Title: Structure and Dynamics of the Trypanosome Plasma Membrane
Abstract: African trypanosomes are the causative agents of sleeping sickness in humans and Nagana in livestock. During their lifecycle the single-celled parasites adapt to the environment of the vector and the host. In the bloodstream of their host, they exhibit a dense coat of GPI-anchored variant surface glycoproteins (VSG). This coat is a central element of the parasite's strategy to evade the host's immune system. Fluidity of the VSG coat is a prerequisite of its proper shielding function. However, the diffusion behavior of the VSGs is limited by the physical properties of their lipid matrix. We have recently introduced super-resolution imaging of intrinsically fast moving flagellates based on cyto-compatible hydrogel embedding. Building on this work we employ leaflet-specific membrane probes and single-molecule fluorescence microscopy to elucidate the structure and dynamics of the plasma membrane in living trypanosomes. Using lipid-anchored eYFP as a probe for the inner membrane leaflet, we found specific domains where the fluorescence either accumulates or appears diluted rather than being homogenously distributed. We hypothesize that this structuring of the membrane is associated with the underlying cytoskeleton. The next steps include employment of a more stable fluorescent label to achieve conclusive information on the dynamic interaction of single probes with the observed domains. Moreover, we aim to track fluorescently labeled lipids in the outer leaflet to gain insight in inter-leaflet coupling in vivo, and we plan a two-color experiment to simultaneously investigate membrane and VSG dynamics.