Membrane protein dynamics in Trypanosomes

The parasite Trypanosoma brucei lives in the bloodstream of their mammalian host, which provides them with access to necessary metabolites, however coming at the cost of being constantly exposed to the immune system. T. brucei uses its highly diffusive, glycosylphosphatidylinositol-anchored variant surface glycoprotein (VSG) coat to efficiently remove compromising antibodies from its surface, hence facilitating immune evasion. Within the cell, efficient and fast sorting of bound and recycling of unbound cargo needs to occur, as it is crucial for the survival of the parasite. New studies using light and electron microscopy revealed a complex, three-dimensional membrane system with overlapping subdomains of classical endosomal markers. Its continuous dynamics however could hardly be addressed with the previously used methods and are still unknown. Using advanced three-dimensional single molecule imaging based on astigmatism, the HALO-tag as a versatile labelling interface and photostable dyes, the speed and behavior of several marker molecules was measured with high spatial and temporal resolution to gain insight into the functional dynamics of the endosomal system in T. brucei. Since the image modalities changed with the introduction of astigmatism, a new analysis workflow was developed, also utilizing recent advancements in neural networks. To facilitate efficient, easy and unbiased data processing, visualization and analysis, ThirdPeak, a software with graphical user interface was developed. After validating its performance and accuracy with VSG diffusion from the cell surface and artificial diffusion data, the dynamics from within the cell were elucidated. Using a labelled membrane-bound marker on the luminal side of the endosomal system revealed slower diffusion compared to the surface, which might be necessary for efficient sorting. Comparing the dynamics of the canonical markers for early, late and recycling endosome, which can be present in the same membrane subdomain, confirmed significant different behaviors and speeds between them, with the early endosome marker being highly dynamic like VSG, while the markers for late and recycling endosome remained mostly immobile, potentially enabling recruitment of further downstream effector proteins. Imaging of a marker for the lysosomal membrane showed a fast, short-lived connection forming between the lysosome and the pocket, potentially documenting a novel lysosomal stress relief mechanism. This work therefore provides a new tool for the analysis of cellular dynamics, spanning the fields of biology, informatics and physics. Using the newly gathered information on the dynamics within the parasite provides further evidence onto existing hypothesis while challenging others. Introducing a significant amount of automatization to the data acquisition will allow for even more efficient and detailed insights, helping to better understand this highly efficient sorting system within the parasite.