Department of Biology, Stanford University, 371 Serra Mall, Stanford, 94305, CA, USA.
Department of Biology, Stanford University, 371 Serra Mall, Stanford, 94305, CA, USA.
Curr Biol. 2021 Jun 7;31(11):2410-2417.e6. doi: 10.1016/j.cub.2021.03.022. Epub 2021 Apr 1.
During mitosis in animal cells, the centrosome acts as a microtubule organizing center (MTOC) to assemble the mitotic spindle. MTOC function at the centrosome is driven by proteins within the pericentriolar material (PCM), however the molecular complexity of the PCM makes it difficult to differentiate the proteins required for MTOC activity from other centrosomal functions. We used the natural spatial separation of PCM proteins during mitotic exit to identify a minimal module of proteins required for centrosomal MTOC function in C. elegans. Using tissue-specific degradation, we show that SPD-5, the functional homolog of CDK5RAP2, is essential for embryonic mitosis, while SPD-2/CEP192 and PCMD-1, which are essential in the one-cell embryo, are dispensable. Surprisingly, although the centriole is known to be degraded in the ciliated sensory neurons in C. elegans, we find evidence for "centriole-less PCM" at the base of cilia and use this structure as a minimal testbed to dissect centrosomal MTOC function. Super-resolution imaging revealed that this PCM inserts inside the lumen of the ciliary axoneme and directly nucleates the assembly of dendritic microtubules toward the cell body. Tissue-specific degradation in ciliated sensory neurons revealed a role for SPD-5 and the conserved microtubule nucleator γ-TuRC, but not SPD-2 or PCMD-1, in MTOC function at centriole-less PCM. This MTOC function was in the absence of regulation by mitotic kinases, highlighting the intrinsic ability of these proteins to drive microtubule growth and organization and further supporting a model that SPD-5 is the primary driver of MTOC function at the PCM.
在动物细胞的有丝分裂过程中，中心体作为微管组织中心（MTOC）组装有丝分裂纺锤体。中心体的 MTOC 功能是由中心粒周围物质（PCM）中的蛋白质驱动的，然而 PCM 的分子复杂性使得难以将 MTOC 活性所需的蛋白质与其他中心体功能区分开来。我们利用 PCM 蛋白在有丝分裂退出过程中的自然空间分离，鉴定了线虫中用于中心体 MTOC 功能的最小蛋白质模块。通过组织特异性降解，我们表明 CDK5RAP2 的功能同源物 SPD-5 对于胚胎有丝分裂是必需的，而在单细胞胚胎中必需的 SPD-2/CEP192 和 PCMD-1 是可有可无的。令人惊讶的是，尽管在秀丽隐杆线虫的纤毛感觉神经元中已知中心体被降解，但我们发现纤毛基部存在“无中心体的 PCM”的证据，并利用该结构作为最小的测试平台来剖析中心体 MTOC 功能。超分辨率成像显示，该 PCM 插入纤毛轴突的内腔，并直接启动向细胞体的树突状微管组装。纤毛感觉神经元中的组织特异性降解揭示了 SPD-5 和保守的微管核因子 γ-TuRC 在无中心体的 PCM 中的 MTOC 功能中的作用，但 SPD-2 或 PCMD-1 则没有作用。这种 MTOC 功能不受有丝分裂激酶的调节，突出了这些蛋白质驱动微管生长和组织的内在能力，并进一步支持了 SPD-5 是 PCM 中 MTOC 功能的主要驱动因素的模型。
