Real time visualization of protein kinase activity in living cells.

A library of fluorescently labeled protein kinase C (PKC) peptide substrates was prepared to identify a phosphorylation-induced reporter of protein kinase activity. The lead PKC substrate displays a 2.5-fold change in fluorescence intensity upon phosphorylation. PKC activity is readily sampled in cell lysates containing the activated PKCs. Immunodepletion of conventional PKCs from the cell lysate eliminates the fluorescence response, suggesting that this peptide substrate is selectively phosphorylated by PKCalpha, beta, and gamma. Finally, living cells microinjected with the peptide substrate exhibit a 2-fold increase in fluorescence intensity upon exposure to a PKC activator. These results suggest that peptide-based protein kinase biosensors may be useful in monitoring the temporal and spatial dynamics of PKC activity in living cells.     

Quantitative fluorescence imaging of protein diffusion and interaction in living cells

Diffusion processes and local dynamic equilibria inside cells lead to nonuniform spatial distributions of molecules, which are essential for processes such as nuclear organization and signaling in cell division, differentiation and migration. To understand these mechanisms, spatially resolved quantitative measurements of protein abundance, mobilities and interactions are needed, but current methods have limited capabilities to study dynamic parameters. Here we describe a microscope based on light-sheet illumination that allows massively parallel fluorescence correlation spectroscopy (FCS) measurements and use it to visualize the diffusion and interactions of proteins in mammalian cells and in isolated fly tissue. Imaging the mobility of heterochromatin protein HP1α (ref. 4) in cell nuclei we could provide high-resolution diffusion maps that reveal euchromatin areas with heterochromatin-like HP1α-chromatin interactions. We expect that FCS imaging will become a useful method for the precise characterization of cellular reaction-diffusion processes.     

Counting nucleosomes in living cells with a combination of fluorescence correlation spectroscopy and confocal imaging

Although methods for light microscopy of chromatin are well established, there are no quantitative data for nucleosome concentrations in vivo. To establish such a method we used a HeLa clone expressing the core histone H2B fused to the enhanced yellow fluorescent protein (H2B-EYFP). Quantitative gel electrophoresis and fluorescence correlation spectroscopy (FCS) of isolated oligonucleosomes show that 5% of the total H2Bs carry the fluorescent tag and an increased nucleosome repeat length of 204 bp for the fluorescent cells. In vivo, the mobility and distribution of H2B-EYFP were studied with a combination of FCS and confocal imaging. With FCS, concentration and brightness of nascent molecules were measured in the cytoplasm, while in the nucleoplasm a background of mobile fluorescent histones was determined by continuous photobleaching. Combining these results allows converting confocal fluorescence images of nuclei into calibrated nucleosome density maps. Absolute nucleosome concentrations in interphase amount up to 250 microM locally, with mean values of 140(+/-28)microM, suggesting that a condensation-controlled regulation of site accessibility takes place at length scales well below 200 nm.     

Interaction of chicken liver basic fatty acid-binding protein with fatty acids: a 13C NMR and fluorescence study

Two different groups of liver fatty acid-binding proteins (L-FABPs) are known: the mammalian type and the basic type. Very few members of this second group of L-FABPs have been characterized and studied, whereas most of the past studies were concerned with the mammalian type. The interactions of chicken liver basic fatty acid-binding protein (Lb-FABP) with 1-(13)C-enriched palmitic acid (PA) and oleic acid (OA) were investigated by (13)C NMR spectroscopy. Samples containing fatty acids (FA) and Lb-FABP at different molar ratios exhibited only a single carboxylate resonance corresponding to bound FA, and showed a binding stoichiometry of 1:1 both for PA and for OA. Fluorescence spectroscopy measurements yielded the same binding stoichiometry for the interaction with cis-parinaric acid [K(d) = 0.38(4) microM]. Competition studies between cis-parinaric acid and the natural ligands indicated a decreasing affinity of chicken Lb-FABP for PA, OA, and retinoic acid (RA). (13)C NMR proved that pH and ionic strength affect complex stability. The carboxyl signal intensity reversibly decreased upon lowering the pH up to 5. The pH dependence of the bound carboxyl chemical shift yielded an apparent pK(a) of 4.8. A decrease of the integrated intensity of the bound carboxylic signal in the NMR spectra was observed while increasing the chloride ion concentration up to 200 mM. This body of evidence indicates that the bound FA is completely ionized at pH 7.4, that its polar head is positioned in a solvent-accessible region, that a FA-protein strong ionic bond is not present, and that high ionic strength causes the release of the bound FA. The reported results show that, insofar as the number of bound ligands and its relative affinity for different FAs are concerned, chicken Lb-FABP is remarkably different from the mammalian liver FABPs, and, within its subfamily, that it is more similar to catfish Lb-FABP while it behaves quite differently from shark or axolotl Lb-FABPs.     

