Description of the Research Program of Dr. J. Paulson
office: HS 417
lab: HS 424
Area of research: The biochemistry of mitosis (cell division); structure of chromosomes and cell nuclei; function and control of post-translational modifications in chromosomal proteins; relationships between mitosis and apoptosis (physiological cell death).
Techniques and equipment used: Mammalian cell culture and cell cycle synchronization, light microscopy, isolation of chromosomes, electrophoresis and chromatography of proteins, enzyme assays.
Minimum experience: General chemistry, some biology or biochemistry.
Preferred experience: Organic chemistry, possibly quantitative analysis, some advanced biology or biochemistry lab work.
1) Specific induction of apoptosis by heat treatment of human cancer cells arrested in mitosis.
We have found that HeLa cells, a human cervical carcinoma cell line, will undergo apoptosis (physiological cell death) when arrested in metaphase of mitosis and then shifted to higher temperature (39 - 42°C). This has been shown by morphological changes in the cells, suppression of those morphological changes by inhibitors of apoptosis, and by apoptosis-specific cleavage of certain proteins. Interphase cells are not affected by the same treatment.
We are now investigating other normal and cancerous human and mouse cell lines and preliminary results suggest that most of them behave similarly to HeLa cells. Future work will focus on testing hypotheses concerning the possible mechanism behind this phenomenon. This work was carried out by undergraduate student Agnes Kecskemeti ('03) and also involves collaboration with Dr. Peter W. Mesner of the University of Wisconsin Whitewater.
2) Identification of the mitotic histone H1 phosphatase in HeLa cells
We have good evidence that the enzyme which dephosphorylates histone H1 at the end of mitosis is Protein Phosphatase 1 (PP1). To prove this, and to learn more about the enzyme, the long term goal is to purify the histone H1 phosphatase. Previously, undergraduate student Chantel Buhrow ('00) developed a convenient enzyme assay for the histone H1 phosphatase (that is, a way to measure the amount of H1 phosphatase activity) and a method to extract the enzyme from chromosomes. Currently, I am collaborating with Dr. Emma Villa-Moruzzi of the University of Pisa in Italy, who has specific antibodies to the different subtypes of PP1. The first step is use those specific antibodies to identify which subtype of the PP1 catalytic subunit is present in chromosomes. (So far it appears to be PP1-delta.) The next step will be to use the antibodies to purify the enzyme from our chromosome extracts by immunoprecipitation. The hope is to identify non-catalytic subunits that regulate the enzyme or are responsible for its localization in chromosomes.
3) Identification of protein phosphatases involved in exit from mitosis in yeast
An enzyme called MPF (for Mitosis Promoting Factor) has been shown to be responsible for initiating the events of mitosis. We have shown, both in human cells and in yeast, that inactivation of MPF causes metaphase-arrested cells to leave mitosis (without segregating their chromosomes or undergoing cytokinesis) and go back to interphase. (The cells will continue through another cell cycle and when they reach the next mitosis they are found to have twice the normal number of chromosomes.) Evidence suggests that protein phosphatases are inactivated (by MPF) at the onset of mitosis and then become active again after MPF is inactivated.
This observation provides a tool to study protein dephosphorylation at the end of mitosis. Based on work done with budding yeast (Saccharomyces cerevisiae ) by Biology/Microbiology Master’s degree student Jason Keaton, we are currently preparing yeast strains with mutations both in MPF and in the yeast PP1 gene (called glc7 ). This will make it possible to test the hypothesis that PP1 is required for dephosphorylation of proteins and exit from mitosis after the inactivation of MPF.
4) Other studies on histone phosphorylation and mitotic chromosome structure
We have made several other interesting observations over the past few years that merit further investigation. First, we found that treatment of cells with inhibitors of protein phosphatases (for example, inhibitors of PP1) induces nuclear envelope breakdown and "prematurely condensed chromosomes" (PCCs) – in other words, a sort of premature mitosis-like state. This happens no matter where the cells are in the cell division cycle, and thus will provide a tool to study aspects of chromosome structure throughout the cell cycle.
Second, we found that during G1-phase and early S-phase, histone H1 does not get phosphorylated in these PCCs. This has some interesting implications and may provide a means to explore the function of histone H1 phosphorylation.
Third, we found that inhibitors of the topoisomerase II do not prevent the formation of PCCs, but they do prevent the individualization of chromosomes. It remains to be worked out, using these inhibitors as tools, whether individualization of chromosomes occurs during chromosome condensation or later, when the chromosomes are separated at anaphase of mitosis.
(1) As mentioned above, I am collaborating with Dr. Peter W. Mesner (University of Wisconsin Whitewater) on studies of apoptosis in metaphase-arrested cells.
(2) I am collaborating with Dr. Emma Villa-Moruzzi (University of Pisa, Italy) to identify and purify the Protein Phosphatase 1 subtype found in mitotic chromosomes.
(3) I am collaborating with Drs. Viesturs Simanis and Andrea Krapp (ISREC, Lausanne, Switzerland) on studies of exit from mitosis in yeast.
(4) I am collaborating with Dr. I. Y. Mahmoud (Emeritus Professor of Biology, University of Wisconsin Oshkosh) and others to characterize the proteins secreted by the oviduct throughout the reproductive cycle of turtles.