Regulating Rho GTPases and their regulators.

Rho GTPases regulate cytoskeletal and cell adhesion dynamics and thereby coordinate a wide range of cellular processes, including cell migration, cell polarity and cell cycle progression. Most Rho GTPases cycle between a GTP-bound active conformation and a GDP-bound inactive conformation to regulate their ability to activate effector proteins and to elicit cellular responses. However, it has become apparent that Rho GTPases are regulated by post-translational modifications and the formation of specific protein complexes, in addition to GTP-GDP cycling. The canonical regulators of Rho GTPases - guanine nucleotide exchange factors, GTPase-activating proteins and guanine nucleotide dissociation inhibitors - are regulated similarly, creating a complex network of interactions to determine the precise spatiotemporal activation of Rho GTPases.

14-3-3 proteins interact with a hybrid prenyl-phosphorylation motif to inhibit G proteins.

Signaling through G proteins normally involves conformational switching between GTP- and GDP-bound states. Several Rho GTPases are also regulated by RhoGDI binding and sequestering in the cytosol. Rnd proteins are atypical constitutively GTP-bound Rho proteins, whose regulation remains elusive. Here, we report a high-affinity 14-3-3-binding site at the C terminus of Rnd3 consisting of both the Cys241-farnesyl moiety and a Rho-associated coiled coil containing protein kinase (ROCK)-dependent Ser240 phosphorylation site. 14-3-3 binding to Rnd3 also involves phosphorylation of Ser218 by ROCK and/or Ser210 by protein kinase C (PKC). The crystal structure of a phosphorylated, farnesylated Rnd3 peptide with 14-3-3 reveals a hydrophobic groove in 14-3-3 proteins accommodating the farnesyl moiety. Functionally, 14-3-3 inhibits Rnd3-induced cell rounding by translocating it from the plasma membrane to the cytosol. Rnd1, Rnd2, and geranylgeranylated Rap1A interact similarly with 14-3-3. In contrast to the canonical GTP/GDP switch that regulates most Ras superfamily members, our results reveal an unprecedented mechanism for G protein inhibition by 14-3-3 proteins.

RhoU forms homo-oligomers to regulate cellular responses.

RhoU is an atypical member of the Rho family of small G-proteins, which has N- and C-terminal extensions compared to the classic Rho GTPases RhoA, Rac1 and Cdc42, and associates with membranes through C-terminal palmitoylation rather than prenylation. RhoU mRNA expression is upregulated in prostate cancer and is considered a marker for disease progression. Here, we show that RhoU overexpression in prostate cancer cells increases cell migration and invasion. To identify RhoU targets that contribute to its function, we found that RhoU homodimerizes in cells. We map the region involved in this interaction to the C-terminal extension and show that C-terminal palmitoylation is required for self-association. Expression of the isolated C-terminal extension reduces RhoU-induced activation of p21-activated kinases (PAKs), which are known downstream targets for RhoU, and induces cell morphological changes consistent with inhibiting RhoU function. Our results show for the first time that the activity of a Rho family member is stimulated by self-association, and this is important for its activity.

Life at the leading edge.

Cell migration requires sustained forward movement of the plasma membrane at the cell's front or "leading edge." To date, researchers have uncovered four distinct ways of extending the membrane at the leading edge. In lamellipodia and filopodia, actin polymerization directly pushes the plasma membrane forward, whereas in invadopodia, actin polymerization couples with the extracellular delivery of matrix-degrading metalloproteases to clear a path for cells through the extracellular matrix. Membrane blebs drive the plasma membrane forward using a combination of actomyosin-based contractility and reversible detachment of the membrane from the cortical actin cytoskeleton. Each protrusion type requires the coordination of a wide spectrum of signaling molecules and regulators of cytoskeletal dynamics. In addition, these different protrusion methods likely act in concert to move cells through complex environments in vivo.

Alzheimer's disease-associated P460L variant of EphA1 dysregulates receptor activity and blood-brain barrier function.

