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The elaboration of polarity is central to organismal development and to the maintenance of functional epithelia. Amongst the controls determining polarity are the PAR proteins, PAR6/aPKCıPAR3, regulating both known and unknown effectors. Here we identify FARP2 as a "RIPR" motif dependent partner and substrate of aPKCıthat is required for efficient polarisation and junction formation. Binding is conferred by a FERM/FA domain-Kinase domain interaction and detachment promoted by aPKCıdependent phosphorylation. FARP2 is shown to promote GTP loading of Cdc42 consistent with upstream regulation of the polarising PAR6-aPKCıcomplex. However, it is shown that aPKCıacts to promote the localised activity of FARP2 through phosphorylation. We conclude that this aPKCı-FARP2 complex formation acts as a positive feedback control to drive polarisation through aPKCıand other Cdc42 effectors [ABSTRACT FROM AUTHOR]

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 Rndinduced 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. [ABSTRACT FROM AUTHOR]

The empirical derivation of PKC (protein kinase C) domain structures and those modelled by homology or imputed from protein behaviour have been extraordinarily valuable both in the elucidation of PKC pathway mechanisms and in the general lessons that extrapolate to other signalling pathways. For PKC family members, there are many domain/subdomain structures and models, covering all of the known domains, variably present in this family of protein serine/threonine kinases (C1, C2, PB1, HR1, kinase domains). In addition to these structures, there are a limited number of complexes defined, including the structure of the PKCe V3-14-3-3 complex. In the context of structure-driven insights into PKC pathways, there are several broadly applicable principles and mechanisms relevant to the operation of and intervention in signalling pathways. These principles have an impact in unexpected ways, from the regulation of membrane targeting, through strategies for pharmacological intervention, to biomarkers. [ABSTRACT FROM AUTHOR]

Summary: 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. [ABSTRACT FROM AUTHOR]

The aPKC [atypical PKC (protein kinase C)] isoforms i and ζ play crucial roles in the formation and maintenance of cell polarity and represent attractive anti-oncogenic drug targets in Ras-dependent tumours. To date, few isoform-specific chemical biology tools are available to inhibit aPKC catalytic activity. In the present paper, we describe the identification and functional characterization of potent and selective thieno[2,3-d]pyrimidine-based chemical inhibitors of aPKCs. A crystal structure of human PKCé kinase domain bound to a representative compound, CRT0066854, reveals the basis for potent and selective chemical inhibition. Furthermore, CRT0066854 displaces a crucial Asn-Phe-Asp motif that is part of the adenosine-binding pocket and engages an acidic patch used by arginine-rich PKC substrates. We show that CRT0066854 inhibits the LLGL2 (lethal giant larvae 2) phosphorylation in cell lines and exhibits phenotypic effects in a range of cell-based assays. We conclude that this compound can be used as a chemical tool to modulate aPKC activity in vitro and in vivo and may guide the search for further aPKC-selective inhibitors. [ABSTRACT FROM AUTHOR]

Overexpression of T-cadherin (T-cad) transcripts occurs in ≈50% of human hepatocellular carcinomas (HCCs). To elucidate T-cad functions in HCC, we examined T-cad protein expression in normal and tumoral human livers and hepatoma cell lines and investigated its influence on invasive potential of HCC using RNA interference silencing of T-cad expression in Mahlavu cells. Whereas T-cad expression was restricted to endothelial cells (EC) from large blood vessels in normal livers, it was up-regulated in sinusoidal EC from 8/15 invasive HCCs. Importantly, in three of them (38%) T-cad was detected in tumor cells within regions in which E-cadherin expression was absent. Among six hepatoma cell lines, only Mahlavu expressed T-cad but not E-cadherin. T-cad exhibited a globally punctuate distribution in quiescent Mahlavu and additionally it concentrated at the leading edge of migrating cells. Matrigel invasion assay revealed that Mahlavu possess a high invasive potential that was significantly inhibited by T-cad silencing. Wound healing and random motility assays demonstrated that inhibition of T-cad expression in Mahlavu significantly reduced their motility. We propose that T-cad expression in tumor cells might occur by cadherin-switching during epithelial-mesenchymal transition and may represent an additional mechanism contributing to HCC metastasis. [ABSTRACT FROM AUTHOR]

WFDC1 is a recently isolated human gene identified as a tumour suppressor gene candidate. It is not known whether alterations in this gene are associated with human cancers. The WFDC1 gene maps in human chromosome 16q24, an area of frequent loss of heterozygosity (LOH) in several tumour types, in particular in hepatocellular carcinoma (HCC). We investigated its role in 46 European HCC by means of the detection of LOH at the WFDC1 locus. We describe here an assay for the detection of loss of heterozygosity at this locus using two dinucleotide repeat polymorphisms identified in WFDC1 introns, with a combined informativity of 86%. LOH was observed in 4/40 informative HCC samples. We further investigated the role of WFDC1 as a tumour suppressor gene candidate in five hepatocellular cell lines and in tumours exhibiting LOH by means of mutation, promoter methylation and gene expression analysis. In HCC samples showing LOH, no mutation of the remaining allele was observed. No significant up or down gene expression was observed in tumour samples comparatively to normal liver and gene expression did not seem related to promoter methylation. These results suggest a minor role, if any, of WFDC1 in hepatocarcinogenesis. However, this approach might be useful for investigating the role of this candidate tumour suppressor gene in other tumour types. [ABSTRACT FROM AUTHOR]

