Mécanismes moléculaires des rhoGDIs (le système rhoGDI3/RhoG/TrioGEF comme modèle d'nvestigation)

Résumé françaisRésumé anglaisORSAY-PARIS 11-BU Sciences (914712101) / SudocSudocFranceF

Late Permain limestones and the Permian-Triassic boundary, new biostratigraphic, palaeobiogeographical and geochemical data in Caucasus and eastern Europe. In ALVARO J.J., Aretz M., Boulvain F., Münnecke A., VACHARD D. & Vennin E. (eds), Palaeozoic Reefs and Bioaccumulations: Climatic and Evolutionary Controls.

International audienc

Clathrin-independent endocytosis, retrograde trafficking, and cell polarity

International audienceSeveral mechanisms allow for cargo internalization into cells within membrane-bound endocytic carriers. How these internalization processes couple to specific pathways of intracellular distribution remains poorly explored. Here, we review uptake reactions that are independent of the conventional clathrin machinery. We discuss how these link to retrograde trafficking from endosomes to the Golgi apparatus and exemplify biological situations in which the polarized secretion capacity of the Golgi apparatus allows for retrograde cargoes to be delivered to specialized areas of the plasma membrane, such as the leading edge of migratory cells or the immunological synapse of immune cells. We also address the evidence that allows to position apicobasal polarity of epithelial cells in this context. The underlying theme is thereby the functional coupling between specific types of endocytosis to intracellular retrograde trafficking for protein cargoes that need to be localized in a highly polarized and dynamic manner to plasmalemmal subdomains

Tumor Delivery of Ultrasound Contrast Agents Using Shiga Toxin B Subunit

The present study demonstrates the targeting of ultrasound contrast agents to human xenograft tumors by exploiting the overexpression of the glycolipid Gb3 in neovasculature. To this end, microbubbles were functionalized with a natural Gb3 ligand, the B subunit of the Shiga toxin (STxB). The targeting of Gb3-expressing tumor cells by STxB microbubbles was first shown by flow cytometry and fluorescence microscopy. A significantly higher proportion of STxB microbubbles were associated with Gb3-expressing tumor cells compared to cells in which Gb3 expression was inhibited. Moreover, ultrasonic imaging of culture plates showed a 12 dB contrast enhancement in average backscattered acoustic intensity on the surface of Gb3-expressing cells compared to Gb3-negative cells. Also, a 18 dB contrast enhancement was found in favor of STxB microbubbles compared to unspecific microbubbles. Microbubble signal intensity in subcutaneous tumors in mice was more than twice as high after the injection of STxB-functionalized microbubbles compared to the injection of unspecific microbubbles. These in vitro and in vivo experiments demonstrated that STxB-functionalized microbubbles bind specifically to cells expressing the Gb3 glycolipid. The cell-binding moieties of toxins thus appear as a new group of ligands for angiogenesis imaging with ultrasound

Site-specific N-glycan profiles of α5β1 integrin from rat liver

Background Information: Like most other cell surface proteins, α5β1 integrin is glycosylated, which is required for its various activities in ways that mostly remain to be determined. Results: Here, we have established the first comprehensive site-specific glycan map of α5β1 integrin that was purified from a natural source, that is, rat liver. This analysis revealed striking site selective variations in glycan composition. Complex bi, tri, or tetraantennary N-glycans were predominant at various proportions at most potential N-glycosylation sites. A few of these sites were nonglycosylated or contained high mannose or hybrid glycans, indicating that early N-glycan processing was hindered. Almost all complex N-glycans had fully galactosylated and sialylated antennae. Moderate levels of core fucosylation and high levels of O-acetylation of NeuAc residues were observed at certain sites. An O-linked HexNAc was found in an EGF-like domain of β1 integrin. The extensive glycan information that results from our study was projected onto a map of α5β1 integrin that was obtained by homology modeling. We have used this model for the discussion of how glycosylation might be used in the functional cycle of α5β1 integrin. A striking example concerns the involvement of glycan-binding galectins in the regulation of the molecular homeostasis of glycoproteins at the cell surface through the formation of lattices or endocytic pits according to the glycolipid-lectin (GL-Lect) hypothesis. Conclusion: We expect that the glycoproteomics data of the current study will serve as a resource for the exploration of structural mechanisms by which glycans control α5β1 integrin activity and endocytic trafficking. Significance: Glycosylation of α5β1 integrin has been implicated in multiple aspects of integrin function and structure. Yet, detailed knowledge of its glycosylation, notably the specific sites of glycosylation, is lacking. Furthermore, the α5β1 integrin preparation that was analyzed here is from a natural source, which is of importance as there is not a lot of literature in the field about the glycosylation of “native” glycoproteins