INTRODUCTION: Genome-wide association studies link susceptibility to late-onset Alzheimer's disease (LOAD) with EphA1. Sequencing identified a non-synonymous substitution P460L as a LOAD risk variant. Other Ephs regulate vascular permeability and immune cell recruitment. We hypothesized that P460L dysregulates EphA1 receptor activity and impacts neuroinflammation. METHODS: EphA1/P460L receptor activity was assayed in isogenic Human Embryonic Kidney (HEK) cells. Soluble EphA1/P460L (sEphA1/sP460L) reverse signaling in brain endothelial cells was assessed by T-cell recruitment and barrier function assays. RESULTS: EphA1 and P460L were expressed in HEK cells, but membrane and soluble P460L were significantly reduced. Ligand engagement induced Y781 phosphorylation of EphA1 but not P460L. sEphA1 primed brain endothelial cells for increased T-cell recruitment; however, sP460L was less effective. sEphA1 decreased the integrity of the brain endothelial barrier, while sP460L had no effect. DISCUSSION: These findings suggest that P460L alters EphA1-dependent forward and reverse signaling, which may impact blood-brain barrier function in LOAD. HIGHLIGHTS: EphA1-dependent reverse signaling controls recruitment of T cells by brain endothelial cells. EphA1-dependent reverse signaling remodels brain endothelial cell contacts. LOAD-associated P460L variant of EphA1 shows reduced membrane expression and reduced ligand responses. LOAD-associated P460L variant of EphA1 fails to reverse signal to brain endothelial cells.

Agricultural intensification and the evolution of host specialism in the enteric pathogen Campylobacter jejuni.

Modern agriculture has dramatically changed the distribution of animal species on Earth. Changes to host ecology have a major impact on the microbiota, potentially increasing the risk of zoonotic pathogens being transmitted to humans, but the impact of intensive livestock production on host-associated bacteria has rarely been studied. Here, we use large isolate collections and comparative genomics techniques, linked to phenotype studies, to understand the timescale and genomic adaptations associated with the proliferation of the most common food-born bacterial pathogen (Campylobacter jejuni) in the most prolific agricultural mammal (cattle). Our findings reveal the emergence of cattle specialist C. jejuni lineages from a background of host generalist strains that coincided with the dramatic rise in cattle numbers in the 20th century. Cattle adaptation was associated with horizontal gene transfer and significant gene gain and loss. This may be related to differences in host diet, anatomy, and physiology, leading to the proliferation of globally disseminated cattle specialists of major public health importance. This work highlights how genomic plasticity can allow important zoonotic pathogens to exploit altered niches in the face of anthropogenic change and provides information for mitigating some of the risks posed by modern agricultural systems.

Rnd3 interacts with TAO kinases and contributes to mitotic cell rounding and spindle positioning.

Rnd3 is an atypical Rho family protein that is constitutively GTP bound, and acts on membranes to induce loss of actin stress fibers and cell rounding. Phosphorylation of Rnd3 promotes 14-3-3 binding and its relocation to the cytosol. Here, we show that Rnd3 binds to the thousand-and-one amino acid kinases TAOK1 and TAOK2 in vitro and in cells. TAOK1 and TAOK2 can phosphorylate serine residues 210, 218 and 240 near the C-terminus of Rnd3, and induce Rnd3 translocation from the plasma membrane to the cytosol. TAOKs are activated catalytically during mitosis and Rnd3 phosphorylation on serine 210 increases in dividing cells. Rnd3 depletion by RNAi inhibits mitotic cell rounding and spindle centralization, and delays breakdown of the intercellular bridge between two daughter cells. Our results show that TAOKs bind, phosphorylate and relocate Rnd3 to the cytosol and that Rnd3 contributes to mitotic cell rounding, spindle positioning and cytokinesis. Rnd3 can therefore participate in the regulation of early and late mitosis and may also act downstream of TAOKs to affect the cytoskeleton.

Hypoxia suppresses myofibroblast differentiation by changing RhoA activity.

