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Cell division – The Wakefield Lab What principles govern the formation of the mitotic spindle? How do they interact to ensure robustness and flexibility to the system? Why is the spindle the shape it is?
early embryos Bioinformatics System perturbation (genetics et al.) Software engineering Mathematical modelling Proteomics Biochemistry qualitative synthetic quantitative analytical What is our approach? Observation (cell biology) • 13 rounds of mitosis within 3 hrs • Maternally produced protein • Syncytial and therefore synchronous • Can collect grammes within a week
msd3 RNAi control msd1 RNAi What proteins that bind MTs are important for cell division? biochemically isolate Drosophila embryonic MAPs Identify via MS and categorise Characterise novel proteins live observation Correlation categorisation Hughes et al., (2008) PLoS Biol
Applying MS data to protein interaction networks Generate extended network of putative MAPs Use MS data to produce a list of interspecies MAPs Rank using PCA/Random forest likelihood statistics Generate interesting hypotheses characterise candidates
Purify via GFP-TRAP Metaphase Anaphase 171 63 49 65 13 7 What proteins are part of mitotic MAP complexes in vivo, and what PTMs are important for their function? metaphase chromosome condensation cytokinesis BOREALIN anaphase SURVIVIN INCENP AURORA B spindle organisation kinetochore:k-MT attachment Mass spec and analyse characterise candidates Identify phosphorylation and SUMOylation sites
Wakefield Lab (Past) Julian Hughes Katherine Fisher (Present) Faisal Khan Pete Jones Dan Hayward Jack Chen Simon Li Acknowledgements Collaborations • Mass Spectrometry Nicole Zitzmann (Oxford) Hannah Florance (Exeter) • Bioinformatics Charlotte Deane (Oxford)
all principles / processes What principles govern the formation of the mitotic spindle? How do they interact to ensure robustness and flexibility to the system? Why is the spindle the shape it is?