The meiotic spindle and chromosomes in oocytes
Hiro Ohkura is a Wellcome Investigator in Science and Professor of Cell Biology at the University of Edinburgh. His group is studying the molecular regulation of meiotic chromosomes and spindle in oocytes using Drosophila as a model system.
Hiro did his PhD study on fission yeast mitosis in Prof Yanagida's lab in Kyoto University, Japan. He then worked as a postdoc in Prof Glover's lab in Dundee University, UK, studying Drosophila mitosis as well as fission yeast mitosis. He was awarded a Wellcome Senior Fellowship in 1997 and has established his lab in the University of Edinburgh, UK.
He held a Wellcome Senior Research Fellowship for 20 years before becoming a Wellcome Investigator in 2018.
Aleksandra Ciszek, Fiona Cullen, Rayane Kaade, Jule Nieken, Gera Pavlova, Emma Peat, Carmela Silva Mason, Emiliya Taskova, Xiang Wang and Li Ern Yap
The meiotic spindle and chromosomes in oocytes
Accurate segregation of chromosomal DNA is essential for life. An error in this process could result in cell death or aneuploidy. Furthermore, chromosome segregation in oocytes is error-prone in humans, and mis-segregation is a major cause of infertility, miscarriages and birth defects. Chromosome segregation in oocytes shares many similarities with those in somatic divisions, but also has notable differences. Distinct features of oocytes potentially hinder accurate chromosome segregation. They include (1) lack of centrosomes, the major microtubule nucleation centres in mitosis, (2) exceptionally large cell volume, and (3) cell cycle arrests at two stages. Oocytes are likely to have specific molecular mechanisms which mitigate negative impacts of these features, but little is known about how oocytes set up the chromosome segregation machinery. Defining the oocyte-specific mechanisms would be crucial to understand error-prone chromosome segregation in human oocytes. Furthermore, it may provide an insight into whether and how cancer cells might gain resistance to antimitotic drugs by activating these pathways.
To understand the molecular pathways which set up the chromosome segregation machinery in oocytes, we take advantage of Drosophila oocytes as a "discovery platform" because of their similarity to mammalian oocytes and suitability for a approach combining Genetics, microscopy and biochemistry. In Drosophila oocytes, as in human oocytes, meiotic chromosomes form a compact cluster called the karyosome within the nucleus. Later, meiotic chromosomes assemble a bipolar spindle without centrosomes in the large volume of the cytoplasm, and establish bipolar attachment. We have identified genes/proteins and regulations specifically important for chromosome organisation and/or spindle formation in oocytes.
Global regulation of spindle-associated proteins is crucial in oocytes due to the absence of centrosomes and their very large cytoplasmic volume, but little is known about how this is achieved beyond involvement of the Ran-importin pathway. We previously uncovered a novel regulatory mechanism in Drosophila oocytes, in which the phospho-docking protein 14-3-3 suppresses microtubule binding of Kinesin-14/Ncd away from chromosomes. To systematically identify microtubuleassociated proteins regulated by 14-3-3 from Drosophila oocytes, proteins from ovary extract were co-sedimented with microtubules in the presence or absence of a 14-3-3 inhibitor. Through quantitative mass-spectrometry, we identified proteins or complexes whose ability to bind microtubules is suppressed by 14-3-3, including the chromosomal passenger complex (CPC), the centralspindlin complex and Kinesin-14/Ncd. We showed that 14-3-3 binds to the disordered region of Borealin, and this binding is regulated differentially by two phosphorylations on Borealin. Mutations at these two phospho-sites compromised normal Borealin localisation and centromere bi-orientation in oocytes, showing that phosphoregulation of 14-3-3 binding is important for Borealin localisation and function.
A. Identification of 14-3-3 regulated microtubule-associated proteins from Drosophila oocytes. Microtubules and their associated proteins were purified from Drosophila ovaries in the presence and absence of a 14-3-3 inhibitor, and analysed by label-free quantitative mass-spectrometry.
B. Volcano plot showing the fold changes of the amounts of each protein detected in microtubule fraction in the presence of the 14-3-3 inhibitor in comparison to its absence. The red box contains 47 proteins that significantly increased their microtubule binding under 14-3-3 inhibition.
C. Upper panel: Diagram of domain organisation of Borealin and Incenp proteins, with three predicted 14-3-3 binding sites (red circles). Lower panel: the sequence surrounding S163 of Borealin has high similarity to the 14-3-3 binding sites of Ncd/Kinesin-14-3-3 binding sites of Ncd/Kinesin-14 and Pav/Kinesin-6.
D. An additional phosphorylation by Aurora B prevents PKD2-phosphorylated MBP-Borealin(113-221) from interacting with GST14-3-3ε. Borealin(113-221) was incubated with human PKD2 kinase alone, human Aurora B kinase alone, both kinases or without kinases, and tested for pull down using GST or GST-14-3-3ε. Repton et al (2022).
Repton, C., Cullen, C. F., Costa, M. F. A., Spanos, C., Rappsilber, J., Ohkura, H. (2022) The phospho-docking protein 14-3-3 regulates microtubule-associated proteins in oocytes including the chromosomal passenger Borealin. bioRxiv. doi: 10.1101/2021.12.16.472885
Barbosa, P., Zhaunova, L., Debilio, S., Steccanella, V., Kelly, V., Ly, T., and Ohkura H. (2021) SCF-Fbxo42 promotes synaptonemal complex assembly by downregulating PP2A-B56. J. Cell Biol. 220, e202009167.
Costa, M. F. A., and Ohkura, H. (2019) The molecular architecture of the meiotic spindle is remodeled during metaphase arrest in oocytes. J. Cell Biol. 218, 2854-2864.