用荧光漂白恢复技术研究竹红菌乙素在小鼠肝癌细胞内的扩散过程

利用竹红菌乙素自身的荧光特征,在ＦＰＲ装置上直接测定了乙素在ＡＨ细胞内的侧向扩散系数和荧光漂白的恢复率。实验结果表明乙素在ＡＨ细胞内的扩散系数Ｄ＝３．２×１０＾－９ｃｍ＾２／ｓ＾－１荧光漂白恢复比率Ｒ＝９７．８％,上述实验说明乙素在细胞膜内与生物大分子之间没有形成共价键形式的结合状态。     

双歧杆菌的完整肽聚糖对小鼠腹腔巨噬细胞缝隙链接的影响

目的探讨双歧杆菌的完整肽聚糖（WPG）对巨噬细胞缝隙连接介导的细胞间通讯（GJIC）的影响。方法首先分离培养昆明小鼠腹腔巨噬细胞,然后以WPG刺激巨噬细胞,再用脂荧光探针标记细胞,最后采用激光共聚焦显微镜结合激光漂白后荧光恢复技术检测反映GJIC变化的荧光恢复程度。结果和对照组相比,以WPG刺激巨噬细胞后,其GJIC的平均荧光恢复率明显增加（P〈0.01）。结论双歧杆菌的WPG可提高巨噬细胞的缝隙连接介导的细胞间通讯。     

Steady state dynamics of intermediate filament networks

We have conducted experiments to examine the dynamic exchange between subunit and polymer of vimentin intermediate filaments (IF) at steady state through the use of xrhodamine-labeled vimentin in fluorescence recovery after photobleaching (FRAP) analysis. The xrhodamine-vimentin incorporated into the endogenous vimentin IF network after microinjection into fibroblasts and could be visualized with a cooled charge-coupled device (CCD) camera and digital imaging fluorescence microscopy. Bar shaped regions were bleached in the fluorescent IF network using a beam from an argon ion laser and the cells were monitored at various times after bleaching to assess recovery of fluorescence in the bleached zones. We determined that bleached vimentin fibers can recover their fluorescence over relatively short time periods. Vimentin fibers in living cells also can exhibit significant movements, but the recovery of fluorescence was not dependent upon movement of fibers. Fluorescence recovery within individual fibers did not exhibit any marked polarity and was most consistent with a steady state exchange of vimentin subunits along the lengths of IF.     

Aurovertin fluorescence changes of the mitochondrial F1-ATPase during multi- and uni-site ATP hydrolysis

The aurovertin-F1 complex was used to monitor fluorescence changes of the mitochondrial adenosine triphosphatase during multi- and uni-site ATP hydrolysis. It is known that the fluorescence intensity of the complex is partially quenched by addition of ATP or Mg2+ and enhanced by ADP (Chang, T., and Penefsky, H. S. (1973) J. Biol. Chem. 248, 2746-2754). In the present study low concentrations of ATP (0.03 mM) induced a marked fluorescence quenching which was followed by a fast fluorescence recovery. This recovery could be prevented by EDTA or an ATP regenerating system. The rate of ATP hydrolysis by the aurovertin-F1 complex and the reversal of the ATP-induced fluorescence quenching were determined in these various conditions. ITP hydrolysis also resulted in fluorescence quenching that was followed by a recovery of fluorescence intensity. Under conditions for single site catalysis, fluorescence quenching was observed upon the addition of ATP. This strongly indicates that fluorescence changes in the aurovertin-F1 complex are due to the binding and hydrolysis of ATP at a catalytic site. Therefore the resulting ADP molecule bound at this catalytic site possibly induces the fluorescence recovery observed.     

