Modelling cell polarization driven by synthetic spatially graded Rac activation

The small GTPase Rac is known to be an important regulator of cell polarization, cytoskeletal reorganization, and motility of mammalian cells. In recent microfluidic experiments, HeLa cells endowed with appropriate constructs were subjected to gradients of the small molecule rapamycin leading to synthetic membrane recruitment of a Rac activator and direct graded activation of membrane-associated Rac. Rac activation could thus be triggered independent of upstream signaling mechanisms otherwise responsible for transducing activating gradient signals. The response of the cells to such stimulation depended on exceeding a threshold of activated Rac. Here we develop a minimal reaction-diffusion model for the GTPase network alone and for GTPase-phosphoinositide crosstalk that is consistent with experimental observations for the polarization of the cells. The modeling suggests that mutual inhibition is a more likely mode of cell polarization than positive feedback of Rac onto its own activation. We use a new analytical tool, Local Perturbation Analysis, to approximate the partial differential equations by ordinary differential equations for local and global variables. This method helps to analyze the parameter space and behaviour of the proposed models. The models and experiments suggest that (1) spatially uniform stimulation serves to sensitize a cell to applied gradients. (2) Feedback between phosphoinositides and Rho GTPases sensitizes a cell. (3) Cell lengthening/flattening accompanying polarization can increase the sensitivity of a cell and stabilize an otherwise unstable polarization.     

YopK of Yersinia pseudotuberculosis controls translocation of Yop effectors across the eukaryotic cell membrane

Introduction of anti-host factors into eukaryotic cells by extracellular bacteria is a strategy evolved by several Gram-negative pathogens. In these pathogens, the transport of virulence proteins across the bacterial membranes is governed by closely related type III secretion systems. For pathogenic Yersinia , the protein transport across the eukaryotic cell membrane occurs by a polarized mechanism requiring two secreted proteins, YopB and YopD. YopB was recently shown to induce the formation of a pore in the eukaryotic cell membrane, and through this pore, translocation of Yop effectors is believed to occur (Håkansson et al ., 1996b). We have previously shown that YopK of Yersinia pseudotuberculosis is required for the development of a systemic infection in mice. Here, we have analysed the role of YopK in the virulence process in more detail. A yopK -mutant strain was found to induce a more rapid YopE-mediated cytotoxic response in HeLa cells as well as in MDCK-1 cells compared to the wild-type strain. We found that this was the result of a cell-contact-dependent increase in translocation of YopE into HeLa cells. In contrast, overexpression of YopK resulted in impaired translocation. In addition, we found that YopK also influenced the YopB-dependent lytic effect on sheep erythrocytes as well as on HeLa cells. A yopK -mutant strain showed a higher lytic activity and the induced pore was larger compared to the corresponding wild-type strain, whereas a strain overexpressing YopK reduced the lytic activity and the apparent pore size was smaller. The secreted YopK protein was found not to be translocated but, similar to YopB, localized to cell-associated bacteria during infection of HeLa cells. Based on these results, we propose a model where YopK controls the translocation of Yop effectors into eukaryotic cells.     

Rac1 activation controls nuclear localization of beta-catenin during canonical Wnt signaling

Canonical Wnt signaling critically regulates cell fate and proliferation in development and disease. Nuclear localization of beta-catenin is indispensable for canonical Wnt signaling; however, the mechanisms governing beta-catenin nuclear localization are not well understood. Here we demonstrate that nuclear accumulation of beta-catenin in response to Wnt requires Rac1 activation. The role of Rac1 depends on phosphorylation of beta-catenin at Ser191 and Ser605, which is mediated by JNK2 kinase. Mutations of these residues significantly affect Wnt-induced beta-catenin nuclear accumulation. Genetic ablation of Rac1 in the mouse embryonic limb bud ectoderm disrupts canonical Wnt signaling and phenocopies deletion of beta-catenin in causing severe truncations of the limb. Finally, Rac1 interacts genetically with beta-catenin and Dkk1 in controlling limb outgrowth. Together these results uncover Rac1 activation and subsequent beta-catenin phosphorylation as a hitherto uncharacterized mechanism controlling canonical Wnt signaling and may provide additional targets for therapeutic intervention of this important pathway.     

