In contrast to the radial microtubule arrays organized by centrosomes in dividing cells, many differentiated cells assemble non-centrosomal microtubule arrays adapted for specific cellular functions, including nuclear positioning and migration, maintenance of tissue architecture and intracellular transportation. In an RNAi screen in C. elegans, we identified NOCA-1 as a novel protein that does not contribute to centrosome-driven embryonic cell divisions but is required to form microtubule arrays in the germline that are essential for organismal fertility. In this dissertation, I focused on NOCA-1 to study the assembly mechanism and cellular function of non- centrosomal microtubule arrays. The noca-1 gene encodes 8 isoforms expressed in a variety of tissues. Two distinct long isoforms control assembly of microtubule arrays in the germline and embryonic epidermis, respectively. In contrast, a short isoform functions in parallel to the microtubule minus end-binding protein Patronin (PTRN-1) to control assembly of microtubule arrays in the post- embryonic epidermis. Evidence for the redundant activity of NOCA-1 and PTRN-1 includes synthetic lethality and early larval stage dye permeability of noca-1[Delta];ptrn- 1[Delta] double mutants, both of which are rescued by selectively expressing PTRN-1 in the post-embryonic epidermis. In support of the genetic interaction, NOCA-1 co-sediments with taxol-stabilized microtubules from worm lysates and purified recombinant NOCA-1, like PTRN-1, binds to microtubule ends in a microtubule-anchoring assay. We conclude that NOCA-1 represents a new class of microtubule end-binding proteins with essential functions of its own, as well as parallel functions with Patronin, in the assembly of non-centrosomal microtubule arrays in multiple C. elegans tissues. Non-centrosomal microtubule arrays in embryonic epidermis contribute to elongation, the process that converts the oval-shaped embryo into an elongated worm. By using two different means of disrupting microtubules, we showed that disrupting microtubules alone does not affect elongation. However, when acto-myosin contractility is compromised by a partial loss-of-function mutant of let-502 (a Rho kinase), disrupting microtubules results in elongation arrests and causes embryonic lethality. Intact microtubules and normal LET-502 activity are required for E-cadherin clustering at adherens junctions and the maturation of hemidesmosomes, possibly explaining the elongation defects we observed. We conclude that microtubules in the embryonic epidermis contribute to elongation, but only is required when actomyosin contractility is reduced

Abscission completes cytokinesis to form the two daughter cells. Although abscission could be organized from the inside out by the microtubule-based midbody or from the outside in by the contractile ring-derived midbody ring, it is assumed that midbody microtubules scaffold the abscission machinery. In this paper, we assess the contribution of midbody microtubules versus the midbody ring in the Caenorhabditis elegans embryo. We show that abscission occurs in two stages. First, the cytoplasm in the daughter cells becomes isolated, coincident with formation of the intercellular bridge; proper progression through this stage required the septins (a midbody ring component) but not the membrane-remodeling endosomal sorting complex required for transport (ESCRT) machinery. Second, the midbody and midbody ring are released into a specific daughter cell during the subsequent cell division; this stage required the septins and the ESCRT machinery. Surprisingly, midbody microtubules were dispensable for both stages. These results delineate distinct steps during abscission and highlight the central role of the midbody ring, rather than midbody microtubules, in their execution.

Precise regulation of microtubule (MT) dynamics is increasingly recognized as a critical determinant of axon regeneration. In contrast to developing neurons, mature axons exhibit noncentrosomal microtubule nucleation. The factors regulating noncentrosomal MT architecture in axon regeneration remain poorly understood. We report that PTRN-1, the C. elegans member of the Patronin/Nezha/calmodulin- and spectrin-associated protein (CAMSAP) family of microtubule minus-end-binding proteins, is critical for efficient axon regeneration in vivo. ptrn-1-null mutants display generally normal developmental axon outgrowth but significantly impaired regenerative regrowth after laser axotomy. Unexpectedly, mature axons in ptrn-1 mutants display elevated numbers of dynamic axonal MTs before and after injury, suggesting that PTRN-1 inhibits MT dynamics. The CKK domain of PTRN-1 is necessary and sufficient for its functions in axon regeneration and MT dynamics and appears to stabilize MTs independent of minus-end localization. Whereas in developing neurons, PTRN-1 inhibits activity of the DLK-1 mitogen-activated protein kinase (MAPK) cascade, we find that, in regeneration, PTRN-1 and DLK-1 function together to promote axonal regrowth.

Non-centrosomal microtubule arrays assemble in differentiated tissues to perform mechanical and transport-based functions. In this study, we identify Caenorhabditis elegans NOCA-1 as a protein with homology to vertebrate ninein. NOCA-1 contributes to the assembly of non-centrosomal microtubule arrays in multiple tissues. In the larval epidermis, NOCA-1 functions redundantly with the minus end protection factor Patronin/PTRN-1 to assemble a circumferential microtubule array essential for worm growth and morphogenesis. Controlled degradation of a γ-tubulin complex subunit in this tissue revealed that γ-tubulin acts with NOCA-1 in parallel to Patronin/PTRN-1. In the germline, NOCA-1 and γ-tubulin co-localize at the cell surface, and inhibiting either leads to a microtubule assembly defect. γ-tubulin targets independently of NOCA-1, but NOCA-1 targeting requires γ-tubulin when a non-essential putatively palmitoylated cysteine is mutated. These results show that NOCA-1 acts with γ-tubulin to assemble non-centrosomal arrays in multiple tissues and highlight functional overlap between the ninein and Patronin protein families.

Centrosomes are composed of a centriolar core surrounded by a pericentriolar material (PCM) matrix that docks microtubule-nucleating γ-tubulin complexes. During mitotic entry, the PCM matrix increases in size and nucleating capacity in a process called centrosome maturation. Polo-like kinase 1 (PLK1) is recruited to centrosomes and phosphorylates PCM matrix proteins to drive their self-assembly, which leads to PCM expansion. Here, we show that in addition to controlling PCM expansion, PLK1 independently controls the generation of binding sites for γ-tubulin complexes on the PCM matrix. Selectively preventing the generation of PLK1-dependent γ-tubulin docking sites led to spindle defects and impaired chromosome segregation without affecting PCM expansion, highlighting the importance of phospho-regulated centrosomal γ-tubulin docking sites in spindle assembly. Inhibiting both γ-tubulin docking and PCM expansion by mutating substrate target sites recapitulated the effects of loss of centrosomal PLK1 on the ability of centrosomes to catalyze spindle assembly.

Dynamic coupling of microtubule ends to kinetochores, built on the centromeres of chromosomes, directs chromosome segregation during cell division. Here, we report that the evolutionarily ancient kinetochore-microtubule coupling machine, the KMN (Knl1/Mis12/Ndc80-complex) network, plays a critical role in neuronal morphogenesis. We show that the KMN network concentrates in microtubule-rich dendrites of developing sensory neurons that collectively extend in a multicellular morphogenetic event that occurs during C. elegans embryogenesis. Post-mitotic degradation of KMN components in sensory neurons disrupts dendritic extension, leading to patterning and functional defects in the sensory nervous system. Structure-guided mutations revealed that the molecular interface that couples kinetochores to spindle microtubules also functions in neuronal development. These results identify a cell-division-independent function for the chromosome-segregation machinery and define a microtubule-coupling-dependent event in sensory nervous system morphogenesis.