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

本子项目是利用由NIH/NCRR资助的中心赠款提供的资源的众多研究子项目之一。子项目和研究者（PI）可能已经从另一个NIH来源获得了主要资金，因此可以在其他CRISP条目中表示。列出的机构是中心的，不一定是研究者的机构。​我们发表了通过连续切片重建和电子断层扫描对秀丽隐杆线虫早期胚胎有丝分裂细胞的描述（Mueller-Reichert et al.， J. Micros. 212:71- 80,2003）。秀丽隐杆线虫的着丝点由覆盖每条染色体的极面无核糖体区组成。每条染色体有10-12个着丝点微管（kmt）终止于该区域。我们没有观察到kmt的捆绑，而是kmt沿着染色体的长度结束，而没有物理接触染色质。正MT端有一个开放的，喇叭形的形态。秀丽隐杆线虫的中心粒由围绕中心管的九个单线态mt组成。这个不寻常的中心粒（直径约150nm）沿其长度包含附属物。在心泡周围材料（PCM）中成核的MT端要么是帽状的（80%），要么是开放的（20%）。开放的MT负端指向染色质，表明动态纺锤体MT具有开放的末端，而aster的更多静态MT可能被封顶。这项工作最近发表（O'Toole et al., JCB 163: 451-456, 2003）。这项研究和未来对野生型细胞的研究将为与有丝分裂纺锤体组装、着丝点结构或中心体组织被破坏的菌株进行比较提供基线。我们正在将这些方法与活单细胞试验相结合，在秀丽隐杆线虫中招募PCM和基于rnai的功能筛选。最近，我们发现了一个新的基因，DCD-1，它是招募PCM到子中心体所必需的。对缺失DCD1的胚胎的分析表明，DCD1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断层扫描进一步验证。

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.