Involvement of Cdc42 small G protein in cell-cell adhesion, migration and morphology of MDCK cells.
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The Rho small G protein family consists of the Rho, Rac, and Cdc42 subfamilies and regulates various cell functions through reorganization of the actin cytoskeleton. We previously showed that the Rho subfamily regulates the formation of stress fibers and focal adhesions whereas the Rac subfamily regulates the E-cadherin-based cell-cell adhesion in MDCK cells. We studied here the function of the Cdc42 subfamily, consisting of two members, Cdc42Hs and G25k, in cell adhesion, migration, and morphology of MDCK cells. For this purpose, we made and used MDCK cell lines stably expressing each of dominant active mutants of Cdc42Hs (sMDCK-Cdc42HsDA) and G25K (sMDCK-G25KDA). Actin filaments at the cell-cell adhesion sites increased in both sMDCK-Cdc42HsDA and -G25KDA cells. Both E-cadherin and beta-catenin, adherens junctional proteins, at the cell-cell adhesion sites also increased in both sMDCK-Cdc42HsDA and -G25KDA cells. Electron microscopic analysis revealed that sMDCK-Cdc42HsDA cells tightly contacted with each other throughout the lateral membranes. Moreover, both the HGF- and TPA-induced disruption of the cadherin-based cell-cell adhesion and the subsequent cell migration were inhibited in both sMDCK-Cdc42HsDA and -G25KDA cells. Co-expression of the dominant negative mutant of Rac1, a member of the Rac subfamily, with the dominant active mutant of Cdc42Hs did not inhibit the increased accumulation of actin filaments at the cell-cell adhesion sites. These results suggest that the Cdc42 subfamily is involved in the cadherin-based cell-cell adhesion in a manner independent of the Rac subfamily. Furthermore, the cells were frequently enveloped by the large multinuclear cells in both sMDCK-Cdc42HsDA and -G25KDA cells. Video microscopic analysis revealed that the cells were engulfed by the large cells during cytokinesis. (+info)
Signaling from Rho to the actin cytoskeleton through protein kinases ROCK and LIM-kinase.
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The actin cytoskeleton undergoes extensive remodeling during cell morphogenesis and motility. The small guanosine triphosphatase Rho regulates such remodeling, but the underlying mechanisms of this regulation remain unclear. Cofilin exhibits actin-depolymerizing activity that is inhibited as a result of its phosphorylation by LIM-kinase. Cofilin was phosphorylated in N1E-115 neuroblastoma cells during lysophosphatidic acid-induced, Rho-mediated neurite retraction. This phosphorylation was sensitive to Y-27632, a specific inhibitor of the Rho-associated kinase ROCK. ROCK, which is a downstream effector of Rho, did not phosphorylate cofilin directly but phosphorylated LIM-kinase, which in turn was activated to phosphorylate cofilin. Overexpression of LIM-kinase in HeLa cells induced the formation of actin stress fibers in a Y-27632-sensitive manner. These results indicate that phosphorylation of LIM-kinase by ROCK and consequently increased phosphorylation of cofilin by LIM-kinase contribute to Rho-induced reorganization of the actin cytoskeleton. (+info)
I-band titin in cardiac muscle is a three-element molecular spring and is critical for maintaining thin filament structure.
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In cardiac muscle, the giant protein titin exists in different length isoforms expressed in the molecule's I-band region. Both isoforms, termed N2-A and N2-B, comprise stretches of Ig-like modules separated by the PEVK domain. Central I-band titin also contains isoform-specific Ig-motifs and nonmodular sequences, notably a longer insertion in N2-B. We investigated the elastic behavior of the I-band isoforms by using single-myofibril mechanics, immunofluorescence microscopy, and immunoelectron microscopy of rabbit cardiac sarcomeres stained with sequence-assigned antibodies. Moreover, we overexpressed constructs from the N2-B region in chick cardiac cells to search for possible structural properties of this cardiac-specific segment. We found that cardiac titin contains three distinct elastic elements: poly-Ig regions, the PEVK domain, and the N2-B sequence insertion, which extends approximately 60 nm at high physiological stretch. Recruitment of all three elements allows cardiac titin to extend fully reversibly at physiological sarcomere lengths, without the need to unfold Ig domains. Overexpressing the entire N2-B region or its NH(2) terminus in cardiac myocytes greatly disrupted thin filament, but not thick filament structure. Our results strongly suggest that the NH(2)-terminal N2-B domains are necessary to stabilize thin filament integrity. N2-B-titin emerges as a unique region critical for both reversible extensibility and structural maintenance of cardiac myofibrils. (+info)
Subcellular localization of GFP-myosin-V in live mouse melanocytes.