Integrin-specific activation of Rac controls progression through the G(1) phase of the cell cycle

Adhesion to fibronectin through the alpha5beta1 integrin enables endothelial cells to proliferate in response to growth factors, whereas adhesion to laminin through alpha2beta1 results in growth arrest under the same conditions. On laminin, endothelial cells fail to translate Cyclin D1 mRNA and activate CDK4 and CDK6. Activated Rac, but not MEK1, PI-3K, or Akt, rescues biosynthesis of cyclin D1 and progression through the G(1) phase. Conversely, dominant negative Rac prevents these events on fibronectin. Mitogens promote activation of Rac on fibronectin but not laminin. This process is mediated by SOS and PI-3K and requires coordinate upstream signals through Shc and FAK. These results indicate that Rac is a crucial mediator of the integrin-specific control of cell cycle in endothelial cells.     

Plasmids of Yersinia pseudotuberculosis

Plasmids with the sizes of 5.7; 51; 70-77; and 120-130 kb were found in six strains among the ten strains collection of Yersinia pseudotuberculosis. The restriction endonucleases analysis. Southern-blot hybridization and physical maps construction were performed for the plasmids. The 70-77 kb plasmids were found to be analogous to the Ca2(+)-dependence plasmid pYVO19 from Yersinia pestis EV76. The difference between the plasmids of this type is in the insertions or deletions located on the similar fragments of the restriction maps. The 51 kb plasmid has no common fragments with the Ca2(+)-dependence plasmids and does not code for virulence properties of the strain harbouring it. No homology is shared by the 5.7 kb plasmid and the 10 kb plasmid from Yersinia pestis EV76. Replicon of the 5.7 kb plasmid has been used to construct the pVS11 vector plasmid.     

Phosphorylation of VE-cadherin controls endothelial phenotypes via p120-catenin coupling and Rac1 activation

To establish the role of vascular endothelial (VE)-cadherin in the regulation of endothelial cell functions, we investigated the effect of phosphorylation of a VE-cadherin site sought to be involved in p120-catenin binding on vascular permeability and endothelial cell migration. To this end, we introduced either wild-type VE-cadherin or Y658 phosphomimetic (Y658E) or dephosphomimetic (Y658F) VE-cadherin mutant constructs into an endothelial cell line (rat fat pad endothelial cells) lacking endogenous VE-cadherin. Remarkably, neither wild-type- nor Y658E VE-cadherin was retained at cell-cell contacts because of p120-catenin preferential binding to N-cadherin, resulting in the targeting of N-cadherin to cell-cell junctions and the exclusion of VE-cadherin. However, Y658F VE-cadherin was able to bind p120-catenin and to localize at adherence junctions displacing N-cadherin. This resulted in an enhanced barrier function and a complete abrogation of Rac1 activation and lamellipodia formation, thereby inhibiting cell migration. These findings demonstrate that VE-cadherin, through the regulation of Y658 phosphorylation, competes for junctional localization with N-cadherin and controls vascular permeability and endothelial cell migration.     

Action of sodium chloride on Yersinia pseudotuberculosis and Yersinia enterocolitica

The bacteriostatic and bactericidal action of sodium chloride on 60 Y. pseudotuberculosis strains, 75 Y. enterocolitica strains and 158 urine-fermenting strains has been studied. A new specific feature of Y. pseudotuberculosis has been revealed: high sensitivity to sodium chloride. The suitability of the sodium chloride test has been shown for the identification of Yersinia and the differentiation of Y. pseudotuberculosis and Y. enterocolitica.     

Community outbreak of Yersinia pseudotuberculosis

An outbreak of Yersinia pseudotuberculosis in Kurashiki, Japan is described. This is the first conclusive report of a community outbreak of this microorganism. A total of 535 pupils, five teachers, and one food attendant contracted the organism. Causative organisms were detected in 19 out of 30 patients. All isolated strains belonged to serotype VA. Out of 653 sera of the pupils, 488 showed elevated agglutinin titers ranging from 1:80 to 1:1,280 or more within a period of 3 months.     

