This research aimed to gain insight into the mechanisms underlying CG exocytosis and their perturbation in the context of immune disorders. To reach this overall objective, the project was carried out through two work packages (WPs):
WP1) Dissecting the function in lymphocyte cytotoxicity of two distinct Munc13-4isoforms
WP2) Gaining insights to the regulation of RE fusion required for subsequent CG release
WP1. Munc13-4 is a crucial regulator of CG exocytosis. Mutations in UNC13D, the gene encoding for Munc13-4, cause a life-threatening hyperinflammatory syndrome, namely familial hemophagocytic lymphocytosis (FHL) type 3. Mutation in other genes encoding for other key factors of lymphocyte cytotoxicity including Perforin, Syntaxin 11 and Munc18-2 account for FHL2, FHL4 and FHL5, respectively. FHL affects about 1 in 50,000 new borns, with 30% of cases depending on mutations affecting Munc13-4. A previous study from Prof. Bryceson’s laboratory based on genetic analysis of FHL3 patients identified a novel alternative isoform of Munc13-4. Interestingly, this isoform originates from a non-coding region of the gene. In this project, the role in the regulation of CG exocytosis of the novel isoform was compared to the conventional Munc13-4. This part of the research was performed in collaboration with the group of Prof. Jens Rettig, Saarland University, Homburg, Germany and Prof. Marina Cavazzana, Imagine Institute, Paris, France. We showed that these isoforms have a similar localization inside the cells and similarly contribute to lymphocyte cytotoxicity. These results have been included in an open access original research article published in June 2020.
WP2. Another key question of this research was to address the role in lymphocyte cytotoxicity of special intracellular organelles, namely recycling endosome (RE). They are small vesicles inside the cells which, as their name suggest, contribute to the transport and recycling of molecules between intracellular organelles and the plasma membrane. A recent study from Prof. Bryceson’s laboratory showed that in cytotoxic lymphocytes, REs contribute to transport towards the plasma membrane of Syntaxin 11, which is a crucial protein for CG exocytosis. The transport and the delivery of Syntaxin 11 to the plasma membrane through REs is regulated by the protein VAMP8. The goal was to identify other important regulators of CG exocytosis, which in addition to Syntaxin 11, are delivered to the plasma membrane through REs regulated by VAMP8 using a high-throughput Mass spectrometry approach. This analytic technique is often used to dissect different components of complex samples. As model of study, a NK-92 cell line that recapitulates the phenotype of the cytotoxic lymphocytes in our body was used. The proposed approach included to genetically manipulate NK-92 to overexpress VAMP8 coupled with a tag. The experimental condition to achieve RE purification from NK-92 were successfully optimized by the researcher. This part of the research is still ongoing. However, the experimental strategy as well as preliminary data were presented by the Researcher during the HERM seminar.
