Dissection of centrosomal γ-TuRC activation pathways controlling microtubule density in interphase cells

Animal microtubule-organizing centers, including the centrosome and the Golgi apparatus, regulate microtubule nucleation and anchoring through the {gamma}-tubulin ring complex ({gamma}-TuRC) and CAMSAP-mediated minus-end stabilization. However, functional redundancy between these pathways has impeded dissection of their contributions to controlling microtubule organization and density. Here, we addressed this problem using combinatorial gene knockouts, protein depletions and Expansion Microscopy. By simultaneously eliminating CAMSAP2 and the {gamma}-TuRC-targeting proteins AKAP450, pericentrin, CDK5RAP2, myomegalin, ninein and AKNA, we generated viable RPE1 cells that lack both Golgi-derived microtubules and {gamma}-TuRC localization within the pericentriolar material and at subdistal appendages. Despite the disruption of these major microtubule-organizing pathways, overall microtubule density was only partially reduced. The remaining microtubules depended on CEP192 and NEDD1, which, together with ch-TOG, can activate {gamma}-TuRC at the centriole wall, in acentriolar cells, and in biochemical reconstitution assays. Our results demonstrate that in the absence of CAMSAP-mediated stabilization, interphase microtubule formation strongly relies on {gamma}-TuRC activation, which occurs through several redundant pathways.