Regulation of Cell–Cell Adhesion by Rac and Rho Small G Proteins in MDCK Cells

The Rho small G protein family, consisting of the Rho, Rac, and Cdc42 subfamilies, regulates various cell functions, such as cell shape change, cell motility, and cytokinesis, through reorganization of the actin cytoskeleton. We show here that the Rac and Rho subfamilies furthermore regulate cell–cell adhesion. We prepared MDCK cell lines stably expressing each of dominant active mutants of RhoA (sMDCK-RhoDA), Rac1 (sMDCK-RacDA), and Cdc42 (sMDCK-Cdc42DA) and dominant negative mutants of Rac1 (sMDCK-RacDN) and Cdc42 (sMDCK-Cdc42DN) and analyzed cell adhesion in these cell lines. The actin filaments at the cell–cell adhesion sites markedly increased in sMDCK-RacDA cells, whereas they apparently decreased in sMDCK-RacDN cells, compared with those in wild-type MDCK cells. Both E-cadherin and β-catenin, adherens junctional proteins, at the cell–cell adhesion sites also increased in sMDCK-RacDA cells, whereas both of them decreased in sMDCK-RacDN cells. The detergent solubility assay indicated that the amount of detergent-insoluble E-cadherin increased in sMDCK-RacDA cells, whereas it slightly decreased in sMDCK-RacDN cells, compared with that in wild-type MDCK cells. In sMDCK-RhoDA, -Cdc42DA, and -Cdc42DN cells, neither of these proteins at the cell–cell adhesion sites was apparently affected. ZO-1, a tight junctional protein, was not apparently affected in any of the transformant cell lines. Electron microscopic analysis revealed that sMDCK-RacDA cells tightly made contact with each other throughout the lateral membranes, whereas wild-type MDCK and sMDCK-RacDN cells tightly and linearly made contact at the apical area of the lateral membranes. These results suggest that the Rac subfamily regulates the formation of the cadherin-based cell– cell adhesion. Microinjection of C3 into wild-type MDCK cells inhibited the formation of both the cadherin-based cell–cell adhesion and the tight junction, but microinjection of C3 into sMDCK-RacDA cells showed little effect on the localization of the actin filaments and E-cadherin at the cell–cell adhesion sites. These results suggest that the Rho subfamily is necessary for the formation of both the cadherin-based cell– cell adhesion and the tight junction, but not essential for the Rac subfamily-regulated, cadherin-based cell– cell adhesion.     

Rho小G蛋白介导的细胞周期调控

Rho小G蛋白作为一个信号分子家族具有多样化的功能, 可以调节细胞骨架重排 、细胞迁移、细胞极性、基因表达、细胞周期调控等. Rho小G蛋白家族对细胞周期 调控的研究主要集中在其对于有丝分裂期细胞的调节作用,包括调节有丝分裂期前 期细胞趋圆化、后期染色体排列及收缩环的收缩作用.近期的研究显示,Rho小G蛋白及其效应分子对于细胞周期G1、S、G2期的调控主要是通过影响细胞周期的正调控因子细胞周期蛋白D1 (cyclin D1) 和负调控因子细胞周期蛋白依赖型激酶相互作用蛋白1及细胞周期蛋白依赖型激酶抑制蛋白27 (p21cip1/p27kip1) 进行的.本文总结了Rho小G蛋白及其效应分子在细胞周期调控,尤其是对G1/S期调控的研究进展,并简要阐述了Rho小G蛋白介导的细胞周期调控异常与癌症发生的关系.     

The Guanine Nucleotide Exchange Factor Tiam1 Affects Neuronal Morphology; Opposing Roles for the Small GTPases Rac and Rho

