Visualizing the surface dynamics on and in Trypanosoma brucei (POP-WS)

Abstract text

Due to its extracellular lifestyle, Trypanosoma brucei is always exposed to the immune system of its host. While being able to switch their predominant variant surface antigen (VSG), their immune evasion strategy also heavily relies on the mobility of their surface coat, which enables them to remove antibodies from their cell surface within seconds via endocytosis. As the fast turnover on the surface must be mirrored in speed for degradation or recycling, we now also turn our attention to the inside of the cell.

We want to visualize the dynamics inside the endosomal network in three dimensions to elucidate how the sorting of membrane proteins is realized in a system, that does not have the time for expensive membrane budding and fusion process.

To measure the intracellular dynamics, we express an insect-stage specific membrane protein (EP1) in the bloodstream form of the parasite, which will stay confined to the endosomal compartments and the flagellar pocket. To selectively mark the protein, we Halo-tagged it. Living cells are labelled using picomoles of the dye, immobilized in hydro gel and imaged at 37°C. With the help of an astigmatic lens, we collect 3D information of our single emitters. The acquired data is processed using SMAP by Jonas Ries and reveals a resolution of 30 nm in x/y and 80 nm in z imaging at 25 Hz. The calculated positions of single emitters are then used to generate tracks using Swift by Ulrike Endersfelder. Those tracks are then analyzed using our custom software based on the SPTAnalysis by Pierre Parutto.

Preliminary data of Atto643-NHS labelled VSG shows that the flagellar membrane can act as a diffusional barrier that can not easily be crossed by surface molecules and that an accumulation of signal could be localized inside the cell in proximity to the flagellar pocket. Overall, the surface dynamics of the VSG of the 3D data seem to be comparable with previously acquired 2D data.

As the comparison of 2D and 3D data lead to a similar diffusion coefficient, the overall workflow for 3D data seems to be suitable. A challenge will arise with the spatially quite confined endosomal system to generate sufficient data for analysis, which will be compensated for by the long-lived Janelia Fluor dyes. In addition, our workflow would allow statistical analysis of the 3D track data, which could change the general understanding of the endocytotic flow and shed light on how the sorting and recycling mechanism really work.