Fluorescence Recovery after Merging a Droplet to Measure the Two-dimensional Diffusion of a Phospholipid Monolayer

We introduce a new method to measure the lateral diffusivity of a surfactant monolayer at the fluid-fluid interface, called fluorescence recovery after merging (FRAM). FRAM adopts the same principles as the fluorescence recovery after photobleaching (FRAP) technique, especially for measuring fluorescence recovery after bleaching a specific area, but FRAM uses a drop coalescence instead of photobleaching dye molecules to induce a chemical potential gradient of dye molecules. Our technique has several advantages over FRAP: it only requires a fluorescence microscope rather than a confocal microscope equipped with high power lasers; it is essentially free from the selection of fluorescence dyes; and it has far more freedom to define the measured diffusion area. Furthermore, FRAM potentially provides a route for studying the mixing or inter-diffusion of two different surfactants, when the monolayers at a surface of droplet and at a flat air/water interface are prepared with different species, independently.     

Recovery from winter depression of photosynthesis in pine and spruce

Summary Recovery from winter depression of photosynthesis was studied in Pinus sylvestris, Pinus conforta and Picea abies by means of chlorophyll fluorescence and gas exchange measurements. During the winter 1986–1987 the fluorescence yield was low and no variable fluorescence was detectable before the end of March. In the field recovery of variable fluorescence/maximum fluorescence (F_v/F_m) during spring was slow for all three species studied. The temperature dependence of recovery was confirmed from measurements of the potential rate of recovery of F_v/F_m at different temperatures in the laboratory. At 20° C, F_v/F_m increased from 0.1 to 0.8 within 3 days. Recovery of F_v/F_m was paralleled by an increase in apparent photon yield. No significant differences could be demonstrated between the studied tree species in potential rate of recovery in the laboratory or in actual recovery in the field.     

Interaction of annexin VI with membranes: highly restricted dissipation of clustered phospholipids in membranes containing phosphatidylethanolamine.

Association of annexin VI with membranes induced extensive clustering of acidic phospholipids as detected by self-quenching of fluorescent-labeled acidic phospholipids [Bazzi, M.D., & Nelsestuen, G.L. (1991) Biochemistry 30, 7961]. The present study examined the rates of protein-induced clustering of acidic phospholipids in membranes containing 10-15% fluorescent-labeled phosphatidic acid dispersed in phosphatidylcholine (PC) or phosphatidylethanolamine (PE). Both membranes supported similar levels of protein-induced fluorescence quenching. With membranes containing PC, protein-membrane association and fluorescence quenching were rapid, and were virtually complete within seconds after the reagents were mixed. Membranes containing PE exhibited rapid protein-membrane association, but showed a fluorescence quenching that was several orders of magnitude slower than membranes containing PC. Calcium chelation resulted in rapid dissociation of protein-membrane complexes. Subsequent recovery of the fluorescence signal of both membranes was virtually complete, but the rate of fluorescence recovery was very different. The recovery was rapid in membranes containing PC, while PE-containing membranes showed slow recovery that approached the rate at which the fluorescent-labeled phosphatidic acid exchanged between vesicles. Thus, the presence of PE appeared to severely restrict dissipation of clustered phospholipids in membranes. Membranes containing PE, N-methyl-PE, N,N-dimethyl-PE, and PC showed successive increases in the rates of fluorescence quenching and recovery, suggesting that hydrogen bonding between head groups was the basis for this property. If the restricted dissipation of phosphatidic acid in PE membranes is a general property, the relative mobility of membrane components and even diffusion on interior cell membranes may be greatly influenced by this phenomenon.     

Binding kinetics of an anti-dinitrophenyl monoclonal Fab on supported phospholipid monolayers measured by total internal reflection with fluorescence photobleaching recovery.