Modulation of Rac1 and ARF6 activation during epithelial cell scattering

Epithelial cell scattering encompasses the dissolution of intercellular junctions, cell-cell dissociation, cell spreading, and motility. The Rac1 and ARF6 GTPases have been shown to regulate one or more of these aforementioned processes. In fact, activated Rac1 has been shown to promote cell-cell adhesion as well as to enhance cell motility, leading to conflicting reports on the effect of Rac1 activation on epithelial cell motility. In this study, we have examined the activation profiles of endogenous Rac1 and ARF6 during the sequential stages of epithelial cell scattering. Using Madin-Darby canine kidney cells treated with hepatocyte growth factor/scatter factor or cell lines stably expressing activated v-Src, we show that Rac1 and ARF6 exhibit distinct activation profiles during cell scattering. We have found that an initial ARF6-dependent decrease in the levels of Rac1-GTP is necessary to induce cell-cell dissociation. This is followed by a steady increase in Rac1 and ARF6 activation and cell migration. In sum, this study documents the progression of ARF6 and Rac1 activities during epithelial cell scattering.     

Comparison of YopE and YopT activities in counteracting host signalling responses to Yersinia pseudotuberculosis infection

Pathogenic Yersinia species share a type III secretion system that translocates Yop effector proteins into host cells to counteract signalling responses during infection. Two of these effectors, YopE and YopT, downregulate Rho GTPases by different mechanisms. Here, we investigate whether YopT and YopE are functionally redundant by dissecting the contribution of these two effectors to the pathogenesis of Yersinia pseudotuberculosis in a mouse infection and tissue culture model. Four days after oral infection, a YopE(+) T (-) strain and a YopE(+) T (+) strain colonized spleens of mice at similar levels, suggesting that YopT is not required for virulence. In contrast, spleen colonization by a YopE(-)T(-) strain was significantly reduced. A YopE(-) T (+) strain colonized spleen at levels comparable to those of the YopE(+) T (-) strain, arguing that YopT can promote virulence in the absence of YopE. Infection of HeLa cells with a YopE(-) T(-)H(-)J(-) strain expressing either YopE or YopT showed that YopE had a stronger antiphagocytic activity than YopT. Expression of YopE strongly inhibited activation of JNK, ERK and NFkappaB, and prevented production of IL-8; whereas YopT moderately inhibited these responses. On the other hand, pore formation was inhibited equally by YopE or YopT. In conclusion, YopE is a potent inhibitor of infection-induced signalling cascades, and YopT can only partially compensate for the loss of YopE.     

Direct isolation of Yersinia enterocolitica and Yersinia pseudotuberculosis from meat

Studies were done to determine the usefulness of dilute alkali (KOH) treatment of meat samples for direct isolation of Yersinia enterocolitica and Yersinia pseudotuberculosis, without enrichment. Virulent Y. enterocolitica and Y. pseudotuberculosis in pork contaminated with 10(2), 10(3), and 10(4) cells per g survived the direct KOH treatment and were never recovered by using KOH postenrichment treatment. From 6 (4.8%) of 125 samples of retail ground pork, four biotype 4 serotype O3 and one biotype 3B serotype O3 strains of Y. enterocolitica and one Y. pseudotuberculosis serotype 4b strain were recovered by using direct KOH treatment without enrichment. As these isolations were attained without using enrichment cultural procedures, they represent an important time-saving alternative to simplify and speed isolation of Yersinia spp. from meat.     

Proline-rich tyrosine kinase 2 and Rac activation by chemokine and integrin receptors controls NK cell transendothelial migration

Protein tyrosine kinase activation is an important requisite for leukocyte migration. Herein we demonstrate that NK cell binding to endothelium activates proline-rich tyrosine kinase 2 (Pyk-2) and the small GTP binding protein Rac that are coupled to integrin and chemokine receptors. Chemokine-mediated, but not integrin-mediated, Pyk-2 and Rac activation was sensitive to pretreatment of NK cells with pertussis toxin, a pharmacological inhibitor of G(i) protein-coupled receptors. Both Pyk-2 and Rac are functionally involved in chemokine-induced NK cell migration through endothelium or ICAM-1 or VCAM-1 adhesive proteins, as shown by the use of recombinant vaccinia viruses encoding dominant negative mutants of Pyk-2 and Rac. Moreover, we found that Pyk-2 is associated with the Rac guanine nucleotide exchange factor Vav, which undergoes tyrosine phosphorylation upon integrin triggering. Finally, we provide direct evidence for the involvement of Pyk-2 in the control of both chemokine- and integrin-mediated Rac activation. Collectively, our results indicate that Pyk-2 acts as a receptor-proximal link between integrin and chemokine receptor signaling, and the Pyk-2/Rac pathway plays a pivotal role in the control of NK cell transendothelial migration.     

