Masahiko Hirano

Direct observation of specific messenger RNA in a single living cell under a fluorescence microscope.

We observed the expression of human c-fos mRNA in a living transfected Cos7 cell under a fluorescence microscope by detecting hybrid formed with two fluorescently labeled oligodeoxynucleotides (oligoDNAs) and c-fos mRNA in the cytoplasm. Two fluorescent oligoDNAs were prepared, each labeled with a fluorescence molecule different from the other. When two oligoDNAs hybridized to an adjacent sequence on the target mRNA, the distance between the two fluorophores became very close and fluorescence resonance energy transfer (FRET) occurred, resulting in changes in fluorescence spectra. To find sequences of high accessibility of c-fos RNA to oligoDNAs, several sites that included loop structures on the simulated secondary structure were selected. Each site was divided into two halves, and the pair of fluorescent oligoDNAs complementary to the sequence was synthesized. Each site was examined for the efficiency of hybridization to c-fos RNA by measuring changes in fluorescence spectra when c-fos RNA was added to the pair of oligoDNAs in solution. A 40 mer specific site was found, and the pair of oligoDNAs for the site were microinjected into Cos7 cells that expressed c-fos mRNA. To block oligoDNAs from accumulating in the nucleus, oligoDNA was bound to a macromolecule (streptavidin) to prevent passage of nuclear pores. Hybridization of the pair of oligoDNAs to c-fos mRNA in the cytoplasm was detected in fluorescence images indicating FRET.

Development of a Time-Resolved Fluorometric Method for Observing Hybridization in Living Cells Using Fluorescence Resonance Energy Transfer

We previously showed that a specific kind of mRNA (c-fos) was detected in a living cell under a microscope by introducing two fluorescently labeled oligodeoxynucleotides, each labeled with donor or acceptor, into the cytoplasm, making them hybridize to adjacent locations on c-fos mRNA, and taking images of fluorescence resonance energy transfer (FRET) (A. Tsuji, H. Koshimoto, Y. Sato, M. Hirano. Y. Sei-Iida, S. Kondo, and K. Ishibashi, 2000, Biophys. J. 78:3260-3274). On the formed hybrid, the distance between donor and acceptor becomes close and FRET occurs. To observe small numbers of mRNA in living cells using this method, it is required that FRET fluorescence of hybrid must be distinguished from fluorescence of excess amounts of non-hybridizing probes and from cell autofluorescence. To meet these requirements, we developed a time-resolved method using acceptor fluorescence decays. When a combination of a donor having longer fluorescence lifetime and an acceptor having shorter lifetime is used, the measured fluorescence decays of acceptors under FRET becomes slower than the acceptor fluorescence decay with direct excitation. A combination of Bodipy493/503 and Cy5 was selected as donor and acceptor. When the formed hybrid had a configuration where the target RNA has no single-strand part between the two fluorophores, the acceptor fluorescence of hybrid had a sufficiently longer delay to detect fluorescence of hybrid in the presence of excess amounts of non-hybridizing probes. Spatial separation of 10-12 bases between two fluorophores on the hybrid is also required. The decay is also much slower than cell autofluorescence, and smaller numbers of hybrid were detected with less interference of cell autofluorescence in the cytoplasm of living cells under a time-resolved fluorescence microscope with a time-gated function equipped camera. The present method will be useful when observing induced expressions of mRNA in living cells.

Dual loading of the fluorescent indicator fura-2 and 2,7-biscarboxyethyl-5(6)-carboxyfluorescein (BCECF) in isolated myocytes

Isolated rat heart myocytes were loaded with both the Ca2+ sensitive fluorescent probe fura-2/AM and the fluorescent pH indicator 2,7-biscarboxyethyl-5(6)-carboxyfluorescein (BCECF/AM). Changes in [Ca2+]i and pHi were measured simultaneously using digitized video fluorescence microscopy. In measurement of [Ca2+]i and pHi, the ratios of dual-loaded cells were not different from single-loaded cells. Using this method, [Ca2+]i and pHi in myocytes were 48 +/- 7 nM and 7.17 +/- 0.05. It is concluded that [Ca2+]i and pHi could be measured simultaneously in isolated myocyte using dual-loading of fura-2 and BCECF.

