Single-molecule fluorescence microscopy demonstrates fast dynamics of the variant surface glycoprotein coat on living trypanosomes
The fluidity of Trypanosoma bruceis dense coat of GPI-anchored variant surface glycoproteins (VSGs) is fundamental for the survival of the parasite. In order to maintain the integrity of the coat, it is recycled on the time scale of a few minutes. This is surprisingly fast as endo- and exocytosis take place in the same small membrane invagination called the flagellar pocket. Here, we present measurements of VSG dynamics on the single-molecule level in living trypanosomes. A large number of short protein trajectories sampling the parasites surface were analysed in two distinct scenarios: diffusion and directed motion. To this end, we employed a previously published algorithm and implemented two extensions to consider rim effects as well as localisations errors inherent to single-mole tracking. Neglect of the latter can have a significant distortive effect on the measured diffusion coefficient; in our case resulting in an underestimation by 20 %. We found large heterogeneity in the local diffusion coefficients and velocities with a surprisingly high average value of [Formula] and [Formula], respectively. To decide on the locally dominant motion mode, we present a guideline based on random walk simulations. We find that VSG dynamics is indeed dominated by diffusion. Complementary simulations on long time scales not accessible in the experiment showed that passive VSG randomisation is fast enough to prevent re-endocytosis newly exocytosed VSGs and to accomplish turnover of the full VSG coat within a few minutes. Author summarySingle-molecule tracking in biological systems often suffers from trajectories being too short to obtain statistically robust decisions on the present motion mode. We faced this issue when investigating the dynamics of the protein surface coat of African trypanosomes. To address the question whether diffusion or directed motion governs coat dynamics, we have adopted an algorithm based on temporal decomposition and spatial binning of an ensemble of single-molecule trajectories. We introduced several extensions to the original approach, including the consideration of localisation errors inherent to single-molecule tracking. This improvement alone already prevented the diffusion coefficient from being underestimated by 20 %. We analysed the coat dynamics in two scenarios, diffusion and directed motion, and offer a decision guideline to identify the locally dominating motion mode. Our extended algorithm is available to the scientific community via GitHub. For the trypanosome surface coat we found that the motion is indeed mainly characterised by diffusion with a surprisingly high diffusion coefficient. This finding solves a long-standing question how the parasite maintains its protein coat.