Proximity labeling at non-centrosomal microtubule-organizing centers reveals VAB-10B and WDR-62 as distinct microtubule regulators

Abstract

SummaryReorganization of microtubules from the centrosome to non-centrosomal subcellular sites is central to cell differentiation. To identify components of non-centrosomal microtubule organizing centers in differentiated cells of a living organism, we developed the biotin ligase-based proximity labeling approach TurboID for use in C. elegans. We identified proteins proximal to the non-centrosomal microtubule minus end protein PTRN-1/Patronin at the apical membrane of epithelial cells, focusing on two conserved proteins: spectraplakin protein VAB-10B and WDR-62, a protein we identify as homologous to vertebrate primary microcephaly disease gene WDR62. We found that WDR-62 and VAB-10B independently regulate the growth and localization of non-centrosomal microtubules and the apical targeting of microtubule minus end proteins. This regulation occurs downstream of cell polarity and in conjunction with actin. Our data suggest a division of labor where microtubule growth and anchoring are regulated by distinct complexes and uncover novel functions of spectraplakins and WDR62 family proteins.

Separable mechanisms drive local and global polarity establishment in the C. elegans intestinal epithelium

“…Minor extensions of PKC-3 away from the apical surface were occasionally observed in Stage 4 and older AFD-1 gut(-) embryos (Fig. S5), similar to those observed in embryos with actin defects (Gobel et al, 2004;Ramalho et al, 2020;Sanchez et al, 2021;Sasidharan et al, 2018;Van Furden et al, 2004). Importantly, this result suggests that while AFD-1 may contribute to the assembly and movement of lateral puncta to the intestinal midline, other mechanisms are sufficient for local and global apical establishment.…”

“…We tagged endogenous PAR-1 with GFP and found that it localized to centrosomes during mitosis prior to Stage 1 (Data not shown) and occasionally at midbodies in Stage 1 embryos (Fig. 1E), consistent with the ability of PAR-1 to bind to microtubules and associate with microtubule-binding proteins (Sanchez (Cohen et al, 2004;Doerflinger et al, 2003;Sanchez et al, 2021). At Stage 1, PAR-1 localized weakly to plasma membranes (Fig.…”

“…S3). While the ZF degradation system does not create a null line, it has been used to robustly deplete ZF-tagged proteins (Abrams and Nance, 2021;Liang et al, 2020;Magescas et al, 2021;Sallee et al, 2021;Sallee et al, 2018;Sanchez et al, 2021) and we see strong depletion.…”

Interactome analysis of Caenorhabditis elegans synapses by TurboID-based proximity labeling

“…However, the authors did not publish interactome or proteomic data for C. elegans. A recent study used TurboID to characterize the interactome of the microtubule-binding protein patronin, but ectopically overexpressed this fusion protein in the largest tissue of the animal, the gut, and did not seek to optimise the protocol (29).…”