The 3D architecture of the Trypanosoma brucei endosomes reveals the structural basis for rapid plasma membrane recycling

Abstract text

Trypanosoma brucei is an extracellular human and livestock pathogen that follows an effective strategy to evade the host immune system. This strategy relies on stochastical changes in the expression of variant surface glycoproteins (VSGs) that cover the entire cell surface of the parasite. Remarkably, trypanosomes recycle one cell surface equivalent within just 12 min and internalize surface bound antibodies within 2 min. Consequently, this parasite requires to have a highly effective endosomal sorting system. Currently, the endosomal system in T. brucei is described as distinct compartments that represent early, late and recycling endosomes. However, this model cannot explain the rapid rate of uptake and recycling in these organisms. Therefore, we analyzed the 3D architecture of the endosomal system.

For this purpose, we used different electron tomography and immuno-electron microscopic methods, as well as immunofluorescence and structured illumination microscopy (SIM) to analyse the 3D anatomy of the endocytic apparatus in T. brucei.

Electron tomograms show that the trypanosome endosomal system is more elaborate than previously thought and differs significantly from the mammalian endosome. Instead of being divided in distinct compartments it seems to consist of few continuous membrane systems. Antibodies against endosomal marker proteins (Rab5 – early endosomes, Rab7 – late endosome, Rab11 – recycling endosome) were used to perform in depth colocalization studies in wide-field microscopy. These analyzes indicate a high level of correlation between the different Rab proteins. To overcome the resolution limit of wide-field microscopy, super resolution microscopy and immuno-electron microscopy was used. All methods provide evidence for the existence of distinct functional endosomal subcompartments on the same membrane system, which questions the textbook picture of separate early, late and recycling endosomes.

Our results suggest that plasma membrane recycling in trypanosomes happens through a quick journey within an "endosomal highway", implying that its speed is diffusion-limited and not by trafficking through physically separated endosomal structures. Thus, we propose that the stream-lined architecture of the trypanosome endosomes is a consequence of the extreme and essential kinetics of plasma membrane recycling in these parasites.