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Biophysics
Cell Biology and Physiology
Molecular Biology
Biophotonics
Developmental Biology
Protein Biochemistry
Biomechanics
Imaging
Structure and Ultrastructure
     


Kao, Joseph P.Y., Ph.D.
Assistant Professor
Department of Physiology
E-mail: jkao@umaryland.edu


Our laboratory is developing photoprobe technology for applications in cell biology. Photoprobes comprise (1) fluorescent indicators that allow us to monitor intracellular processes in living cells and (2) photochemical probes that allow us to perturb or manipulate intracellular processes by flashing cells with pulses of light. Two current examples suffice to illustrate the general approach.

Because calcium ions are a ubiquitous second messenger in all cells, it is desirable to monitor the dynamics of Ca2+ regulation in living cells. One family of fluorescent indicators under development, dubbed "fura-X", is shown in the accompanying figure. The basic structure of fura-X is that of a fluorescent Ca2+ indicator. However, judicious choice of the functional group X can enhance uptake of the indicator by cells, improve retention of the indicator in the cytosol or, most importantly, allow the cell to target the indicator to specific subcellular locations such as the nucleus, the mitochondria, the cytosolic face of the plasma membrane, or the cytoskeleton. Such site-specific indicator targeting would yield unique information about subcellular Ca2+ dynamics that could not be obtained through any other technique.

Often it is inadequate merely to monitor a biological process. The ability to manipulate the process while it is being observed can be even more valuable because frequently, by observing the effect of such manipulations, casually related steps in the biological process can be elucidated. Deliberate perturbation of cells can be accomplished by using a class of photochemical probes called "caged" compounds. Caged compounds are photosensitive molecules in which the presence of a photolabile masking group abolishes the biological activity of an effector molecule. However, when the caged molecule is exposed to a flash of ultraviolet light, the masking or "caged" group can be photochemically removed to restore the bioactivity of the effector molecule. Using caged compounds allows bioactive substances to be photogenerated in situ rapidly. The light-induced reaction by which the intercellular messenger, carbon monoxide (CO), is photoreleased from our "caged CO" is shown in the accompanying scheme. To date, we can also use light flashes to release neurotransmitters (glutamate and GABA), modulate an intracellular Ca2+ pump, and raise intracellular free Ca2+ concentration.

Our ultimate aim is to devise a large array of techniques whereby both the observation and manipulation of cells can be accomplished through the use of light.

Rossi, F.M., M. Margulis, C-M., Tang, and J.P.Y. Kao. 1997. M-Nmoc-glutamate: A new caged glutamate with high chemical stability and low pre-photolysis activity. J. Biol. Chem. 272:32933-32939.

Moore, K.A., A.S. Cohen, R. Bangalore, J.P.Y. Kao, and D. Weinreich. 1998. Ca2+-induced Ca2+ relase mediates a slow post-spike hyperpolarization in rabbit vagal afferent neurones. J. Neurophysiol. 79:688-694.



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