Renal filtration and reabsorption of GFP in <Emphasis Type="Italic">Rana temporaria</Emphasis>: Effect of arginine-vasotocin

The renal tubular uptake of green fluorescent protein (GFP) in frog Rana temporaria was studied by laser confocal microscopy. The specific green fluorescence was revealed in cells of proximal tubules 30 min after intravenous GFP injection. The GFP fluorescence was located predominantly in the apical part of the cytoplasm in the form of intensively fluorescing vesicles. The GFP injections increased dose-dependently the GFP tubular uptake. This was confirmed by the quantitative assessment of intensity of the specific fluorescence, its relative vesicular density, and by correlation analysis. Preliminary administration of arginine-vasotocin into the dorsal lymphatic sack decreased significantly the GFP absorption. The effect of arginine-vasotocin was inhibited by pretreatment with an antagonist of vasopressin V_i-receptors. The results of this work together with literature data allow believing that a decrease in the GFP absorption in the frog kidney under effect of arginine-vasotocin is due to a fall of the AVT-dependent glomerular filtration rate and to a decrease in the input of protein into the lumen of tubules. The action of arginine-vasotocin seems to be mediated via the V_i-like receptors of preglomerular blood vessels.     

Effect of different proteins on reabsorption of yellow fluorescent protein in the kidney of the brown frog <Emphasis Type="Italic">Rana temporaria</Emphasis>

Protein reabsorption in the proximal tubules (PT) of the frog kidney was studied by immunohistochemistry, fluorescent and confocal microscopy. The yellow fluorescent protein (YFP) was introduced in combination with other proteins. Reabsorption of YFP co-injected with lysozyme or the green fluorescent protein (GFP) was indistinguishable from that of YFP injection alone. Preliminary lysozyme injection did not change YFP absorption in contrast to YFP uptake reduced after GFP pretreatment. Lysozyme loading for 4 days led to a significant reduction in YFP absorption. The results show that receptor-mediated endocytosis in the frog kidney depends on the molecular nature of absorbable ligands, conditions of their competitive absorption and lysosomal accumulation in PT epithelial cells.     

Renal filtration and reabsorption of GFP in frog Rana temporaria: effect of arginine-vasotocin

The renal tubular uptake of green fluorescent protein (GFP) in frog Rana temporaria was studied by laser confocal microscopy. The specific green fluorescence was revealed in the proximal tubule cells 30 min after intravenous GFP injection. The GFP fluorescence was distributed predominantly in the apical part of the cytoplasm in the form of the intensively fluorescing vesicles. The GFP injections increased dose-dependently the GFP tubular uptake. This was confirmed by the quantitative assessment of intensity of the specific fluorescence, its relative vesicular density, and by correlation analysis. Preliminary administration of arginine vasotocin into the dorsal lymphatic sac decreased significantly the GFP absorption. The effect of arginine vasotocin was inhibited by pretreatment a vasopressin V1-receptor antagonist. These results suggest that a decrease in the GFP absorption is due to a fall of the AVT-dependent glomerular filtration rate and consequently a decrease in the filtered GFP amount. The effect of arginine vasotocin on the GFP absorption seems to be mediated via the V1-like receptors of preglomerular blood vessels.     

Sex-related differences in the handling of fluorescent ovalbumin by the proximal tubule of the rat kidney

Summary Sex-dependent protein handling in the rat renal tubular system was studied both qualitatively and quantitatively using the method of direct fluorescent protein tracing. The protein tracer, fluorescent ovalbumin, was synthesized by conjugating hen ovalbumin with fluorescein isothiocyanate (FITC), and the fluorescence characteristics of fluores-ceinthiocarbamyl (FTC)-ovalbumin conjugates with different degrees of labelling were studied. Heavily labelled tracer was intravenously injected into male and female rats, and both kidneys were perfused; the right kidney was then homogenized and used for quantitative fluorometric measurements, while the left kidney was perfusion fixed and prepared for fluorescence mciroscopy. The tubular reabsorption of fluorescent ovalbumin was studied 4 min and 10 min after the injection of different doses (1.4, 7.0 and 14.0 mg/kg body weight) of the tracer, and the tubular catabolism was investigated in animals killed 60 and 120 min after the injection. Fluorescence microscopy demonstrated that, in both sexes and regardless of the dose administered and the time after injection, specifically fluorescent protein or its degradation products was only present in the epithelial cells of the proximal tubule. With regard to sex-dependent differences in protein handling, fluorometry indicated that at 4 min (7.0 mg) and at 10 min (all doses) after injection, female animals had reabsorbed more fluorescent protein than males. With regard to the catabolic phase, both the fluorescence microscopy and the fluorometric results showed that the female rats had degraded the fluorescent tracer at a significantly higher rate than males. The results are discussed in connection with sex-dependent proteinuria in rats.In honour of Prof. P. van DuijnSupported by the Deutsche Forschungsgemeinschaft (SFB 105)     

