Admin supplemental funds to purchase a Cherry Biotech CherryTemp temperature-controlled stage for live cell imaging of temperature-sensitive C. elegans mutants with oocyte meiotic spindle defects.

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Project Summary Oocyte meiosis ends with two rounds of cell division that produce an egg with a haploid genome. While mitosis uses bipolar spindles that assemble using two centrosomes, oocytes assemble bipolar spindles in the absence of centrosomes. While the assembly and function of mitotic spindles have been studied extensively, acentrosomal oocyte spindle assembly remains poorly understood, and C. elegans provides an appealing model system for investigating the mechanisms that govern this process. Moreover, our understanding of oocyte spindle assembly remains incomplete in all widely used model systems, with live cell imaging only recently beginning to shed light on the dynamics of spindle assembly, and somewhat piecemeal genetic studies having provided a partial but incomplete catalogue of the important players. We propose a systematic and quantitative analysis of oocyte meiotic spindle assembly and spindle bipolarity, using a valuable collection of temperature-sensitive mutants we have isolated. We also will use RNA interference to identify the genes and pathways that operate during oocyte spindle assembly in C. elegans, along with live-cell fluorescent video-microscopy and genome editing to provide a more complete and mechanistic understanding of this fundamentally important process. In 2014, we published a manuscript describing our initial work on oocyte spindle pole assembly in Molecular Biology of the Cell, and in 2015 another manuscript in The Journal of Cell Biology and describing our evidence that the microtubule depolymerase KLP- 7/MCAK acts through kinetochores to promote the coalescence of oocyte spindle poles. These results, together with our identification of additional temperature-sensitive mutants with oocyte spindle defects and a thorough survey of the literature covering the most widely used model systems, provide the foundation for the specific aims we now propose to advance our understanding of acentrosomal oocyte meiotic spindle assembly. Even though mitotic spindles rely extensively on centrosomes to organize bipolar spindles, oocyte meiosis and somatic mitosis share the use of acentrosomal pathways for nucleating and organizing microtubules. Thus investigating oocyte spindle assembly will improve our understanding the abnormal mitotic proliferations that result in cancer. Moreover, human oocytes are remarkably prone to errors in spindle assembly and often produce aneuploid oocytes that upon fertilization develop abnormally. A better understanding of oocyte meiotic spindle assembly is therefore relevant to human fertility and development.