The transdifferentiation of epithelial cells into motile mesenchymal cells, a process known as epithelial-mesenchymal transition (EMT), is integral in development, wound healing and stem cell behaviour, and contributes pathologically to fibrosis and cancer progression. This switch in cell differentiation and behaviour is mediated by key transcription factors, including SNAIL, zinc-finger E-box-binding (ZEB) and basic helix-loop-helix transcription factors, the functions of which are finely regulated at the transcriptional, translational and post-translational levels. The reprogramming of gene expression during EMT, as well as non-transcriptional changes, are initiated and controlled by signalling pathways that respond to extracellular cues. Among these, transforming growth factor-β (TGFβ) family signalling has a predominant role; however, the convergence of signalling pathways is essential for EMT.

Molecular mechanisms of epithelial–mesenchymal transition

The actin cytoskeleton mediates a variety of essential biological functions in all eukaryotic cells. In addition to providing a structural framework around which cell shape and polarity are defined, its dynamic properties provide the driving force for cells to move and to divide. Understanding the biochemical mechanisms that control the organization of actin is thus a major goal of contemporary cell biology, with implications for health and disease. Members of the Rho family of small guanosine triphosphatases have emerged as key regulators of the actin cytoskeleton, and furthermore, through their interaction with multiple target proteins, they ensure coordinated control of other cellular activities such as gene transcription and adhesion.

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Rho GTPases and the Actin Cytoskeleton

Normal tissue cells are generally not viable when suspended in a fluid and are therefore said to be anchorage dependent. Such cells must adhere to a solid, but a solid can be as rigid as glass or softer than a baby's skin. The behavior of some cells on soft materials is characteristic of important phenotypes; for example, cell growth on soft agar gels is used to identify cancer cells. However, an understanding of how tissue cells-including fibroblasts, myocytes, neurons, and other cell types-sense matrix stiffness is just emerging with quantitative studies of cells adhering to gels (or to other cells) with which elasticity can be tuned to approximate that of tissues. Key roles in molecular pathways are played by adhesion complexes and the actinmyosin cytoskeleton, whose contractile forces are transmitted through transcellular structures. The feedback of local matrix stiffness on cell state likely has important implications for development, differentiation, disease, and regeneration.

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Tissue Cells Feel and Respond to the Stiffness of Their Substrate

Cell migration is a highly integrated multistep process that orchestrates embryonic morphogenesis; contributes to tissue repair and regeneration; and drives disease progression in cancer, mental retardation, atherosclerosis, and arthritis. The migrating cell is highly polarized with complex regulatory pathways that spatially and temporally integrate its component processes. This review describes the mechanisms underlying the major steps of migration and the signaling pathways that regulate them, and outlines recent advances investigating the nature of polarity in migrating cells and the pathways that establish it.

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Cell migration: integrating signals from front to back.

Rho GTPases are molecular switches that control a wide variety of signal transduction pathways in all eukaryotic cells. They are known principally for their pivotal role in regulating the actin cytoskeleton, but their ability to influence cell polarity, microtubule dynamics, membrane transport pathways and transcription factor activity is probably just as significant. Underlying this biological complexity is a simple biochemical idea, namely that by switching on a single GTPase, several distinct signalling pathways can be coordinately activated. With spatial and temporal activation of multiple switches factored in, it is not surprising to find Rho GTPases having such a prominent role in eukaryotic cell biology.

Rho GTPases in cell biology.

p160(ROCK) is a protein serine/threonine kinase that binds to GTP-Rho and is activated by this binding. We have recently found that the expression of p160(ROCK) induces focal adhesions and stress fibres in HeLa cells, whereas a dominant-negative form of this kinase suppresses Rho-induced formation of these structures, suggesting that this kinase is a downstream target of Rho in this process [Ishizaki, Naito, Fujisawa, Maekawa, Watanabe, Saito and Narumiya (1997) FEBS Lett. 404, 118-124]. To find out the mode of action of p160(ROCK), we developed immunoblotting with an anti-p160(ROCK) antibody and investigated the subcellular localization of p160(ROCK) during platelet aggregation. In resting human platelets, more than 90% of p160(ROCK) was present in the Triton X-100-soluble fraction. When platelets were stimulated with thrombin, approx. 10% of p160(ROCK) was translocated to the Triton X-100-insoluble fraction. This translocation was detected as early as 20 s after stimulation and reached a maximum at 5 min; it was suppressed by the addition of EDTA or an Arg-Gly-Asp-Ser peptide (RGDS), both of which inhibit integrin alphaIIbbeta3-mediated platelet aggregation. Using [32P]Pi-loaded platelets, we found that p160(ROCK) was phosphorylated in response to stimulation by thrombin. This phosphorylation, however, was not affected by the addition of EDTA and RGDS. These results suggest that p160(ROCK) translocates to cytoskeleton in a manner dependent on integrin ligation and works in an early stage of cytoskeletal reorganization in thrombin-stimulated platelets.

