Visualizing the subcellular localization of RHOB-GTP and GTPase-Effector complexes using a split-GFP/nanobody labelling assay.

Small GTPases are highly regulated proteins that control essential signaling pathways through the activity of their effector proteins. Among the RHOA subfamily, RHOB regulates peculiar functions that could be associated with the control of the endocytic trafficking of signaling proteins. Here, we used an optimized assay based on tripartite split-GFP complementation to localize GTPase-effector complexes with high-resolution. The detection of RHOB interaction with the Rhotekin Rho binding domain (RBD) that specifically recognizes the active GTP-bound GTPase, is performed in vitro by the concomitant addition of recombinant GFP1-9 and a GFP nanobody. Analysis of RHOB-RBD complexes localization profiles combined with immunostaining and live cell imaging indicated a serum-dependent reorganization of the endosomal and membrane pool of active RHOB. We further applied this technology to the detection of RHO-effector complexes that highlighted their subcellular localization with high resolution among the different cellular compartments.

Impact of Regorafenib on Endothelial Transdifferentiation of Glioblastoma Stem-like Cells.

Glioblastomas (GBM) are aggressive brain tumours with a poor prognosis despite heavy therapy that combines surgical resection and radio-chemotherapy. The presence of a subpopulation of GBM stem cells (GSC) contributes to tumour aggressiveness, resistance and recurrence. Moreover, GBM are characterised by abnormal, abundant vascularisation. Previous studies have shown that GSC are directly involved in new vessel formation via their transdifferentiation into tumour-derived endothelial cells (TDEC) and that irradiation (IR) potentiates the pro-angiogenic capacity of TDEC via the Tie2 signalling pathway. We therefore investigated the impact of regorafenib, a multikinase inhibitor with anti-angiogenic and anti-tumourigenic activity, on GSC and TDEC obtained from irradiated GSC (TDEC IR+) or non-irradiated GSC (TDEC). Regorafenib significantly decreases GSC neurosphere formation in vitro and inhibits tumour formation in the orthotopic xenograft model. Regorafenib also inhibits transdifferentiation by decreasing CD31 expression, CD31+ cell count, pseudotube formation in vitro and the formation of functional blood vessels in vivo of TDEC and TDEC IR+. All of these results confirm that regorafenib clearly impacts GSC tumour formation and transdifferentiation and may therefore be a promising therapeutic option in combination with chemo/radiotherapy for the treatment of highly aggressive brain tumours.

Long-Read Genome Sequence of the Sugar Beet Rhizosphere Mycoparasite Pythium oligandrum.

Pythium oligandrum is a soil born free living oomycete able to parasitize fungi and oomycetes prey, including important plant and animals pathogens. Pythium oligandrum can colonize endophytically the root tissues of diverse plants where it induces plant defenses. Here we report the first long-read genome sequencing of a P. oligandrum strain sequenced by PacBio technology. Sequencing of genomic DNA loaded onto six SMRT cells permitted the acquisition of 913,728 total reads resulting in 112X genome coverage. The assembly and polishing of the genome sequence yielded180 contigs (N50 = 1.3 Mb; L50 = 12). The size of the genome assembly is 41.9 Mb with a longest contig of 2.7 Mb and 15,007 predicted protein-coding genes among which 95.25% were supported by RNAseq data, thus constituting a new Pythium genome reference. This data will facilitate genomic comparisons of Pythium species that are commensal, beneficial or pathogenic on plant, or parasitic on fungi and oomycete to identify key genetic determinants underpinning their diverse lifestyles. In addition comparison with plant pathogenic or zoopathogenic species will illuminate genomic adaptations for pathogenesis toward widely diverse hosts.