Mechanism of Mitotic Chromosome Condensation (funded by the Wellcome Trust and Cancer Research UK)

Mitotic chromosome segregation requires proper chromosome assembly and the phosphorylation of a number of chromosomal proteins. The Aurora B protein kinase and its binding partners INCENP and survivin have a critical role in this process. We are using both biochemical and imaging based approaches to study the function and regulation of the Aurora B protein complex.

Our biochemical analysis is based on mitotic chromosome eluates containing the chromosomal complement of protein kinases and other factors in soluble form. We have previously shown that a complex containing the Aurora B protein kinase is the major mitotic chromosome-associated histone H3 kinase and that protein phosphatase 1 plays a critical role in regulating Aurora B (Murnion et al, 2001). We are currently dissecting the mechanism of activation of aurora-B, focussing most closely of the role of INCENP. We are also identifying new components of the Aurora B complex and new substrates for the complex within chromosomes.

To complement our biochemical analyses, we are using digital imaging to study the dynamics of the Aurora B complex in living cells. In collaboration with the Owen-Hughes Lab, we have also developed assays based on measurements of fluorescence energy transfer (FRET) to detect cell cycle dependent changes in chromatin structure. These assays are being used to probe the interior structure of the mitotic chromosome. A critical question in these studies is the effect of histone H3 phosphorylation in chromatin structure and chromosome condensation. We have previously shown that histone H3 phosphorylation changes chromatin flexibility (Murnion et al, 2001). We are determining the structural basis for this change using our FRET assays.

These assays depend on the collection of quantitatively accurate image data from fluorescence microscopes. We have therefore determined signal-to-noise limits in different modes of fluorescence microscopy (Swedlow et al., 2002). We have also characterized the use of deconvolution methods in fluorescence microscopy (Wallace et al, 2001).