The invasion-inducing T-lymphoma invasion and metastasis 1 (Tiam1) protein functions as a guanine nucleotide exchange factor (GEF) for the small GTPase Rac1. Differentiation-dependent expression of Tiam1 in the developing brain suggests a role for this GEF and its effector Rac1 in the control of neuronal morphology. Here we show that overexpression of Tiam1 induces cell spreading and affects neurite outgrowth in N1E-115 neuroblastoma cells. These effects are Rac-dependent and strongly promoted by laminin. Overexpression of Tiam1 recruits the α6β1 integrin, a laminin receptor, to specific adhesive contacts at the cell periphery, which are different from focal contacts. Cells overexpressing Tiam1 no longer respond to lysophosphatidic acid– induced neurite retraction and cell rounding, processes mediated by Rho, suggesting that Tiam1-induced activation of Rac antagonizes Rho signaling. This inhibition can be overcome by coexpression of constitutively active RhoA, which may indicate that regulation occurs at the level of Rho or upstream. Conversely, neurite formation induced by Tiam1 or Rac1 is further promoted by inactivating Rho. These results demonstrate that Rac- and Rho-mediated pathways oppose each other during neurite formation and that a balance between these pathways determines neuronal morphology. Furthermore, our data underscore the potential role of Tiam1 as a specific regulator of Rac during neurite formation and illustrate the importance of reciprocal interactions between the cytoskeleton and the extracellular matrix during this process.     

EphA2 Engages Git1 to Suppress Arf6 Activity Modulating Epithelial Cell–Cell Contacts

ADP-ribosylation factor (Arf) 6 activity is crucially involved in the regulation of E-cadherin–based cell–cell adhesions. Erythropoietin-producing hepatocellular carcinoma (Eph)-family receptors recognize ligands, namely, ephrins, anchored to the membrane of apposing cells, and they mediate cell–cell contact-dependent events. Here, we found that Arf6 activity is down-regulated in Madin-Darby canine kidney cells, which is dependent on cell density and calcium ion concentration, and we provide evidence of a novel signaling pathway by which ligand-activated EphA2 suppresses Arf6 activity. This EphA2-mediated suppression of Arf6 activity was linked to the induction of cell compaction and polarization, but it was independent of the down-regulation of extracellular signal-regulated kinase 1/2 kinase activity. We show that G protein-coupled receptor kinase-interacting protein (Git) 1 and noncatalytic region of tyrosine kinase (Nck) 1 are involved in this pathway, in which ligand-activated EphA2, via its phosphorylated Tyr594, binds to the Src homology 2 domain of Nck1, and then via its Src homology 3 domain binds to the synaptic localizing domain of Git1 to suppress Arf6 activity. We propose a positive feedback loop in which E-cadherin–based cell–cell contacts enhance EphA-ephrinA signaling, which in turn down-regulates Arf6 activity to enhance E-cadherin–based cell–cell contacts as well as the apical-basal polarization of epithelial cells.     

A Safeguard Mechanism Regulates Rho GTPases to Coordinate Cytokinesis with the Establishment of Cell Polarity

The spatiotemporal control of cell polarity is crucial for the development of multicellular organisms and for reliable polarity switches during cell cycle progression in unicellular systems. A tight control of cell polarity is especially important in haploid budding yeast, where the new polarity site (bud site) is established next to the cell division site after cell separation. How cells coordinate the temporal establishment of two adjacent polarity sites remains elusive. Here, we report that the bud neck associated protein Gps1 (GTPase-mediated polarity switch 1) establishes a novel polarity cue that concomitantly sustains Rho1-dependent polarization and inhibits premature Cdc42 activation at the site of cytokinesis. Failure of Gps1 regulation leads to daughter cell death due to rebudding inside the old bud site. Our findings provide unexpected insights into the temporal control of cytokinesis and describe the importance of a Gps1-dependent mechanism for highly accurate polarity switching between two closely connected locations.     

Dismantling Cell–Cell Contacts during Apoptosis Is Coupled to a Caspase-dependent Proteolytic Cleavage of β-Catenin

Cell death by apoptosis is a tightly regulated process that requires coordinated modification in cellular architecture. The caspase protease family has been shown to play a key role in apoptosis. Here we report that specific and ordered changes in the actin cytoskeleton take place during apoptosis.

