Identification of a bipartite focal adhesion localization signal in RhoU/Wrch-1, a Rho family GTPase that regulates cell adhesion and migration

International audienceBackground information. Rho GTPases are important regulators of cytoskeleton dynamics and cell adhesion. RhoU/ Wrch-1 is a Rho GTPase which shares sequence similarities with Rac1 and Cdc42 (cell division cycle 42), but has also extended N-and C-terminal domains. The N-terminal extension promotes binding to SH3 (Src homology 3)-domain-containing adaptors, whereas the C-terminal extension mediates membrane targeting through palmitoyl-ation of its non-conventional CAAX box. RhoU/Wrch-1 possesses transforming activity, which is negatively regulated by its N-terminal extension and depends on palmitoylation. Results. In the present study, we have shown that RhoU is localized to podosomes in osteoclasts and c-Src-expressing cells, and to focal adhesions of HeLa cells and fibroblasts. The N-terminal extension and the palmitoyl-ation site were dispensable, whereas the C-terminal extension and effector binding loop were critical for RhoU targeting to focal adhesions. Moreover, the number of focal adhesions was reduced and their distribution changed upon expression of activated RhoU. Conversely, RhoU silencing increased the number of focal adhesions. As RhoU was only transiently associated with adhesion structures, this suggests that RhoU may modify adhesion turnover and cell migration rate. Indeed, we found that migration distances were increased in cells expressing activated RhoU and decreased when RhoU was knocked-down. Conclusions. Our data indicate that RhoU localizes to adhesion structures, regulates their number and distribution and increases cell motility. It also suggests that the RhoU effector binding and C-terminal domains are critical for these functions

Bone resorption by osteoclasts involves fine tuning of RHOA activity by its microtubule-associated exchange factor GEF-H1

Bone health is controlled by the balance between bone formation by osteoblasts and degradation by osteoclasts. A disequilibrium in favor of bone resorption leads to osteolytic diseases characterized by decreased bone density. Osteoclastic resorption is dependent on the assembly of an adhesion structure: the actin ring, also called podosome belt or sealing zone, which is composed of a unique patterning of podosomes stabilized by microtubules. A better understanding of the molecular mechanisms regulating the crosstalk between actin cytoskeleton and microtubules network is key to find new treatments to inhibit bone resorption. Evidence points to the importance of the fine tuning of the activity of the small GTPase RHOA for the formation and maintenance of the actin ring, but the underlying mechanism is not known. We report here that actin ring disorganization upon microtubule depolymerization is mediated by the activation of the RHOA-ROCK signaling pathway. We next show the involvement of GEF-H1, one of RHOA guanine exchange factor highly expressed in osteoclasts, which has the particularity of being negatively regulated by sequestration on microtubules. Using a CRISPR/Cas9-mediated GEF-H1 knock-down osteoclast model, we demonstrate that RHOA activation upon microtubule depolymerization is mediated by GEF-H1 release. Interestingly, although lower levels of GEF-H1 did not impact sealing zone formation in the presence of an intact microtubule network, sealing zone was smaller leading to impaired resorption. Altogether, these results suggest that a fine tuning of GEF-H1 through its association with microtubules, and consequently of RHOA activity, is essential for osteoclast sealing zone stability and resorption function

Identification and characterization of a triacylglycerol lipase in Arabidopsis homologous to mammalian acid lipases

AbstractTriacylglycerol (TAG) lipases have been thoroughly characterized in mammals and microorganisms. By contrast, very little is known on plant TAG lipases. An Arabidopsis cDNA called AtLip1 (At2g15230), which exhibits strong homology to lysosomal acid lipase, was found to drive the synthesis of an active TAG lipase when expressed in the baculovirus system. The lipase had a maximal activity at pH 6 and the specific activity was estimated to be about 45μmolmin−1mg−1 protein using triolein as a substrate. Knock-out mutant analysis showed no phenotype during germination indicating that this enzyme is fully dispensable for TAG storage breakdown during germination. Northern blot analyses indicated that the transcript is present in all tissues tested

An Intermittent Live Cell Imaging Screen for siRNA Enhancers and Suppressors of a Kinesin-5 Inhibitor

Kinesin-5 (also known as Eg5, KSP and Kif11) is required for assembly of a bipolar mitotic spindle. Small molecule inhibitors of Kinesin-5, developed as potential anti-cancer drugs, arrest cell in mitosis and promote apoptosis of cancer cells. We performed a genome-wide siRNA screen for enhancers and suppressors of a Kinesin-5 inhibitor in human cells to elucidate cellular responses, and thus identify factors that might predict drug sensitivity in cancers. Because the drug's actions play out over several days, we developed an intermittent imaging screen. Live HeLa cells expressing GFP-tagged histone H2B were imaged at 0, 24 and 48 hours after drug addition, and images were analyzed using open-source software that incorporates machine learning. This screen effectively identified siRNAs that caused increased mitotic arrest at low drug concentrations (enhancers), and vice versa (suppressors), and we report siRNAs that caused both effects. We then classified the effect of siRNAs for 15 genes where 3 or 4 out of 4 siRNA oligos tested were suppressors as assessed by time lapse imaging, and by testing for suppression of mitotic arrest in taxol and nocodazole. This identified 4 phenotypic classes of drug suppressors, which included known and novel genes. Our methodology should be applicable to other screens, and the suppressor and enhancer genes we identified may open new lines of research into mitosis and checkpoint biology

Tensins are versatile regulators of Rho GTPase signalling and cell adhesion: Regulation of Rho GTPases signalling by Tensins

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Cibler l’activité de l’ostéoclaste

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The Evolutionary Landscape of Dbl-Like RhoGEF Families: Adapting Eukaryotic Cells to Environmental Signals

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Using a Modified Yeast Two-Hybrid System to Screen for Chemical GEF Inhibitors

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Éditorial

Nègre Anne, Blangy Michel. Éditorial. In: Diplômées, n°206, 2003. Les Olympes de la parole. pp. 146-147