Sex-related differences in the handling of fluorescent ovalbumin by the proximal tubule of the rat kidney

Sex-dependent protein handling in the rat renal tubular system was studied both qualitatively and quantitatively using the method of direct fluorescent protein tracing. The protein tracer, fluorescent ovalbumin, was synthesized by conjugating hen ovalbumin with fluorescein isothiocyanate (FITC), and the fluorescence characteristics of fluoresceinthiocarbamyl (FTC)-ovalbumin conjugates with different degrees of labelling were studied. Heavily labelled tracer was intravenously injected into male and female rats, and both kidneys were perfused; the right kidney was then homogenized and used for quantitative fluorometric measurements, while the left kidney was perfusion fixed and prepared for fluorescence microscopy. The tubular reabsorption of fluorescent ovalbumin was studied 4 min and 10 min after the injection of different doses (1.4, 7.0 and 14.0 mg/kg body weight) of the tracer, and the tubular catabolism was investigated in animals killed 60 and 120 min after the injection. Fluorescence microscopy demonstrated that, in both sexes and regardless of the dose administered and the time after injection, specifically fluorescent protein or its degradation products was only present in the epithelial cells of the proximal tubule. With regard to sex-dependent differences in protein handling, fluorometry indicated that at 4 min (7.0 mg) and at 10 min (all doses) after injection, female animals had reabsorbed more fluorescent protein than males. With regard to the catabolic phase, both the fluorescence microscopy and the fluorometric results showed that the female rats had degraded the fluorescent tracer at a significantly higher rate than males. The results are discussed in connection with sex-dependent proteinuria in rats.     

Receptor-Mediated Endocytosis of Lysozyme in Renal Proximal Tubules of the Frog Rana Temporaria

The mechanism of protein reabsorption in the kidney of lower vertebrates remains insufficiently investigated in spite of raising interest to the amphibian and fish kidneys as a useful model for physiological and pathophysiological examinations. In the present study, we examined the renal tubular uptake and the internalization rote of lysozyme after its intravenous injection in the wintering frog Rana temporaria using immunohisto- and immunocytochemistry and specific markers for some endocytic compartments. The distinct expression of megalin and cubilin in the proximal tubule cells of lysozyme-injected frogs was revealed whereas kidney tissue of control animals showed no positive immunoreactivity. Lysozyme was detected in the apical endocytic compartment of the tubular cells and colocalized with clathrin 10 min after injection. After 20 min, lysozyme was located in the subapical compartment negative to clathrin (endo-somes), and intracellular trafficking of lysozyme was coincided with the distribution of megalin and cubilin. However, internalized protein was retained in the endosomes and did not reach lysosomes within 30 min after treatment that may indicate the inhibition of intra-cellular trafficking in hibernating frogs. For the first time, we provided the evidence that lysozyme is filtered through the glomeruli and absorbed by receptor-mediated clathrin-dependent endocytosis in the frog proximal tubule cells. Thus, the protein uptake in the amphibian mesonephros is mediated by megalin and cubilin that confirms a critical role of endocytic receptors in the renal reabsorption of proteins in amphibians as in mammals.Key words: Endocytic receptor, frog, kidney, lysozyme, protein uptake, proximal tubule     

Tubular protein uptake pattern in the frog model (<Emphasis Type="Italic">Rana temporaria</Emphasis>): The effect of previous protein loading