In this context, we have dissected one of the first hallmarks in cell death, represented by the severing of contacts among neighboring cells. More specifically, we provide demonstration for the mechanism that could contribute to the disassembly of cytoskeletal organization at cell–cell adhesion. In fact, β-catenin, a known regulator of cell–cell adhesion, is proteolytically processed in different cell types after induction of apoptosis. Caspase-3 (cpp32/apopain/yama) cleaves in vitro translated β-catenin into a form which is similar in size to that observed in cells undergoing apoptosis. β-Catenin cleavage, during apoptosis in vivo and after caspase-3 treatment in vitro, removes the amino- and carboxy-terminal regions of the protein. The resulting β-catenin product is unable to bind α-catenin that is responsible for actin filament binding and organization. This evidence indicates that connection with actin filaments organized at cell–cell contacts could be dismantled during apoptosis. Our observations suggest that caspases orchestrate the specific and sequential changes in the actin cytoskeleton occurring during cell death via cleavage of different regulators of the microfilament system.

     

Two New Ypt GTPases Are Required for Exit From the Yeast trans-Golgi Compartment

Small GTPases of the Ypt/rab family are involved in the regulation of vesicular transport. These GTPases apparently function during the targeting of vesicles to the acceptor compartment. Two members of the Ypt/rab family, Ypt1p and Sec4p, have been shown to regulate early and late steps of the yeast exocytic pathway, respectively. Here we tested the role of two newly identified GTPases, Ypt31p and Ypt32p. These two proteins share 81% identity and 90% similarity, and belong to the same protein subfamily as Ypt1p and Sec4p. Yeast cells can tolerate deletion of either the YPT31 or the YPT32 gene, but not both. These observations suggest that Ypt31p and Ypt32p perform identical or overlapping functions. Cells deleted for the YPT31 gene and carrying a conditional ypt32 mutation exhibit protein transport defects in the late exocytic pathway, but not in vacuolar protein sorting. The ypt31/ 32 mutant secretory defect is clearly downstream from that displayed by a ypt1 mutant and is similar to that of sec4 mutant cells. However, electron microscopy revealed that while sec4 mutant cells accumulate secretory vesicles, ypt31/32 mutant cells accumulate aberrant Golgi structures. The ypt31/32 phenotype is epistatic to that of a sec1 mutant, which accumulates secretory vesicles. Together, these results indicate that the Ypt31/32p GTPases are required for a step that occurs in the transGolgi compartment, between the reactions regulated by Ypt1p and Sec4p. This step might involve budding of vesicles from the trans-Golgi. Alternatively, Ypt31/ 32p might promote secretion indirectly, by allowing fusion of recycling vesicles with the trans-Golgi compartment.     

Stimulation of phospholipase C-beta2 by the Rho GTPases Cdc42Hs and Rac1.

Neutrophils contain a soluble guanine-nucleotidebinding protein, made up of two components with molecular masses of 23 and 26 kDa, that mediates stimulation of phospholipase C-beta2 (PLCbeta2). We have identified the two components of the stimulatory heterodimer by amino acid sequencing as a Rho GTPase and the Rho guanine nucleotide dissociation inhibitor LyGDI. Using recombinant Rho GTPases and LyGDI, we demonstrate that PLCbeta2 is stimulated by guanosine 5'-O-(3-thiotriphosphate) (GTP[S])-activated Cdc42HsxLyGDI, but not by RhoAxLyGDI. Stimulation of PLCbeta2, which was also observed for GTP[S]-activated recombinant Rac1, was independent of LyGDI, but required C-terminal processing of Cdc42Hs/Rac1. Cdc42Hs/Rac1 also stimulated PLCbeta2 in a system made up of purified recombinant proteins, suggesting that this function is mediated by direct protein-protein interaction. The Cdc42Hs mutants F37A and Y40C failed to stimulate PLCbeta2, indicating that the Cdc42Hs effector site is involved in this interaction. The results identify PLCbeta2 as a novel effector of the Rho GTPases Cdc42Hs and Rac1, and as the first mammalian effector directly regulated by both heterotrimeric and low-molecular-mass GTP-binding proteins.     

