Elucidating Zip1's role in chromosome segregation

All sexually reproducing organisms use a specialised cell cycle division (meiosis) to generate gametes (e.g. sperm and egg cells in animals, pollen and megaspores in plants, and spores in fungi). During meiosis, a single round of DNA replication is followed by two consecutive nuclear divisions that result in four haploid gametes. The first meiotic division is special in that the equivalent parental ('homologous') chromosomes pair, recombine and eventually segregate away from each other. Crossover recombination is particularly important in many organisms. The physical exchange of DNA and the proteins associated with DNA allow the homologous chromosome pairs to attach to the spindle in a bipolar fashion. Failure of homologous pairs to receive a crossover is associated with inaccurate chromosome numbers in the resulting gametes, leading to chromosomal abnormalities in the offspring such as Down's syndrome and infertility in humans. Since there is variation in the number of crossovers an average cell receives, mechanisms that cope with homologous chromosome pairs without a crossover ('non-exchange' pairs) are important to the genomic integrity of gametes. Some organisms- such as Drosophila- have mechanisms that are capable of ensuring accurate segregation of several non-exchange pairs. In budding yeast, non-exchange pairs do not segregate randomly suggesting that alternative mechanisms for segregation are functioning. Recently, budding yeast was used to model human female infertility, hypothesised to be composed of 'two hits'. The first of these is the failure of a chromosome pair to receive a crossover and the second, is the failure of the spindle checkpoint. Therefore, understanding and identifying factors involved in the accurate segregation of non-exchange pairs ('distributive disjunction') is important. We have identified the first non-spindle checkpoint protein that promotes distributive disjunction in budding yeast. Zip1 aids non-exchange pairs to segregate appropriately at the first meiotic division in the absence of an inhibitor, Msh4. Furthermore, elevated temperature is also required for Zip1 to exert its effect. How Zip1 ensures distributive disjunction is not unknown and we therefore propose several experiments to elucidate and understand how Zip1 acts. For example, does Zip1 help centromeres to stay together thus ensuring that the chromosomes are attached properly to the meiotic spindle? Or does Zip1 play another role, possibly unrelated to any other known role of Zip1? Our experiments combine approaches from molecular biology, microscopy, and genetics to understand how this protein functions.

The reciprocal exchange (crossing over) of homologous chromosomes has long been thought to be important in ensuring accurate disjunction of homologs at the first meiotic division. In budding yeast and many other organisms, crossovers are promoted by the Msh4 epistasis group. This group includes the evolutionarily highly conserved Msh4 and Msh5 proteins in addition to proteins (Zip1, Zip2, and Zip3) required for the formation of the synaptonemal complex, a tripartite proteinaceous structure. In budding yeast and also mammals, the formation of the SC and crossovers are interdependent insofar that mutants that do not form the SC (e.g. zip1) also do not form crossovers and vice versa. How important are crossovers in ensuring proper disjunction? In some organisms such as Drosophila males, homologs disjoin accurately despite lacking crossovers ('distributive disjunction'). Other organisms such as budding yeast also appear to use distributive disjunction, however, since its description in 1986, only a single gene, the spindle checkpoint protein MAD3, has been implicated in this mechanism. We have recently identified the first non-spindle checkpoint protein in budding yeast that promotes distributive disjunction. Zip1 is required for this process, and its role appears to be independent of its function in crossing over and SC formation. To elucidate Zip1's role in chromosome segregation, we will test whether Zip1 may have a role in holding centromeres together in a process that may be similar to and/or mimic its recently discovered centromere coupling function. We have also observed that Zip1 is on the metaphase spindle in msh4 cells, suggesting that Zip1's role in distributive disjunction may be distinct from its role in centromere coupling. How and why Zip1 localises to the metaphase spindles will also be investigated. Finally, we are interested in understanding how the spindle checkpoint interacts with Zip1 to ensure distributive segregation.