Fibroblasts show a high range of phenotypic plasticity, including transdifferentiation into myofibroblasts. Myofibroblasts are responsible for generation of the contraction forces that are important for wound healing and scar formation. Overactive myofibroblasts, by contrast, are involved in abnormal scarring. Cell stretching and extracellular signals such as transforming growth factor ß can induce the myofibroblastic program, whereas microenvironmental conditions such as reduced tissue oxygenation have an inhibitory effect. We investigated the effects of hypoxia on myofibroblastic properties and linked this to RhoA activity. Hypoxia reversed the myofibroblastic phenotype of primary fibroblasts. This was accompanied by decreased αSMA (ACTA2) expression, alterations in cell contractility, actin reorganization and RhoA activity. We identified a hypoxia-inducible induction of ARHGAP29, which is critically involved in myocardin-related transcription factor-A (MRTF-A) signaling, the differentiation state of myofibroblasts and modulates RhoA activity. This novel link between hypoxia and MRTF-A signaling is likely to be important for ischemia-induced tissue remodeling and the fibrotic response.This article has an associated First Person interview with the first author of the paper.

Identification of Mycoplasma species and related organisms from ruminants in England and Wales during 2005-2019.

BACKGROUND: Mycoplasma species have been associated with economically important diseases affecting ruminants worldwide and include contagious bovine pleuropneumonia (CBPP), contagious caprine pleuropneumonia (CCPP) and contagious agalactia, listed by the World Organisation for Animal Health (OIE). The Mycoplasma Team at the Animal and Plant Health Agency provides an identification service for Mycoplasma and Ureaplasma species of veterinary importance to the United Kingdom (UK), supporting the detection of new and emerging pathogens, as well as contributing to the surveillance of endemic, and the OIE listed diseases exotic to the UK. Mycoplasma and other Mollicutes species were identified from diagnostic samples from farmed ruminants in England and Wales using a combination of culture and 16S rRNA gene-based PCR-denaturing gradient gel electrophoresis, submitted between 2005 and 2019. RESULTS: A total of 5578 mollicutes identifications, which include mycoplasmas and the related acholeoplasmas and ureaplasmas, were made from farmed ruminant animals during the study period. Throughout the study period, the pathogen Mycoplasma bovis was consistently the most frequently identified species, accounting for 1411 (32%) of 4447 molecular identifications in cattle, primarily detected in the lungs of pneumonic calves, followed by joints and milk of cattle showing signs of arthritis and mastitis, respectively. M. bovirhinis, M. alkalescens, M. dispar, M. arginini and Ureaplasma diversum, were also common. Mixed species, principally M. bovis with M. alkalescens, M. arginini or M. bovirhinis were also prevalent, particularly from respiratory samples. The non-cultivable blood-borne haemoplasmas Candidatus 'Mycoplasma haemobos' and Mycoplasma wenyonii were identified from cattle, with the latter species most often associated with milk-drop. M. ovipneumoniae was the predominant species identified from sheep and goats experiencing respiratory disease, while M. conjunctivae preponderated in ocular samples. The UK remains free of the ruminant mycoplasmas listed by OIE. CONCLUSIONS: The continued high prevalence of M. bovis identifications confirms its ongoing dominance and importance as a significant pathogen of cattle in England and Wales, particularly in association with respiratory disease. M. ovipneumoniae has seen a general increase in prevalence in recent years, notably in coughing lambs and should therefore be considered as a primary differential diagnosis of respiratory disease in small ruminants.

Bcl-3 promotes multi-modal tumour cell migration via NF-κB1 mediated regulation of Cdc42.

A key challenge in the implementation of anti-metastatics as cancer therapies is the multi-modal nature of cell migration, which allows tumour cells to evade the targeted inhibition of specific cell motility pathways. The nuclear factor-kappaB (NF-κB) co-factor B-cell lymphoma 3 (Bcl-3) has been implicated in breast cancer cell migration and metastasis, yet it remains to be determined exactly which cell motility pathways are controlled by Bcl-3 and whether migrating tumour cells are able to evade Bcl-3 intervention. Addressing these questions and the mechanism underpinning Bcl-3's role in this process would help determine its potential as a therapeutic target. Here we identify Bcl-3 as an upstream regulator of the two principal forms of breast cancer cell motility, involving collective and single-cell migration. This was found to be mediated by the master regulator Cdc42 through binding of the NF-κB transcription factor p50 to the Cdc42 promoter. Notably, Bcl-3 depletion inhibited both stable and transitory motility phenotypes in breast cancer cells with no evidence of migratory adaptation. Overexpression of Bcl-3 enhanced migration and increased metastatic tumour burden of breast cancer cells in vivo, whereas overexpression of a mutant Bcl-3 protein, which is unable to bind p50, suppressed cell migration and metastatic tumour burden suggesting that disruption of Bcl-3/NF-κB complexes is sufficient to inhibit metastasis. These findings identify a novel role for Bcl-3 in intrinsic and adaptive multi-modal cell migration mediated by its direct regulation of the Rho GTPase Cdc42 and identify the upstream Bcl-3:p50 transcription complex as a potential therapeutic target for metastatic disease.

