Developing an in vivo MT nucleation assay to investigate g-tubulin independent centrosomal MT nucleation (360G-Wellcome-207230_Z_17_Z)

Centrosomes are major microtubule organising centres (MTOCs) in animal cells. During mitosis they recruit large numbers of gamma-tubulin ring complexes (g-TuRCs), which nucleate and anchor the microtubules required for spindle formation. Recent work in the Conduit lab has surprisingly shown that centrosomes lacking g-TuRCs can still organise microtubules. Nevertheless, it remains unclear if these microtubules are generated at centrosomes, or generated in the cytoplasm and then anchored at centrosomes. I aim to establish an in vivo microtubule nucleation assay to test these alternative possibilities. Drosophila larval brains, which are highly mitotically active, will be dissected from either wild-type flies or from mutant flies where the centrosomes lack g-TuRCs. They will be cooled on ice for 40 minutes in order to depolymerise all microtubules and then transferred to 25 degrees and chemically fixed at different timepoints. The brains will be stained for microtubules, centrosomes and mitotic DNA using antibodies already available in the Conduit lab and images will be taken on a confocal microscope. The location and intensity of new microtubule growth will be assessed. If the g-TuRC negative centrosomes do nucleate microtubules, the assay will be used to test candidate proteins for their role in centrosomal non-g-TuRC mediated microtubule nucleation.