| Project Detail |
Microtubule-organizing centers (MTOCs) are key eukaryotic structures for microtubule nucleation and organization. Many MTOCs contain centrioles, organelles with two critical functions. i) In mitosis, they localize to the pole of the mitotic spindle aiding its organization. ii) After cell division, centrioles are repurposed as basal bodies at the plasma membrane where they are essential for cilia/flagella formation. Defects in centriole/basal body (CBB) repurposing can have devastating consequences and lead e.g. to infertility in human sperm. CBB functions correlate with subcellular localization, but is the spatiotemporal control required for this organelle to fulfill two distinct functions? How did the ancestral CBB evolve these functions? And why are different MTOCs not present simultaneously in a cell? To tackle these questions, I will first analyze and compare CBB repurposing strategies during the cell cycle of the green alga Chlamydomonas rheinhardtii with those in the complex life cycle of the acellular slime mold Physarum polycephalum, two evolutionary distant eukaryotes. To do so, I will develop and apply a correlative microscopy workflow in which time-lapse imaging of cells is followed by cryo-fixation and expansion microscopy (Cryo-ExM) combined with Stimulated Emission Depletion (STED). Using transient overexpression and laser ablation, I will interfere with spatial and temporal control of CBB repurposing to elucidate the role of subcellular location of this bifunctional organelle. Further, I will study how cells cope with several different MTOCs at a time, likely competing for tubulin. This project will provide a novel, high-end correlative live-cell and super-resolution imaging pipeline, which can be easily adapted for various species and cellular structures. Understanding the different strategies of CBB repurposing that evolved will have broad implications as a basis to understand CBB defects in the human cell cycle and during spermatogenesis. |