Mammalian Rho GTPases: new insights into their functions from in vivo studies.

Rho GTPases are key regulators of cytoskeletal dynamics and affect many cellular processes, including cell polarity, migration, vesicle trafficking and cytokinesis. These proteins are conserved from plants and yeast to mammals, and function by interacting with and stimulating various downstream targets, including actin nucleators, protein kinases and phospholipases. The roles of Rho GTPases have been extensively studied in different mammalian cell types using mainly dominant negative and constitutively active mutants. The recent availability of knockout mice for several members of the Rho family reveals new information about their roles in signalling to the cytoskeleton and in development.

RhoBTB1 interacts with ROCKs and inhibits invasion.

RhoBTB1 is an atypical Rho GTPase with two BTB domains in addition to its Rho domain. Although most Rho GTPases regulate actin cytoskeletal dynamics, RhoBTB1 is not known to affect cell shape or motility. We report that RhoBTB1 depletion increases prostate cancer cell invasion and induces elongation in Matrigel, a phenotype similar to that induced by depletion of ROCK1 and ROCK2. We demonstrate that RhoBTB1 associates with ROCK1 and ROCK2 and its association with ROCK1 is via its Rho domain. The Rho domain binds to the coiled-coil region of ROCK1 close to its kinase domain. We identify two amino acids within the Rho domain that alter RhoBTB1 association with ROCK1. RhoBTB1 is a substrate for ROCK1, and mutation of putative phosphorylation sites reduces its association with Cullin3, a scaffold for ubiquitin ligases. We propose that RhoBTB1 suppresses cancer cell invasion through interacting with ROCKs, which in turn regulate its association with Cullin3. Via Cullin3, RhoBTB1 has the potential to affect protein degradation.

Inhibition of Rho-associated kinases suppresses cardiac myofibroblast function in engineered connective and heart muscle tissues.

Cardiac fibrosis is a hallmark of heart failure for which there is no effective pharmacological therapy. By genetic modification and in vivo inhibitor approaches it was suggested that the Rho-associated kinases (ROCK1 and ROCK2) are involved in pro-fibrotic signalling in cardiac fibroblasts and that they may serve as targets for anti-fibrotic therapies. We demonstrate that simultaneous inhibition of ROCK1 and ROCK2 strongly interfered with tissue formation and their biomechanical properties in a model of engineered connective tissue (ECT), comprised of cardiac fibroblasts and collagen. These effects were observed with both rat and human ECT. Inhibitors of different chemistries, including the isoquinoline inhibitors Fasudil and H1152P as well as the pyrazol-phenyl inhibitor SR-3677, showed comparable effects. By combined treatment of ECT with TGF-ß and H1152P, we could identify ROCK as a mediator of TGF-ß-dependent tissue stiffening. Moreover, expression analyses suggested that lysyl oxidase (LOX) is a downstream target of the ROCK-actin-MRTF/SRF pathway and inhibition of this pathway by Latrunculin A and CCG-203971 showed similar anti-fibrotic effects in the ECT model as ROCK inhibitors. In line with the collagen crosslinking function of LOX, its inhibition by ß-aminopropionitrile resulted in reduced ECT stiffness, but let tissue compaction unaffected. Finally, we show that ROCK inhibition also reduced the compaction and stiffness of engineered heart muscle tissues. Our results indicate that pharmacological inhibition of ROCK has a strong anti-fibrotic potential which is in part due to a decrease in the expression of the collagen crosslinking enzyme lysyl oxidase.