Rho GTPases Are Involved in the Regulation of NF-κB by Genotoxic Stress

A common cellular response to genotoxic agents and inflammatory cytokines is the activation of NF-κB. Here, we addressed the question of whether small GTPases of the Rho family are involved in the stimulation of NF-κB signaling by genotoxic agents or TNFα in HeLa cells. Inhibition of isoprenylation of Rho proteins by use of the HMG-CoA reductase inhibitor lovastatin attenuated UV-, doxorubicin-, and TNFα-induced degradation of IκBα as well as drug-stimulated DNA binding activity of NF-κB. Furthermore, NF-κB-regulated gene expression stimulated by either UV irradiation or treatment with TNFα was abrogated by lovastatin pretreatment. This indicates that isoprenylated regulatory proteins participate in the regulation of NF-κB by DNA-damaging agents as well as by TNFα. Specific blockage of Rho signaling by Clostridium difficile toxin B attenuated UV- and doxorubicin-induced activation of NF-κB, but did not affect stimulation of NF-κB by TNFα. Obviously, signaling to NF-κB by genotoxic and nongenotoxic stimuli occurs via different molecular mechanisms, either involving Rho GTPases or not. Based on the data, we suggest Rho GTPases to be essentially required for genotoxic stress-induced signaling to NF-κB.     

Pathogenicity island-dependent activation of Rho GTPases Rac1 and Cdc42 in Helicobacter pylori infection

Helicobacter pylori has been identified as the major aetiological agent in the development of chronic gastritis and duodenal ulcer, and it plays a role in the development of gastric carcinoma. Attachment of H. pylori to gastric epithelial cells leads to nuclear and cytoskeletal responses in host cells. Here, we show that Rho GTPases Rac1 and Cdc42 were activated during infection of gastric epithelial cells with either the wild-type H. pylori or the mutant strain cagA. In contrast, no activation of Rho GTPases was observed when H. pylori mutant strains (virB7 and PAI) were used that lack functional type IV secretion apparatus. We demonstrated that H. pylori-induced activation of Rac1 and Cdc42 led to the activation of p21-activated kinase 1 (PAK1) mediating nuclear responses, whereas the mutant strain PAI had no effect on PAK1 activity. Activation of Rac1, Cdc42 and PAK1 represented a very early event in colonization of gastric epithelial cells by H. pylori. Rac1 and Cdc42 were recruited to the sites of bacterial attachment and are therefore probably involved in the regulation of local and overall cytoskeleton rearrangement in host cells. Finally, actin rearrangement and epithelial cell motility in H. pylori infection depended on the presence of a functional type IV secretion system encoded by the cag pathogenicity island (PAI).     

Tyrosine Phosphorylation and Src Family Kinases Control Keratinocyte Cell–Cell Adhesion

In their progression from the basal to upper differentiated layers of the epidermis, keratinocytes undergo significant structural changes, including establishment of close intercellular contacts. An important but so far unexplored question is how these early structural events are related to the biochemical pathways that trigger differentiation. We show here that β-catenin, γ-catenin/plakoglobin, and p120-Cas are all significantly tyrosine phosphorylated in primary mouse keratinocytes induced to differentiate by calcium, with a time course similar to that of cell junction formation. Together with these changes, there is an increased association of α-catenin and p120-Cas with E-cadherin, which is prevented by tyrosine kinase inhibition. Treatment of E-cadherin complexes with tyrosine-specific phosphatase reveals that the strength of α-catenin association is directly dependent on tyrosine phosphorylation. In parallel with the biochemical effects, tyrosine kinase inhibition suppresses formation of cell adhesive structures, and causes a significant reduction in adhesive strength of differentiating keratinocytes. The Fyn tyrosine kinase colocalizes with E-cadherin at the cell membrane in calcium-treated keratinocytes. Consistent with an involvement of this kinase, fyn-deficient keratinocytes have strongly decreased tyrosine phosphorylation levels of β- and γ-catenins and p120-Cas, and structural and functional abnormalities in cell adhesion similar to those caused by tyrosine kinase inhibitors. Whereas skin of fyn−/− mice appears normal, skin of mice with a disruption in both the fyn and src genes shows intrinsically reduced tyrosine phosphorylation of β-catenin, strongly decreased p120-Cas levels, and important structural changes consistent with impaired keratinocyte cell adhesion. Thus, unlike what has been proposed for oncogene-transformed or mitogenically stimulated cells, in differentiating keratinocytes tyrosine phosphorylation plays a positive role in control of cell adhesion, and this regulatory function appears to be important both in vitro and in vivo.     