The continental coastal waters of the Eastern Channel, from Normandy to Hauts-de-France, are subject to the major influence of unbalanced nutrient inputs from inflowing rivers. Several episodes of harmful algal blooms (HABs) compromising fishing and shellfish farming activities have been observed at the coast. For a better understanding of how the land-to-sea aquatic continuum functions, the GRAFS-RIVERSTRAHLER river biogeochemical model was implemented to cover the watersheds of 11 rivers flowing into this area (including the Seine) and chained with the ecological marine ECO-MARS3D model, applied to the French Northern coastal zone. Human activities strongly impact on the functioning of coastal ecosystems. Specifically, for these fertile soils of Northern France, intensive agricultural nitrogen (N) deliveries in excess over silica (Si) and phosphorus (P), essentially of diffuse origin, are potentially responsible for coastal eutrophication. Phosphorous is today equally supplied by diffuse and point sources, after a drastic reduction of inputs from wastewater treatment plants since the 2000s, and is better balanced regarding Si, as shown by the indicators of coastal eutrophication potential (P-ICEP versus N-ICEP). However, despite this drastic P reduction, HABs still appear repeatedly. Exploration of several scenarios of agro-food chain reorganization shows that (i) further progress in urban wastewater treatment to fully comply with current European regulations will not result in a significant reduction of nutrient fluxes to the sea, hence including HABs, and (ii) radical structural changes in agriculture, based on generalization of long and diversified organic crop rotations, reconnection of crop and livestock farming and changes in the human diet have the capacity to significantly reduce nutrient flows, coastal eutrophication and HABs. [ABSTRACT FROM AUTHOR]

The population dynamics of different Pseudo-nitzschia species, along with particulate domoic acid (pDA) concentrations, were studied from May 2012 to December 2013 in the Bay of Seine (English Channel, Normandy). While Pseudo-nitzschia spp. blooms occurred during the two years of study, Pseudo-nitzschia species diversity and particulate domoic acid concentrations varied greatly. In 2012, three different species were identified during the spring bloom ( P. australis , P. pungens and P. fraudulenta ) with high pDA concentrations (∼1400 ng l −1 ) resulting in shellfish harvesting closures. In contrast, the 2013 spring was characterised by a P. delicatissima bloom without any toxic event. Above all, the results show that high pDA concentrations coincided with the presence of P. australis and with potential silicate limitation (Si:N < 1), while nitrate concentrations were still replete. The contrasting environmental conditions between 2012 and 2013 highlight different environmental controls that might favour the development of either P. delicatissima or P. australis . This study points to the key role of Pseudo-nitzschia diversity and cellular toxicity in the control of particulate domoic acid variations and highlights the fact that diversity and toxicity are influenced by nutrients, especially nutrient ratios. [ABSTRACT FROM AUTHOR]

Summary Atypical protein kinase C (aPKC) is a key apical-basal polarity determinant and Par complex component. It is recruited by Par3/Baz (Bazooka in Drosophila ) into epithelial apical domains through high-affinity interaction. Paradoxically, aPKC also phosphorylates Par3/Baz, provoking its relocalization to adherens junctions (AJs). We show that Par3 conserved region 3 (CR3) forms a tight inhibitory complex with a primed aPKC kinase domain, blocking substrate access. A CR3 motif flanking its PKC consensus site disrupts the aPKC kinase N lobe, separating P-loop/αB/αC contacts. A second CR3 motif provides a high-affinity anchor. Mutation of either motif switches CR3 to an efficient in vitro substrate by exposing its phospho-acceptor site. In vivo, mutation of either CR3 motif alters Par3/Baz localization from apical to AJs. Our results reveal how Par3/Baz CR3 can antagonize aPKC in stable apical Par complexes and suggests that modulation of CR3 inhibitory arms or opposing aPKC pockets would perturb the interaction, promoting Par3/Baz phosphorylation. [ABSTRACT FROM AUTHOR]

During 2002–2005, a new container terminal in the commercial harbour of Le Havre, named “Port2000”, was built on the northern flank of the Seine estuary, northern France. This extension is already known to have modified the estuary current and sediment dynamics, as well as reducing biomass of the suprabenthos assemblage, for the whole downstream part of the system. However, studies on other biotic communities were largely inconclusive, and an ecosystem-wide analysis was still lacking. Here, we performed a before/after study of ecosystem dynamics of the different habitats of the Seine estuary, using a Linear Inverse Modelling technique (LIM-MCMC) to estimate all flows occurring in the food web. Ecological Network Analysis indices were calculated, summarising ecosystem functioning traits and giving indications about the habitat health status. Results showed that the southern flank (FS, Fosse Sud ) exhibits all characteristics to be considered as the least stressed habitat of the estuary: system activity and functional specialisation of flows were stable between periods, ecosystem recycling processes and detrital dynamics were also stable; an increase in trophic specialisation (decrease in system omnivory) was the only change confirming a general ecological succession. The northern flank (FN, Fosse Nord ), where the actual terminal was built, showed a food web with increased importance of lower trophic levels (increased detritivory and carbon recycling), increased stability and flow efficiency, but possibly regressed to a previous step in ecological succession. In the central navigation channel (CH), patterns of network indices were overall inconclusive and the general image is one of a constantly shifting food web, a condition possibly caused by the year-round dredging activities. The functioning of the Seine estuary – especially of FN and FS – seems to have been modified by the combination of harbour construction and the related mitigation measures. Network indices partially captured this combination of changes and, although not fully operational yet, they are promising tools to comply with the European Union mandate of defining ecosystem health status. [ABSTRACT FROM AUTHOR]

