Michael E. Geusz
Department of Biological Sciences, Bowling Green State University
Our lab examines:
1. The role of circadian timing in multiple biological processes important in disease prevention and new medical treatments
2. Advanced imaging techniques for monitoring gene expression in living cells and organisms
The physiology and behavior of many organisms undergo daily cycles with the help of internal biological pacemakers that keep time by maintaining circadian rhythms in the body. This circadian system synchronizes internal timing to external 24-hour cycles of light and dark while coordinating rhythmic processes within multiple organs and tissues. Circadian pacemakers have been identified in nearly all organs of mammals, and the neural pacemaker in the suprachiasmatic nucleus (SCN) of the hypothalamus appears to be a dominant node within these timing networks. One current question is how the SCN and other circadian pacemakers interact to remain in phase with each other and with the daily cycles of the environment. Additional important questions concern the role of circadian timing in cancer cell proliferation.
Circadian rhythms in adult neurogenesis
Our recent report in PLOS One describes circadian rhythms in the core clock gene mPer1 during neurogenesis in vitro (Malik et al., 2015, in press).
We also identified for the first time circadian rhythms in gene expression within tumorsphere cultures made cancer stem cells (Sharma et al., 2014; Sharma and Geusz, 2013). Rhythms were identified through bioluminescence imaging of tumorspheres expressing a fusion protein of firefly luciferase and the circadian clock protein mPER2 expressed under control by the mPer2 gene promoter. We also characterized and imaged the cancer stem cells in C6 rat glioma cell cultures by developing a method based on a fluorescent Hoechst dye (Sharma et al., 2014). Ongoing experiments are characterizing circadian properties of cancer stem cells in various tumorigenic cell lines.
Previously, we used luciferase bioluminescence imaging to detect mPer1 gene expression in the stroma of Lewis lung carcinoma (LLC) tumors in live mice (Geusz et al., 2010). Current projects are evaluating whether the circadian clock influences how tumor stromal cells support cancer cell growth.
Using several human esophageal cancer cell lines, we characterized how cancer stem cells have elevated sensitivity to the anti-cancer effects of curcurmin, an ingredient of the spice turmeric (Almanaa et al., 2012). We also identified effects of curcumin on LLC cells and how light can act as a photosensitizer with curcumin (Yan et al., 2012). A new method was developed to identify cancer stem cells in cell cultures based on their elevated aldehyde dehydrogenase activity so that ongoing cell physiology can be imaged in live cancer stem cells (Almanaa et al., 2013).
Neuronal circadian clocks
By imaging luciferase bioluminescence in brain slice cultures we were able to identify circadian oscillations in mper1 gene expression in the mesencephalic trigeminal nucleus of the midbrain of mice (Hiler et al., 2008). We are determining how these cells compare with the better understood circadian clock cells of the hypothalamic SCN. Because they are considerably larger than SCN neurons they may provide advantages for imaging studies.
Methods for imaging gene expression in live mice
We have developed techniques for imaging circadian rhythms in the clock cells of the SCN (Geusz, 2001; Sigworth et al., 2003) and in the tissues of live mice (Collaco and Geusz, 2003 ; Collaco et al., 2005, Hiler al, 2006). Several immediate-early genes are expressed in the SCN. The immediate-early gene c-fos is an effective marker for neural activity and is expressed in the SCN when animals are exposed to light that shifts the phase of their circadian system. These phase shifts serve to keep the circadian clocks synchronized with the daily cycles of the environment. Using a transgenic mouse with the human c-fos promoter controlling the firefly luciferase gene (fos::luc) we are imaging c-fos expression patterns in intact animals and brain slice cultures.
We are also imaging a second transgenic mouse to identify where in the body cells are most permissive for reactivating herpes virus infections and whether the circadian clock plays a role. These mice contain the luciferase gene controlled by the promoter and enhancer of the human cytomegalovirus major immediate-early gene. This gene's induction is required for reactivation of the virus from the latent state. The transgenic mice were crossed with the hairless albino mouse strain HRS/J to facilitate imaging of deep tissues (Collaco and Geusz, 2003). Because the mice can be imaged repeatedly to follow ongoing or induced gene expression, fewer animals are needed than with other methods to monitor cellular responses, such as mRNA assays.
Multi-microelectrode recordings from neurons in culture
Many neurons can be recorded simultaneously through gold microelectrodes embedded in the bottom of culture dishes. This method is particularly well suited for the long-term recordings needed for measuring circadian rhythms in circadian clock neurons (Herzog et al, 1997 ; Nunemaker et al., 2001).
Hiler, D.J., Greenwald, M.L., and Geusz, M.E. (2006) Imaging gene expression in live transgenic mice after providing luciferin in drinking water. Photochemical and Photobiological Sciences. 5: 1082-5.
Collaco AM, Rahman S, Dougherty EJ, Williams BB, Geusz ME. (2005) Circadian regulation of a viral gene promoter in live transgenic mice expressing firefly luciferase. Molecular Imaging and Biology Oct 20:1-9
Geusz, M. E. (2001) Bioluminescence imaging of gene expression in living cells and tissues. In: Methods in Cellular Imaging. Basics of Fluorescence, Fluorophores, Microscopy and Detectors. A. Periasamy, (ed.) Oxford Univ. Press, New York, pp. 395-408.
Sigworth, L. A., Chandler, T. R., Liao, L., and Geusz, M. E. (2001). Luciferase imaging reveals distinct patterns of gene regulation in live brain slices. In: Bioluminescence & Chemiluminescence : Proceedings of the 11th International Symposium on Bioluminescence Chemiluminescence : Asilomar Conference Grounds, Pacific Grove, Monterey, California, USA : 6-10 September 2000. J.F. Case et al . (eds.) World Scientific, New Jersey pp. 185-188.
Nunemaker, C.S., DeFazio, R.A., Geusz, M.E., Herzog, E.D., Pitts, G.R., Moenter, S.M. (2001) Long-term recordings of networks of immortalized gonadotropin-releasing hormone neurons reveal spontaneous episodic patterns of action potential firing. J Neurophysiol 86: 86-93.
Geusz, M.E., Fletcher C., Block, G.D., Straume, M., Copeland, N.G., Jenkins, N.A., Kay, S.A., Day, R.N. (1997) Long term monitoring of circadian rhythms in c fos gene expression from suprachiasmatic nucleus cultures. Current Biology 7: 758-766.