How Ninein Coordinates a Microtubule-Organizing Center in the Drosophila Larval Fat Body

The centrosome, comprised of a pair of centrioles, is the best-studied microtubule-organizing center (MTOC). However, after utilizing the centrosome to organize the mitotic spindle during cell division, differentiated cells will often reassign microtubule (MT)-organizing function to a site apart from the centrosome, creating a non-centrosomal MTOC (ncMTOC). Ninein (Nin) is a microtubule anchor at the subdistal appendages of mother centrioles and the pericentriolar material (PCM) of centrosomes that also functions to organize MTs at ncMTOCs. In humans, mutations in the NIN gene have been linked to Seckel syndrome, an inherited developmental disorder, and an increased risk for colorectal cancer. In this work, I characterize Nin using Drosophila melanogaster (fruit flies) as a model organism. Nin is not essential in flies, but it does localize to the periphery of the centrosome as well as to ncMTOCs in specialized, differentiated cell types such as the wing epithelia, muscle, and fat body, pointing to a role for Nin in supporting non-centrosomal MT organization. In this work, I dissect the protein domains, key interactions, and epistatic relationships involving Nin at the Drosophila larval fat body cells' perinuclear ncMTOC. I have defined domains of Nin responsible for its localization to the ncMTOC on the fat body cell nuclear surface and localization within the nucleus. I have confirmed the interaction seen in human cells between Drosophila Dynein light intermediate chain (Dlic) and Nin and have mapped this interaction to an N-terminal domain. Additionally, I have mapped an ensconsin (ens) binding domain to Nin and shown that this domain is sufficient to create a robust, juxtanuclear MTOC together with ens. Altogether, these findings reveal novel features of Nin function and its regulation of a ncMTOC.