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

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

4-Chloro­benzoyl-meso-octa­methyl­calix[2]pyrrolidino[2]pyrrole: an acyl chloride derivative of a partially reduced calix[4] pyrrole

In the title compound, C35H47ClN4O, the two pyrrolidine rings have envelope conformations. The conformation of the macrocycle is stabilized by N—H⋯N hydrogen bonds and a C—H⋯N inter­action. The benzoyl ring is inclined to the adjacent pyrrole ring by 11.66 (11)°, with a centroid–centroid distance of 3.7488 (13) Å. In the crystal, molecules are linked by N—H⋯O hydrogen bonds into helical chains propagating in [010] and C—H⋯O and C—H⋯π interactions are also observed

Aboriginal tourism in northern Canada : how collaborative research can improve community engagement in tourism projects

Meeting: Celebrating Dialogue : An International SAS2 Forum, November 3, 2008, Carleton University, Ottawa, ON, CASituated in the James Bay area, the Cree Nation is comprised of 15,000 people in nine communities spread out over 350,000 square kilometers. The project’s goal is to position Eeyou Istchee (the traditional territory and homeland of the Cree) as a Cree tourism destination for external markets. Participants were able to share knowledge, build bridges, develop links between communities and operators, and work together at the regional level. Community Tourism Officers were provided with tools they can use independently at the community level and for other meetings (Band Council)

Towards a Structural Comprehension of Bacterial Type VI Secretion Systems: Characterization of the TssJ-TssM Complex of an Escherichia coli Pathovar

Type VI secretion systems (T6SS) are trans-envelope machines dedicated to the secretion of virulence factors into eukaryotic or prokaryotic cells, therefore required for pathogenesis and/or for competition towards neighboring bacteria. The T6SS apparatus resembles the injection device of bacteriophage T4, and is anchored to the cell envelope through a membrane complex. This membrane complex is composed of the TssL, TssM and TagL inner membrane anchored proteins and of the TssJ outer membrane lipoprotein. Here, we report the crystal structure of the enteroaggregative Escherichia coli Sci1 TssJ lipoprotein, a two four-stranded β-sheets protein that exhibits a transthyretin fold with an additional α-helical domain and a protruding loop. We showed that TssJ contacts TssM through this loop since a loop depleted mutant failed to interact with TssM in vitro or in vivo. Biophysical analysis of TssM and TssJ-TssM interaction suggest a structural model of the membrane-anchored outer shell of T6SS. Collectively, our results provide an improved understanding of T6SS assembly and encourage structure-aided drug design of novel antimicrobials targeting T6SS

The structure of the ternary Eg5–ADP–ispinesib complex

The human kinesin Eg5 is responsible for bipolar spindle formation during early mitosis. Inhibition of Eg5 triggers the formation of monoastral spindles, leading to mitotic arrest that eventually causes apoptosis. There is increasing evidence that Eg5 constitutes a potential drug target for the development of cancer chemotherapeutics. The most advanced Eg5-targeting agent is ispinesib, which exhibits potent antitumour activity and is currently in multiple phase II clinical trials. In this study, the crystal structure of the Eg5 motor domain in complex with ispinesib, supported by kinetic and thermodynamic binding data, is reported. Ispinesib occupies the same induced-fit pocket in Eg5 as other allosteric inhibitors, making extensive hydrophobic interactions with the protein. The data for the Eg5-ADP-ispinesib complex suffered from pseudo-merohedral twinning and revealed translational noncrystallographic symmetry, leading to challenges in data processing, space-group assignment and structure solution as well as in refinement. These complications may explain the lack of available structural information for this important agent and its analogues. The present structure represents the best interpretation of these data based on extensive data-reduction, structure-solution and refinement trials