Picosecond rotation of small polar fluorophores in the cytosol of sea urchin eggs.

A new fluorescence method to measure viscosity in cell cytosol [Fushimi, K., & Verkman, A. S. (1991) J. Cell Biol. 112, 719-725] has been applied to determine fluid-phase viscosity in sea urchin eggs. Freshly harvested eggs from Lytechinus pictus were loaded with the dyes 2,7-bis(2-carboxyethyl)-5-(and-6-)carboxyfluorescein (BCECF), 6-carboxyfluorescein (6CF), fluorescein, or calcein. Fluorescence lifetimes and anisotropy decay were measured in single eggs by multiharmonic, frequency-domain microfluorometry using a 1-2-micron focused laser spot and 25x air objective. In calibration solutions consisting of glycerol in pH 8 buffered sea water, probe lifetime was single exponential and probe rotation was isotropic with a single correlation time which increased linearly with viscosity in the range 1-3.6 cP. In eggs at 22 degrees C, there were single lifetimes (in nanoseconds) of 3.6 (BCECF), 3.4 (6CF), 3.2 (fluorescein), and 3.3 (calcein). Probe rotation in eggs had two components, a fast component (in picoseconds, mean +/- SE, 10-18 eggs) of 568 +/- 39 (BCECF), 311 +/- 21 (6CF), 313 +/- 15 (fluorescein), and 516 +/- 44 (calcein) and a slow component of 10-40 ns. The fractional amplitude of the fast component, corresponding to unbound dye, was 0.72-0.81. Apparent viscosities of fluid-phase cytoplasm (centipoises) given by the four different probes were in good agreement: 2.3 +/- 0.2 (BCECF), 2.1 +/- 0.1 (6CF), 2.5 +/- 0.1 (fluorescein), and 2.3 +/- 0.2 (calcein). The viscosity in cytosol of sea urchin eggs (2.1-2.5 cP) is thus relatively low, yet significantly greater than that of water (1 cP) or cytosol in cultured fibroblasts (1.2-1.4 cP).(ABSTRACT TRUNCATED AT 250 WORDS)     

Dual probe fluorescence monitoring of intracellular free calcium during ischemia in mouse heart by using continuous compensation for pH dependence of the dissociation constant of Fura-2, and the interference of myoglobin

Mitochondrial damage is the main source of cellular injury upon ischemia-reperfusion, and calcium loading has been implicated in this phenomenon. The use of optical probes for calcium monitoring of the intact heart is hampered by internal filter effects of intracellular hemoproteins, endogenous fluorescence, and their sensitivity to pH. We describe here a method for measurement of intracellular free calcium in isolated myoglobin-deficient perfused mouse hearts under conditions of large intracellular pH fluctuations by simultaneous fluorescence monitoring of the calcium-probe Fura-2 and the pH probe BCECF through dual wavelength excitation of both probes. In myoglobin-containing mouse heart endogenous chromophores interfere with Fura-2 fluorometry. It is shown that a paradoxical decrease in Fura-2 fluorescence occurs during ischemia in isolated mouse hearts. Simultaneous recording of BCECF fluorescence (calibrated against pH measurement with phosphorus NMR) and data reduction based on continual recalculation of the apparent dissociation constant of the calcium-probe complex revealed that a marked increase in intracellular free calcium occurs, and that the Fura-2 fluorescence decrease was caused by an increase in dissociation constant due to intracellular acidification. Intracellular free calcium rose almost linearly during a 20-min period of ischemia and returned to basal values rapidly upon the commencement of perfusion.     

Dual probe fluorescence monitoring of intracellular free calcium during ischemia in mouse heart by using continuous compensation for pH dependence of the dissociation constant of Fura-2, and the interference of myoglobin