Fluorescence photobleaching recovery with total internal reflection illumination (TIR-FPR) has been used to measure the dissociation kinetics of a fluorescein-labeled anti-dinitrophenyl monoclonal Fab specifically bound to supported monolayers composed of a mixture of dipalmitoylphosphatidylcholine and dinitrophenyl-conjugated dipalmitoylphosphatidylethanolamine. The fluorescence recovery curves were not monoexponential; when analyzed as a sum of two exponentials, the rates and fractional recoveries were approximately 1 s-1 (approximately 50%) and approximately 0.1 s-1 (approximately 30%). The data did not change as a function of the Fab solution concentration, indicating that the fluorescence recovery curves were not influenced by the rate of diffusion in bulk solution. Also, the recovery curves were independent of the size of the illuminated area, indicating that surface diffusion did not significantly contribute to the rate and shape of the fluorescence recovery. The measured off rates and apparent association constant (1.6 x 10(5) M-1) were analyzed with the theoretical formalism for a proposed mechanism that accounts for the nonmonoexponential kinetics.     

Phase topology and percolation in two-component lipid bilayers: a monte Carlo approach.

Monte Carlo simulations of fluorescence recovery after photobleaching (FRAP) experiments on two-component lipid bilayers systems in the solid-fluid phase coexistence region were carried out to study the geometry and size of fluid domains in these bilayers. The gel phase was simulated by superposable elliptical domains, which were either of predetermined dimensions, increasing in number with increasing gel phase fraction, or of predetermined number, increasing in dimensions with increasing gel phase fraction. The simulations were done from two perspectives: 1) a time-independent analysis of fractional fluorescence recovery as a function of fractional fluid phase in the system; and 2) a time-dependent analysis of fractional fluorescence recovery as a function of time at a given fraction of fluid phase in the system. The time-dependent simulations result in recovery curves that are directly comparable to experimental FRAP curves and provide topological and geometrical models for the coexisting phases that are consistent with the experimental result.     

Reversible photobleaching of enhanced green fluorescent proteins

Color variants of green fluorescent protein (GFP) are increasingly used for multicolor imaging, fluorescence resonance energy transfer (FRET), and fluorescence recovery after photobleaching (FRAP). Here we show that experimental settings commonly used in these imaging experiments may induce an as yet uncharacterized reversible photobleaching of fluorescent proteins, which is more pronounced at acidic pH. Whereas the reversible photobleaching spectrum of eCFP corresponds to its absorption spectrum, reversible photobleaching spectra of yellow variants resemble absorption spectra of their protonated states. Fluorescence intensities recover spontaneously with time constants of 25-58 s. The recovery of eCFP can be further accelerated by illumination. The resulting steady-state fluorescence reflects a variable equilibrium between reversible photobleaching, spontaneous recovery, and light-induced recovery. These processes can cause significant artifacts in commonly applied imaging techniques, photobleach-based FRET determinations, and FRAP assays.     

Comparison of two fluorescence immunoassay methods for the detection of endocrine disrupting chemicals in water

We describe two fluorescence immunoassays capable of detecting endocrine disrupting compounds in waste water. The first fluorescence method is a heterogeneous assay using total internal reflection fluorescence (TIRF) detection. The second method is a homogeneous assay that utilizes energy transfer (ETIA). Both fluorescence immunoassays are compared with respect to detection principle and ability to quantify the model analytes estrone, estradiol, and ethinylestradiol in a complex matrix regarding recovery rates and limits of detection. Calibrations were performed for the three analytes using both fluorescence methods. Limits of detection between 0.01 and 0.85 microg/l are achieved. In addition, measurements in synthetic waste water spiked with the analytes were performed. Both immunoassays allow the detection in waste water with recovery rates in the range of 70-112%.     

Demonstration of phycobilisome mobility by the time- and space-correlated fluorescence imaging of a cyanobacterial cell

The cell-wide mobility of PBSs was confirmed by synchronously monitoring the fluorescence recovery after photobleaching (FRAP) and the fluorescence loss in photobleaching (FLIP). On the other hand, a fluorescence recovery was still observed even if PBSs were immobile (PBSs fixed on the membranes by betaine and isolated PBSs fixed on the agar plate) or PBS mobility was unobservable (cell wholly bleached). Furthermore, it was proved that some artificial factors were involved not only in FRAP but also in FLIP, including renaturation of the reversibly denatured proteins, laser scanning-induced fluorescence loss and photo-damage to the cell. With consideration of the fast renaturation component in fluorescence recovery, the diffusion coefficient was estimated to be tenfold smaller than that without the component. Moreover, it was observed that the fluorescence intensity on the bleached area was always lower than that on the non-bleached area, even after 20 min, while it should be equal if PBSs were mobile freely. Based on the increasing proportion of the PBSs anti-washed to Triton X-100 (1%) with prolonged laser irradiation to the cells locked in light state 1 by PBQ, it was concluded that some PBSs became immobile due to photo-linking to PSII.     