QUANTIFICATION OF INTRACELLULAR FREE SODIUM IONS BY USING A NEW FLUORESCENT INDICATOR, SODIUM-BINDING BENZOFURAN ISOPHTHALATE IN GUINEA PIG MYOCYTES

Isolated guinea pig myocytes were loaded with the Na(+)-sensitive fluorescent probe, sodium-binding benzofuran isophthalate (SBFI). The 340/380 nm fluorescence ratios were measured with fluorescence microscopy. The distribution of intracellular Na+ concentration ([Na+]i) was homogenous, and the mean resting [Na+]i was 8.4 +/- 0.5 mM. There was a significant relationship (r = 0.66, p less than 0.001) between elevation of [Na+]i and shortening of longitudinal length of the cells, during the perfusion of 100 microM strophanthidin. It is concluded that this method is suitable for measuring [Na+]i in isolated myocytes.

Distribution of functional glutamate receptors in cultured embryonic Drosophila myotubes revealed using focal release of L-glutamate from caged compound by laser.

During the formation of neuromuscular junctions in Drosophila embryos, glutamate receptors undergo a drastic change in distribution. To study the underlying mechanism of this developmental process, it is desirable to map the distribution of functional receptors with accurate spatial resolution. Since glutamate receptors desensitize within several milliseconds, the agonist must be applied rapidly. To fulfil these requirements we used laser stimulation of a caged compound to release L-glutamate at a focal spot. Since the glutamate receptor channel is permeable to Ca2+, we assayed the change in internal Ca2+ concentration using a Ca2+ indicator, fluo-3. Using this approach, we mapped the distribution of functional glutamate receptors in cultured embryonic Drosophila myotubes and myoblasts. Consistent with previous immunofluorescence studies using an antibody against a glutamate receptor subunit, a large increase of internal Ca2+ concentration was observed when laser stimulation was located close to some nuclei in the myotube. No change was detected when the laser stimulus was applied over any regions of the myoblasts. No increase of the internal Ca2+ concentration in myotubes was observed when the external solution contained either glutamate at a desensitizing concentration (1 mM) or a glutamate receptor channel blocker, argiotoxin (1 microg/ml). These results indicate that a rise in intracellular Ca2+ concentration can be used to show the distribution of the functional receptor on the muscle surface membrane.

Changes in the intracellular concentration of Ca2+ in NG108-15 cells induced by the photorelease of second messengers near the plasma membrane

The nemoprotective effects of hypothermia have been well recognized, and hypothermia has recently become a useful1 therapy for prevention of brain ischemia or injury. But the mechanism of neuroprotection by hypothermia has not been well clarified. We investigated the effects of temperature on the electrical activity by intracellular recording of pyramidal neuron (CA3), using guinea pig hippocampal slice. Hypothermia reduced EPSP slope in a temperature dependent manner, but did not influence on EPSP amplitude down to 20 “C With regard to spikes generated by presynaptic stimulation or direct current injection, hypothermia did not change the level of critical threshold for spike generation and spike amplitude regardless of progressive prolongation of spike duration and 1,+..,,... . . ..+r. ,.,.,1:..* l-l.,,, “,.*:..:~:_^^_ ..^_^ A r..ll.. . ..l.-.. AL.. ._^_^ & ..__ . ..^^ __:___I A_ -<or _~~_~ c-.rv. ,arE;,,tiy WILlI W”l,ll& 111~i~si Q~LIVIII~ L~UYC;IC;U luuy w11e1r rue rnupcra~ure was rarseu 10 3)~ L even rrom 13 L, These results indicate that synaptic function of neurons is reversibly attenuated with hypothermia , but the properties of excitability and membrane potential of neurons are well preserved under deep hypothermia.

Changes in Cell Morphology, [Ca2+]i and pHi During Metabolic Inhibition in Isolated Myocytes of Diabetic Rats Using Dual-Loading of Fura-2 and BCECF

Diabetes Mellitus (DM) has been shown to be associated with heart failure in the absence of atherosclerosis (1,2), suggesting a diabetic cardiomyopathy (3,4). The mechanism of heart failure due to the diabetic cardiomyopathy remains to be elucidated. The small vessel disease (5) and the abnormalities of subcellular mechanisms such as myosin ATPase (6) and myosin isoenzymes (7), have been reported in DM myocardium. Recently, abnormalities of Ca2+ metabolism have been reported in DM myocardium, which showed decreased Ca2+-ATPase of sarcoplasmic reticulum (SR) (8,9) and sarcolemma (10). It has also been reported that the activity of Na+/Ca2+-exchange was lower in DM myocardium (11). Previous reports have suggested the possibility of the Ca2+ overload in diabetic cardiomyopathy (11).