Building Dendrite Architecture via Microtubule Nucleation - Microtubules (MTs) are a major component of the eukaryotic cytoskeleton, and their formation and activity are a primary driver of cellular architecture. MT formation in the cell requires the presence of a universal MT nucleation module, made up of the γ-tubulin ring complex (γ-TuRC), which acts as a template for tubulin assembly into MTs, and its co-nucleator, XMAP215. The formation of specialized MT-based structures relies on biochemical pathways that recruit and activate γ-TuRC at specific sites. Previously, it was thought that all MTs originated from centrosomes. However, we now know that this is not correct and that there are numerous other MT organizing centers (MTOCs). The mitotic spindle is an excellent example of MT assembly, and will be the focus of Aim 1 in the proposed K99. In acentrosomal spindles, chromosomes act as the primary drivers of MT nucleation through a gradient of activated RanGTP. Downstream of Ran, the augmin complex recruits γ-TuRC to form new MTs from pre-existing ones in a pathway called branching MT nucleation. Branching MT nucleation amplifies MT mass while conserving polarity and we now know that branching MT nucleation is responsible for generating the bulk of MTs in the spindle. The pressing question driving K99 Aim 1 is how, precisely, does a spindle emerge from branched MT networks? In this aim, I will use in vitro reconstitution to determine how branched MTs undergo nucleation near chromosomes during early spindle assembly, and how these branched networks mature into a spindle. My research will elucidate how single MTs that are a mere 25 nm in diameter self-organize into a micron- scale structure that performs essential functions of the cell. Then, in Aim 2 of the K99, I will uncover which spindle assembly factors are used to build other MT-based structures. Specifically, I will perform a targeted RNAi screen using Drosophila dendritic arborization (da) neurons, which display highly elaborate patterning, as a model system to determine which acentrosomal MT nucleation effectors have a role in dendrite formation. Finally, in the R00 phase of my research, I will determine how MTs are nucleated in dendrites throughout development, and how this nucleation contributes to dendrite patterning observed in mature neurons. To do this, in R00 Aim 1, I will use super-resolution imaging to precisely quantify where MTOCs appear in dendrites and explore how MTOCs execute MT nucleation using acentrosomal pathways. In Aim 2 of the R00 section, I will perform proximity labeling and use expansion microscopy with super-resolution imaging to define the nanoscale protein architecture of dendritic MTOCs, yielding key insights about the role of MT nucleation in driving dendrite morphogenesis, which ultimately gives rise to neuronal function. Each phase of my research will prepare me to run an independent research lab, with the goal of using my skills to substantially advance understanding of cellular architecture and how architecture both gives rise to and controls cellular functions and processes.
