Department of Biology, Stanford University, 371 Serra Mall, Stanford, CA 94305, USA.
Department of Biology, Stanford University, 371 Serra Mall, Stanford, CA 94305, USA; Department of Chemistry, Massachusetts Institute of Technology, Cambridge, MA, USA; Departments of Genetics and Chemistry, Stanford University, Stanford, CA, USA.
Curr Biol. 2021 Aug 23;31(16):3586-3600.e11. doi: 10.1016/j.cub.2021.06.021. Epub 2021 Jul 8.
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. elegans. 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.
微管是具有极性的细胞内聚合物，在细胞中发挥着关键作用，包括运输、极性和细胞分裂。在真核细胞类型中，微管采用不同的细胞内组织形式来适应这些不同的功能，这些功能由特定的细胞位点（称为微管组织中心，MTOC）协调。超过 50 年的 MTOC 生物学研究主要集中在中心体上；然而，大多数分化细胞利用非中心体的 MTOC（ncMTOC）来将微管组织成不同的排列，这对于细胞功能至关重要。为了鉴定必需的 ncMTOC 成分，我们开发了基于生物素连接酶的 TurboID 接近标记方法，用于 C. elegans。我们鉴定了与肠上皮细胞顶端 ncMTOC 中的微管负端蛋白 PTRN-1/Patronin 接近的蛋白质，重点关注两种保守蛋白： spectrin 蛋白 VAB-10B/MACF1 和 WDR-62，我们将其鉴定为与脊椎动物原发性小头畸形疾病蛋白 WDR62 同源的蛋白。VAB-10B 和 WDR-62 与中心体不相关，而是特异性调节非中心体微管和微管负端蛋白的顶端靶向。VAB-10B 的耗竭导致微管定位错误和微管核化复合物 γ-微管蛋白环复合物（γ-TuRC）的定位延迟，而 WDR-62 的缺失减少了动态微管的数量并破坏了 γ-TuRC 的定位。这种调节发生在细胞极性之后，并与肌动蛋白结合。由于这是 WDR62 家族蛋白非中心体作用的首次报道，我们扩展了这种重要疾病蛋白的基本细胞生物学作用。我们的研究鉴定了必需的 ncMTOC 成分，并提出了一种分工，其中微管的生长和定位受到明显的调节。
