3-D FINE STRUCTURE OF CENTROSOMES IN WT AND SPD-6 (RNAI) EMBRYOS OF C ELEGANS

该子项目是利用NIH/NCRR资助的中心赠款提供的资源的许多研究子项目之一。子项目和研究者（PI）可能从另一个NIH来源获得了主要资金，因此可以在其他CRISP条目中表示。所列机构为中心，不一定是研究者所在机构。秀丽隐杆线虫是研究细胞分裂分子机制的极好模型系统。我们已经发表了一个描述有丝分裂细胞在早期C。通过连续切片重建和电子断层摄影术（Mueller-Reichert等，J. Micros. 212：71-80，2003）。 C.线虫的染色体由无核糖体区组成，这些无核糖体区覆盖了每条染色体的极面。每条染色体有10-12个着丝粒微管（kMT）终止于该区域。我们没有观察到kMT的捆绑，而是kMT沿着染色体的长度沿着结束，而没有物理接触染色质。正MT端具有开放的扩口形态。梭elegans中心粒由9个单态MT围绕一个中心管形成。这个异常的中心粒（直径约150 nm）含有沿着其长度的附属物。MT末端，成核在pericentriolar材料（PCM）是加盖（80%）或开放（20%）。开放的MT负末端朝向染色质定位，表明动态纺锤体MT具有开放末端，而紫菀的更多静态MT可能被加帽。这项工作最近已经发表（O 'Toole et al.，JCB 163：451-456，2003）。这项和未来的野生型细胞的研究将提供一个基线，与有丝分裂纺锤体组装，动粒结构或中心体组织已被破坏的菌株进行比较。我们正在将这些方法与活单细胞测定结合用于PCM的募集和基于RNAi的C.优雅最近，我们发现了一个新的基因，DCD-1，这是需要招募PCM的女儿中心体。对缺失DCD-1的胚胎的分析表明，DCD-1的功能对于新形成的中心粒积累PCM是必要的。这些结果现在将通过EM层析成像进行进一步测试。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Caenorhabditis elegans is an excellent model system for studying the molecular machinery underlying cell division. We have published a description of mitotic cells in early C. elegans embryos by serial section reconstruction and electron tomography (Mueller-Reichert et al., J. Micros. 212:71-80, 2003). Kinetochores in C. elegans consists of ribosome-free zones that cover the poleward-face of each chromosome. 10-12 kinetochore microtubules (kMTs) per chromosome terminate in this zone. We observed no bundling of kMTs, rather the kMTs end along the length of the chromosome without physically touching the chromatin. The plus MT ends have an open, flared morphology. The C. elegans centriole is formed from nine singlet MTs surrounding a central tube. This unusually centriole (diameter ~150nm) contains appendages along its length. MT ends, nucleated in the pericentriolar material (PCM) are either capped (80%) or open (20%). The open MT minus ends are positioned towards the chromatin, indicating that dynamic spindle MTs have open ends, while more static MTs of the aster might be capped. This work has recently been published (O'Toole et al., JCB 163: 451-456, 2003). This and future study of wild-type cells will provide a baseline for comparisons with strains in which mitotic spindle assembly, kinetochore structure or centrosome organization have been disrupted. We are applying these methods in combination with live single-cell assays for the recruitment of PCM and with an RNAi-based functional screen in C. elegans. Recently, we identified a novel gene, DCD-1, that is required to recruit PCM to daughter centrosomes. Analysis of embryos depleted of DCD-1 suggest that DCD1 function is necessary for the newly formed centriole to accumulate PCM. These results will now be further tested by EM tomography.