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Class-V myosins are two-headed actin-based mechanoenzymes that function in the transport and subcellular localization of organelles and possibly in the outgrowth of cellular processes. To determine which domains of myosin-V are involved in intracellular localization of this motor protein, we have expressed fusions of the green fluorescent protein with segments from two distinct myosin-V heavy chains. The expression patterns of constructs encoding four different domains of chick brain myosin-Va were compared to a single construct encoding the globular tail region of mouse myosin-Vb. In transfected mouse melanocytes, expression of the NH(2)-terminal head (catalytic domain) of chick brain myosin-Va codistributed with actin filaments throughout the cytoplasm. A similar construct encoding the myosin-Va head with the associated neck (light chain binding sites), also codistributed with actin filaments. The GFP-head-neck peptide was also highly concentrated in the tips of filopodia in B16 melanocytes wild type for myosin-Va (MYO5a gene), but was concentrated throughout the entire filopodia of S91-6 melanocytes derived from dilute mice with mutations in the MYO5a gene. Evidence is also presented that the globular tail of myosin-Va, but not myosin-Vb, targets this motor molecule to the centrosome as confirmed by colocalization in cells stained with antibodies to (gamma)-tubulin. Expression of the GFP-myosin-Va globular tail causes displacement of endogenous myosin-V from centrosomes as visualized by immunolabeling with antibodies to the head domain of myosin-V. Treatment with the microtubule-disrupting drug nocodazole markedly reduces myosin-V staining at the centrosome. In contrast, there was no detectable diminution of myosin-V staining at the centrosome in cells treated with the actin filament-disrupting drug cytochalasin D. Thus, while localization of the myosin-V motor domain to actin-rich regions is consistent with conventional models of actomyosin-based motility, localization to the centrosome occurs in the complete absence of the myosin-V motor domain and is dependent on intact microtubules. (+info)
Differential activation of focal adhesion kinase, Rho and Rac by the ninth and tenth FIII domains of fibronectin.
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Fibronectins are widely expressed extracellular matrix ligands that are essential for many biological processes. Fibronectin-induced signaling pathways are elicited in diverse cell types when specific integrin receptors bind to the ninth and tenth FIII domains, FIII9-10. Integrin-mediated signal transduction involves activation of signaling pathways of the growth factor-dependent Ras-related small GTP-binding proteins Rho and Rac, and phosphorylation of focal adhesion kinase. We have dissected the requirement of FIII9 and FIII10 for Rho and Rac activity and phosphorylation of focal adhesion kinase in BHK fibroblasts and Swiss 3T3 cells. We demonstrate that FIII10 supports cell attachment but does not induce phosphorylation of focal adhesion kinase. In Swiss 3T3 cells, growth factor-independent phosphorylation of focal adhesion kinase and downstream adhesion events are dependent upon the presence of FIII9 in the intact FIII9-10 pair, whereas FIII10-mediated focal adhesion kinase phosphorylation requires a synergistic signal from growth factors. Furthermore, FIII10 is able to elicit cellular responses mediated by Rho, but not Rac, whereas FIII9-10 can elicit both Rho- and Rac-mediated responses. We propose that activation of specific integrin subunits by the FIII10 and FIII9-10 ligands elicits distinct signaling events. This may represent a general molecular mechanism for activation of receptor-specific signaling pathways by a multi-domain ligand. (+info)
Activation of the small GTPase Cdc42 by the inflammatory cytokines TNF(alpha) and IL-1, and by the Epstein-Barr virus transforming protein LMP1.