Mitochondrial damage is the main source of cellular injury upon ischemia–reperfusion, and calcium loading has been implicated in this phenomenon. The use of optical probes for calcium monitoring of the intact heart is hampered by internal filter effects of intracellular hemoproteins, endogenous fluorescence, and their sensitivity to pH.We describe here a method for measurement of intracellular free calcium in isolated myoglobin-deficient perfused mouse hearts under conditions of large intracellular pH fluctuations by simultaneous fluorescence monitoring of the calcium-probe Fura-2 and the pH probe BCECF through dual wavelength excitation of both probes. In myoglobin-containing mouse heart endogenous chromophores interfere with Fura-2 fluorometry.It is shown that a paradoxical decrease in Fura-2 fluorescence occurs during ischemia in isolated mouse hearts. Simultaneous recording of BCECF fluorescence (calibrated against pH measurement with phosphorus NMR) and data reduction based on continual recalculation of the apparent dissociation constant of the calcium-probe complex revealed that a marked increase in intracellular free calcium occurs, and that the Fura-2 fluorescence decrease was caused by an increase in dissociation constant due to intracellular acidification. Intracellular free calcium rose almost linearly during a 20-min period of ischemia and returned to basal values rapidly upon the commencement of perfusion.     

Estimation of matrix pH in isolated heart mitochondria using a fluorescent probe

Isolated heart mitochondria hydrolyze the acetoxymethyl esters of the Ca2+-sensitive fluorescent probe fura-2 and the pH-sensitive 2',7'-biscarboxyethyl-5(6)-carboxyfluorescein (BCECF). The resulting charged forms of the probes are retained in the mitochondrial matrix and appear well-suited for the estimation of pCa and pH in this compartment. The mitochondria esterase activity is stimulated by Ca2+, inhibited by butacaine and quinine, and shows an alkaline pH optimum. The esterase has a similar affinity for the two probes (Km about 1.5 microM) and a somewhat higher Vmax for BCECF. Intramitochondrial pH can be determined by recording the ratio of the fluorescence of matrix BCECF at its excitation maximum of 509 nm to that at 450 nm, an excitation wavelength that is unresponsive to pH. A calibration plot relating the fluorescence ratio to pH is constructed using detergent-lysed mitochondria and the excitation maximum of 500 nm for BCECF in aqueous solution. Estimates of matrix pH by BCECF fluorescence in its useful range (pH 6 to 8) agree well with values obtained using the distribution of 5,5-dimethyl-2,4-oxazolidenedione. In protocols in which the fluorescence with excitation at 450 nm does not vary, a direct recording of BCECF fluorescence with excitation at 509 nm can be used to follow the kinetics of matrix pH changes.     

A fluorescence dequenching method for monitoring exocytotic membrane fusion in fertilization of single sea urchin eggs

A method for monitoring exocytotic membrane fusion by using a fluorescent membrane probe is presented. The method is based on the relief from concentration-dependent self-quenching (dequenching) of fluorescence of 5-N-(octadecanoyl)aminofluorescein (AF18), an amphiphilic derivative of fluorescein. The validity and usefulness of this method were shown by the following results: 1) self-quenching of AF18 fluorescence occurred in the plasma membrane of unfertilized eggs of a sea urchin, Pseudocentrotus depressus, which were heavily stained with the fluorescent dye; 2) dequenching of AF18 fluorescence occurred upon fertilization in normal eggs but not in EGTA-injected eggs; 3) Ca²⁺ induced both AF18 fluorescence dequenching and cortical granule disappearance in the isolated sea urchin egg cortex; and 4) simultaneous measurements of the intracellular Ca²⁺ concentration ([Ca²⁺]_i) and dequenching of AF18 fluorescence by using a simple one-excitation and two-emission wavelength system.     

Changes in intracellular acidic compartments in sea urchin eggs after activation

Acridine orange (AO) was used as a vital probe for looking at acidic intracellular compartments in sea urchin eggs. This weak base is concentrated by acidic compartments, shifting its fluorescence from green to red due to the formation of dye aggregates. Fertilization or parthenogenetic activation with ionophore A23187 resulted in the appearance of orange fluorescent granules of sizes ranging from 1 to 2 microns at the cortical region of the egg. In one species of sea urchin (Lytechinus pictus), these granules migrate inward before cell division and associate with the forming mitotic apparatus. Treatments that discharge the transmembrane pH gradient (NH4Cl, nigericin, monensin, and acidic external pH) eliminate the orange fluorescence, indicating they are acidic compartments. Spectrofluorimetric measurements showed a decrease in monomer fluorescence accompanying egg activation which is reversible by similar treatments as seen with the fluorescence microscopic observations. Stratified eggs which were subsequently fertilized had acidic granules concentrated at the centripetal pole. This allowed the electron microscopic identification of the granules and showed they are present in the unfertilized egg, although not able to concentrate the AO. Activation of eggs in the absence of Na+ prevented the cytoplasmic alkalinization and also inhibited the appearance of acidic granules. The results indicate that the internal pH rises after egg activation triggers the acidification of these granules. Their possible functions, as in intracellular pH regulation, are discussed.     

