How to make a microtubule organizing center - Project Summary: The eukaryotic cytoskeleton is a highly dynamic, yet, at the same time, highly organized network of protein filaments, the most important being actin and microtubules (MTs). MTs are essential for defining cellular shape and structure, as well as trafficking membrane-bound vesicles, organelles, and, during cell division, chromosomes. To perform these functions, the cell must tightly regulate the formation of new MTs via the process of MT nucleation. Cells primarily regulate MT nucleation by controlling the universal MT nucleator, the γ-tubulin ring complex (γ-TuRC). To initiate MT nucleation, γ-TuRC is recruited to specialized regions known as MT organizing centers (MTOCs), where γ-TuRC is activated and spatially oriented so that the resulting MT has the appropriate polarity. Many of the first discovered MTOCs are also the most complex, including the basal body, which generates flagellar MTs, and the centrosome, which generates many spindle MTs. These classical MTOCs are composed of up to hundreds of unique proteins organized into complicated large-scale structures. This makes them poorly suited to uncover MTOC function: namely, how MTOCs recruit, orient, and activate γ-TuRC. To will illuminate these three basic principles by investigating γ-TuRC regulation in two model essential, yet simple MTOCs: in the K99 phase, the vertebrate MT branch site and, in the R00 phase, the nuclear plaque of model excavate Trypanosoma brucei. I will use a combination of reconstitution experiments and cryo-electron tomography to uncover how these divergent, model MTOCs use shared principles to regulate γ-TuRC. My results will both help the research community understand MTOC function, as well as provide new therapeutic avenues to treat infection by parasitic excavates, an emerging global health threat. As proposed, this research plan will allow me to successfully pivot from my postdoctoral work with Dr. Petry, an expert on vertebrate MT nucleation, to establish my own, independent research focus on excavate MT nucleation and launch as an independent investigator. In addition, I have established a mentor team for the K99 phase of the proposal to train me in key technical areas that I require for the independent R00 phase. First, I will master a TIRF-based assay pioneered in Dr. Petry’s lab that allows visualization of microtubule nucleation at the single molecule level. Second, which I will learn cryo-electron tomography, a key technique of my proposal, in collaboration with world experts Dr. Andreas Hoenger and Dr. Martin Beck. In learning this technique, I will benefit from my mentorship team of Dr. Petry, Dr. Hoenger, Dr. Beck, and the cutting-edge vibrant structural biology community at Princeton as a whole. Finally, I have recruited Dr. Chris de Graffenried, an expert in excavate biology, to serve as a scientific mentor as I transition into this new model system. Through the training phase of the proposal and beyond, I will learn from my mentor team how to successfully lead a research group that fearlessly leverages a broad range of techniques and disciplines to answer critical scientific questions, and will, in the R00 phase, establish this group at a leading U.S. institution.
