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G.E. Folkers

Scientific background

Control of the cell cycle regulation is critically dependent on the activation and inactivation of cyclin-cyclin dependent kinase complexes. This is mediated by cell cycle dependent control of cyclin expression but also the timed degradation of cyclins at specific stage of the cell cycle is important. Ubiquitination of proteins is well known to initiate protein degradation and also in cell cycle regulation a key role is demonstrated for the ubiquitination pathway (). Next to ubiquitin also a related protein small ubiquitin-like modifier (Sumo) is playing an important role in cell cycle progression (). Interestingly evidence is accumulating that addition of a sumo group to key cell cycle progression proteins such as anaphase promoting complex lead to activation, while the subsequent ubiquitination of the sumoylated protein inactivates the complex.

Project description

In the group we like to understand how ubiquitin and sumo can regulate various stages of cell-cycle progression. We study ubiquitination in vivo using NMR spectroscopy and like to understand how structural changes of the proteins involved regulate these processes. To enable that we electroporate stable isotope labelled proteins (13C and or 15N) into cells and look at the changes in the structural properties of these proteins throughout cell cycle amongst other situations. While electroporating Sumo or Sumo without its N-terminal tail we noticed that the latter was more stable in vivo and led to uncharacterized cell division effects leading to polyploidy and later cell death. We would like to understand the role of Sumo in cell cycle regulation, in this research project we intend to optimize the methods to synchronize cells at different stages of the cell cycle in combination with the electroporation of Sumo proteins. By analysing various stages of the cell-cycle using immunofluorescence in the presence or absence of Sumo proteins we would like to understand at which stage of the cell-cycle the sumo-dependent changes take place and how this contributes to cell cycle regulation

Research proposal

This study will generate modified bacterial Sumo expression plasmid, express these plasmids in E.coli and purify the protein using standard purification methods and if needed label the protein with a fluorescent tag. The (fluorescent) proteins will be electroporated into eukaryotic cells. By inspecting cell morphology during life cell immunofluorescence microscopy following removal of various cell cycle blocking reagents, we intend to find out at which stage of the cell cycle the modified Sumo proteins interfere with normal cell-cycle progression.

Methods used

• Recombinant DNA technology including PCR, cloning, sequencing
• Protein expression, purification and labelling of proteins
• Tissue culture
• Transient transfection and electroporation of proteins
• Light microscopy and immunofluorescence microscopy