Distribution of Membrane-Associated Calcium in Fertilized Sea Urchin Eggs during Mitosis*

The distribution of membrane-associated calcium in dividing sea urchin eggs was examined with chlortetracycline as a fluorescent chelate probe. The fluorescence of bound chlortetracycline in fertilized eggs was initially evenly distributed, but began to gather around the nucleus in prophase, and formed a dumb-bell shaped condensation enclosing the mitotic apparatus by metaphase. During anaphase and telophase, the fluorescence was observed in kinetochore-to-pole regions of the spindle, with little fluorescence in the interkinetochore region. The astral regions showed intense fluorescence. The distribution of the chlortetracycline-fluorescence coincided with that of ER-like membranes seen in electron micrographs. The distribution of the fluorescence was obscure and the birefringence of spindles disappeared on perfusion on perfusion of the cells in metaphase with 1 mM tetracaine, which is known to displace membrane-bound calcium. These results suggest that intracellular free calcium ions are sequestered in the membrane system associated with the mitotic apparatus during mitosis.     

Mapping of fluorescence anisotropy in living cells by ratio imaging. Application to cytoplasmic viscosity.

Steady-state and time-resolved fluorescence properties of probes incorporated into living cells give information about the microenvironment near the probe. We have extended studies of spatially averaged fluorescence anisotropy (r) by using an epifluorescence microscope, equipped with excitation and emission polarizers and an image analysis system, to map r of nonoriented fluorophores incorporated into cultured cells. With this imaging system, r for reflected light or glycogen scattering solutions was greater than 0.98. Measurement of r over the range 0.01-0.35 for fluorophores in bulk solution and in thin capillary tubes placed side-by-side gave values equivalent to r measured by cuvette fluorometry. Cytoplasmic viscosity (eta) in Madin-Darby canine kidney (MDCK) cells and Swiss 3T3 fibroblasts was examined from anisotropy images and time-resolved fluorescence decay of the cytoplasmic probes 2,7-bis-carboxyethyl-5 (and 6)-carboxy-fluorescein (BCECF) and indo-1. Nanosecond lifetimes and anisotropy decay were measured using a pulsed light source and gated detector interfaced to the epifluorescence microscope. Anisotropy images of BCECF in MDCK cells revealed two distinct regions of r: one from the cytoplasm (r = 0.144 +/- 0.008) and a second appearing at late times from the interstitial region (r = 0.08 +/- 0.03), representing BCECF trapped beneath the tight junctions. Anisotropy values, taken together with intracellular life-times and the calibration between r and eta/tau f for water/glycerol mixtures, gave eta values of 10-13 cP at 23 degrees C. These values assume little fluorophore binding to intracellular components and are therefore upper limits to cytoplasmic viscosity. These data establish a new methodology to map anisotropy in intact cells to examine the role of fluidity in cellular physiology.     

Continuous measurement of the cytoplasmic pH in Lactococcus lactis with a fluorescent pH indicator

The cytoplasmic pH of Lactococcus lactis was studied with the fluorescent pH indicator 2',7'-bis-(2-carboxyethyl)-5 (and-6)-carboxyfluorescein (BCECF). A novel method was applied for loading bacterial cells with BCECF, which consists of briefly treating a dense cell suspension with acid in the presence of the probe. This results in a pH gradient, which drives accumulation of the probe in the cytoplasm. After neutralization the probe was well retained in cells stored on ice. BCECF-loaded cells were metabolically active, and were able to generate a pH gradient upon energization. The probe leaks out slowly at elevated temperatures. Efflux is stimulated upon energization of the cells, and is most likely catalyzed by an active transport system. It is a first-order process, and the rate constant could be deduced from the decrease of the fluorescence signal in periods of constant intracellular pH. This allowed a correction of the fluorescence signal for efflux of the probe. After calibration the cytoplasmic pH could be calculated from efflux-corrected fluorescence traces.     

