Kyle R. Gee

Modulation of mitochondrial function by endogenous Zn2+ pools

Recent evidence suggests that intracellular Zn2+ accumulation contributes to the neuronal injury that occurs in epilepsy or ischemia in certain brain regions, including hippocampus, amygdala, and cortex. Although most attention has been given to the vesicular Zn2+ that is released into the synaptic space and may gain entry to postsynaptic neurons, recent studies have highlighted pools of intracellular Zn2+ that are mobilized in response to stimulation. One such Zn2+ pool is likely bound to cytosolic proteins, like metallothioneins. Applying imaging techniques to cultured cortical neurons, this study provides novel evidence for the presence of a mitochondrial pool distinct from the cytosolic protein or ligand-bound pool. These pools can be pharmacologically mobilized largely independently of each other, with Zn2+ release from one resulting in apparent net Zn2+ transfer to the other. Further studies found evidence for complex and potent effects of Zn2+ on isolated brain mitochondria. Submicromolar levels, comparable to those that might occur on strong mobilization of intracellular compartments, induced membrane depolarization (loss of Δψm), increases in currents across the mitochondrial inner membrane as detected by direct patch clamp recording of mitoplasts, increased O2 consumption and decreased reactive oxygen species (ROS) generation, whereas higher levels decreased O2 consumption and increased ROS generation. Finally, strong mobilization of protein-bound Zn2+ appeared to induce partial loss of Δψm, suggesting that movement of Zn2+ between cytosolic and mitochondrial pools might be of functional significance in intact neurons.

Measuring zinc in living cells. A new generation of sensitive and selective fluorescent probes.

New fluorescent indicators with nanomolar to micromolar affinities for Zn(2+) have been synthesized in wavelengths from UV to the far red. The UV light-excited indicators are ratiometric. The visible wavelength indicators are non-ratiometric and exhibit large and pH-independent fluorescence increases with increasing zinc concentrations, with little to no sensitivity to physiologically relevant Ca(2+) concentrations. Experiments in neuronal and non-neuronal cell cultures show the new indicators to retain their sensitivity to and selectivity for zinc after conversion to cell-permeable forms.

Activation and inactivation of Ca2+ release by NAADP+

Nicotinic acid adenine dinucleotide phosphate (NAADP) is a recently identified metabolite of NADP that is as potent as inositol trisphosphate (IP) and cyclic ADP-ribose (cADPR) in mobilizing intracellular Ca2 in sea urchin eggs and microsomes (Clapper, D. L., Walseth, T. F., Dargie, P. J., and Lee, H. C.(1987) J. Biol. Chem. 262, 9561-9568; Lee, H. C., and Aarhus, R.(1995) J. Biol. Chem. 270, 2152-2157). The mechanism of Ca release activated by NAADP and the Ca stores it acts on are different from those of IP and cADPR. In this study we show that photolyzing caged NAADP in intact sea urchin eggs elicits long term Ca oscillations. On the other hand, uncaging threshold amounts of NAADP produces desensitization. In microsomes, this self-inactivation mechanism exhibits concentration and time dependence. Binding studies show that the NAADP receptor is distinct from that of cADPR, and at subthreshold concentrations, NAADP can fully inactivate subsequent binding to the receptor in a time-dependent manner. Thus, the NAADP-sensitive Ca release process has novel regulatory characteristics, which are distinguishable from Ca release mediated by either IP or cADPR. This battery of release mechanisms may provide the necessary versatility for cells to respond to diverse signals that lead to Ca mobilization.

Synthesis of novel fluorinated coumarins: Excellent UV-light excitable fluorescent dyes

Two new fluorinated fluorescent dyes, 6,8-difluoro-7-hydroxy-4-methylcoumarin (Marina Blue) and 3-carboxy-6,8-difluoro-7-hydroxycoumarin (Pacific Blue), exhibit excellent photophysical properties among a series of novel fluorinated 7-hydroxycoumarins. Most of these fluorinated coumarins have quantum yields (0.63 to 0.89) equal to or higher than that of the parent compound (0.63), which, in combination with their lower pKaS and higher photostability, make them superior fluorescent dyes for use as reporter molecules in biological systems.

A new mitochondrial fluorescent zinc sensor

A novel cationic fluorescent zinc (Zn2+) indicator (RhodZin-3) with nanomolar affinity for Zn2+ has been synthesized. RhodZin-3 exhibits large pH-independent fluorescence increases in the orange region of the visible wavelength spectrum with increasing zinc concentrations, and no sensitivity to physiologically relevant Ca2+ concentrations. Experiments in neuronal cell cultures show that RhodZin-3 effectively localizes into mitochondria and detects changes of intramitochondrial free Zn2+ ([Zn2+]m).