The effect of increasing protein load on subsequent receptor-mediated protein uptake was studied in the kidney of the common frog Rana temporaria L. Results of in vivo experiments were analyzed in fixed kidney sections using fluorescent or confocal microscopy and immunohistochemistry. Lysozyme was used for daily tubular loading in short-term experiments. Reabsorption of yellow fluorescent protein (YFP) in the proximal tubule (PT) was tested 60 min after introduction into the dorsal lymphatic sac. YFP uptake decreased progressively with increasing duration of lysozyme preload from 2 to 4 days. Lysozyme loading and single protein injections did not change the morphological characteristics of frog glomeruli and PTs, as shown by light and electron microscopy and morphometric analysis. Cessation of loading led to a decrease in the amount of lysozyme accumulated in PT cells. Reduced YFP uptake gradually recovered after cessation of the 4-day load. Restoration of YFP reabsorption was accompanied by increasing expression of endocytic receptors, megalin and cubilin. Based on the data obtained, the frog model can be successfully used for studying both morphological and functional changes in the nephron caused by tubular or glomerular proteinuria and molecular mechanisms involved in the process of renal protein reabsorption.     

Renal clearance of absorbed intact GFP in the frog and rat intestine

Intestine absorption of intact green fluorescent protein (GFP) and its following accumulation in the renal proximal tubule cells after its intragastric administration have been established by confocal microscopy in the rat and frog. Reabsorbed GFP was revealed in the endosomes and lysosomes of the proximal tubule cells by the methods of GFP photooxidation and immunofluorescent microscopy. The GFP intestine absorption rate and GFP accumulation in the kidney were significantly higher in the frog than in the rat. No specific fluorescence was revealed in the liver and colon cells after the GFP intragastric administration. The data obtained indicate the ability of the small intestine in the frog and rat to absorb intact proteins and an important role of the kidney in exogenous protein metabolism.     

Homo-FRET microscopy in living cells to measure monomer-dimer transition of GFP-tagged proteins

Fluorescence anisotropy decay microscopy was used to determine, in individual living cells, the spatial monomer-dimer distribution of proteins, as exemplified by herpes simplex virus thymidine kinase (TK) fused to green fluorescent protein (GFP). Accordingly, the fluorescence anisotropy dynamics of two fusion proteins (TK27GFP and TK366GFP) was recorded in the confocal mode by ultra-sensitive time-correlated single-photon counting. This provided a measurement of the rotational time of these proteins, which, by comparing with GFP, allowed the determination of their oligomeric state in both the cytoplasm and the nucleus. It also revealed energy homo-transfer within aggregates that TK366GFP progressively formed. Using a symmetric dimer model, structural parameters were estimated; the mutual orientation of the transition dipoles of the two GFP chromophores, calculated from the residual anisotropy, was 44.6 +/- 1.6 degrees, and the upper intermolecular limit between the two fluorescent tags, calculated from the energy transfer rate, was 70 A. Acquisition of the fluorescence steady-state intensity, lifetime, and anisotropy decay in the same cells, at different times after transfection, indicated that TK366GFP was initially in a monomeric state and then formed dimers that grew into aggregates. Picosecond time-resolved fluorescence anisotropy microscopy opens a promising avenue for obtaining structural information on proteins in individual living cells, even when expression levels are very low.     

Characterization of the activity of a plastid-targeted green fluorescent protein in Arabidopsis.

In Arabidopsis thaliana the PALE CRESS (PAC) gene product is required for both chloroplast and cell differentiation. Transgenic Arabidopsis plants expressing a translational fusion of the N-terminal part of the PAC protein harboring the complete plastid-targeting sequence and the green fluorescent protein (GFP) exhibit high GFP fluorescence. Detailed analyses based on confocal imaging of various tissues and cell types revealed that the PAC-GFP fusion protein accumulates in chloroplasts of mature stomatal guard cells. The GFP fluorescence within the guard cell chloroplasts is not evenly distributed and appears to be concentrated in suborganellar regions. GFP localization studies demonstrate that thin tubular projections emanating from chloroplasts and etioplasts often connect the organelles with each other. Furthermore, imaging of non-green and etiolated tissue further revealed that GFP fluorescence is present in proplastids, etioplasts, chromoplasts, and amyloplasts. Even photobleaching of carotenoid-free plastids does not affect PAC-GFP accumulation in the organelles of the guard cells indicating that the protein translocation machinery is functional in all types of plastids. The specific accumulation of GFP in guard cell chloroplasts, their tubular connections, the translocation of the precursor polypeptide into the different types of organelles, as well as the use of a plastid-targeted GFP protein as a versatile marker is discussed in the context of previously described observations.     

