Gene targeting tools for tissue-specific protein purification and analysis of the LIN-5 NuMA cell-division complex

A human body consists of an estimated hundred trillion cells, of which a substantial fraction needs to be renewed every day. This makes cell division one of the most critical processes in human development and health. Successful cell division requires coordination between chromosome segregation and cytoplasmic cleavage, so that each daughter cell receives an intact genome. In addition, the cell cleavage plane needs to be coordinated with cell- and tissue polarity to generate cells with proper fates. This coordination is particularly important during asymmetric cell divisions that create different cell types and maintain stem cell populations. To divide asymmetrically, the mitotic spindle needs to be positioned with respect to the axis of cell- or tissue polarity, so that cell cleavage generates cells at specific positions, e.g. with respect to a stem cell niche, or with unequal amounts of fate determinants. How polarity cues instruct the position of the spindle, and how this is coordinated with cell-cycle progression, are currently major questions in the fields of development and stem cell research. Many insights in the regulation of human cell division come from studies in genetic model systems. Such studies have identified a large number of protein complexes with cell division and polarity functions, often with protein kinases as the key regulators. This includes an evolutionarily conserved complex of LIN-5/GPR/Gα in C. elegans, Mud/Pins/Gα in Drosophila and NuMA/LGN/Gα in humans, which controls the position of the spindle and cell cleavage plane. It remains unknown how the function of this complex is regulated to control the position of the spindle and cell-cleavage plane in coordination with chromosome segregation during symmetric and asymmetric cell division. Given the complexity of the regulatory networks involved, a deeper understanding of cell division requires multidisciplinary approaches that combine sophisticated genetic, biochemical, and cell biological strategies. Our collaboration with the state of the art proteomics group of Prof. Albert Heck uniquely allows us to combine quantitative mass spectrometry, live imaging and C. elegans genetics. This has recently resulted in the discovery of a mechanism by which the division plane is controlled by cell polarity during asymmetric division of the C. elegans zygote (see: preliminary results). Similar multidisciplinary approaches are needed to analyze the more complex cell types and tissues with levels of regulation that differ from the one-cell egg. As an attractive ?next level? model, we have started to analyze the stem cell-like division pattern of epithelial seam cells in the C. elegans epidermis. These cells go through rounds of anterior-posterior oriented symmetric and asymmetric divisions at reproducible times of development. We have identified genes that control the seam cell divisions, including lin-5, and have developed methods for detailed live imaging of seam cell divisions throughout larval development. The goal of the current project is to develop and apply methods for sophisticated biochemical analysis of protein phosphorylation and complex formation in a tissue specific manner. We propose to use the latest homologous recombination techniques, in order to insert a ?GFP-biotinylation? tag into endogenous genes. We will combine this with tissue specific expression of the BirA biotinylation enzyme, purify biotinylated proteins with streptavidin beads, followed by analysis with mass spectrometry. As the first application, we will use this strategy to examine global as well as tissue-specific regulation of LIN-5 in intestinal and skin epithelia, during symmetric and asymmetric cell divisions. In addition, we will use the MosTIC gene conversion technique to generate phosphoresistant and phosphomimetic alleles of the critical lin-5 phosphorylation sites, and examine the exact functional consequences by live-cell imaging in different tissues. Our detailed analysis will lead to a deeper understanding of normal cell division, asymmetric stem-cell division, and division defects that contribute to cancer.