basal membrane with ZO-1 and Rho-kinase. Thus, we suggest that tight junctions serve as attachment points for the actomyosin ring during wound closure and that Rho-kinase is required for localization and activation of the contractile ring.

Epithelial cell-cell interactions require localized adhesive interactions between E-cadherin on opposing membranes and the activation of downstream signaling pathways that affect membrane and actin dynamics. However, it is not known whether E-cadherin engagement and activation of these signaling pathways are locally coordinated or whether signaling is sustained or locally down-regulated like other receptor-mediated pathways. To obtain high spatiotemporal resolution of immediate-early signaling events upon E-cadherin engagement, we used laser tweezers to place beads coated with functional E-cadherin extracellular domain on cells. We show that cellular E-cadherin accumulated rapidly around beads, reaching a sustained plateau level in 1-3 min. Phosphoinositides and Rac1 co-accumulated with E-cadherin, reached peak levels with E-cadherin, but then rapidly dispersed. Both E-cadherin and Rac1 accumulated independently of Rac1 GTP binding/hydrolysis, but these activities were required for Rac1 dispersal. E-cadherin accumulation was dependent on membrane dynamics and actin polymerization, but actin did not stably co-accumulate with E-cadherin; mathematical modeling showed that diffusionmediated trapping could account for the initial E-cadherin accumulation. We propose that initial E-cadherin accumulation requires active membrane dynamics and involves diffusion-mediated trapping at contact sites; to propagate further contacts, phosphatidylinositol 3-kinase and Rac1 are transiently activated by E-cadherin engagement and initiate a new round of membrane dynamics, but they are subsequently suppressed at that site to allow maintenance of weak E-cadherin mediated adhesion.Members of the cadherin family of Ca 2ϩ -dependent cell adhesion proteins initiate cell adhesion by trans pairing of cadherins on opposing cell surfaces (1, 2) and then stabilize adhesion by accumulating in the plane of the membrane (3-6) by a process thought to involve the actin cytoskeleton. Live cell analysis of asynchronous cell-cell contacts indicates that initial interactions occur opportunistically between opposing lamellipodia formed as cells crawl. Subsequent expansion of the contact occurs through interactions between additional lamellipodia confined to the perimeter of the contact, whereas lamellipodia activity generally decreases within the contact (6 -8). However, many questions remain. It is unclear whether E-cadherin engagement results in or is the consequence of downstream activation of membrane and actin dynamics, whether E-cadherin accumulation is an active process driven by actin assembly, or how lamellipodia activity is locally regulated during cell-cell adhesion.Actin and membrane dynamics mediated by Rho family GTPases have been implicated in regulating lamellipodia activity and cadherin adhesion (5, 9 -12). Reports show that Rac1 is activated upon E-cadherin adhesion (9, 13, 14) and co-localized with E-cadherin after 30 min (11), and that products of PI 4 3-kinase (8, 15-17), PI(3,4)-bisphosphate and PI(3,4,5)-triphosphate (PI phosphat...

Summary Interactions between epithelial cells are mediated by adherens junctions that are dynamically regulated during development. Here we show that the turnover of β-catenin is increased at cell interfaces that are targeted for disassembly during Drosophila axis elongation. The Abl tyrosine kinase is concentrated at specific planar junctions and is necessary for polarized β-catenin localization and dynamics. abl mutant embryos have decreased β-catenin turnover at shrinking edges, and these defects are accompanied a reduction in multicellular rosette formation and axis elongation. Abl promotes β-catenin phosphorylation on the conserved tyrosine 667 and expression of an unphosphorylatable β-catenin mutant recapitulates the defects of abl mutants. Notably, a phosphomimetic β-cateninY667E mutation is sufficient to increase β-catenin turnover and rescues axis elongation in abl deficient embryos. These results demonstrate that the asymmetrically localized Abl tyrosine kinase directs planar polarized junctional remodeling during Drosophila axis elongation through the tyrosine phosphorylation of β-catenin.