Isolation of peroxisome-defective CHO mutant cells using green fluorescent protein

The authors constructed a recombinant green fluorescent protein (GFP) (PTS-GFP), which carries peroxisome targeting signal (PTS1 or PTS2) as an additional sequence, by polymerase chain reaction. The gene encoding for the recombinant GFP was constructed into an eukaryotic expression vector, and stable transformant of CHO cell expressing PTS-GFP was isolated, following the transfection of the plasmid encoding for the GFP. Each expressed PTS-GFP appeared to be localized in peroxisomes, because the GFP was observed in cellular structures, as was catalase. The observation proposed a visual screening procedure for isolating peroxisome-defective mutant. Following an enrichment of mutant cells by use of 9-(1′-pyrene)nonanol/ultraviolet irradiation (P9OH/UV) method, five peroxisome-defective mutants were isolated by pursuing the fluorescent signals from GFP. Two mutants (SK24 and SK32) were isolated from CHO cells expressing PTS1-GFP, and three mutants (PT13, PT32, and PT54) were isolated from cells expressing PTS2-GFP. Four mutants, except for PT13, showed cytosolic distributions of both PTS-GFP and catalase. On the other hand, mutant PT13 showed a cytosolic distribution on PTS2-GFP, but a peroxisomal distribution on catalase. Cell fusion analysis between SK24 mutant and other mutants indicated that PT54 mutant is in the same complementation group (CG) as SK24, but that SK32, PT13, and PT32 mutants are in different complementation group(s) from SK24.     

Expression and analysis of green fluorescent proteins in human embryonic kidney cells by capillary electrophoresis

The green fluorescent protein (GFP) has attracted much interest as a reporter for gene expression. In this paper, application of capillary electrophoresis with laser-induced fluorescent (CE-LIF) for quantitation of green fluorescence protein in cellular extracts and single cells is investigated. The S65T mutant form of GFP protein was successfully expressed in human embryonic kidney (HEK293) cells, and its production was confirmed by fluorescence microscopy and CE-LIF. The mass limit of detection for the mutant S65T was 5.3 x 10(-20) mol, which was better than that for the wild-type GFP by a factor of six. Detection of a small amount of GFP is difficult by conventional techniques such as fluorescent microscopy due to interference from cell autofluorescence at low GFP concentrations. The HEK293 cells were transfected with the GFP plasmid that produced S65T-GFP. Transient production of S65T protein was detected 2 h after the transfection and reached a maximum after 48 h. The protein concentration began to decrease significantly after 96 h. Single cell analysis of HEK293 cells after transfection with GFP plasmid indicate a nonuniform production of S65T-GFP protein among cells.     

Visualization of Cu²⁺ uptake and release in plant cells by fluorescence lifetime imaging microscopy

A principal objective in life sciences is the visualization of biochemical processes. Fluorescence-based techniques are widely used to demonstrate transport of relevant substances across cellular membranes. In this paper we report a novel noninvasive, real-time fluorescence lifetime imaging microscopy method for visualizing uptake and release of divalent copper ions (Cu(2+) ) in vivo. For this purpose, we employed a green fluorescent protein (GFP) form able to change its fluorescence lifetime upon Cu(2+) binding. We demonstrate that this technique is selective for Cu(2+) . We show the reversible decrease of the fluorescence lifetime of GFP from 2.2 to 1.6 ns in Escherichia coli and from 1.8 to 1.3 ns in root cells of Arabidopsis after the addition of Cu(2+) . Cu(2+) uptake of epidermal tobacco cells leads to a drop of the GFP lifetime from 2.5 to 2.2 ns. In summary, the spatially resolved visualization of Cu(2+) distribution in vivo is demonstrated in prokaryote and eukaryote cells.     

