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.

Real-time monitoring of in vitro transcriptional RNA synthesis using fluorescence resonance energy transfer.

We have developed a novel method for real-time monitoring of RNA synthesis in in vitro transcription reactions using fluorescence resonance energy transfer (FRET). Two 15mer DNAs, either of which was labeled with Bodipy493/503 as a donor or Cy5 as an acceptor, were prepared. When the two fluorescent DNAs hybridized to adjacent locations on Xenopus: elongation factor 1-alpha (xelf1-alpha) RNA, the distance between the two fluorophores became very close, causing FRET to occur and resulting in changes in fluorescence spectra. A high accessibility 30mer site of xelf1-alpha RNA was found and excess amounts of a pair of donor and acceptor DNA probes that were complementary to the site were added to the in vitro transcription reaction solution. Changes in fluorescence spectra were observed in response to progression of xelf1-alpha RNA synthesis that showed that the fluorescent probes hybridized to the synthesized RNA. Furthermore, when probes hybridizing to the synthesized xelf1-alpha RNA with less efficiency were used to monitor the reaction, spectral changes in response to RNA synthesis were also observed. This result suggests that the probes hybridized to synthesizing RNA molecules before they folded to form secondary structure and that there is no need to select sites on the RNA for the probes, which is required for probes hybridizing to folded RNA molecules.

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.

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.

Restricted photorelease of biologically active molecules near the plasma membrane.

An evanescent wave of ultraviolet light was successfully used to release biologically active molecules from caged compounds in living cells. The evanescent wave was generated by the total internal reflection in a limited region near the plasma membrane attached to the illuminated interface. At first, the photolysis efficiency of the evanescent wave of ultraviolet laser light was studied using caged glutamic acid in vitro. Then, caged Ca2+ introduced in the living cultured cell was similarly photolyzed by the evanescent wave and the resulting elevations of the concentration of intracellular Ca2+ in the proximity of the plasma membrane and in the cytosol were observed with a simultaneously introduced fluorescent calcium indicator. Inositol trisphosphate can also be photoreleased near the plasma membrane, which enables study of the temporal and spatial pathways of signal transduction. The method developed here provides a useful tool for studying signal transduction near the plasma membrane in a living cell.

Immunohistochemical demonstration of embryonic expression of an odor receptor protein and its zonal distribution in the rat olfactory epithelium.

Using an antibody raised against an odor receptor protein, we investigated immunohistochemically the spatial distribution in the embryonic and adult rat olfactory epithelium of the olfactory receptor neurons that express the odor receptor protein. In adults, the immunoreactive olfactory receptor neurons were intermingled with immuno-negative receptor neurons, but were mostly restricted within a circumferential zone located in the lateral part of the epithelium. The immunoreactive olfactory receptor neurons were observed as early as embryonic day 14, with a strong tendency to localize in the lateral part of the epithelium. These results indicate that both selection of the odor receptor protein by individual olfactory receptor neurons and zonal segregation of the odor receptor protein expression occur early in embryonic development of the olfactory system.

R2D5 antigen: a calcium-binding phosphoprotein predominantly expressed in olfactory receptor neurons.

R2D5 is a mouse monoclonal antibody that labels rabbit olfactory receptor neurons. Immunoblot analysis showed that mAb R2D5 recognizes a 22-kD protein with apparent pI of 4.8, which is abundantly contained in the olfactory epithelium and the olfactory bulb. We isolated cDNA for R2D5 antigen and confirmed by Northern analysis and neuronal depletion technique that R2D5 antigen is expressed predominantly, but not exclusively, in olfactory receptor neurons. Analysis of the deduced primary structure revealed that R2D5 antigen consists of 189 amino acids with calculated M(r) of 20,864 and pI of 4.74, has three calcium-binding EF hands, and has possible phosphorylation sites for Ca2+/calmodulin-dependent protein kinase II (CaM kinase II) and cAMP-dependent protein kinase (A kinase). Using the bacterially expressed protein, we directly examined the biochemical properties of R2D5 antigen. R2D5 antigen binds Ca2+ and undergoes a conformational change in a manner similar to calmodulin. R2D5 antigen is phosphorylated in vitro by CaM kinase II and A kinase at different sites, and 1.81 and 0.80 mol of Pi were maximally incorporated per mol of R2D5 antigen by CaM kinase II and A kinase, respectively. Detailed immunohistochemical study showed that R2D5 antigen is also expressed in a variety of ependymal cells in the rabbit central nervous system. Aside from ubiquitous calmodulin, R2D5 antigen is the first identified calcium-binding protein in olfactory receptor neurons that may modulate olfactory signal transduction. Furthermore our results indicate that olfactory receptor neurons and ependymal cells have certain signal transduction components in common, suggesting a novel physiological process in ependymal cells.

