lucifer-yellow and 6-carboxyfluorescein

Topics: Animals; Cells, Cultured; Dextrans; Endocytosis; Fluoresceins; Fluorescent Dyes; Histocytochemistry; Isoquinolines; Organelles; Pinocytosis; Receptors, Cell Surface

The outer hair cell motor protein, prestin, which resides exclusively in the cell's lateral membrane, underlies the mammal's exquisite sense of hearing. Here we show that photoexposure of the commonly used dyes Lucifer yellow, 6-carboxy-fluorescein, and 4-(2-[6-(dioctylamino)-2-naphthalenyl]ethenyl)-1-(3-sulfopropyl)-pyridinium (di-8-ANEPPS), that are in contact with the cell's lateral membrane can photo-inactivate the motor irreversibly, as evidenced by reduction in prestin's gating charge displacement or non-linear capacitance. Furthermore, utilizing restricted fiber optic illumination of the lateral membrane, we show that whole-cell, non-linear capacitance is depleted beyond that expected for an immobile population in the exposed area. These data indicate that lateral diffusion of prestin occurs within the cell's lateral plasma membrane.

Topics: Animals; Cell Membrane; Fluoresceins; Fluorescence Recovery After Photobleaching; Fluorescent Dyes; Guinea Pigs; Hair Cells, Auditory, Outer; Isoquinolines; Models, Chemical; Molecular Motor Proteins; Proteins; Pyridinium Compounds

To investigate gap-junctional intercellular communication (GJIC) in LNCaP and DU145 human prostate cancer cells.. Normal rat liver F344 (WB1) cells were used as positive controls. Functional GJIC was inspected using either the scrape-loading/dye transfer (SL/DT) method or fluorescence recovery after photobleaching (FRAP) analysis. In the former, GJIC activity was expressed as a measure of the extent of diffusion of Lucifer Yellow after cell monolayers were scraped using a surgical blade and exposed to dye for a few minutes at room temperature. In the latter, cells were incubated for 15 minutes at 37 degrees C with 5,6-carboxyfluorescein diacetate dye and the dye transfer visualized by photobleaching individual cells with a 488-nm laser and monitoring the recovery of fluorescence using a laser cytometer.. The preliminary results obtained indicate that neither LNCaP nor DU145 cells have functional GJIC, while, as expected, WB1 cells show unimpaired GJIC activity. Equivalent results were consistently obtained using either SL/DT or the FRAP approach. However, using FRAP analysis, DU145 cells only showed weak recovery of fluorescence after a total observation interval of 15 minutes.. The present data, though preliminary, suggest that disruption of GJIC may play a role in development of malignancy in the human prostate.

Topics: Animals; Cell Communication; Fluoresceins; Fluorescent Dyes; Gap Junctions; Humans; Isoquinolines; Male; Microscopy, Confocal; Microscopy, Fluorescence; Prostatic Neoplasms; Rats; Tumor Cells, Cultured

Scanning microphotolysis (Scamp), a recently developed photobleaching technique, was used to analyze the transport of two small organic anions and one inorganic cation through single pores formed in human erythrocyte membranes by the channel-forming toxin aerolysin secreted by Aeromonas species. The transport rate constants of erythrocyte ghosts carrying a single aerolysin pore were determined to be (1.83 +/- 0.43) x 10(-3) s-1 for Lucifer yellow, (0.33 +/- 0.10) x 10(-3) s-1 for carboxyfluorescein, and (8.20 +/- 2.30) x 10(-3) s-1 for Ca2+. The radius of the aerolysin pore was derived from the rate constants to be 19-23 A, taking steric hindrance and viscous drag into account. The size of the Ca2+ rate constant implies that at physiological extracellular Ca2+ concentrations (> 1 mM) the intracellular Ca2+ concentration would be elevated to the critical level of > 1 microM in much less than a second after formation of a single aerolysin pore in the plasma membrane. Thus changes in the levels of Ca2+ or other critical intracellular components may be more likely to cause cell death than osmotic imbalance.

Topics: Aeromonas; Bacterial Toxins; Biophysical Phenomena; Biophysics; Calcium; Cell Death; Cell Membrane Permeability; Erythrocyte Membrane; Fluoresceins; Fluorescent Dyes; Humans; In Vitro Techniques; Ion Channels; Ion Transport; Isoquinolines; Kinetics; Models, Biological; Molecular Structure; Photolysis; Pore Forming Cytotoxic Proteins

Term pregnant human myometrial cells in whole mounts were microinjected by pressure with the fluorescent probes Lucifer Yellow and carboxyfluorescein. Tissues obtained from acute and elective sections displayed weak dye-coupling when injected with Lucifer Yellow. Injection of carboxyfluorescein into cells from the elective sections resulted in a more extensive dye-coupling than that observed with Lucifer Yellow. These results indicate that term pregnant human myometrial cells are metabolically coupled before labor and carboxyfluorescein is superior to Lucifer Yellow in detecting the coupling.

