Immune cells rely on information conveyed by fragments of intracellular components being presented on major histocompatibility complex (MHC) molecules on the cell surface. Many pathogens are too large to be recognized directly by immune cells, and must first be digested into smaller fragments (peptides) that can be presented by specialized antigen-presenting cells (APCs), such as dendritic cells (DCs) and macrophages.
Cancer immunotherapy attempts to harness the power and specificity of the immune system to treat tumors. The molecular identification of cancer-specific antigens has allowed the development of antigen-specific personalized immunotherapy. Due to their properties, DCs are often called 'nature's adjuvants' and thus have become a leading vehicle for antigen delivery.
MHC class II associated invariant
chain (Ii) or CD74 (Cluster of Differentiation 74) is a protein essential for proper formation and trafficking of MHC class II to the antigen loading compartment and thus efficient antigen presentation. Ii induces a delay in endosomal maturation and an enlargement of early endosomes dependent on its cytoplasmic domain. This function is independent of the normal early endosomal fusion machinery comprising the small GTPase Rab5, PI3 kinase and the tethering factor EEA1.
All membrane fusion events in eukaryotic cells are mediated by SNARE (soluble NSF attachment protein receptor) proteins. Four different SNARE-domains (Qa, Qb, Qc and R) are needed to form a functional complex. We have recently performed an siRNA screen to identify SNAREs and SNARE-regulating proteins involved in Ii-induced endosome enlargement.
The aim of this master project is to characterize the specific SNARE complex in Ii-positive endosomes and its functional significance in antigen presentation. The master student will study the localization and interaction of SNARE proteins by immunofluorescence, expression of fluorescent protein-tagged SNARE proteins and confocal microscopy, depletion of target proteins, biochemistry, immunprecipitaions etc. The experimental set up is also based on antigen presenting cell lines where invariant chain is KO by CRISPR/Cas9 and cells where the different fluorescent Rabs are inserted by the same method.
Methods:
The project will take place in the group of Oddmund Bakke who is also head of the NorMIC Oslo imaging platform. The student will the in particular learn several state-of-the-art microscopy techniques image processing and quantitative image analysis that are very much in demand.
The project will also involve additional techniques of molecular biology and cell biology, including mammalian cell culture, gene manipulations, protein expression and purification, and immunoprecipitation +++ and is particularly suited for a motivated candidate that have a wish to continue working with cellular/molecular research and imaging. The project is a basis for the further development of a cancer immunotherapy vaccine (W?lchli S et al.Eur J Immunol. 2014 Mar;44(3):774-84. doi: 10.1002/eji.201343671) and Kucera et al 2020, https://www.biorxiv.org/content/10.1101/2020.05.13.091579v1 ) and recent work on the SNARES:
Margiotta A, Frei DM, Sendstad IH, Janssen L, Neefjes J, Bakke O. Invariant chain regulates endosomal fusion and maturation through an interaction with the SNARE Vti1b. J Cell Sci. 2020 Oct 9;133(19):jcs244624. doi: 10.1242/jcs.244624. PMID: 32907852.
Methods in imaging are also well described in:
Skjeldal FM, Haugen LH, Mateus D, Frei DM, R?dseth AV, Hu X, Bakke O. De novo formation of early endosomes during Rab5-to-Rab7a transition. J Cell Sci. 2021 Apr 15;134(8):jcs254185. doi: 10.1242/jcs.254185. Epub 2021 Apr 27. PMID: 33737317; PMCID: PMC8106955.
Oddmund Bakke, professor, Main Supervisor
phone: +47 922855787/95851479. email: oddmund.bakke@ibv.uio.no
Frode Skjeldal, Daily supervisor
22854361/40218114 frode.skjeldal@ibv.uio.no
Laboratory:
NorMIC-Oslo Imaging Platform,
3rd floor Kristine Bonnevies hus
Department of Biosciences, University of Oslo