Biogenesis of the Trypanosoma brucei subpellicular microtubule array

PROJECT SUMMARY The trypanosomatids cause a broad range of severe human illnesses across the entire world. The success of these parasites stems in large part from their ability to adapt their cellular morphology to suit the environments within their mammalian and insect hosts. The extensive range of observed cellular morphologies rely on a set of microtubules that underlie the cell surface, known as the subpellicular array. These microtubules are heavily crosslinked and remarkably stable, but very little is known about how the array maintains its organization or how it duplicates during cell division. During a recent proximity-dependent biotinylation screen in Trypanosoma brucei, we identified two proteins that are essential for shaping the array and assuring that it is duplicated correctly during cell division. The first, an orphan kinesin named Kinesin Localized to the Ingressing Furrow (KLIF), is essential for the segregation of the array into two distinct units at the end of cell division. KLIF is a very effective microtubule bundler in vitro, which suggests that its primary function is to organize microtubules within the array to form a new cell posterior by gathering microtubule plus-ends into a pole. The other, called Posterior And Ventral Edge Protein 1 (PAVE1) is a component of microtubule crosslinks present at the posterior portion of the array and is essential for tapering the array to produce the parasite’s distinctive shape. This proposal will use these proteins to understand how the subpellicular array is assembled and maintains its shape. In Aim 1, the precise track KLIF takes as it ingresses along the furrow will be established using super- resolution and live-cell microscopy. We will study the KLIF RNAi phenotype using EM and live-cell imaging to determine the specifics of the microtubule organizing defect. Full-length KLIF will be expressed to test its oligomerization state and function. In Aim 2, the microtubule-binding properties of PAVE1 and its interacting partners will be studied using biophysical approaches. PAVE1 preference for microtubule plus ends at the cell posterior will be probed using a pulse-chase strategy in conjunction with treatments that alter microtubule dynamics. In Aim 3, immunoprecipitation and proximity-dependent biotinylation will be employed to map the interacting partners of both KLIF and PAVE1 so that the pathways involved in subpellicular array biogenesis can be established. This work will further the fundamental understanding of how trypanosomatids establish and transmit their complex cellular morphologies, which are essential parts of their biology. Pathways involved in these processes that are unique and essential may be potential targets for further drug design.