We are trying to understand the central but poorly understood organelle that organizes the cytoplasm in eukaryotic cells. Our unique approach to the study of the centrosome emphasizes the dissection of the centrosome subunit as a multi-protein complex that binds to the minus end of a single microtubule. As our model system for such a study, we are using the early Drosophila embryo, whose centrosomal mechanisms can be safely assumed to be essentially identical to those of a human cell. Drosophila has the following advantages: large quantities of embryos are easily obtained; the syncytial early embryos are large, robust, single cells that are easily microinjected with proteins; these embryos have a highly organized cytoplasm, with easily observable, rapid changes in spatial organization caused by the centrosome; and powerful genetic analyses of centrosome function are possible. In the past grant period, we have discovered and extensively characterized three Drosophila centrosomal proteins (and cDNAs): DMAP60, DMAP190, and the l4OkD LK6 protein kinase, as well as partially characterizing others. In addition, we have complete cDNA clones for two proteins suspected to be intimately involved with the actin-induced membrane-furrowing that is caused by the centrosome: a 134kD actin-binding protein called ABP8, and the product of a maternal-effect mutant gene that we have named scrambled. The current proposal has four specific aims: 1) to scale up and refine our existing Drosophila embryo centrosome purification to obtain biochemical quantities of active centrosomes of sufficient purity to identify a full complement of centrosomal proteins; 2) to dissect the structure and function of a complex of three Drosophila centrosomal proteins studied in our laboratory (DMAP190, DMAP60, and gamma-tubulin) and determine the individual contributions of its components to the microtubule binding observed for the entire complex; 3) to use both biochemical and genetic approaches to decipher the role of the LK6 protein kinase in the regulation of centrosome function; and 4) to identify the function of the ABP8 and scrambled proteins in an attempt to decipher how actin filament-induced membrane furrowing is linked to the centrosome.
| Liu, Ling; Su, Xiaoyang; Quinn 3rd, William J et al. (2018) Quantitative Analysis of NAD Synthesis-Breakdown Fluxes. Cell Metab 27:1067-1080.e5 |
| Groen, Aaron C; Mitchison, Timothy J (2016) Purification and Fluorescent Labeling of Tubulin from Xenopus laevis Egg Extracts. Methods Mol Biol 1413:35-45 |
| Özlü, Nurhan; Qureshi, Mohammad H; Toyoda, Yusuke et al. (2015) Quantitative comparison of a human cancer cell surface proteome between interphase and mitosis. EMBO J 34:251-65 |
| Coughlin, Margaret; Groen, Aaron C; Mitchison, Timothy J (2014) Electron microscopy of microtubule cytoskeleton assembly in vitro. Methods Mol Biol 1117:259-71 |
| Mitchison, Timothy J; Nguyen, Phuong; Coughlin, Margaret et al. (2013) Self-organization of stabilized microtubules by both spindle and midzone mechanisms in Xenopus egg cytosol. Mol Biol Cell 24:1559-73 |
| Kueh, Hao Yuan; Niethammer, Philipp; Mitchison, Timothy J (2013) Maintenance of mitochondrial oxygen homeostasis by cosubstrate compensation. Biophys J 104:1338-48 |
| Hu, Chi-Kuo; Ozlu, Nurhan; Coughlin, Margaret et al. (2012) Plk1 negatively regulates PRC1 to prevent premature midzone formation before cytokinesis. Mol Biol Cell 23:2702-11 |
| Hu, Chi-Kuo; Coughlin, Margaret; Mitchison, Timothy J (2012) Midbody assembly and its regulation during cytokinesis. Mol Biol Cell 23:1024-34 |
| Zhang, Xin; Wang, Wenchao; Bedigian, Anne V et al. (2012) Dopamine receptor D3 regulates endocytic sorting by a Prazosin-sensitive interaction with the coatomer COPI. Proc Natl Acad Sci U S A 109:12485-90 |
| Maiuri, Paolo; Terriac, Emmanuel; Paul-Gilloteaux, Perrine et al. (2012) The first World Cell Race. Curr Biol 22:R673-5 |
Showing the most recent 10 out of 70 publications