Prohibitins Are Required for Cancer Cell Proliferation and Adhesion

Prohibitin 1 (PHB1) is a highly conserved protein that together with its homologue prohibitin 2 (PHB2) mainly localizes to the inner mitochondrial membrane. Although it was originally identified by its ability to inhibit G1/S progression in human fibroblasts, its role as tumor suppressor is debated. To determine the function of prohibitins in maintaining cell homeostasis, we generated cancer cell lines expressing prohibitin-directed shRNAs. We show that prohibitin proteins are necessary for the proliferation of cancer cells. Down-regulation of prohibitin expression drastically reduced the rate of cell division. Furthermore, mitochondrial morphology was not affected, but loss of prohibitins did lead to the degradation of the fusion protein OPA1 and, in certain cancer cell lines, to a reduced capability to exhibit anchorage-independent growth. These cancer cells also exhibited reduced adhesion to the extracellular matrix. Taken together, these observations suggest prohibitins play a crucial role in adhesion processes in the cell and thereby sustaining cancer cell propagation and survival.     

Myosin Light Chain–activating Phosphorylation Sites Are Required for Oogenesis in Drosophila

The Drosophila spaghetti squash (sqh) gene encodes the regulatory myosin light chain (RMLC) of nonmuscle myosin II. Biochemical analysis of vertebrate nonmuscle and smooth muscle myosin II has established that phosphorylation of certain amino acids of the RMLC greatly increases the actin-dependent myosin ATPase and motor activity of myosin in vitro. We have assessed the in vivo importance of these sites, which in Drosophila correspond to serine-21 and threonine-20, by creating a series of transgenes in which these specific amino acids were altered. The phenotypes of the transgenes were examined in an otherwise null mutant background during oocyte development in Drosophila females.

Germ line cystoblasts entirely lacking a functional sqh gene show severe defects in proliferation and cytokinesis. The ring canals, cytoplasmic bridges linking the oocyte to the nurse cells in the egg chamber, are abnormal, suggesting a role of myosin II in their establishment or maintenance. In addition, numerous aggregates of myosin heavy chain accumulate in the sqh null cells. Mutant sqh transgene sqh-A20, A21 in which both serine-21 and threonine-20 have been replaced by alanines behaves in most respects identically to the null allele in this system, with the exception that no heavy chain aggregates are found. In contrast, expression of sqh-A21, in which only the primary phosphorylation target serine-21 site is altered, partially restores functionality to germ line myosin II, allowing cystoblast division and oocyte development, albeit with some cytokinesis failure, defects in the rapid cytoplasmic transport from nurse cells to cytoplasm characteristic of late stage oogenesis, and some damaged ring canals. Substituting a glutamate for the serine-21 (mutant sqh-E21) allows oogenesis to be completed with minimal defects, producing eggs that can develop normally to produce fertile adults. Flies expressing sqh-A20, in which only the secondary phosphorylation site is absent, appear to be entirely wild type. Taken together, this genetic evidence argues that phosphorylation at serine-21 is critical to RMLC function in activating myosin II in vivo, but that the function can be partially provided by phosphorylation at threonine-20.

     

Cross-talk between Rho and Rac GTPases drives deterministic exploration of cellular shape space and morphological heterogeneity

One goal of cell biology is to understand how cells adopt different shapes in response to varying environmental and cellular conditions. Achieving a comprehensive understanding of the relationship between cell shape and environment requires a systems-level understanding of the signalling networks that respond to external cues and regulate the cytoskeleton. Classical biochemical and genetic approaches have identified thousands of individual components that contribute to cell shape, but it remains difficult to predict how cell shape is generated by the activity of these components using bottom-up approaches because of the complex nature of their interactions in space and time. Here, we describe the regulation of cellular shape by signalling systems using a top-down approach. We first exploit the shape diversity generated by systematic RNAi screening and comprehensively define the shape space a migratory cell explores. We suggest a simple Boolean model involving the activation of Rac and Rho GTPases in two compartments to explain the basis for all cell shapes in the dataset. Critically, we also generate a probabilistic graphical model to show how cells explore this space in a deterministic, rather than a stochastic, fashion. We validate the predictions made by our model using live-cell imaging. Our work explains how cross-talk between Rho and Rac can generate different cell shapes, and thus morphological heterogeneity, in genetically identical populations.     