Correcting for artifacts in complex aqueous solutions when using the pH-sensitive dye 2',7'-bis-(2-carboxyethyl)- 5-(and -6)carboxyfluorescein

The pH-sensitive fluorescent indicator dye 2', 7'-bis-(2-carboxyethyl)-5-(and -6)carboxyfluorescein (BCECF) is routinely used to measure intracellular pH within cells. Surprisingly, no studies have been performed to see if various solution parameters modulate the fluorescence intensity of BCECF even though viscosity artifacts have been reported for particular Ca2+ selective dyes. In this report we demonstrate that even minor increases in the concentration of a number of different agents significantly decrease the excitation fluorescence intensity at two wavelengths routinely used to determine solution pH. Solution viscosity was varied using a number of different agents including glycerol, sucrose, polyethylene glycol, polyvinylpyrrolidone, and methylcellulose. In general, there was a detectable and significant decrease in the maximum fluorescence excitation ratio as the viscosity was increased, although the effect was more dramatic with Newtonian solutions than with non-Newtonian solutions. This same general effect was seen at pH 6.5, 7.0, and 7.3, a range of pH levels where BCECF is found to be particularly sensitive. To correct for these artifactually low values we used different combinations of excitation wavelengths to determine which could be used to accurately measure pH while minimizing the artifact. Choosing excitation wavelengths so that excitation ratios were collected at 470 and 435 nm allowed a significant signal to quantitatively measure pH while the artifact was nearly abolished.     

Different routes lead to apoptosis in unfertilized sea urchin eggs

Results obtained in various species, from mammals to invertebrates, show that arrest in the cell cycle of mature oocytes is due to a high ERK activity. Apoptosis is stimulated in these oocytes if fertilization does not occur. Our previous data suggest that apoptosis of unfertilized sea urchin eggs is the consequence of an aberrant short attempt of development that occurs if ERK is inactivated. They contradict those obtained in starfish, another echinoderm, where inactivation of ERK delays apoptosis of aging mature oocytes that are nevertheless arrested at G1 of the cell cycle as in the sea urchin. This suggests that the cell death pathway that can be activated in unfertilized eggs is not the same in sea urchin and in starfish. In the present study, we find that protein synthesis is necessary for the survival of unfertilized sea urchin eggs, contrary to starfish. We also compare the effects induced by Emetine, an inhibitor of protein synthesis, with those triggered by Staurosporine, a non specific inhibitor of protein kinase that is widely used to induce apoptosis in many types of cells. Our results indicate that the unfertilized sea urchin egg contain different mechanisms capable of leading to apoptosis and that rely or not on changes in ERK activity, acidity of intracellular organelles or intracellular Ca and pH. We discuss the validity of some methods to investigate cell death such as measurements of caspase activation with the fluorescent caspase indicator FITC-VAD-fmk or acidification of intracellular organelles, methods that may lead to erroneous conclusions at least in the sea urchin model.     

Alteration of lipid organization following fertilization of sea urchin eggs

The fluorescent probe merocyanine 540 was used to examine the organization of the lipids in the external leaflet of the plasma membrane after fertilization of sea urchin eggs. These lipids in unfertilized eggs are closely packed, as evidenced by their inability to bind the dye, whereas in fertilized eggs and cells of embryos up to at least the gastrula stage, the membrane becomes more loosely organized, and stains with bright ring fluorescence. Induction of late fertilization events with ammonia failed to induce this change in staining behavior. Sperm components are not required to induce this alteration since parthenogenetically activated eggs stained. However, treatment of eggs with procaine, which specifically inhibits the early event of cortical granule fusion, was effective in suppressing staining. These results indicate that cortical granule fusion after fertilization results in a change in the organization of the lipids of the plasma membrane of sea urchin eggs.     

Subcellular Localization of Catalase in Sea Urchin (Tetrapigus niger) Gametes: Implications for Peroxisome Biogenesis

Peroxisomes are essential subcellular organelles that appear to be derived from pre-existing organelles. To test the presence of peroxisomes in sea urchin (Tetrapigus niger) sperm and eggs, we performed biochemical and morphological experiments to evaluate the subcellular distribution of catalase as the typical peroxisomal marker. In sea urchin sperm, we found that catalase is localized in the cell cytosol. In contrast, sea urchin eggs contain sedimentable catalase, presumably contained in peroxisome-like structures detected by immunomicroscopy and by cytochemistry. Our results show, for the first time, evidence for the presence of peroxisome-like structures in sea urchin eggs and provide evidence for the peroxisome biogenesis hypothesis by division of pre-existing organelles.     

