Regulation of Key Cell Cycle Events

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The Rhind lab studies questions of cellular growth control and organization. We are interested in how cells make organizational decisions without obvious external input; in effect, how cells create order out of chaos. The two particular questions that we are currently focusing on are how cells decide when to divide, in order to maintain cell-size homeostasis, and how cells decide when to activate individual DNA replication origins, in order to regulate the spatial and temporal organization of DNA replication. The mechanism of cell-size regulation is an enduring mystery in cell biology. The ability to maintain size homeostasis is an essential cellular function, and regulation of proper cell size is a key feature of differentiation, required for cellular function from tiny platelets to enormous oocytes. Moreover, loss of size homeostasis is a characteristic of many cancers and tissue-degenerative diseases. Although recent progress has been made on specific mechanisms in several systems, there is still no system in which the problem is well understood. We study cell size in fission yeast because it affords a well-characterized, evolutionarily conserved and experimentally tractable system. We are investigating the hypothesis that cell size in fission yeast is controlled by the size-dependent expression of two mitotic activators: Cdc13 and Cdc25. We have shown that both Cdc13 and Cdc25 levels increase in proportion to cell size, and propose that this size-dependent expression prevents cells from entering mitosis until they reach their correct size for division. We are currently working to test that hypothesis directly and to understand how these two proteins are expressed in size-dependent manners. DNA replication timing is a fundamental aspect of nuclear metabolism. Well-organized replication timing is essential for timely completion of replication and thus stable genome inheritance. Replication timing is also correlated with, and has been proposed to regulate, gene expression, chromatin modification, chromosome structure, and genome evolution. Dysregulated replication timing is a feature of cancer cells and contributes to their genome instability and pathological gene-expression phenotypes. Replication timing is regulated by the timing of replication origin firing, which is in turn regulated by the probability of firing, which varies between origins. To investigate the regulation of origin firing, we have pioneered single-molecule origin mapping techniques that allow us to measure both the location and firing probability of origins across the genome. Our results and the results of others that have shown how the heterogeneity of origin firing at the single cell level is averaged over the population to produce emergent replication timing patterns. It is therefore critical to understand how the probability of origin firing is regulated. We are pursuing this question in both budding yeast, which affords well-defined and well-characterized origins for specific mechanistic experiments, and mammalian cells, which allow us to apply what we learn to more complicated genomes and developmental pathways.