Specific contributions of the small GTPases Rho, Rac, and Cdc42 to Dbl transformation.

Dbl is a representative prototype of a growing family of oncogene products that contain the Dbl homology/pleckstrin homology elements in their primary structures and are associated with a variety of neoplastic pathologies. Members of the Dbl family have been shown to function as physiological activators (guanine nucleotide exchange factors) of the Rho-like small GTPases. Although the expression of GTPase-defective versions of Rho proteins has been shown to induce a transformed phenotype under different conditions, their transformation capacity has been typically weak and incomplete relative to that exhibited by dbl-like oncogenes. Moreover, in some cases (e.g. NIH3T3 fibroblasts), expression of GTPase-defective Cdc42 results in growth inhibition. Thus, in attempting to reconstitute dbl-induced transformation of NIH3T3 fibroblasts, we have generated spontaneously activated ("fast-cycling") mutants of Cdc42, Rac1, and RhoA that mimic the functional effects of activation by the Dbl oncoprotein. When stably expressed in NIH3T3 cells, all three mutants caused the loss of serum dependence and showed increased saturation density. Furthermore, all three stable cell lines were tumorigenic when injected into nude mice. Our data demonstrate that all three Dbl targets need to be activated to promote the full complement of Dbl effects. More importantly, activation of each of these GTP-binding proteins contributes to a different and distinct facet of cellular transformation.     

Role of the small Rho GTPases Rac1 and Cdc42 in host cell invasion of Campylobacter jejuni

Host cell invasion of the food-borne pathogen Campylobacter jejuni is one of the primary reasons of tissue damage in humans but molecular mechanisms are widely unclear. Here, we show that C. jejuni triggers membrane ruffling in the eukaryotic cell followed by invasion in a very specific manner first with its tip followed by the flagellar end. To pinpoint important signalling events involved in the C. jejuni invasion process, we examined the role of small Rho family GTPases. Using specific GTPase-modifying toxins, inhibitors and GTPase expression constructs we show that Rac1 and Cdc42, but not RhoA, are involved in C. jejuni invasion. In agreement with these observations, we found that internalization of C. jejuni is accompanied by a time-dependent activation of both Rac1 and Cdc42. Finally, we show that the activation of these GTPases involves different host cell kinases and the bacterial fibronectin-binding protein CadF. Thus, CadF is a bifunctional protein which triggers bacterial binding to host cells as well as signalling leading to GTPase activation. Collectively, our results suggest that C. jejuni invade host target cells by a unique mechanism and the activation of the Rho GTPase members Rac1 and Cdc42 plays a crucial role in this entry process.     

Acute loss of Cell–Cell Communication Caused by G Protein–coupled Receptors: A Critical Role for c-Src

Gap junctions mediate cell–cell communication in almost all tissues, but little is known about their regulation by physiological stimuli. Using a novel single-electrode technique, together with dye coupling studies, we show that in cells expressing gap junction protein connexin43, cell–cell communication is rapidly disrupted by G protein–coupled receptor agonists, notably lysophosphatidic acid, thrombin, and neuropeptides. In the continuous presence of agonist, junctional communication fully recovers within 1–2 h of receptor stimulation. In contrast, a desensitization-defective G protein–coupled receptor mediates prolonged uncoupling, indicating that recovery of communication is controlled, at least in part, by receptor desensitization. Agonist-induced gap junction closure consistently follows inositol lipid breakdown and membrane depolarization and coincides with Rho-mediated cytoskeletal remodeling. However, we find that gap junction closure is independent of Ca²⁺, protein kinase C, mitogen-activated protein kinase, or membrane potential, and requires neither Rho nor Ras activation. Gap junction closure is prevented by tyrphostins, by dominant-negative c-Src, and in Src-deficient cells. Thus, G protein–coupled receptors use a Src tyrosine kinase pathway to transiently inhibit connexin43-based cell–cell communication.