Fluorescence staining of living cells with fluorescamine

Summary The interaction of fluorescamine with living plant and animal cells was investigated to determine which subcellular structures and molecular species might react with the dye and to assess its effects on cell viability and function.Plasma and nuclear membranes ofXenopus erythrocytes, mitochondria of sea urchin sperm, growing apices of Timothy root hairs, and various organelles ofNitella andEuglena were labelled as judged by fluorescence microscopy. Cytoplasmic fluorescence was particulate inNitella and easily displaced by moderate centrifugal fields in sea urchin eggs. Chloroplasts and nuclei isolated from cells labelledin vivo exhibited fluorescamine dependent fluorescence.Reaction seemed to have little or no effect on cell viability (Euglena) photoautotrophic growth (Euglena), cell motility (sperm), fertilizability (sperm or egg), embryonic development (sea urchin), or cytoplasmic streaming (Nitella, Timothy).Quantitative fluorometric analysis of thein vivo reactants in sperm indicated a reaction preference for phospholipid over protein compared to control cells dissociated in SDS prior to labelling. The bulk of labelled lipid was phosphatidylethanolamine.These results suggest that fluorescamine is a true vital dye which can label the cell surface as well as penetrate deeply within cells to label a variety of organelles. The distribution of fluorescence and results of chemical analysis suggest thatin vivo the dye may preferentially react with membrane.     

Simultaneous measurement of water volume and pH in single cells using BCECF and fluorescence imaging microscopy

Regulation and maintenance of cell water volume and intracellular pH (pHi) are vital functions that are interdependent; cell volume regulation affects, and is in turn affected by, changes in pHi. Disruption of either function underlies various pathologies. To study the interaction and kinetics of these two mechanisms, we developed and validated a quantitative fluorescence imaging microscopy method to measure simultaneous changes in pHi and volume in single cells loaded with the fluorescent probe BCECF. CWV is measured at the excitation isosbestic wavelength, whereas pHi is determined ratiometrically. The method has a time resolution of <1 s and sensitivity to osmotic changes of approximately 1%. It can be applied in real time to virtually any cell type attached to a coverslip, independently of cellular shape and geometry. Calibration procedures and algorithms developed to transform fluorescence signals into changes in cell water volume (CWV) and examples of applications are presented.     

Evidence for probenecid-sensitive organic anion transporters on polarized thyroid cells in culture

Epithelial thyroid cells in primary cultures loaded with BCECF/AM rapidly released the impermeant fluorescent dye BCECF (bis(carboxyethyl)carboxyfluorescein) in the incubation medium. Cells organized into follicles rapidly cleared BCECF (80% within 10 min) whereas fluorescence microscopy did not show any fluorescence in the follicular cavity. Cells organized into monolayers on plastic exported BCECF into the medium (70% within 40 min) whereas fluorescence microscopy showed intense fluorescence under the domes. BCECF efflux was blocked by probenecid, one of the known inhibitors of organic anion transporters, with similar efficiency in both structures. Maximal and half-maximal effects were respectively observed for 5 mM and 0.4 mM probenecid. The polarity of BCECF efflux was studied by using monolayers on collagen-coated Nuclepore filters: 85% of BCECF released was found in the basal compartment and 15% in the apical compartment. These findings suggested that thyroid cells in culture expressed a transport mechanism for the anionic form of BCECF. Furthermore, the observed activation of the Na+/H+ exchanger by probenecid suggested that the presence of this blocker did not overcome problems arising in the use of BCECF as intracellular pH indicator for thyroid cells.     