A European inter-laboratory trial to evaluate the performance of three serological methods for diagnosis of Mycoplasma bovis infection in cattle using latent class analysis.

BACKGROUND: Mycoplasma bovis (M. bovis) is an emerging bovine pathogen, leading to significant economic losses in the livestock industry worldwide. Infection can result in a variety of clinical signs, such as arthritis, pneumonia, mastitis and keratoconjunctivitis, none of which are M. bovis-specific. Laboratory diagnosis is therefore important. Serological tests to detect M. bovis antibodies is considered an effective indicator of infection in a herd and often used as a herd test. Combined with clinical judgement, it can also be used to implement control strategies and/or to estimate the disease prevalence within a country. However, due to lack of harmonisation of approaches to testing, and serological tests used by different laboratories, comparisons of prevalence data between countries is often difficult. A network of researchers from six European countries designed and participated in an inter-laboratory trial, with the aim of evaluating the sensitivity (Se) and specificity (Sp) of two commercially available ELISA tests (ID Screen® ELISA (IDvet) and BIO K302 ELISA (BIO-X Diagnostics)) for diagnosis of M. bovis infection. Each laboratory received a blinded panel of bovine sera and tested independently, according to manufacturer's instructions. Western blot analyses (WB) performed by one of the participating laboratories was used as a third diagnostic test in the statistical evaluation of Se and Sp values using latent class analysis. RESULTS: The Se of WB, the ID Screen® ELISA and the BIO K302 ELISA were determined to be 91.8, 93.5 and 49.1% respectively, and corresponding Sp of the three tests were 99.6, 98.6 and 89.6%, respectively. CONCLUSIONS: The present study is, to our knowledge, the first to present an inter-laboratory comparison of the BIO K302 ELISA and the ID Screen® ELISA. Based on our results, the ID Screen® ELISA showed high consistency with WB and performed with higher precision and accuracy than the BIO K302 ELISA.

A European interlaboratory trial to evaluate the performance of different PCR methods for Mycoplasma bovis diagnosis.

BACKGROUND: Several species-specific PCR assays, based on a variety of target genes are currently used in the diagnosis of Mycoplasma bovis infections in cattle herds with respiratory diseases and/or mastitis. With this diversity of methods, and the development of new methods and formats, regular performance comparisons are required to ascertain diagnostic quality. The present study compares PCR methods that are currently used in six national veterinary institutes across Europe. Three different sample panels were compiled and analysed to assess the analytical specificity, analytical sensitivity and comparability of the different PCR methods. The results were also compared, when appropriate, to those obtained through isolation by culture. The sensitivity and comparability panels were composed of samples from bronchoalveolar fluids of veal calves, artificially contaminated or naturally infected, and hence the comparison of the different methods included the whole workflow from DNA extraction to PCR analysis. RESULTS: The participating laboratories used i) five different DNA extraction methods, ii) seven different real-time and/or end-point PCRs targeting four different genes and iii) six different real-time PCR platforms. Only one commercial kit was assessed; all other PCR assays were in-house tests adapted from published methods. The analytical specificity of the different PCR methods was comparable except for one laboratory where Mycoplasma agalactiae was tested positive. Frequently, weak-positive results with Ct values between 37 and 40 were obtained for non-target Mycoplasma strains. The limit of detection (LOD) varied from 10 to 103 CFU/ml to 103 and 106 CFU/ml for the real-time and end-point assays, respectively. Cultures were also shown to detect concentrations down to 102 CFU/ml. Although Ct values showed considerable variation with naturally infected samples, both between laboratories and tests, the final result interpretation of the samples (positive versus negative) was essentially the same between the different laboratories. CONCLUSION: With a few exceptions, all methods used routinely in the participating laboratories showed comparable performance, which assures the quality of diagnosis, despite the multiplicity of the methods.

An RNAi screen of Rho signalling networks identifies RhoH as a regulator of Rac1 in prostate cancer cell migration.

