Aquaporin 4 (AQP4) is a channel protein that facilitates water transport through cell membranes and is arranged in orthogonal arrays in these membranes. Autoantibodies against AQP4 play a key role in the autoimmune disease neuromyelitis optica. This is currently incurable and causes symptoms such as visual disturbances and convulsions. For later investigations of the interaction of autoantibodies and AQP4, membranes with AQP4 arrays are isolated and analysed in this project. Here we show approaches for the isolation of AQP4 membrane pieces and how different cell disruption steps contribute to the improvement of the isolation. The membranes are extracted from HEK293 cells expressing fluorescently labelled AQP4-RFP (red fluorescent protein). The membrane fraction is to be isolated by centrifugation, applied to planar substrates and then imaged in high resolution using fluorescence microscopy (FM) and atomic force microscopy (AFM). However, this proved to be demanding and there were problems with contamination by cellular debris. Therefore, an alternative approach to obtain AQP4 membranes, the isolation of so-called Giant Plasma Membrane Vesicles (GPMVs), was carried out. This allowed further knowledge to be gained about the coating, distribution and number of AQP4 in the isolated membrane material. It was also possible to successfully bind antibodies to the isolated AQP4 membranes. The successful recording of the antibody binding confirmed that the optimisation steps are going in the right direction and that further improvements to the methods and therapies for the treatment of NMO can be developed and achieved on the basis of these research results.
Date of Award | 2024 |
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Original language | German (Austria) |
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Supervisor | Andreas Karner (Supervisor) |
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Modellsysteme zur Charakterisierung des Clusterings von Antikörpern
Gaderer, J. S. (Author). 2024
Student thesis: Master's Thesis