Absolute Quantification of Drug Vector Delivery to the Cytosol

International audienceMacromolecular drugs inefficiently cross membranes to reach their cytosolic targets. They require drug delivery vectors to facilitate their translocation across the plasma membrane or escape from endosomes. Optimization of these vectors has however been hindered by the difficulty to accurately measure cytosolic arrival. We have developed an exceptionally sensitive and robust assay for the relative or absolute quantification of this step. The assay is based on benzylguanine and biotin modifications on a drug delivery vector of interest, which allow, respectively, for selective covalent capture in the cytosol with a SNAP-tag fusion protein and for quantification at picomolar sensitivity. The assay was validated by determining the absolute numbers of cytosolic molecules for two drug delivery vectors: the B-subunit of Shiga toxin and the cell-penetrating peptide TAT. We expect this assay to favor delivery vector optimization and the understanding of the enigmatic translocation process

A new delivery system for auristatin in STxB-drug conjugate therapy

International audienc

Local IFNα enhances the anti-tumoral efficacy of systemic anti-PD1 to prevent tumor relapse

International audienceBackground Tumor relapse constitutes a major challenge for anti-tumoral treatments, including immunotherapies. Indeed, most cancer-related deaths occur during the tumor relapse phase. Methods We designed a mouse model of tumor relapse in which mice transplanted with E7 + TC1 tumor cells received a single therapeutic vaccination of STxB-E7+IFNα. Unlike the complete regression observed after two vaccinations, such a treatment induced a transient shrinkage of the tumor mass, followed by a rapid tumor outgrowth. To prevent this relapse, we tested the efficacy of a local administration of IFNα together with a systemic therapy with anti-PD1 Ab. The immune response was analyzed during both the tumor regression and relapse phases. Results We show that, during the regression phase, tumors of mice treated with a single vaccination of STxB-E7 + IFNα harbor fewer activated CD8 T cells and monocytes than tumors doomed to fully regress after two vaccinations. In contrast, the systemic injection of an anti-PD1 Ab combined with the peri-tumoral injection of IFNα in this time frame promotes infiltration of activated CD8 T cells and myeloid cells, which, together, exert a high cytotoxicity in vitro against TC1 cells. Moreover, the IFNα and anti-PD1 Ab combination was found to be more efficient than IFNα or anti-PD1 used alone in preventing tumor relapse and was better able to prolong mice survival. Conclusions Together, these results indicate that the local increase of IFNα in combination with an anti-PD1 therapy is an effective way to promote efficient and durable innate and adaptive immune responses preventing tumor relapse

In Vivo Tumor Targeting by the B-Subunit of Shiga Toxin

Delivery of drugs to the appropriate target cells would improve efficacy and reduce potential side effects. The nontoxic B-subunit of the intestinal pathogen-produced Shiga toxin (STxB) binds specifically to the glycosphingolipid Gb 3 , overex-pressed in membranes of certain tumor cells, and enters these cells through the retrograde pathway. Therefore, STxB binding to Gb 3 receptors may be useful for cell-specific vectorization or imaging purposes. Here we labeled STxB with a fluorophore to evaluate its potential as an in vivo cell-specific targeting reagent in two different models of human colorectal carcinoma. Fluorescent STxB was administered systemically to xenografted nude mice, and its biodistribution was studied by optical imaging. The use of fluorescent STxB allowed the combination of the macroscopic observations with analyses at the cellular level using confocal microscopy. After administration, the fluorescent STxB was slowly eliminated by renal excretion. However, it accumulated in the tumor area. Furthermore, STxB was demonstrated to enter the Gb 3 -expressing tumoral cells, as well as the epithelial cells of the neovascularization and the monocytes and macrophages surrounding the xenografts

In Vivo Tumor Targeting by the B-Subunit of Shiga Toxin

Delivery of drugs to the appropriate target cells would improve efficacy and reduce potential side effects. The nontoxic B-subunit of the intestinal pathogen-produced Shiga toxin (STxB) binds specifically to the glycosphingolipid Gb 3 , overex-pressed in membranes of certain tumor cells, and enters these cells through the retrograde pathway. Therefore, STxB binding to Gb 3 receptors may be useful for cell-specific vectorization or imaging purposes. Here we labeled STxB with a fluorophore to evaluate its potential as an in vivo cell-specific targeting reagent in two different models of human colorectal carcinoma. Fluorescent STxB was administered systemically to xenografted nude mice, and its biodistribution was studied by optical imaging. The use of fluorescent STxB allowed the combination of the macroscopic observations with analyses at the cellular level using confocal microscopy. After administration, the fluorescent STxB was slowly eliminated by renal excretion. However, it accumulated in the tumor area. Furthermore, STxB was demonstrated to enter the Gb 3 -expressing tumoral cells, as well as the epithelial cells of the neovascularization and the monocytes and macrophages surrounding the xenografts