Postdiarrheal arthropathy of Yersinia pseudotuberculosis.

Two patients with acute gastroenteritis in whom polyarthritis subsequently developed were found to have positive serologic results for Yersinia pseudotuberculosis. With resolution of the arthropathy the antibody titres decreased. While the patient without the histocompatibility antigen HLA-B27 had an acute, self-limited arthritis, the patient with this antigen had a more chronic arthritis. Serologic typing and stool culture for Y. pseudotuberculosis should be done in cases of postdysenteric arthritis.     

The serodiagnosis of human infections with Yersinia enterocolitica and Yersinia pseudotuberculosis

The techniques of sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting were evaluated for the serodiagnosis of human infections with Yersinia enterocolitica and Yersinia pseudotuberculosis. Lipopolysaccharide (LPS) was prepared from strains comprising four serogroups of Y. enterocolitica and five serogroups of Y. pseudotuberculosis, tested against 200 sera submitted to the Laboratory of Enteric Pathogens for routine serodiagnosis, and shown to contain antibodies to Yersinia LPS by agglutination. Forty four sera were found to contain antibodies that bound to one of the LPS preparations used in the immunoassay. Thirty five of the sera contained antibodies to the LPS of Y. enterocolitica O3, whilst three contained antibodies to the LPS of Y. enterocolitica O5, 27 and Y. enterocolitica O9 LPS respectively. Two sera had antibodies to the LPS of Y. pseudotuberculosis II and a single serum contained antibodies to Y. pseudotuberculosis IV. The SDS-PAGE-immunoblotting procedure described proved to be a reliable procedure for the serodiagnosis of infections with Y. enterocolitica and Y. pseudotuberculosis.     

Effect of acidin-pepsin on Yersinia pseudotuberculosis

The effect of acidin-pepsin solution at a concentration of 1 : 100 on newly isolated Yersinia cultures has been tested in three series of experiments. Acidin-pepsin has been found to exert a bactericidal effect on Yersinia and to induce the appearance of involutionary forms and large rod-shaped Yersinia cells with a better capacity for survival. The surviving Yersinia cells do not pass their resistance to acidin-pepsin on to their progeny and show low invasiveness and pathogenicity in white mice.     

Sprouty regulates cell migration by inhibiting the activation of Rac1 GTPase

Sprouty (SPRY) protein negatively modulates fibroblast growth factor and epidermal growth factor actions. We showed that human SPRY2 inhibits cell growth and migration in response to serum and several growth factors. Using rat intestinal epithelial (IEC-6) cells, we investigated the involvement of the Rho family of GTPases, RhoA, Rac1, and cdc42 in SPRY2-mediated inhibition of cell migration and proliferation. The ability of TAT-tagged SPRY2 to inhibit proliferation and migration of IEC-6 cells transfected with constitutively active mutants of RhoA(G14V), Rac1(G12V), and cdc42 (F28L) was determined. Constitutively active RhoA(G14V), Rac1(G12V), or cdc42(F28L) did not protect cells from the anti-proliferative actions of TAT-SPRY2. The ability of TAT-hSPRY2 to inhibit migration was not altered by of RhoA(G14V) and cdc42(F28L). However, Rac1(G12V) obliterated the ability of SPRY2 to inhibit cell autonomous or serum-induced migration. Also, the activation of endogenous Rac1 was attenuated by TAT-SPRY2. Thus, SPRY2 mediates its anti-migratory actions by inhibiting Rac1 activation.     

Direct isolation of Yersinia enterocolitica and Yersinia pseudotuberculosis from meat.

Studies were done to determine the usefulness of dilute alkali (KOH) treatment of meat samples for direct isolation of Yersinia enterocolitica and Yersinia pseudotuberculosis, without enrichment. Virulent Y. enterocolitica and Y. pseudotuberculosis in pork contaminated with 10(2), 10(3), and 10(4) cells per g survived the direct KOH treatment and were never recovered by using KOH postenrichment treatment. From 6 (4.8%) of 125 samples of retail ground pork, four biotype 4 serotype O3 and one biotype 3B serotype O3 strains of Y. enterocolitica and one Y. pseudotuberculosis serotype 4b strain were recovered by using direct KOH treatment without enrichment. As these isolations were attained without using enrichment cultural procedures, they represent an important time-saving alternative to simplify and speed isolation of Yersinia spp. from meat.