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.
| Giddings Jr, Thomas H; Morphew, Mary K; McIntosh, J Richard (2017) Preparing Fission Yeast for Electron Microscopy. Cold Spring Harb Protoc 2017: |
| Zhao, Xiaowei; Schwartz, Cindi L; Pierson, Jason et al. (2017) Three-Dimensional Structure of the Ultraoligotrophic Marine Bacterium ""Candidatus Pelagibacter ubique"". Appl Environ Microbiol 83: |
| Brown, Joanna R; Schwartz, Cindi L; Heumann, John M et al. (2016) A detailed look at the cytoskeletal architecture of the Giardia lamblia ventral disc. J Struct Biol 194:38-48 |
| Saheki, Yasunori; Bian, Xin; Schauder, Curtis M et al. (2016) Control of plasma membrane lipid homeostasis by the extended synaptotagmins. Nat Cell Biol 18:504-15 |
| Höög, Johanna L; Lacomble, Sylvain; Bouchet-Marquis, Cedric et al. (2016) 3D Architecture of the Trypanosoma brucei Flagella Connector, a Mobile Transmembrane Junction. PLoS Negl Trop Dis 10:e0004312 |
| Park, J Genevieve; Palmer, Amy E (2015) Properties and use of genetically encoded FRET sensors for cytosolic and organellar Ca2+ measurements. Cold Spring Harb Protoc 2015:pdb.top066043 |
| McCoy, Kelsey M; Tubman, Emily S; Claas, Allison et al. (2015) Physical limits on kinesin-5-mediated chromosome congression in the smallest mitotic spindles. Mol Biol Cell 26:3999-4014 |
| Höög, Johanna L; Lötvall, Jan (2015) Diversity of extracellular vesicles in human ejaculates revealed by cryo-electron microscopy. J Extracell Vesicles 4:28680 |
| Marc, Robert E; Anderson, James R; Jones, Bryan W et al. (2014) The AII amacrine cell connectome: a dense network hub. Front Neural Circuits 8:104 |
| Weber, Britta; Tranfield, Erin M; Höög, Johanna L et al. (2014) Automated stitching of microtubule centerlines across serial electron tomograms. PLoS One 9:e113222 |
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