Transport of virally expressed green fluorescent protein through the secretory pathway in tobacco leaves is inhibited by cold shock and brefeldin A

Potato virus X (PVX) has been used as an expression vector to target the green fluorescent protein (GFP) from the jellyfish Aequorea victoria to the endoplasmic reticulum (ER) of tobacco (Nicotiana clevelandii L.) leaves. Expression of free GFP resulted in strong cytoplasmic fluorescence with organelles being imaged in negative contrast. Translocation of GFP into the lumen of the ER was mediated by the use of the sporamin signal peptide. Retention of GFP in the ER was facilitated by the splicing of the ER retrieval/retention tetrapeptide, KDEL to the carboxy terminus of GFP. Fluorescence of GFP was restricted to a labile cortical network of ER tubules with occasional small lamellae and to streaming trans-vacuolar strands. Secretion of ER-targeted GFP was inhibited both by cold shock and low concentrations of the secretory inhibitor brefeldin A. However, both prolonged cold and prolonged incubation in brefeldin A resulted in the recovery of secretory capability. In leaves infected with the GFP-KDEL construct, high concentrations of brefeldin A induced the tubular network of cortical ER to transform into large lamellae or sheets which reverted to the tubular network on removal of the drug. Received: 8 October 1998 / Accepted: 16 November 1998     

Cellular trafficking and photochemical internalization of cell penetrating peptide linked cargo proteins: a dual fluorescent labeling study

Initial cellular uptake of cell penetrating peptide (CPP) linked macromolecules is usually endosomal, with passage from endosome to cytosol a major limitation to efficient delivery. To gain a better understanding of the passage of the CPP-linked proteins, we studied the uptake and localization of CPP-linked proteins that contained two different forms of fluorescent markers, GFP protein and chemically conjugated tetramethylrhodamine, in living cells. Rhodamine labeled TAT-GFP was internalized in multiple cell lines including HEK293, N18-RE-105, hippocampal slices, and human neural progenitor cells and showed predominantly endosomal localization of both fluorescent markers. Cytosolic localization of some rhodamine label was detected to suggest that some of the GFP label had exited from the endosome. However, quantification of the distribution of the rhodamine and GFP label indicated that the protein location was primarily endosomal and that the distribution of TAT-GFP was not significantly different than that of an exclusively endosomal localized exogenous protein (tetanus toxin fragment C - TTC). As a result, photochemical internalization (PCI) was evaluated and caused a significant quantitative redistribution of cellular fluorescence of rhodamine and GFP labels to demonstrate increased cytosolic delivery of GFP. While rhodamine-labeled TAT-GFP showed cytosolic delivery with exposure to specific wavelengths of fluorescent illumination, a similarly labeled GFP fusion protein containing the membrane binding domain of TTC did not mediate PCI in N18-RE-105 cells.     

Construction of a virulent, green fluorescent protein-tagged Yersinia ruckeri and detection in trout tissues after intraperitoneal and immersion challenge

A green fluorescent protein (GFP) expressing strain of Yersinia ruckeri was created by the transposition of a Tn10-GFP-kan cassette into the genome of Y. ruckeri Strain YRNC10. The derivative, YRNC10-gfp, was highly GFP fluorescent, retained the gfp-km marker in the absence of kanamycin selection, and exhibited in vitro growth kinetics similar to those of the wild type strain. YRNC10-gfp colonized and caused mortality in immersion and intraperitoneally challenged rainbow trout Oncorhynchus mykiss, although it was modestly attenuated compared to the wild type strain. The distribution and location of YRNC10-gfp in infected fish was visualized by epifluorescence microscopy. Abundant extracellular bacteria and a small number of intracellular bacteria were observed in the kidney, spleen and peripheral blood. To determine the percentage of trout cells containing intracellular bacteria, GFP fluorescence was measured by flow cytometry. A small population of GFP positive leukocytes was detected in peripheral blood (1.6%), spleen (1.1%) and anterior kidney (0.4%) tissues. In summary, this is the first report of the construction of a virulent, GFP-tagged Y. ruckeri, which may be a useful model for detecting and imaging the interactions between an aquatic pathogen and the natural salmonid host.     

