During cell division daughter cells must receive one and only one copy of each and every chromosome. To achieve this, all chromosomes must be duplicated; the duplicated products must be recognized as being equivalent, i.e. sister chromatids, so that they can be segregated to the two daughter cells. The identity of the sister chromatids is maintained by the presence of sister chromatid cohesion, which is dissolved only after all duplicated chromosomes have formed bipolar spindle attachments. Various regulatory mechanisms act to ensure that sister chromatids separate in a timely manner; failure to do so is likely to result in chromosome missegregation, leading to cellular abnormalities. In fact, one of the leading causes of cancerous transformations is a defect in proper chromosome segregation during mitosis. We are studying the regulation of chromosome segregation and nuclear division in budding yeast. One of the key regulators of chromosome segregation in yeast is a protein called Pds1p (an ortholog of the protein securin in higher eukaryotes). Pds1p is at the epicenter of a key regulatory process, known as the spindle assembly checkpoint, which inhibits chromosome segregation until proper chromosome-spindle attachments are formed. Pds1p is an inhibitor of a protease, called separase, which is needed to separate sister chromatids during mitosis. Using Pds1p as a starting point, we previously conducted several studies that led to the discovery of several novel proteins and processes associated with mitosis, two of which were characterized further in the past year: 1. Apq12p, a novel protein involved in cell cycle regulation. In a previous study (Sarin et al, Genetics 2004) we searched for novel functions that are needed for chromosome segregation. Since Pds1p is an essential component of the spindle checkpoint pathway, and because the inactivation of this checkpoint sensitizes cells to mutations that negatively affect the chromosome segregation process, we looked for mutations that result in cell death when combined with a deletion of the PDS1 gene. Through this screen we isolated APQ12 that codes for an ER-associated protein of unknown function. The involvement of the ER protein in cell cycle regulation is intriguing, as this is one of the first examples that links ER function with cell cycle progression. Our initial findings suggest that Apq12p affects cyclin levels and is involved in the correct positioning of the cell division septum. We are currently trying to determine the molecular function of Apq12 and to identify interacting proteins that may be involved in the Apq12 pathway. 2. Nuclear positioning in budding yeast. An unexpected role for separase, discovered by us in 2004 (Ross and Cohen-Fix, Dev Cell 2004) is the control of nuclear positioning during mitosis. We found that separase activates a pathway that promotes the migration of the nuclear pole containing the mother-bound chromosomes, toward the cortex of the mother cell. In an attempt to identify the underlying mechanism of this process, we screened through a collection of mutants to determine if any were defective in this mother-bound force. One of the mutations isolated was in the ASE1 gene, which codes for a spindle associated protein. The previously known function of Ase1p, a homolog of the mammalian PRC1, is to stabilize the spindle midzone. We are currently determining how Ase1p is involved in nuclear positioning. Specifically, our experiments address how Ase1 is regulated and whether the involvement of Ase1p in nuclear positioning is distinct from its role in stabilizing the spindle midzone. We are also studying the underlying mechanism and structures that control nuclear shape. It is noteworthy that abnormalities in nuclear shape are one of the hallmarks of cancerous cells, but the consequences of shape change, and its relevance to the etiology of the disease, are unknown. To gain insights into how nuclear shape maintained, we used a yeast mutant strain that exhibits an abnormally shaped nucleus. This strain is defective the in regulation of phospholipids biosynthesis, through a mutation in the SPO7 gene, there by causing nuclear membrane proliferation. Importantly, however, the expansion of the nuclear membrane is not uniform but is confined to the region that is associated with the yeast nucleolus. Our investigations have led us to propose that the yeast nuclear membrane has distinct domains that differ in their susceptibility to membrane expansion. Furthermore, our findings suggest that there is a structure that restricts nuclear membrane expansion in the nuclear domain that contains the bulk of the chromosomal DNA. This structure, which is likely to be essential for maintaining nuclear shape, is the subject of ongoing investigations.
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