Glioma stem cells invasive phenotype at optimal stiffness is driven by MGAT5 dependent mechanosensing.

BACKGROUND: Glioblastomas stem-like cells (GSCs) by invading the brain parenchyma, remains after resection and radiotherapy and the tumoral microenvironment become stiffer. GSC invasion is reported as stiffness sensitive and associated with altered N-glycosylation pattern. Glycocalyx thickness modulates integrins mechanosensing, but details remain elusive and glycosylation enzymes involved are unknown. Here, we studied the association between matrix stiffness modulation, GSC migration and MGAT5 induced N-glycosylation in fibrillar 3D context. METHOD: To mimic the extracellular matrix fibrillar microenvironments, we designed 3D-ex-polyacrylonitrile nanofibers scaffolds (NFS) with adjustable stiffnesses by loading multiwall carbon nanotubes (MWCNT). GSCs neurosphere were plated on NFSs, allowing GSCs migration and MGAT5 was deleted using CRISPR-Cas9. RESULTS: We found that migration of GSCs was maximum at 166 kPa. Migration rate was correlated with cell shape, expression and maturation of focal adhesion (FA), Epithelial to Mesenchymal Transition (EMT) proteins and (β1,6) branched N-glycan binding, galectin-3. Mutation of MGAT5 in GSC inhibited N-glycans (β1-6) branching, suppressed the stiffness dependence of migration on 166 kPa NFS as well as the associated FA and EMT protein expression. CONCLUSION: MGAT5 catalysing multibranched N-glycans is a critical regulators of stiffness induced invasion and GSCs mechanotransduction, underpinning MGAT5 as a serious target to treat cancer

Trypanosoma brucei glycosomal glyceraldehyde-3-phosphate dehydrogenase genes are stage-regulated at the transcriptional level.

Regions 5' of the glycosomal glyceraldehyde-3-phosphate dehydrogenase (gGAPDH) gene from Trypanosoma brucei were tested for their ability to promote chloramphenicol acetyl-transferase (CAT) expression on reintroduction by electroporation into the parasite. Deletion analysis mapped the gGAPDH promoter to within 403 nts of the start of translation. A transcription initiation site was mapped at around -190 nts from the ATG start codon by RNase protection and by primer extension. The higher expression of gGAPDH in bloodstream T. brucei, compared to procyclic (insect) forms, was largely attributed to differences in promoter activity. The gGAPDH promoter gave rise to relatively high CAT signals upon transfection into bloodstream T. brucei and relatively low signals in procyclic T. brucei, compared with levels resulting from transfection with the procyclic acidic repetitive protein (PARP) promoter. In addition, RNase protection data showed a higher level of gGAPDH primary transcripts in bloodstream. T. brucei. The PARP mini-exon addition region abolished transient CAT expression directed by either the gGAPDH or PARP promoters in bloodstream T. brucei implying that transplicing can be a point of stage-specific gene regulation

The actin gene promoter of Trypanosoma brucei.

The actin genes of Trypanosoma brucei are transcribed constitutively during the parasite life-cycle, by a polymerase sensitive to alpha-amanitin. The start region of the actin gene transcription unit was mapped by virtue of the accumulation of promoter-proximal transcripts which occurs following moderate UV irradiation. This region, located about 4 kilobases upstream from the genes, was able to direct transient expression of the bacterial Chloramphenicol Acetyl Transferase (CAT) gene in both bloodstream and procyclic forms of the parasite. The essential region of the promoter was defined by deletion, and appeared to be within 600 bp upstream from the putative transcription start site. It does not share significant homology with the other trypanosome promoters described so far (VSG, procyclin, rDNA), which all direct alpha-amanitin resistant transcription

Premier pas vers la caractérisation du mécanisme d'action et de la toxicité des phostines sur des modèles de gliomes in vitro et in vivo.

National audienc

Validation d'une preuve de concept : les phostines comme agents antiprolifératifs

National audienc

In vitro guide RNA/mRNA chimaera formation in Trypanosoma brucei RNA editing.

The post-transcriptional processing of various mitochondrial transcripts in kinetoplastids, kRNA editing, adds and removes uridines, producing mature messenger RNAs. This editing seems to be directed by 'guide' RNAs (gRNAs) which are complementary to portions of the mature message. The editing mechanism has been proposed to entail transesterification. Detection of chimaeric gRNA-mRNA molecules, intermediates predicted by transesterification, support this model. We report here the in vitro formation of such chimaeras where endogenous gRNAs are covalently linked to added synthetic mRNA. Addition of gel-purified gRNAs to the standard reaction mix increases chimaera formation. This increase is not observed when the gRNA 3'-hydroxyl group is chemically modified, identifying this terminal hydroxyl as the reactive group. These results provide the first experimental evidence for an in vitro RNA editing event and support the involvement of transesterification as a chemical mechanism