Cancer cells interact with endothelial cells during the process of metastatic spreading. Here, we use a small interfering RNA screen targeting Rho GTPases in cancer cells to identify Cdc42 as a critical regulator of cancer cell-endothelial cell interactions and transendothelial migration. We find that Cdc42 regulates β1 integrin expression at the transcriptional level via the transcription factor serum response factor (SRF). β1 integrin is the main target for Cdc42-mediating interaction of cancer cells with endothelial cells and the underlying extracellular matrix, as exogenous β1 integrin expression was sufficient to rescue the Cdc42-silencing phenotype. We show that Cdc42 was required in vivo for cancer cell spreading and protrusion extension along blood vessels and retention in the lungs. Interestingly, transient Cdc42 depletion was sufficient to decrease experimental lung metastases, which suggests that its role in endothelial attachment is important for metastasis. By identifying β1 integrin as a transcriptional target of Cdc42, our results provide new insight into Cdc42 function. [ABSTRACT FROM AUTHOR]

Summary: Little is known of the intracellular machinery that controls the motility of newborn neurons. We have previously shown that the proneural protein Neurog2 promotes the migration of nascent cortical neurons by inducing the expression of the atypical Rho GTPase Rnd2. Here, we show that another proneural factor, Ascl1, promotes neuronal migration in the cortex through direct regulation of a second Rnd family member, Rnd3. Both Rnd2 and Rnd3 promote neuronal migration by inhibiting RhoA signaling, but they control distinct steps of the migratory process, multipolar to bipolar transition in the intermediate zone and locomotion in the cortical plate, respectively. Interestingly, these divergent functions directly result from the distinct subcellular distributions of the two Rnd proteins. Because Rnd proteins also regulate progenitor divisions and neurite outgrowth, we propose that proneural factors, through spatiotemporal regulation of Rnd proteins, integrate the process of neuronal migration with other events in the neurogenic program. [ABSTRACT FROM AUTHOR]

Background: To establish a new experimental model of human hepatocellular carcinoma by orthotopic implantation of tumoral cells with its subsequent removal, to generate and modulate circulating tumoral cells. Materials and methods: Three human hepatoma cell lines (HepG2, PLC/PRF, and Mahlavu) were orthotopically implanted under the Glisson''s capsule of the left lateral lobe of the liver in a total of 56 non-obese diabetic/severe combined immunodeficiency mice. Tumor removal was performed 30 d after injection, and a laparotomy without tumor removal was done in control mice. Generation of circulating cells was monitored by flow cytometry using fluorescein isothiocyanate-conjugated anti-HLA antibody. Results: In 26 mice implanted with Mahlavu cells, 20 developed a unique tumor allowing a resection (77%), which was technically feasible in 80% of cases. The overall perioperative mortality was 30% (3/10) after resection; no mortality was observed in the control group. The circulating tumoral cells decreased dramatically after resection of the tumor as compared with control mice. Conclusion: This new model is feasible and may be an interesting useful tool to study the hepatocellular carcinoma metastatic process and is consistent with the human clinical practice. [Copyright &y& Elsevier]

Abstract: The application of the new Water Framework Directive (WFD) of the European Union will require a dense and frequent monitoring of chlorophyll-a near the coast. Not counting the transitional water bodies located in the vicinity of estuaries, not less than seventy four coastal water bodies have to be monitored along the coast of the French Atlantic continental shelf and the English Channel. All the available data have to be gathered to implement a comprehensive monitoring scheme. To this purpose, we evaluate the capacity of ocean colour imagery to complete the conventional in situ data set collected in coastal networks. Satellite-derived chlorophyll-a concentration is obtained by the application of a coastal Look-Up-Table to water-leaving radiance of the Sea-viewing Wide Field Instrument Sensor (SeaWiFS) for the 1998–2004 period. Seven years of satellite-derived and in situ chlorophyll-a concentrations are compared at seven representative stations of different water bodies. These comparisons show that the satellite products are reliable in most of the situations studied and throughout the seasons. Then the satellite imagery is used to classify the coastal waters following the eutrophication risk criterion of the WFD. This classification is made according to the percentile-90 of chlorophyll-a calculated during the productive season, from March to October. Despite a lack of sensor coverage over a small fraction of the near shore waters, this work shows that the satellite monitoring can considerably ease the application of the WFD. [Copyright &y& Elsevier]