BACKGROUND: Cell migration is essential for development and tissue repair, but it also contributes to disease. Rho GTPases regulate cell migration, but a comprehensive analysis of how each Rho signalling component affects migration has not been carried out. RESULTS: Through an RNA interference screen, and using a prostate cancer cell line, we find that approximately 25% of Rho network components alter migration. Some genes enhance migration while others decrease basal and/or hepatocyte growth factor-stimulated migration. Surprisingly, we identify RhoH as a screen hit. RhoH expression is normally restricted to haematopoietic cells, but we find it is expressed in multiple epithelial cancer cell lines. High RhoH expression in samples from prostate cancer patients correlates with earlier relapse. RhoH depletion reduces cell speed and persistence and decreases migratory polarity. Rac1 activity normally localizes to the front of migrating cells at areas of dynamic membrane movement, but in RhoH-depleted cells active Rac1 is localised around the whole cell periphery and associated with membrane regions that are not extending or retracting. RhoH interacts with Rac1 and with several p21-activated kinases (PAKs), which are Rac effectors. Similar to RhoH depletion, PAK2 depletion increases cell spread area and reduces cell migration. In addition, RhoH depletion reduces lamellipodium extension induced by PAK2 overexpression. CONCLUSIONS: We describe a novel role for RhoH in prostate cancer cell migration. We propose that RhoH promotes cell migration by coupling Rac1 activity and PAK2 to membrane protrusion. Our results also suggest that RhoH expression levels correlate with prostate cancer progression.

The STRIPAK complex components FAM40A and FAM40B regulate endothelial cell contractility via ROCKs.

BACKGROUND: Endothelial cells provide a barrier between blood and tissues, which is regulated to allow molecules and cells in out of tissues. Patients with cerebral cavernous malformations (CCM) have dilated leaky blood vessels, especially in the central nervous system. A subset of these patients has loss-of-function mutations in CCM3. CCM3 is part of the STRIPAK protein complex that includes the little-characterized proteins FAM40A and FAM40B. RESULTS: We show here that FAM40A and FAM40B can interact with CCM3. Knockdown of CCM3, FAM40A or FAM40B in endothelial cells by RNAi causes an increase in stress fibers and a reduction in loop formation in an in vitro angiogenesis assay, which can be reverted by inhibiting the Rho-regulated ROCK kinases. FAM40B depletion also increases endothelial permeability. CONCLUSIONS: These results demonstrate the importance of the FAM40 proteins for endothelial cell physiology, and suggest that they act as part of the CCM3-containing STRIPAK complex.

Rnd3-induced cell rounding requires interaction with Plexin-B2.

Rnd proteins are atypical members of the Rho GTPase family that induce actin cytoskeletal reorganization and cell rounding. Rnd proteins have been reported to bind to the intracellular domain of several plexin receptors, but whether plexins contribute to the Rnd-induced rounding response is not known. Here we show that Rnd3 interacts preferentially with plexin-B2 of the three plexin-B proteins, whereas Rnd2 interacts with all three B-type plexins, and Rnd1 shows only very weak interaction with plexin-B proteins in immunoprecipitations. Plexin-B1 has been reported to act as a GAP for R-Ras and/or Rap1 proteins. We show that all three plexin-B proteins interact with R-Ras and Rap1, but Rnd proteins do not alter this interaction or R-Ras or Rap1 activity. We demonstrate that plexin-B2 promotes Rnd3-induced cell rounding and loss of stress fibres, and enhances the inhibition of HeLa cell invasion by Rnd3. We identify the amino acids in Rnd3 that are required for plexin-B2 interaction, and show that mutation of these amino acids prevents Rnd3-induced morphological changes. These results indicate that plexin-B2 is a downstream target for Rnd3, which contributes to its cellular function.

Endothelial cell-cell adhesion and signaling.

Endothelial cells line blood vessels and provide a dynamic interface between the blood and tissues. They remodel to allow leukocytes, fluid and small molecules to enter tissues during inflammation and infections. Here we compare the signaling networks that contribute to endothelial permeability and leukocyte transendothelial migration, focusing particularly on signals mediated by small GTPases that regulate cell adhesion and the actin cytoskeleton. Rho and Rap GTPase signaling is important for both processes, but they differ in that signals are activated locally under leukocytes, whereas endothelial permeability is a wider event that affects the whole cell. Some molecules play a unique role in one of the two processes, and could therefore be targeted to selectively alter either endothelial permeability or leukocyte transendothelial migration.