Making a spindle in the right place

Supervisors: Professor Hiro Ohkura, Professor Carol MacKintosh

Project description:

Genes must be passed on accurately from cell to cell and from parents to children. Failure to do so can be a cause or contributing factor in human illnesses, such as cancer or reproductive/birth defects. During eukaryotic cell divisions, cells dramatically change their organisation.  DNA carrying genes is packaged into chromosomes and a bipolar spindle made of microtubules and microtubule-associated proteins is assembled to segregate the chromosomes.  Spatial regulation of microtubule-associated proteins (MAPs) is important for forming a proper bipolar spindle and accurate chromosome segregation.  This is especially critical in oocytes, as they lack centrosomes and have an exceptionally large volume (Ohkura, 2015).

This project aims to uncover spatial regulation of MAPs at the molecular level.  The Ran-importin system is well-documented to spatially regulate MAPs in mitotic cells and oocytes, but lines of evidence suggest the presence of alternative systems.  We recently identified a novel mechanism which reads a spatial cue to activate a MAP (kinesin-14) only around chromosomes in oocytes, which involves 14-3-3 and Aurora B (Beaven et al, 2017).

The first objective of the project is to comprehensively identify MAPs which are controlled by the 14-3-3 protein using combination of genetics, biochemistry, bioinformatics and microscopy.  14-3-3 binding proteins will be affinity-purified from mitotic and ovary extract, and the 14-3-3 interactomes will be identified by quantitative mass-spectrometry.  This will be cross-referenced with the microtubule interactomes which our lab has already defined from Drosophila and humans (Syred et al, 2013).

The second objective is to understand the molecular mechanism of spatial regulation using a combination of in vivo, in vitro and in silico analysis.  To understand the functions of 14-3-3 targets, in vivo RNAi or CRISPR will be used to deplete the proteins in cells and ovaries, and the spindle morphology and chromosome alignment will be examined by immunostaining and live-imaging.  The student will mainly use Drosophila oocytes and cells, but also mammalian cells to gain insight into conservation.  Spatial regulation will be reconstituted in vitro using pure components, and tested by in silico mathematical modelling.

The third objective is to test an interplay between Ran-importin and Aurora B-14-3-3, and the presence of yet unidentified pathways for spatial regulation of spindle proteins.  The pathways will be disrupted individually and simultaneously, and tested for the localisation of various crucial spindle proteins in oocytes.

Genes must be passed on accurately from cell to cell and from parents to children. Failure to do so can be a cause or contributing factor in human illnesses, such as cancer or reproductive/birth defects. During eukaryotic cell divisions, cells dramatically change their organisation.  DNA carrying genes is packaged into chromosomes and a bipolar spindle made of microtubules and microtubule-associated proteins is assembled to segregate the chromosomes.  Spatial regulation of microtubule-associated proteins (MAPs) is important for forming a proper bipolar spindle and accurate chromosome segregation.  This is especially critical in oocytes, as they lack centrosomes and have an exceptionally large volume (Ohkura, 2015).

     This project aims to uncover spatial regulation of MAPs at the molecular level.  The Ran-importin system is well-documented to spatially regulate MAPs in mitotic cells and oocytes, but lines of evidence suggest the presence of alternative systems.  We recently identified a novel mechanism which reads a spatial cue to activate a MAP (kinesin-14) only around chromosomes in oocytes, which involves 14-3-3 and Aurora B (Beaven et al, 2017).

     The first objective of the project is to comprehensively identify MAPs which are controlled by the 14-3-3 protein using combination of genetics, biochemistry, bioinformatics and microscopy.  14-3-3 binding proteins will be affinity-purified from mitotic and ovary extract, and the 14-3-3 interactomes will be identified by quantitative mass-spectrometry.  This will be cross-referenced with the microtubule interactomes which our lab has already defined from Drosophila and humans (Syred et al, 2013).

The second objective is to understand the molecular mechanism of spatial regulation using a combination of in vivo, in vitro and in silico analysis.  To understand the functions of 14-3-3 targets, in vivo RNAi or CRISPR will be used to deplete the proteins in cells and ovaries, and the spindle morphology and chromosome alignment will be examined by immunostaining and live-imaging.  The student will mainly use Drosophila oocytes and cells, but also mammalian cells to gain insight into conservation.  Spatial regulation will be reconstituted in vitro using pure components, and tested by in silico mathematical modelling.

The third objective is to test an interplay between Ran-importin and Aurora B-14-3-3, and the presence of yet unidentified pathways for spatial regulation of spindle proteins.  The pathways will be disrupted individually and simultaneously, and tested for the localisation of various crucial spindle proteins in oocytes.

References:

R Beaven, RN Bastos, C Spanos, P Romé, CF Cullen, J Rappsilber, R Giet, G Goshima and H Ohkura (2017) 14-3-3 regulation of Ncd reveals a new mechanism for targeting proteins to the spindle in oocytes. J. Cell Biol. in press.

H Ohkura (2015) Meiosis: An overview of key differences from mitosis. Cold Spring Harb Perspect Biol. 7: a015859.

HM Syred, J Welburn, J Rappsilber and H Ohkura (2013) Cell cycle regulation of microtubule interactomes: multi-layered regulation is critical for the interphase/mitosis transition. Mol. Cell. Proteomics 12: 3135-3147

 

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