E.H.W. Pap

RATIO-FLUORESCENCE MICROSCOPY OF LIPID OXIDATION IN LIVING CELLS USING C11-BODIPY581/591

A ratio‐fluorescence assay was developed for on‐line localization and quantification of lipid oxidation in living cells. The assay explores the oxidative sensitivity of C11‐BODIPY581/591. Upon oxidation, the fluorescence of this fluorophore shifts from red to green. The probe incorporates readily into cellular membranes and is about twice as sensitive to oxidation as arachidonic acid. Using confocal microscopy, the cumene hydroperoxide‐induced oxidation of C11‐BODIPY581/591 was visualized at the sub‐cellular level in rat‐1 fibroblasts. Pre‐loading of the cells with tocopherol retarded this oxidation. The data demonstrate that C11‐BODIPY581/591 is a valuable tool to quantify lipid oxidation and anti‐oxidant efficacy in single cells.

Peroxisomes in human fibroblasts have a basic pH.

eroxisomes are single-membrane-bound organelles found in nearly all eukaryotic cells. These organelles have a central function in lipid metabolism, including the beta-oxidation of verylong-chain and branched-chain fatty acids and the biosynthesis of ether phospholipids and cholesterol. Another characteristic of peroxisomes is their ability to degrade hydrogen peroxide by catalase. A deficiency in one or more peroxisomal enzymes has been linked to at least 20 (often lethal) disorders, showing the key role of this organelle in normal functioning of the human body. Peroxisomes are fragile structures that easily lose their integrity upon isolation. This poses a serious problem for studying these organelles in vitro and explains why our knowledge about the properties of the peroxisomal membrane, including the change in pH across it, is limited. In vivo, peroxisomes have been shown to be closed structures that are impermeable to NADH and NADPH, implying the existence of NADP redox shuttles. Here we study the pH in peroxisomes by targeting a pH-sensitive fluorescent reporter group to these organelles in living fibroblasts. We attained specific targeting by conjugating the fluorophore to a membrane-permeable peptide that contains a type-I peroxisome-targeting sequence (PTS1; amino-acid sequence AKL). Using this peptide probe, we establish that peroxisomes of human fibroblasts have a pH of 8.2 ± 0.3. Fibroblasts from RCDP (rhizomelic form of chondrodysplasia punctata) type 1 patients with severe mutations in the PEX7 protein, which result in an isolated defect in peroxisomal import of proteins with a PTS2 sequence, are still capable of importing the probe into peroxisomes, but have a pH of 6.5 ± 0.3. We covalently linked the pH-sensitive (5and 6-)carboxySNAFL-2 moiety to the PTS1-containing heptapeptide acetylCKGGAKL-COOH at the lysine near the amino terminus. This peptide probe (SNAFL-2–PTS1) was rapidly taken up into the cells and a punctate pattern of fluorescence was found, indicative of a peroxisomal localization. To confirm that these structures are indeed peroxisomes, we used a fixable analogue (BODIPY–PTS1) in co-localization studies with Cy5-labelled antibodies against different peroxisomal proteins (Fig. 1a–c). The probe was targeted only towards the peroxisomes (Fig. 1b). Further evidence that the probe was incorporated into peroxisomes came from studies of human fibroblasts with defects in peroxisomal import of PTS1bearing proteins. This probe was not incorporated into peroxiP

Endoplasmic reticulum resident proteins of normal human dermal fibroblasts are the major targets for oxidative stress induced by hydrogen peroxide.

The membrane-permeable fluorescein-labelled tyramine conjugate (acetylTyrFluo) was used to identify the proteins of normal human dermal fibroblasts most susceptible to oxidation by hydrogen peroxide [Van der Vlies, Wirtz and Pap (2001) Biochemistry 40, 7783-7788]. By exposing the cells to H(2)O(2) (0.1 mM for 10 min), TyrFluo was covalently linked to target proteins. TyrFluo-labelled and [(35)S]Met-labelled cell lysates were mixed and subjected to two-dimensional PAGE. After Western blotting the (35)S-labelled proteins were visualized by autoradiography and the TyrFluo-labelled proteins by using anti-fluorescein antibody. The TyrFluo-labelled proteins were matched with the (35)S-labelled proteins and identified by comparison with our mastermap of proteins. Protein disulphide isomerase (PDI), IgG-binding protein (BiP), calnexin, endoplasmin and glucose-regulated protein 58 (endoplasmic reticulum protein 57/GRP58) were identified as targets of oxidation. All these proteins reside in the endoplasmic reticulum and are part of the protein folding machinery. In agreement, confocal laser scanning microscopy showed co-localization of TyrFluo-labelled proteins and the KDEL receptor ERD-2, a marker for the endoplasmic reticulum.