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Integrin-dependent translocation of p160ROCK to cytoskeletal complex in thrombin-stimulated human platelets.

Rho-associated coiled-coil-forming protein serine/threonine kinase (ROCK) consisting of two isoforms, ROCK-I and ROCK-II, functions downstream of the small GTPase Rho for assembly of actomyosin bundles. To examine the role of ROCK isoforms in vivo, we previously generated and examined mice deficient in each of the two isoforms individually. Here, we further examined the in vivo role of ROCK isoforms by generating mice deficient in both isoforms. Cross-mating of ROCK-I(+/-) ROCK-II(+/-) double heterozygous mice showed that all of the ROCK-I(-/-) ROCK-II(-/-) homozygous mice die in utero before 9.5 days post-coitum (dpc) and ROCK-I(-/-) ROCK-II(+/-) homo-heterozygous or ROCK-I(+/-) ROCK-II(-/-) hetero-homozygous mice die during a period from 9.5 to 12.5 dpc, whereas mice of other genotypes survive until 12.5 dpc with the expected Mendelian ratio. All of the ROCK-I(+/-) ROCK-II(-/-) or ROCK-I(-/-) ROCK-II(+/-) mice showed impaired body turning and defective vascular remodeling in the yolk sac. Impairment of vascular remodeling was also observed in wild-type embryos treated ex vivo with a ROCK inhibitor, Y-27632. These results suggest that ROCK isoforms function redundantly during embryogenesis and play a critical role in vascular development.

/pdf/impaired-vascular-remodeling-in-the-yolk-sac-of-embryos-34xc82xmq1.pdf

Impaired vascular remodeling in the yolk sac of embryos deficient in ROCK-I and ROCK-II

Recruitment of specific molecules to a specific membrane site is essential for communication between specialized membranous organelles. In the present study, we identified IQGAP1 as a novel GDP-bound-Rab27a-interacting protein. We found that IQGAP1 interacts with GDP-bound Rab27a when it forms a complex with GTP-bound Cdc42. We also found that IQGAP1 regulates the endocytosis of insulin secretory membranes. Silencing of IQGAP1 inhibits both endocytosis and the glucose-induced redistribution of endocytic machinery, including Rab27a and its binding protein coronin 3. These processes can also be inhibited by disruption of the trimeric complex with dominant negative IQGAP1 and Cdc42. These results indicate that activation of Cdc42 in response to the insulin secretagogue glucose recruits endocytic machinery to IQGAP1 at the cell periphery and regulates endocytosis at this membrane site.

Activated Cdc42-Bound IQGAP1 Determines the Cellular Endocytic Site

Protective effect of hydrogen sulfide on pancreatic beta-cells

The small GTPase Rho regulates cell morphogenesis through remodeling of the actin cytoskeleton. While Rho is overexpressed in many clinical cancers, the role of Rho signaling in oncogenesis remains unknown. mDia1 is a Rho effector producing straight actin filaments. Here we transduced mouse embryonic fibroblasts from mDia1-deficient mice with temperature-sensitive v-Src and examined the involvement and mechanism of the Rho-mDia1 pathway in Src-induced oncogenesis. We showed that in v-Src-transduced mDia1-deficient cells, formation of actin filaments is suppressed, and v-Src in the perinuclear region does not move to focal adhesions upon a temperature shift. Consequently, membrane translocation of v-Src, v-Src-induced morphological transformation, and podosome formation are all suppressed in mDia1-deficient cells with impaired tyrosine phosphorylation. mDia1-deficient cells show reduced transformation in vitro as examined by focus formation and colony formation in soft agar and exhibit suppressed tumorigenesis and invasion when implanted in nude mice in vivo. Given overexpression of c-Src in various cancers, these findings suggest that Rho-mDia1 signaling facilitates malignant transformation and invasion by manipulating the actin cytoskeleton and targeting Src to the cell periphery.

Toshimasa Ishizaki

Papers

Integrin-dependent translocation of p160ROCK to cytoskeletal complex in thrombin-stimulated human platelets.

Impaired vascular remodeling in the yolk sac of embryos deficient in ROCK-I and ROCK-II

Activated Cdc42-Bound IQGAP1 Determines the Cellular Endocytic Site

Protective effect of hydrogen sulfide on pancreatic beta-cells

mDia1 targets v-Src to the cell periphery and facilitates cell transformation, tumorigenesis, and invasion