Proximity labeling reveals non-centrosomal microtubule-organizing center components required for microtubule growth and localization

Abstract

Microtubules are polarized intracellular polymers that play key roles in the cell, including in transport, polarity, and cell division. Across eukaryotic cell types, microtubules adopt diverse intracellular organization to accommodate these distinct functions coordinated by specific cellular sites called microtubule-organizing centers (MTOCs). Over 50 years of research on MTOC biology has focused mainly on the centrosome; however, most differentiated cells employ non-centrosomal MTOCs (ncMTOCs) to organize their microtubules into diverse arrays, which are critical to cell function. To identify essential ncMTOC components, we developed the biotin ligase-based, proximity-labeling approach TurboID for use in C.\xa0elegans. We identified proteins proximal to the microtubule minus end protein PTRN-1/Patronin at the apical ncMTOC of intestinal epithelial cells, focusing on two conserved proteins: spectraplakin protein VAB-10B/MACF1 and WDR-62, a protein we identify as homologous to vertebrate primary microcephaly disease protein WDR62. VAB-10B and WDR-62 do not associate with the centrosome and instead specifically regulate non-centrosomal microtubules and the apical targeting of microtubule minus-end proteins. Depletion of VAB-10B resulted in microtubule mislocalization and delayed localization of a microtubule nucleation complex ɣ-tubulin ring complex (γ-TuRC), while loss of WDR-62 decreased the number of dynamic microtubules and abolished γ-TuRC localization. This regulation occurs downstream of cell polarity and in conjunction with actin. As this is the first report for non-centrosomal roles of WDR62 family proteins, we expand the basic cell biological roles of this important disease protein. Our studies identify essential ncMTOC components and suggest a division of labor where microtubule growth and localization are distinctly regulated.