Fluorescence dynamics of diphenyl-1,3,5-hexatriene-labeled phospholipids in bilayer membranes.

A comparative study of the dynamical fluorescence properties of three phosphatidylcholines having a diphenyl-1,3,5-hexatriene (DPH) group attached at different depths from the head group incorporated into membrane vesicles has been carried out. The probes were covalently attached to the sn-2 position of the glycerol part of the phosphatidylcholine via either carboxyl, ethyl or propanoyl links. The vesicles were composed of either dimyristoylphosphatidylcholine or dipalmitoylphosphatidylcholine. The experimental time-resolved polarized fluorescence data of the probes were analysed by two different methods: maximum entropy and global analysis. Distributed fluorescence lifetimes and correlation times of the DPH derivatives were obtained with the maximum entropy method. All DPH derivatives exhibited a bimodal distribution of fluorescence lifetimes with a dependence of the lifetime peak positions on the lipid phase, confirming previous data in the literature. The anisotropic rotational dynamics of the DPH moieties in the membranes could be described by several distributed correlation times. In the fluid phase of the membrane the residual anisotropy of free DPH became very small in contrast with those of the other probes, indicating that restriction of probe rotation is mainly imposed by the molecular geometry of the lipid probes. A two-dimensional analysis using the maximum entropy method demonstrated that both rotational correlation times were associated with the same set of fluorescence lifetimes. Global analysis of the data sets according to the general rotational diffusion model yielded weighted orientational distributions. Unexpectedly, a component of the DPH moiety oriented parallel to the membrane surface was obtained in the orientational distributions of the DPH lipids (as was reported earlier for DPH and TMA-DPH), which seems at variance with the geometric constraints imposed by the headgroups.

Peptide-based targeting of fluorophores to peroxisomes in living cells

Peptides carrying different fluorophores can be designed to incorporate spontaneously into living cells when added to the medium. By incorporating the peroxisome-targeting sequence PTS1, the peptide is recognized by the protein-import machinery of peroxisomes and, as a result, can accumulate in these organelles. Depending on the cell type, an inhibitor of the multidrug-resistance protein might be required to ensure strong accumulation. In this update, we discuss the potential of these peptide-linked fluorophores in solving issues related to organelle function and dynamics.

Targeted fluorescent probes in peroxisome function.

Fluorescent peptides form a new generation of analytical tools for visualizing intracellular processes and molecular interactions at the level of single cells. The peptide-based reporters combine the sensitivity of fluorescence detection with the information specificity of amino acid sequences. Recently we have succeeded in targeting a fluorescent heptapeptide (acetyl-CKGGAKL) carrying a peroxisomal targeting signal (PTS1) to peroxisomes in intact cells. The fluorophores conjugated to the PTS1-peptide were fluorescein, BODIPY and the pH-sensitive SNAFL-2. When added to cells, these fluorescent peptides were internalized at 37 °C and typically visible in the cell after 15 min or less. Cells lacking an active peroxisomal protein import system, as in the case of Zellweger syndrome, were stained diffusely throughout the cell. Uptake of the peptide probes was not inhibited at 4 °C or when the cells were depleted of ATP. Under these conditions translocation to peroxisomes was blocked. This indicates that the uptake by cells is diffusion-driven and not an active process. Using the SNAFL-2-PTS1 peptide, we established by ratio-imaging that peroxisomes of human fibroblasts have an internal pH of 8.2. The concurrent pH gradient over the peroxisomal membrane was dissipated when an ionophore (CCCP) was added. In fibroblasts of chondrodysplasia punctata patients with defects in the peroxisomal import of proteins carrying a PTS2 sequence, import of the PTS1-peptide probe into peroxisomes appeared normal, but these peroxisomes have a pH of 6.8 equal to that of the cytosol. Coupling different fluorophores to the PTS1-peptide offers the possibility of determining in time and space as to how peroxisomes function in living cells.