Protein tyrosine kinase-dependent release of intracellular calcium in the sea urchin egg

The aminoguanide, methylglyoxal bis(guanylhydrazone) (MGBG), was shown to stimulate phosphorylation of RR-SRC, a synthetic protein tyrosine kinase (PTK) substrate, and different levels of tyrosyl phosphorylation of endogenous proteins in a sea urchin egg membrane-cortex preparation. Stimulating protein tyrosine kinase activity in the sea urchin egg stimulated intracellular Ca2+ release, because microinjection of 1-5 mM of MGBG into unfertilized eggs triggered a transient rise in intracellular Ca2+ activity ([Ca2+]i) after a brief latent period. Pretreating eggs with PTK-specific inhibitors, genistein or tyrphostin B42, significantly inhibited the MGBG-induced rise in [Ca2+]i. Methylglyoxal bis(guanylhydrazone) stimulation of PTK activities in the unfertilized sea urchin egg appeared to trigger Ca2+ release through phospholipase C (PLC)-dependent inositol 1,4,5-trisphosphate (InsP3) production. The MGBG-induced Ca2+ response could be suppressed in eggs preloaded with the InsP3 receptor antagonist, heparin, and was reduced in eggs pretreated with U73122, a PLC inhibitor. However, the response was unchanged in eggs treated with nicotinamide, an inhibitor of ADP-ribosyl cyclase, or nifedipine, an inhibitor of nicotinic acid adenine dinucleotide phosphate activity. These results suggest that MGBG may be useful as a chemical agonist of PTK in sea urchin eggs and allow direct testing of the PTK requirement for the transient rise in [Ca2+]i in sea urchin eggs during fertilization. Although genistein was observed to significantly delay the onset, the sperm-induced Ca2+ response in PTK inhibitor-loaded eggs otherwise appeared normal. Therefore, it was concluded that sea urchin eggs contain a PTK-dependent pathway that can mediate intracellular Ca2+ release, but PTK activity does not appear to be required for the fertilization response.     

Cross fertilization between sea urchin eggs and oyster spermatozoa

Interphylum crossing was examined between sea urchin eggs (Temnopleurus hardwicki) and oyster sperm (Crassostrea gigas). The eggs could receive the spermatozoa with or without cortical change. The fertilized eggs that elevated the fertilization envelope began their embryogenesis. Electron microscopy revealed that oyster spermatozoa underwent acrosome reaction on the sea urchin vitelline coat, and their acrosomal membrane fused with the egg plasma membrane after the appearance of an intricate membranous structure in the boundary between the acrosomal process and the egg cytoplasm. Oyster spermatozoa penetrated sometimes into sea urchin eggs without stimulating cortical granule discharge and consequently without fertilization envelope formation. The organelles derived from oyster spermatozoa seemed to be functionally inactive in the eggs whose cortex remained unchanged.     

Localization of tropomyosin in sea urchin eggs

Localization of tropomyosin in sea urchin eggs was investigated immunohistochemically. A rabbit antiserum against tropomyosin prepared from lantern muscle of the sea urchin was used for the indirect immunofluorescence staining of unfertilized and fertilized eggs. The tropomyosin-specific fluorescence was observed at the peripheral region beneath the plasma membrane, mitotic apparatus and contractile ring. The mitotic apparatus isolated from sea urchin eggs was also stained with the anti-tropomyosin serum.     

Flow cytometric measurement of cytoplasmic pH: a critical evaluation of available fluorochromes

Three pH-sensitive fluorochromes-4-methyl-umbelliferone(4MU),2, 3-dicyano-hydroquinone (DCH), and 2',7'-bis(carboxyethyl)-5,6-carboxy fluorescein (BCECF)--were evaluated for their resolution, range, and stability of cellular fluorescence. Flow cytometric techniques for determining cytoplasmic pH (pHi) have been fully described for 4MU and DCH; BCECF has previously been used for fluorimetric estimation of pHi, and was adapted to flow cytometry. For each fluorochrome, the ratio of fluorescence intensity at two wavelengths gives a measure of pHi, which may be calibrated by obtaining the fluorescence ratios for cells suspended in buffers of varying pH in the presence of a proton ionophore. Reliable calibration proved difficult using 4MU, partly because of poor retention within cells. Both DCH and BCECF could be calibrated using a fluorescence ratio and had resolutions of 0.2 and 0.4 pH units, respectively. The fluorescence of DCH is so strongly pH dependent that there were practical difficulties in its use over a wide pH range; however, pHi measurements are possible between pH 6.0 and pH 7.5 using either DCH or BCECF. Substantial dye leakage was found for 4MU and, to a lesser extent, DCH, while BCECF was retained by cells for up to 2 hours. Despite its lower resolution BCECF had a usable range of more than 1.5 pH units and this coupled with its stable fluorescence and excitation at 488 nm rather than UV suggests a wide application.