Rho GDP dissociation inhibitor-mediated disruption of Rho GTPase activity impairs lens fiber cell migration, elongation and survival

To explore the role of the Rho GTPases in lens morphogenesis, we overexpressed bovine Rho GDP dissociation inhibitor (RhoGDIα), which serves as a negative regulator of Rho, Rac and Cdc42 GTPase activity, in a lens-specific manner in transgenic mice. This was achieved using a chimeric promoter of δ-crystallin enhancer and αA-crystallin, which is active at embryonic day 12. Several individual transgenic (Tg) lines were obtained, and exhibited ocular specific phenotype comprised of microphthalmic eyes with lens opacity. The overexpression of bovine RhoGDIα disrupted membrane translocation of Rho, Rac and Cdc42 GTPases in Tg lenses. Transgenic lenses also revealed abnormalities in the migration pattern, elongation and organization of lens fibers. These changes appeared to be associated with impaired organization of the actin cytoskeleton and cell-cell adhesions. At E14.5, the size of the RhoGDIα Tg lenses was larger compared to wild type (WT) and the central lens epithelium and differentiating fibers exhibited an abnormal increase of bromo-deoxy-uridine incorporation. Postnatal Tg eyes, however, were much smaller in size compared to WT eyes, revealing increased apoptosis in the disrupted lens fibers. Taken together, these data demonstrate a critical role for Rho GTPase-dependent signaling pathways in processes underlying morphogenesis, fiber cell migration, elongation and survival in the developing lens.     

Control of developmental networks by Rac/Rho small GTPases: How cytoskeletal changes during embryogenesis are orchestrated

Small GTPases in the Rho family act as major nodes with functions beyond cytoskeletal rearrangements shaping the Caenorhabditis elegans embryo during development. These small GTPases are key signal transducers that integrate diverse developmental signals to produce a coordinated response in the cell. In C. elegans, the best studied members of these highly conserved Rho family small GTPases, RHO‐1/RhoA, CED‐10/Rac, and CDC‐42, are crucial in several cellular processes dealing with cytoskeletal reorganization. In this review, we update the functions described for the Rho family small GTPases in spindle orientation and cell division, engulfment, and cellular movements during C. elegans embryogenesis, focusing on the Rho subfamily Rac. Please also see the video abstract here     

Small Rho GTPases are important for acinus formation in a human salivary gland cell line

Rho GTPases participate in a wide variety of signal transduction pathways regulating the actin cytoskeleton, gene expression, cellular migration and proliferation. The aim of this study was to evaluate the role of Rho GTPases in signal transduction pathways during acinus formation in a human salivary gland (HSG) cell line initiated by extracellular matrix (ECM; Matrigel) alone or in combination with epidermal growth factor, basic fibroblast growth factor and lysophosphatidic acid (LPA). Immunohistochemical and Western blotting analyses showed that HSG cells contained RhoA, RhoB, Rac1 and Cdc42 proteins. All growth factors enhanced the effects of ECM on acinus formation, in a pathway dependent on PI3-kinase and Rho GTPases. The role of ROCK, a major RhoA effector, seemed limited to cortical actin polymerization. LPA stimulated cell migration and acinus formation in a PI3-kinase-independent pathway. The results suggest that Rho proteins are important for epithelial-mesenchymal interactions during salivary gland development.This work was supported by FAPESP (grant numbers: 97/09507-6, 01/09047-2).     

Model of coupled transient changes of Rac, Rho, adhesions and stress fibers alignment in endothelial cells responding to shear stress

Interactions of cell adhesions, Rho GTPases and actin in the endothelial cells' response to external forces are complex and not fully understood, but a qualitative understanding of the mechanosensory response begins to emerge. Here, we formulate a mathematical model of the coupled dynamics of cell adhesions, small GTPases Rac and Rho and actin stress fibers guiding a directional reorganization of the actin cytoskeleton. The model is based on the assumptions that the interconnected cytoskeleton transfers the shear force to the adhesion sites, which in turn transduce the force into a chemical signal that activates integrins at the basal surface of the cell. Subsequently, activated and ligated integrins signal and transiently de-activate Rho, causing the disassembly of actin stress fibers and inhibiting the maturation of focal complexes into focal contacts. Focal complexes and ligated integrins activate Rac, which in turn enhances focal complex assembly. When Rho activity recovers, stress fibers re-assemble and promote the maturation of focal complexes into focal contacts. Merging stress fibers self-align, while the elevated level of Rac activity at the downstream edge of the cell is translated into an alignment of the cells and the newly forming stress fibers in the flow direction. Numerical solutions of the model equations predict transient changes in Rac and Rho that compare well with published experimental results. We report quantitative data on early alignment of the stress fibers and its dependence on cell shape that agrees with the model.     

PAK1 and PAK2 have different roles in HGF-induced morphological responses

Hepatocyte growth factor (HGF) stimulates dissociation of epithelial cells (scattering) and cell migration. Several Rho GTPases are required for HGF-induced scattering. PAK1 and PAK2 are members of the p21-activated kinase (PAK) family of serine/threonine kinases, and are activated by the Rho GTPases Rac and Cdc42. Here we investigate the contributions of PAK1 and PAK2 to HGF-induced motile response. HGF stimulates phosphorylation of PAK1 and PAK2. Knockdown of PAK1 inhibits HGF-stimulated migration and loss of cell–cell junctions in DU145 prostate carcinoma cells, whereas knockdown of PAK2 enhances loss of cell–cell junctions and increases lamellipodium extension but does not affect migration speed. On the other hand, in PC3 prostate carcinoma cells, which lack cell–cell junctions, knockdown of PAK1 or PAK2 reduces HGF-stimulated migration. PAK2 knockdown increases phosphorylation of PAK1, indicating that PAK2 provides a negative feedback on PAK1. We hypothesise that PAK2 acts in part via PAK1 to regulate HGF-induced scattering.     

Decreased transport of leptin across the blood–brain barrier in rats lacking the short form of the leptin receptor

Leptin is produced in adipose tissue in the periphery, but its satiety effect is exerted in the CNS that it reaches by a saturable transport system across the blood–brain barrier (BBB). The short form of the leptin receptor has been hypothesized to be the transporter, with impaired transport of leptin being implicated in obesity. In Koletsky rats, the splice variant that gives rise to the short form of the leptin receptor contains a point mutation that results in marked obesity. We studied the transport of leptin across the BBB in Koletsky rats and found it to be significantly less than in their lean littermates. By contrast, Sprague–Dawley rats matched in weight to each of these two groups showed no difference in the blood–to–brain influx of leptin. HPLC showed that most of the leptin crossing the BBB in rats remained intact and capillary depletion showed that most of the leptin reached the parenchyma of the brain. The results indicate that the short form of the leptin receptor is involved in the transport of leptin across the BBB.