Hardwick, Kevin

Harrington, Lea

Sochaj, Alicja Maria

Biotechnology and Biological Sciences Research Council (BBSRC)

2013-09-13T09:27:17Z

2013-09-13T09:27:17Z

2013-06-29

The accurate transmission of genetic material in the germline is of vital importance to the offspring since the entire organism is affected. It is therefore critical that chromosomes are faithfully distributed during the formation of gametes in a specialised form of cell division known as meiosis. Errors in meiotic cell divisions are a frequent cause of infertility, miscarriages, and birth defects in humans. The meiotic chromatin undergoes dynamic rearrangements during these divisions that are poorly understood at the molecular level. In prophase of the first meiotic division, the chromatin of Drosophila melanogaster oocytes detaches from the nuclear envelope to form a compact spherical cluster known as karyosome. It was previously shown that the karyosome is required for faithful chromosome segregation, but knowledge about its formation and maintenance is limited. I wish to understand how karyosome formation is regulated and identified genes important for karyosome formation in a genome-wide cytological screen of Drosophila melanogaster oocytes. The screen comprised 3,916 candidate genes expressed in ovaries, of which 209 genes showed karyosome defects upon knockdown. I found that genes encoding mitochondrial proteins, including electron transport chain components, are overrepresented amongst genes whose knockdown results in severe and reproducible karyosome defects. Interestingly, mitochondrial dysfunction induced a distinct karyosome defect characterised by three individual chromatin clusters in proximity to the nuclear envelope. Furthermore, my studies revealed that mitochondrial dysfunction not only impairs karyosome formation, but also karyosome maintenance throughout mid-oogenesis and synaptonemal complex dynamics. I asked how mitochondrial dysfunction triggers karyosome defects and aimed at further mechanistical insights. I found that mitochondrial dysfunction forces a low percentage of oocytes into apoptotic cell death, but karyosome defects occur independent of apoptosis. The knockdown of ATP synthase subunits induced the distinct karyosome defects observed upon mitochondrial dysfunction, suggesting a direct link between the karyosome phenotype and reduced levels of cellular ATP. I further determined the dependence of observed karyosome abnormalities on meiotic checkpoint activation. My work thus identified a set of genes with reproducible and checkpoint-independent karyosome defects upon knockdown. The uncharacterised function of these genes in karyosome formation and maintenance remains to be investigated and future research will pave the way for a better understanding of the karyosome at a molecular level. Furthermore, I established a link between mitochondrial dysfunction and a karyosome phenotype characterised by chromatin attachment to the nuclear envelope. The functional role of mitochondria is an increasingly important consideration in both male and female fertility. My study therefore provides a novel insight, considering that mechanistical details on how mitochondrial diseases link to infertility are sparse.

http://hdl.handle.net/1842/7763

en

The University of Edinburgh

Meadows, J. C., Shepperd, L. A., Vanoosthuyse, V., Lancaster, T. C., Sochaj, A. M., Buttrick, G. J., Hardwick, K. G., and Millar, J. B. A. (2011). Spindle checkpoint silencing requires association of PP1 to both Spc7 and kinesin-8 Motors. Developmental Cell 20, 739–750.

Shepperd, L. A., Meadows, J. C., Sochaj, A. M., Lancaster, T. C., Zou, J., Buttrick, G. J., Rappsilber, J., Hardwick, K. G., and Millar, J. B. A. (2012). Phosphodependent recruitment of Bub1 and Bub3 to Spc7/KNL1 by Mph1 kinase maintains the spindle checkpoint. Current Biology 22, 891–899.

Zich, J., Sochaj, A. M., Syred, H. M., Milne, L., Cook, A. G., Ohkura, H., Rappsilber, J., and Hardwick, K. G. (2012). Kinase activity of fission yeast mph1 is required for Mad2 and Mad3 to stably bind the anaphase promoting complex. Current Biology 22, 296–301.

mitotic checkpoint

mitosis

fission yeast.

Dissecting roles and regulation of the fission yeast kinetochore protein Spc7

Thesis or Dissertation

Doctoral

PhD Doctor of Philosophy