Demonstration of phycobilisome mobility by the time- and space-correlated fluorescence imaging of a cyanobacterial cell

The cell-wide mobility of PBSs was confirmed by synchronously monitoring the fluorescence recovery after photobleaching (FRAP) and the fluorescence loss in photobleaching (FLIP). On the other hand, a fluorescence recovery was still observed even if PBSs were immobile (PBSs fixed on the membranes by betaine and isolated PBSs fixed on the agar plate) or PBS mobility was unobservable (cell wholly bleached). Furthermore, it was proved that some artificial factors were involved not only in FRAP but also in FLIP, including renaturation of the reversibly denatured proteins, laser scanning-induced fluorescence loss and photo-damage to the cell. With consideration of the fast renaturation component in fluorescence recovery, the diffusion coefficient was estimated to be tenfold smaller than that without the component. Moreover, it was observed that the fluorescence intensity on the bleached area was always lower than that on the non-bleached area, even after 20 min, while it should be equal if PBSs were mobile freely. Based on the increasing proportion of the PBSs anti-washed to Triton X-100 (1%) with prolonged laser irradiation to the cells locked in light state 1 by PBQ, it was concluded that some PBSs became immobile due to photo-linking to PSII.     

Photoinhibition of Photosynthesis and its Recovery in Red Algae

In different marine red algae (Chondrus crispus, Delesseria sanguinea, Membranoptera alata, Phycodrys rubens, Phyllophora truncata, Polyneura hilliae) photoinhibition of photosynthesis has been investigated by means of both fluorescence and oxygen measurements. Measurements of absolute oxygen production show that photoinhibition causes a decline in the initial slope and in the rate of bending of the fluence rate-response curve (i.e. the photosynthetic efficiency at non-saturating fluence rates), as well as a decline in the photosynthetic capacity (Pm) at saturating fluence rates. Fluorescence data (Fv/Fm) were consistent with the results of oxygen measurements. Under excessive light photoinhibition protects photosynthesis against photo-damage in red algae. However, an increase in the initial fluorescence (Fo) after photoinhibitory treatment indicates that it could not prevent photodamage entirely. Action spectra of photoinhibition demonstrate that the main photoinhibition site in Polyneura hiliae is PS II, because far red light absorbed by PS I was ineffective. The strong increase of Fo in the blue wavelength range and the slight and partial recovery in weak blue light indicate that blue light especially causes photodamage. Recovery of photosynthesis requires dim white light conditions. Experiments with monochromatic light also show a wavelength dependence of recovery. Moreover, the recovery of photosynthesis after a photoinhibitory treatment is strongly temperature dependent, indicating participation of enzymatic processes. The comparison of fluorescence and oxygen measurement of the recovery shows different results in some species. The rate of oxygen production in red control light increased immediately after photoinhibited algae were exposed to weak light conditions. Surprisingly, the ratio of variable to maximum fluorescence (Fv/Fm) of Phyllophora truncata and the maximum fluorescence (Fm) of Polyneura hilliae show first a delay of the recovery under weak light conditions. Thus, in recovery experiments fluorescence and oxygen data are not quite consistent.     