Coding of odor molecules by mitral/tufted cells in rabbit olfactory bulb. II. Aromatic compounds.

1. Recordings of extracellular spike responses were made from single mitral/tufted cells in the ventromedial region of the main olfactory bulb of urethan-chloralose-anesthetized rabbits. Using periodic artificial inhalations, the olfactory epithelium was stimulated with series of aromatic and aliphatic compounds systematically varying in molecular conformation. 2. Analysis of response specificity of single mitral/tufted cells for alkylbenzenes indicated that the length of the hydrocarbon side chain attached to the benzene ring plays a role in determining the specificity of excitatory spike responses. 3. For a panel of isomeric (ortho-, meta-, and para-positions) disubstituted benzenes, single mitral/tufted cells tended to be activated selectively by one or two specific structural isomer(s). For a panel that contained both alkylbenzenes and disubstituted benzenes, single mitral/tufted cells were activated by subsets of odor molecules having similar conformations. These observations suggest that the overall conformation of the aromatic compounds plays an important role in determining tuning specificity of individual mitral/tufted cells. 4. For a panel of monosubstituted benzenes with various functional groups, single mitral/tufted cells in the ventromedial region tended to be activated not only by molecules having a hydrocarbon side chain (alkylbenzenes), but also by those having a methoxy group (--O--CH3), a bromine (--Br), or a chlorine (--Cl). However, most of the neurons were not activated by those having an amino group (--NH2), a hydroxy group (--OH), nor a carboxyl group (--COOH). 5. Examination with an expanded panel of stimulus odor molecules that included both aromatic and aliphatic compounds indicated that single mitral/tufted cells show excitatory spike responses to a range of odor molecules (molecular receptive range) having similar conformations. Different mitral/tufted cells in the ventromedial region typically showed different molecular receptive ranges. 6. In mitral/tufted cells with relatively high spontaneous discharges, single neurons in the ventromedial region showed inhibitory responses to subsets of odor molecules in addition to the excitatory response to other subsets of odor molecules. The odor molecules that caused inhibitory responses in single mitral/tufted cells showed molecular conformations resembling each other. 7. The present results together with previous studies indicate that determination of the molecular receptive range properties (both excitatory extent and inhibitory extent) of single mitral/tufted cells is a useful method for characterizing individual bulbar neurons. These results further support the hypothesis that conformational parameters of ligand odor molecules play a key role in sensory processing in the main olfactory bulb.

Distribution of putative odour receptor proteins in olfactory epithelium.

To investigate immunohistochemically the spatial localization of putative odour receptor proteins, we synthesized a polypeptide which corresponds to a region of a putative odour receptor protein, I3, and raised an antibody to the peptide. In rat olfactory epithelium, the antibody specifically recognized cilia of a small subset of olfactory receptor neurons, suggesting that the odour receptor protein is localized selectively in the cilia. Olfactory receptor neurons having immunoreactive cilia were distributed sparsely throughout the epithelium. This suggests that receptor neurons expressing a similar odour receptor protein are probably distributed similarly in the epithelium.

Postnatal development of cholecystokinin-like immunoreactivity and its mRNA level in rat brain regions.

Developmental changes of preprocholecystokinin mRNA (CCK mRNA) and cholecystokinin-like immunoreactivity (CCK-LI) were examined in rat brain regions (frontal cortex, colliculi, hippocampus, striatum, and cerebellum) using RNA dot blot assays with cholecystokinin (CCK) cDNA and radioimmunoassay, respectively. The CCK-LI levels in all regions examined were very low at birth. Excluding the cerebellum, the levels in these regions increased postnatally and reached adult values at 28 days of age. In contrast to CCK-LI, CCK mRNA levels changed dramatically during development. A considerable amount of CCK mRNA was detected in the frontal cortex and hippocampus at birth. The changes in the level of CCK mRNA in the frontal cortex and colliculi paralleled those of CCK-LI, including a rapid increase from 7 to 14 days of age. The synthesis of CCK mRNA preceded the appearance of CCK-LI. CCK mRNA levels in the hippocampus and striatum exhibited a transient increase, with a peak at 14 days of age. In the adult brain, the CCK mRNA levels were high in the frontal cortex, moderate in the hippocampus and colliculi, and low in the striatum. The cerebellum contained only a negligible amount of CCK mRNA during development. The relatively high level of CCK-LI compared with the low level of CCK mRNA in the striatum supports the idea that most of the striatal CCK-LI is supplied from extrastriatal regions.(ABSTRACT TRUNCATED AT 250 WORDS)