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Cdc42, a Rho-family GTPase, has been implicated in several signal transduction pathways, including organization of the actin cytoskeleton, activation of the c-Jun N-terminal MAP kinase (JNK) and stimulation of the nuclear transcription factor kappa B (NF(kappa)B). We report here that exposure of fibroblasts to the inflammatory cytokines tumor necrosis factor (alpha) (TNF(alpha)) and interleukin-1 (IL-1) triggers the activation of Cdc42 leading first to filopodia formation and subsequently to Rac and Rho activation. Inhibition of Cdc42 completely suppresses cytokine-induced actin polymerization, but not activation of JNK or NF(kappa)B. The latent membrane protein 1 of Epstein-Barr virus, LMP1, is thought to mimic constitutively activated TNF family receptors. When expressed in fibroblasts, LMP1 stimulates Cdc42-dependent filopodia formation as well as JNK and NF(kappa)B activation. Using LMP1 mutants, we show that activation of Cdc42 and JNK/NF(kappa)B occur through distinct pathways and that Cdc42 activation is independent of LMP1's interaction with TRADD and TRAF proteins. (+info)
Differential effects of ovariectomy on calcium activation of cardiac and soleus myofilaments.
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The hypothesis that ovarian sex hormone deficiency affects cardiac myofilament activation was tested. Chemically skinned ventricular trabeculae and single soleus muscle fibers were prepared from 10- and 14-wk ovariectomized and control rats. Tension-pCa (-log [Ca(2+)]) relations of left ventricular trabeculae and soleus fibers were compared to test whether thin filament proteins are potential sites of modulated activation. Trabeculae from ovariectomized rats exhibited a significant increase in Ca(2+) sensitivity with no change in maximal tension-generating ability. In contrast, soleus fibers demonstrated no shift in Ca(2+) sensitivity but generated significantly less maximal tension. No changes in thin filament protein isoform expression or loss of thin filament proteins were apparent in the trabeculae or soleus fibers from ovariectomized rats. Although not directly tested, our results are consistent with a possible modulation of regulatory proteins (e.g., cardiac troponin I) to account for the observed change in myofilament responsiveness of hearts from ovariectomized rats. Other possible mechanisms for the altered myocardial Ca(2+) sensitivity after ovariectomy are discussed. (+info)
In vivo, villin is required for Ca(2+)-dependent F-actin disruption in intestinal brush borders.
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Villin is an actin-binding protein localized in intestinal and kidney brush borders. In vitro, villin has been demonstrated to bundle and sever F-actin in a Ca(2+)-dependent manner. We generated knockout mice to study the role of villin in vivo. In villin-null mice, no noticeable changes were observed in the ultrastructure of the microvilli or in the localization and expression of the actin-binding and membrane proteins of the intestine. Interestingly, the response to elevated intracellular Ca(2+) differed significantly between mutant and normal mice. In wild-type animals, isolated brush borders were disrupted by the addition of Ca(2+), whereas Ca(2+) had no effect in villin-null isolates. Moreover, increase in intracellular Ca(2+) by serosal carbachol or mucosal Ca(2+) ionophore A23187 application abolished the F-actin labeling only in the brush border of wild-type animals. This F-actin disruption was also observed in physiological fasting/refeeding experiments. Oral administration of dextran sulfate sodium, an agent that causes colonic epithelial injury, induced large mucosal lesions resulting in a higher death probability in mice lacking villin, 36 +/- 9.6%, compared with wild-type mice, 70 +/- 8.8%, at day 13. These results suggest that in vivo, villin is not necessary for the bundling of F-actin microfilaments, whereas it is necessary for the reorganization elicited by various signals. We postulate that this property might be involved in cellular plasticity related to cell injury. (+info)