Refolding of pyridoxine-5'-P oxidase assisted by GroEL

The unfolding of brain pyridoxine-5'-P oxidase by guanidinium chloride has been investigated at equilibrium. Circular dichroism, fluorescence spectroscopy and gel exclusion chromatography were used to monitor the unfolding process. The enzyme dissociates reversibly into monomers, but the fluorescence properties of the cofactor FMN are not restored upon dilution with potassium phosphate buffer (pH 7.4). Spontaneous refolding leads to 20% recovery of the catalytic activity. Addition of GroEL to the renaturing buffer accelerates the recovery of catalytic activity that approaches a level of 80% with respect to the native enzyme. The rate of recovery of catalytic activity assisted by GroEL parallels the rate of FMN fluorescence quenching, suggesting that structural rearrangements of the catalytic domain is the last step to take place in the refolding process.     

Progressive and spatially differentiated stability of microtubules in developing neuronal cells

The establishment of neural circuits requires both stable and plastic properties in the neuronal cytoskeleton. In this study we show that properties of stability and lability reside in microtubules and these are governed by cellular differentiation and intracellular location. After culture for 3, 7, and 14 d in nerve growth factor-containing medium, PC-12 cells were microinjected with X-rhodamine-labeled tubulin. 8-24 h later, cells were photobleached with a laser microbeam at the cell body, neurite shaft, and growth cone. Replacement of fluorescence in bleached zones was monitored by digital video microscopy. In 3-d cultures, fluorescence recovery in all regions occurred by 26 +/- 17 min. Similarly, in older cultures, complete fluorescence recovery at the cell body and growth cone occurred by 10-30 min. However, in neurite shafts, fluorescence recovery was markedly slower (71 +/- 48 min for 7-d and 201 +/- 94 min for 14-d cultures). This progressive increase in the stability of microtubules in the neurite shafts correlated with an increase of acetylated microtubules. Acetylated microtubules were present specifically in the neurite shaft and not in the regions of fast microtubule turnover, the cell body and growth cone. During the recovery of fluorescence, bleached zones did not move with respect to the cell body. We conclude that the microtubule component of the neuronal cytoskeleton is differentially dynamic but stationary.     

Spruce photosynthesis: possibility of early damage diagnosis due to exposure to magnesium or potassium deficiency

Summary The effects of exposure to magnesium or potassium deficiency on spruce needle photosynthesis were studied before symptoms of damage were visible, and recovery was followed. Water content, pigment content and O₂ evolution rate were chosen as parameters of physiological activity in addition to chlorophyll fluorescence. In the case of exposure to Mg deficiency, the first symptoms detected by all the physiological parameters were observed in older needles. An effect on chlorophyll fluorescence [decrease in fluorescence index (R_fd) and photochemical quenching (q_Q), and increase in non-photochemical quenching (q_E)] was detected in younger needles at the beginning of the deficiency treatment with little effect on the other physiological parameters [decrease in water content to 96.5%, and in photosynthetic rate expressed over dry weight (PSDW) to 90.7%]. No recovery was observed. After a K-deficiency exposure, both kinds of needles were affected, but a high recovery rate was observed, especially in the current-year needles, where a complete recovery from damage was detected by fluorescence and confirmed later by visual inspection. Finally, these results indicate the possibility of an important early diagnosis of damage in forest decline and suggest that chlorophyll fluorescence can be used, at least in the laboratory, as a qualitative test of plant health.     

Exchangeability of alpha-actinin in living cardiac fibroblasts and muscle cells

We have investigated the exchangeability of alpha-actinin in various structures of cultured chick cardiac fibroblasts and muscle cells using fluorescent analogue cytochemistry in combination with fluorescence recovery after photobleaching. Living cells were microinjected with tetramethylrhodamine-labeled alpha-actinin, which became localized in cellular structures. Small areas of labeled structures were then photobleached with a laser pulse, and the subsequent recovery of fluorescence was monitored with an image intensifier coupled to an image-processing system. In fibroblasts, fluorescence recovery was studied in stress fibers and in adhesion plaques. Bleached spots in adhesion plaques generally attained complete recovery within 20 min; whereas complete recovery in stress fibers occurred within 30 to 60 min. In muscle cells, alpha-actinin became localized in the Z-lines of sarcomeres, in punctate structures, and in apparently continuous bundle-like structures. Fluorescence recovery in Z-lines, punctate structures, and some bundle-like structures was extremely slow. Complete recovery did not occur within the 6- to 7-h observation period. However, some bundle-like structures recovered completely within 60 min, a rate similar to that of stress fibers in fibroblasts. These results indicate that fluorescently labeled alpha-actinin is more stably associated with structures in muscle cells than in fibroblasts. In addition, different structures within the same cell can display different alpha-actinin exchangeabilities which, in muscle cells, could be developmentally related.     