Topics: Cell Communication; Female; Fluoresceins; Fluorescent Dyes; Gap Junctions; Humans; Isoquinolines; Labor, Obstetric; Membrane Potentials; Microinjections; Myometrium; Pregnancy

Gap junctional communication provides a mechanism for regulating multicellular activities by allowing the exchange of small diffusible molecules between neighboring cells. The diversity of gap junction proteins may exist to form channels that have different permeability properties. We report here that induction of terminal differentiation in mouse primary keratinocytes by calcium results in a specific switch in gap junction protein expression. Expression of alpha 1 (connexin 43) and beta 2 (connexin 26) gap junction proteins is down-modulated, whereas that of beta 3 (connexin 31) and beta 4 (connexin 31.1) proteins is induced. Although both proliferating and differentiating keratinocytes are electrically coupled, there are significant changes in the permeability properties of the junctions to small molecules. In parallel with the changes in gap junction protein expression during differentiation, the intercellular transfer of the small dyes neurobiotin, carboxyfluorescein, and Lucifer yellow is significantly reduced, whereas that of small metabolites, such as nucleotides and amino acids, proceeds unimpeded. Thus, a switch in gap junction protein expression in differentiating keratinocytes is accompanied by selective changes in junctional permeability that may play an important role in the coordinate control of the differentiation process.

Topics: Animals; Animals, Newborn; Biotin; Calcium; Cell Differentiation; Cells, Cultured; Connexins; Electric Conductivity; Electrophysiology; Fluoresceins; Fluorescent Dyes; Immunoblotting; Intercellular Junctions; Isoquinolines; Keratinocytes; Mice; Mice, Inbred Strains

This paper describes the early morphological and physiological development of pyramidal neurons in layer 5 of the rat visual cortex in relation to the targets chosen by their axons. Cells were prelabeled by retrograde transport from the superior colliculus or the contralateral visual cortex and intracellularly injected either in fixed slices or after recording in living slices. In the adult, corticotectal cells have thick apical dendrites with an extensive terminal arborization extending into layer 1, and fire characteristic bursts of action potentials when injected with a depolarizing current; interhemispheric cells have slender apical dendrites that terminate without a terminal tuft, usually in layer 2/3, and they display a more regular firing pattern (Kasper et al.: J Comp Neurol, this issue, 339:459-474). At embryonic day E18 (when axons of the two classes of cells are already taking different routes towards their targets) and E21, pyramidal-like cells throughout the cortical plate all have similar soma-dendritic morphology, with spindle-shaped cell bodies and few, short basal dendrites but apical dendrites that all end in distinct tufts in the marginal zone. At postnatal day P3, after the axons of both cell classes have reached their targets, all pyramidal neurons in layer 5 still have distinct terminal arborizations in layer 1, though they vary in complexity and extent. The somata are now more mature (round to ovoid in shape), and the basal dendritic tree has extended. As early as P5, all cells studied could be clearly classified as tufted or untufted (considerably earlier than previously reported; Koester and O'Leary: J Neurosci 12:1382, '92), and these features correlated precisely with the projection target, as in the adult. Measurement showed that although interhemispheric cells lose their terminal tufts, in general the trunks of their apical dendrites do not withdraw but continue to grow, at roughly the same rate as those of corticotectal cells. The two classes of layer 5 pyramidal neurons differentiate from each other in three distinct phases: pathway selection by axons precedes the loss of the apical tuft by interhemispheric cells, and these morphological characteristics are established 10 days before the onset of burst-firing in corticotectal cells. These three steps may be guided by different molecular signals.

Topics: Action Potentials; Animals; Axons; Dendrites; Electrophysiology; Female; Fluoresceins; Histocytochemistry; In Vitro Techniques; Isoquinolines; Lysine; Membrane Potentials; Pregnancy; Pyramidal Cells; Rats; Visual Cortex; Visual Pathways

We describe a new, simple and reliable technique to fill molluscan neurones from their cut axons with sufficient fluorescent dye for photoinactivation experiments. The fluorescent dye 5(6)-carboxyfluorescein (5-CF) travels quickly up the nerves of the gastropod mollusc, Lymnaea stagnalis into the buccal ganglia and fills the cell bodies in 1-3 h. 5-CF filled neurones can be located in the intact ganglia with low intensity blue light. Impalement shows that they are alive and show normal resting, action and synaptic potentials. Intense laser light (wavelength 442 nm, intensity 0.5 MW.m-2) kills all the 5-CF filled cells in less than 5 min in laboratory reared snails. Unstained neurones are not killed. 5-CF fills neurones quicker than Lucifer yellow (LY) when the dye is applied axonally. Neurones stained with Lucifer yellow do not contain sufficient dye to be killed with 5 min laser illumination, but this irradiation reduces the membrane resistance to less than 25%.

Topics: Animals; Axons; Brain Mapping; Central Nervous System; Esophagus; Evoked Potentials; Fluoresceins; Fluorescent Dyes; Ganglia; Isoquinolines; Lasers; Lymnaea; Neurons; Photic Stimulation

Dye-coupling in an in vitro preparation of the supporting cells of the guinea-pig organ of Corti was evaluated by use of the fluorescent dyes, Lucifer Yellow, fluorescein and 6 carboxyfluorescein. Despite the presence of good electrical coupling in Hensen cells (coupling ratios greater than 0.6) the spread of Lucifer yellow was inconsistent. Hensen cells are very susceptible to photoinactivation, i.e., cell injury upon illumination of intracellular dye; and this in conjunction with Lucifer Yellow's charge and K+-induced precipitability may account for its variability of spread. Fluorescein and 6 carboxyfluorescein, on the other hand, spread more readily and to a greater extent than Lucifer Yellow, often spreading to cell types other than those of Hensen. Dye spread is rapid, occurring within a few minutes. These results suggest that molecules of metabolic importance also may be shared by the supporting cells of the organ of Corti.

Topics: Animals; Cell Membrane; Fluorescein; Fluoresceins; Fluorescent Dyes; Guinea Pigs; Isoquinolines; Membrane Potentials; Organ of Corti