Centrosomes are the major and best-understood microtubule-organizing centers (MTOCs) in animal cells, yet in most differentiated cells non-centrosomal MTOCs (ncMTOCs) redirect MT organization instead of the centrosome. The sheer diversity of ncMTOCs reflects the different functions of various cell types and the subcellular locations and mechanisms of MT assembly that serve those roles. Understanding the molecular architecture of these ncMTOCs, the mechanisms of MT assembly they employ, and the functions they serve to the diverse cell types they serve remains an important frontier in cell biology and disease. The few ncMTOCs that have been deciphered reveal architectures and mechanisms of MT assembly as diverse as the functions they serve for the varied cell types. In this proposal we expand on a project to define the functions of a perinuclear ncMTOC in fat body cells which serves the cell by providing nuclear positioning and is essential for retrograde endosomal trafficking to support the vital secretory roles of this cell type. We will determine the unique molecular mechanisms of MT assembly by two distinct protein complexes that control MT assembly/nucleation from this ncMTOC. Specifically, this proposal aims to further define the structure of the fat body ncMTOC on the nuclear surface, and to define the mechanisms of MT assembly by Patronin and Ninein and how they cooperate with the microtubule polymerase Minispindles (Msps) and other partners to nucleate MTs. The outcomes of this project will be a definitive and novel understanding of the molecular and physiological functions of the fat body ncMTOC, revealing potentially novel disease etiologies for the proteins involved and the processes that they regulate. Moreover, as a molecular MT nucleation paradigm that is independent of the widespread microtubule nucleator gamma-tubulin, we will define at least one new mechanism to generate MTs at MTOCs.