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

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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.