Flow tagging velocimetry in incompressible flow using photo-activated nonintrusive tracking of molecular motion (PHANTOMM)

We report the development of a new optical flow tagging velocimetry technique for hydrodynamic flows. The method utilizes highly water-soluble caged dye Photo-Activated Fluorophores (PAFs) which serve as fluorescent tracers, with essentially indefinite lifetime. Demonstration experiments are presented in a bench-top poiseulle flow and a 5,000 gallon water channel facility. Results of experiments designed to quantify critical optical characteristics of the caged dye PAFs are also presented, as is a comparison with other, similar, optical velocimetry approaches.

Monitoring presynaptic calcium dynamics in projection fibers by in vivo loading of a novel calcium indicator.

Fluorometric calcium measurements have revealed presynaptic residual calcium (Ca(res)) to be an important regulator of synaptic strength. However, in the mammalian brain, it has not been possible to monitor Ca(res) in fibers that project from one brain region to another. Here, we label neuronal projections by injecting dextran-conjugated calcium indicators into brain nuclei in vivo. Currently available dextran conjugates distort Ca(res) due to their high affinity for calcium. Therefore, we synthesized a low-affinity indicator, fluo-4 dextran, that can more accurately measure the amplitude and time course of Ca(res). We then demonstrate the utility of fluo-4 dextran by measuring Ca(res) at climbing fiber presynaptic terminals. This method promises to facilitate the study of many synapses in the mammalian CNS, both in brain slices and in vivo.

Caged Nicotinic Acid Adenine Dinucleotide Phosphate SYNTHESIS AND USE

Nicotinic acid adenine dinucleotide phosphate (NAADP) is a metabolite of NADP with Ca2+ mobilizing activity. The Ca2+ release mechanism activated by NAADP as well as the Ca2+ stores that it acts on are different from those activated by either cyclic ADP-ribose or inositol 1,4,5-trisphosphate (IP3) (Lee, H. C., and Aarhus, R. (1995) J. Biol. Chem. 270, 2152-2157). In order to demonstrate unambiguously that NAADP can mobilize Ca2+ stores in live cells, a caged analog was synthesized by reacting NAADP with 1-(2-nitrophenyl)diazoethane. Anion exchange high pressure liquid chromatography (HPLC) was used to purify one particular caged form from the mixture of products. Phosphate analyses following specific enzymatic cleavage indicate that the caging group is on the 2′-phosphate. This is confirmed by 31P NMR spectroscopy, showing that the 2′-phosphate of the caged compound exhibits an altered chemical shift of −2.6 ppm as compared with 2.3 ppm determined for the 2′-phosphate of NAADP. Caged NAADP had no Ca2+ releasing activity at a concentration as high as 1 μM when tested on sea urchin egg microsomes. After photolysis, it released Ca2+, was effective in nanomolar range, and was indistinguishable from authentic NAADP. The regeneration of NAADP after photolysis was also confirmed by HPLC analyses. The analog is particularly susceptible to UV and can be efficiently photolyzed using a spectrofluorimeter. To demonstrate its utility in live cells, caged NAADP was microinjected into sea urchin eggs. Photolysis effectively regenerated NAADP and activated Ca2+ oscillations in the eggs. Removal of external Ca2+ did not prevent the Ca2+ oscillations but only delayed the second Ca2+ peak by about 45 s, indicating that the oscillations are due to release from internal stores and not caused by Ca2+ influx. A mechanism based on sensitization of the Ca2+ release by Ca2+ loading is proposed to account for the Ca2+ oscillation observed.

Caged Q-rhodamine dextran: a new photoactivated fluorescent tracer.

An amine-reactive caged rhodamine was synthesized and conjugated to aminodextran. The resulting tracer was injected into a single cell zebrafish embryo, and a portion of the tracer was photolyzed in a single cell after development. The resulting fluorescent cell was imaged by fluorescence microscopy through a single round of cell division.

Site-specific protein labeling using PRIME and chelation-assisted click chemistry

This protocol describes an efficient method to site-specifically label cell-surface or purified proteins with chemical probes in two steps: probe incorporation mediated by enzymes (PRIME) followed by chelation-assisted copper-catalyzed azide-alkyne cycloaddition (CuAAC). In the PRIME step, Escherichia coli lipoic acid ligase (LplA) site-specifically attaches a picolyl azide (pAz) derivative to a 13-aa recognition sequence that has been genetically fused onto the protein of interest. Proteins bearing pAz are chemoselectively derivatized with an alkyne-probe conjugate by chelation-assisted CuAAC in the second step. We describe herein the optimized protocols to synthesize pAz to perform PRIME labeling and to achieve CuAAC derivatization of pAz on live cells, fixed cells and purified proteins. Reagent preparations, including synthesis of pAz probes and expression of LplA, take 12 d, whereas the procedure for performing site-specific pAz ligation and CuAAC on cells or on purified proteins takes 40 min–3 h.