Research objectives and content
This proposal concerns the regulation of mitosis. The activity of the mitotic kinase cdc2 is tightly controlled by different regulatory proteins. An important aspect is where these factors are in the cell. Cyclins, cdks and cdc25s all comprise multigene families where one major difference between the family members is their subcellular localization. We are using Green Fluorescent Protein (GFP) as a marker to follow the localization of cell cycle proteins in living cells. We have made chimeras of GFP and different cell cycle regulators. Single cells expressing the chimeras are being followed through the cell cycle by time-lapse video-microscopy. Using this technique, I wish to investigate further the activation of cdc2 by cyclin B 1 binding and cdc25 phophatase activity. This also involves the use of mutants that are deficient in localization and/or function.
Training content (objective, benefit and expected impact)
In Jonathon Pines' laboratory, advanced new technology is used to study protein dynamics in living cells. I would like to become familiar with these techniques i.e. fluorescence-video microscopy, confocal microscopy and image processing. I will also use microinjection, in vitro mutagenesis and antisense technology. These methods, together with the use of GFP as fluorescent marker has numerous applications in cell biology and will potentially be very useful in my future career.