Single-molecule fluorescence microscopy demonstrates fast dynamics of the variant surface glycoprotein coat on living trypanosomes

Abstract

The fluidity of Trypanosoma brucei ′s 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 parasite's 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 D = 1.00 μm 2/s and v = 1.99 μm/s, 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.

ThirdPeak: A flexible tool designed for the robust analysis of two- and three-dimensional (single-molecule) tracking data

“…Table 1). The diffusion coefficients through fitting to the cumulative squared jump distance distribution and are in good agreement with previously determined 2D diffusion data 14 (Sup. Fig.…”

Continuous endosomes form functional subdomains and orchestrate rapid membrane trafficking in trypanosomes

“…This lipid anchoring enables the highly dynamic behaviour of the VSG coat ( Bartossek et al, 2017 ), further facilitated by N -glycans that insulate VSG molecules, thereby minimizing protein–protein interactions ( Hartel et al, 2016 ). VSG molecules exhibit diffusion on the plasma membrane with coefficients that enable fast randomization ( Hartel et al, 2016 ; Schwebs et al, 2022 ). This rapid VSG diffusion is precisely balanced with membrane recycling kinetics, with one surface equivalent being internalized and recycled within 12 min ( Engstler et al, 2004 ).…”

Continuous endosomes form functional subdomains and orchestrate rapid membrane trafficking in trypanosomes

“…This lipid anchoring enables the highly dynamic behaviour of the VSG coat (Bartossek et al, 2017), further facilitated by N-glycans that insulate VSG molecules, thereby minimizing protein-protein interactions (Hartel et al, 2016). VSG molecules exhibit diffusion on the plasma membrane with coefficients that enable fast randomization (Hartel et al, 2016; Schwebs et al, 2022). This rapid VSG diffusion is precisely balanced with membrane recycling kinetics, with one surface equivalent being internalized and recycled within 12 minutes (Engstler et al, 2004).…”