Quantitative fluorescence analysis of the adsorption of lysozyme to phospholipid vesicles

Experiments directed to measure the interaction of lysozyme with liposomes consisting of phosphatidylcholine (PC) and phosphatidylserine (PS) have been conducted by monitoring both protein and lipid fluorescence and fluorescence anisotropy of the protein. The binding of lysozyme to the unilamellar vesicles was quantified using a novel method of analysis in which the fractional contribution at moderate binding conditions is determined from either total fluorescence decay or anisotropy decay curves of tryptophan at limiting binding conditions. In the energy transfer experiments PC and PS lipids labelled with two pyrene acyl chains served as energy acceptors of the excited tryptophan residues in lysozyme. The binding was strongly dependent on the molar fraction of negatively charged PS in neutral PC membranes and on the ionic strength. Changes in the tryptophan fluorescence decay characteristics were found to be connected with long correlation times, indicating conformational rearrangements induced by binding of the protein to these lipid membranes. The dynamics of membrane bound protein appeared to be dependent on the physical state of the membrane. Independent of protein fluorescence studies, formation of a protein-membrane complex can also be observed from the lipid properties of the system. The interaction of lysozyme with di-pyrenyl-labelled phosphatidylserine in anionic PS/PC membranes resulted in a substantial decrease of the intramolecular excimer formation, while the excimer formation of dipyrenyl-labelled phosphatidylcholine in neutral PC membranes barely changed in the presence of lysozyme.

Reorientational properties of fluorescent analogues of the protein kinase C cofactors diacylglycerol and phorbol ester

The reorientational properties of the fluorescently labelled protein kinase C (PKC) cofactors diacylglycerol (DG) and phorbol ester (PMA) in vesicles and mixed micelles have been investigated using time-resolved polarised fluorescence. The sn-2 acyl chain of DG was replaced by diphenylhexatriene- (DPH) propionic acid, while a dansyl labelled analogue of phorbol ester was used. The extent of ordering of DPH-DG in vesicles turned out to be slightly different from that of the control choline lipid DPH-PC. Addition of PKC to vesicles containing 30 mole% brain PS considerably slowed down the DPH-DG anisotropy decay. This was not observed when DPH-DG was replaced by DPH-PC. Analysis of the fluorescence anisotropy decays of these DPH-lipids in micelles polyoxyethylene-9-laurylether mixed with 10 mole% of the essential phosphatidylserine allowed estimation of their lateral diffusion, orientation distribution and reorientational dynamics within the micelles. Addition of PKC resulted in a significantly slower decay of the fluorescence anisotropy of both DPH-DG and DPH-PC even in the absence of calcium, indicating a calcium independent complexation of PKC with the PS containing micelles. Addition of calcium resulted in a further reduction of the decay of anisotropy of DPH-DG but not of DPH-PC indicating that the Ca2+ dependent immobilisation is cofactor-specific. Similar specific interactions with PKC resulted in a slower decay of dansylated PMA when calcium and PS were present.

ERYTHROCYTE BAND 3 PROTEIN STRONGLY INTERACTS WITH PHOSPHOINOSITIDES

85% of the phosphorus coisolated with band 3 protein during separation of the intrinsic proteins of the human erythrocyte membrane by zonal electrophoresis in high concentrations of acetic acid was found to be derived from phosphoinositides, mainly phosphatidylinositol 4,5‐bisphosphate. When native band 3 protein and pyrene‐labelled phospholipids were present in micelles of the nonionic detergent nonaethyleneglycol lauryl ether, strong resonance energy transfer was observed between the tryptophan residues and phosphatidylinositol 4,5‐bisphosphate and, to a smaller degree, phosphatidylinositol 4‐phosphate. We conclude that band 3 protein strongly interacts with phosphoinositides, in particular with phosphatidylinositol 4,5‐bisphosphate.