Diffusion, patching, and capping of stearoylated dextrans on 3T3 cell plasma membranes

Fluorescence-labeled trinitrophenylated stearoylated dextrans have been used as controllable analogues of cell membrane proteins on model membranes and on a variety of natural cell membranes. This paper reports their behavior on 3T3 mouse fibroblast plasma membranes. Spatial distribution on the membrane was studied by fluorescence microscopy, and molecular mobility was measured by fluorescence photobleaching recovery. At concentrations from 10(2) to 3 X 10(3) molecules/micron2 essentially homogeneous fluorescence was observed after treatment with these stearoyldextrans in culture. Diffusion coefficients and fractional recovery of fluorescence after photobleaching were cvoncentration independent. For 3 X 10(3) molecules/micron2 we found at 23 degrees C D = (3.0 +/- 1.8) X 10(-10) cm2/s with 65 +/- 17% recovery and at 37 degrees C D = (7.0 +/- 5.0) X 10(-10) cm2/s without a change of the fractional recovery. Cross-linking with antibodies stopped diffusion on a macroscopic scale and sometimes induced patching, mottling (defined as the development of gaps in the fluorescence layer), and capping (defined as the confinement of the fluorescence to less than 50% of the cell). Capping required approximately 3 h at 37 degrees C and was inhibited by metabolic poisons and cytochalasin B. These drugs did not affect stearoyldextran diffusion or fractional recovery. Colchicine, which did not dramatically affect capping, slowed diffusion two- to threefold but did not affect fractional recovery. The antibody inhibition of the diffusion of stearoyldextrans precedent to capping did not affect the diffusion of a lipid probe or fluorescein isothiocyanate labeled membrane proteins. When the trinitrophenylated stearoyldextran was cleared from most of the surface by capping and the surface subsequently relabeled with stearoyldextran, the diffusion coefficient and fractional recovery of the second label were identical with those of the first label prior to capping. Thus, capping does not clear an immobilizing factor from the membrane.     

Total internal reflection/fluorescence photobleaching recovery study of serum albumin adsorption dynamics.

The total internal reflection/fluorescence photobleaching recovery (TIR/FPR) technique (Thompson et al. 1981. Biophys. J. 33:435) is used to study adsorbed bovine serum albumin dynamics at a quartz glass/aqueous buffer interface. Adsorbed fluorescent labeled protein is bleached by a brief flash of the evanescent wave of a focused totally internally reflected laser beam. The rates of adsorption/desorption and surface diffusion determine the subsequent fluorescence recovery. The protein surface concentration is low enough to be proportional to the observed fluorescence and high enough to insure that the observed recovery rates arise mainly from adsorbed rather than bulk protein dynamics. The photobleaching recovery curves for rhodamine-labeled bovine serum albumin reveal both an irreversibly bound state and a multiplicity of reversibly bound states. The relative amount of reversible to irreversible adsorption increases with increasing bulk protein concentration. Since the adsorbed protein concentration appears to be too high to pack into a homogeneous surface monolayer, the wide range of desorption rates possibly results from multiple layers of protein on the surface. Comparison of the fluorescence recovery curves obtained with various focused laser beam widths suggests that some of the reversibly bound bovine serum albumin molecules can surface diffuse. Aside from their relevance to the surface chemistry of blood, these results demonstrate the feasibility of the TIR/FPR technique for measuring molecular dynamics on solid surfaces.