Effects of melatonin and natural and synthetic analogues of arginine vasotocin on plasma prolactin levels in adult male rats

A significant elevation in plasma prolactin was observed 10 min following the intravenous injection of 100 microgram of melatonin into either estrogen-progesterone (EP) primed or into nonsteroid-treated male rats. 60 min postinjection in the EP primed rat, the groups treated with 100 microgram or 10 mg of melatonin had signficantly elevated plasma prolactin levels while no effect was observed with these same doses in the nonsteroid-treated rats. Compared to diluent-treated controls, a significant elevation in plasma prolactin was observed at 10, 20 and 60 min following the intravenous injection of either 1 microgram arginine vasotocin (AVT) or 1 mg melatonin into EP primed male rats. A consistent rise in plasma prolactin was also evident after the injection of 1 microgram of either arginine vasopressin, lysine vasopressin or AVT. Oxytocin had no effect on plasma prolactin values. The intravenous administration of 1 microgram of (deamino-1,6 dicarba, 8-arginine)-vasotocin caused a significant elevation of plasma prolactin 10 and 20 min after injection. However, the injection of another analogue of AVT, (4-leucine, 8-arginine)-vasotocin, had no effect on prolactin release at the time points measured.     

Green fluorescent protein photobleaching: a model for protein damage by endogenous and exogenous singlet oxygen

Characterization of protein damage during photosensitization of chlorin e6-treated cells was performed using the green fluorescent protein (GFP). The GFP-chromophore damage caused by singlet oxygen was studied in COS 7 kidney cells and E. coli bacteria following light irradiation. Electron spin resonance (ESR) revealed the generation of endogenous singlet oxygen (1O2) by photoactivated GFP, an effect similar to that produced by the exogenous photosensitizer chlorin e6. A light dose-dependent photobleaching effect of GFP was pronounced at low pH or upon photosensitization with chlorin e6. However, the 1O2 quenchers beta-carotene and sodium azide minimized GFP photo-bleaching. Gel electrophoresis of photosensitized GFP followed by fluorescence multi-pixel spectral imaging revealed the binding of chlorin e6 to GFP, affecting the photobleaching efficacy. Fluorescence multi-pixel spectral imaging of GFP-transfected COS 7 cells demonstrated the presence of GFP in the cytoplasm and nucleus, while chlorin e6 was found to be concentrated in the perinuclear vesicles. Exposure of the cells to light induced GFP photobleaching in the close vicinity of chlorin e6 vesicles. We conclude that photoactivated GFP generates endogenous 1O2, inducing chromophore damage, which can be enhanced by the cooperation of exogenous chlorin e6.     

Green fluorescent protein as a marker in transgenic mice

Green fluorescent protein (GFP) found in Aequorea victoria absorbs blue light and emits green fluorescence without exogenous substrates or co-factors. We studied the possibility of using the GFP as a marker in mammals. Transgenic mice were produced using the GFP coding sequence, ligated with the chicken beta-actin promoter. Green fluorescence was observed in muscle, pancreas, kidney, heart and other organs in all the three transgenic mouse lines. Detection of the transgenic mouse was possible by observing a tail or fingers of new born pups under a fluorescent microscope. The marker also enabled us to detect localized expression of the transgene in intact tissues without preliminary steps. It was also demonstrated that the GFP expression could be quantified by measuring the fluorescence in tissue extracts.     

High expression of green fluorescent protein in Pichia pastoris leads to formation of fluorescent particles

Wild type gene for green fluorescent protein (GFP) was stably integrated into the Pichia pastoris genome and yielded an expression level of over 40% of total cellular protein. The high cytoplasmic concentration of fluorescent (properly folded and processed) GFP caused the formation of fluorescent spherical structures, which could be observed by fluorescence or confocal microscopy after controlled permeabilization of the yeast cells with 0.2% N-lauroyl sarcosine (NLS). Fluorescent GFP particles were also isolated after removal of the cell wall and found to be quite resistant to 0.2% N-lauroyl sarcosine. SDS-PAGE analysis of the isolated fluorescent particles revealed the presence of an 80 kDa protein (alcohol oxidase) and GFP (30%). We conclude that GFP is able to enter spontaneously into the peroxisomes and is inserted into densely packed layers of alcohol oxidase. Consequently, the formation of similar fluorescent particles can also be expected in other organisms when using high-level expression systems. As GFP is widely used in fusion with other proteins as a reporter for protein localization and for many other applications in biotechnology, care must be taken to avoid false interpretations of targeting or trafficking mechanisms inside the cells. In addition, when whole cells or cytoplasmic fractions are used for the quantitative determination of GFP levels, incorrect and misleading values of GFP could be obtained due to the formation of fluorescent particles containing material inside which is not available for fluorescence measurements.