Involvement of arginine in the binding of heme and fatty acids to fatty acid-binding protein from bovine liver

Fatty acid-binding protein from bovine liver but not from bovine heart binds hematin in a saturable manner with high affinity. This property is not confined to a particular isoform as both, pI 6.0- and pI 7.0 L-FABP, bind hematin similarly. In competition experiments hematin and oleic acid could replace each other demonstrating that they share at least parts of the same binding site. Common structural features, i.e. the presence of carboxylic groups and of hydrophobic carbon chains led to the hypothesis that both ligands interact similarly with L-FABP. This was supported by the decrease of binding affinity for either ligand upon modification with phenylglyoxal. Modification in the presence of fatty acid revealed the protection of one of the two arginines of L-FABP. By peptide mapping and Edman degradation Arg¹²² was identified as the counterpart of the fatty acids carboxylic group.     

Very long chain n-3 and n-6 polyunsaturated fatty acids bind strongly to liver fatty acid-binding protein

Synthesis of n-3 and n-6 very long chain-PUFAs (VLC-PUFAs) from 18-carbon essential fatty acids is differentially regulated. The predominant product arising from n-3 fatty acids is docosahexaenoic acid (22:6n-3), with the liver serving as the main site of production. The synthetic pathway requires movement of a 24-carbon intermediate from the endoplasmic reticulum to peroxisomes for retroconversion to 22:6n-3. The mechanism of this intra-organelle flux is unknown, but could be binding-protein facilitated. We thus investigated binding of a series of previously untested VLC-PUFAs to liver fatty acid-binding protein (L-FABP). Three fluorometric assays were employed, all of which showed strong binding (K(d)' approximately 10(-8) to 10(-7) M) of 20-, 22-, and 24-carbon n-3 PUFAs to L-FABP. In contrast, synthesis of the predominant n-6 PUFA product, arachidonic acid, does not require intra-organelle transport. However, we found that n-6 VLC-PUFAs bound to L-FABP with affinities (K(d)' approximately 10(-8) to 10(-7) M) comparable to their n-3 counterparts.Although these results raise the possibility that L-FABP may participate in the cytoplasmic processing of n-3 and n-6 VLC-PUFAs, there is no evidence on the basis of binding affinities that L-FABP accounts for differences in the predominant products formed by the n-3 and n-6 PUFA metabolic pathways.     

Interaction of the adipocyte fatty acid-binding protein with the hormone-sensitive lipase: regulation by fatty acids and phosphorylation

Adipocyte fatty acid-binding protein (AFABP/aP2) forms a physical complex with the hormone-sensitive lipase (HSL) and AFABP/aP2-null mice exhibit reduced basal and hormone-stimulated lipolysis. To identify the determinants affecting the interaction fluorescence resonance energy transfer (FRET) imaging was used in conjunction with a mutagenesis strategy to evaluate the roles AFABP/aP2 fatty acid binding and HSL phosphorylation have in complex formation as well as determine the HSL binding site on AFABP/aP2. The nonfatty acid binding mutant of AFABP/aP2 (R126Q) failed to form a FRET-competent complex with HSL either under basal or forskolin-stimulated conditions, indicating that lipid binding is required for association. Once bound to HSL and on the surface of the lipid droplet, YFP-AFABP/aP2 (but not YFP-HSL) exhibited energy transfer between the fusion protein and BODIPY-C12-labeled triacylglycerol. Serine to alanine mutations at the two PKA phosphorylation sites of HSL (659 and 660), or at the AMPK phosphorylation sites (565), blocked FRET between HSL and AFABP/aP2. Substitution of isoleucine for lysine at position 21 of AFABP/aP2 (K21I), but not 31 (K31I), resulted in a non-HSL-binding protein indicating that residues on helix alphaI of AFABP/aP2 define a component of the HSL binding site. These results indicate that the ligand-bound form of AFABP/aP2.interacts with the activated, phosphorylated HSL and that the association is likely to be regulatory; either delivering FA to inhibit HSL (facilitating feedback inhibition) or affecting multicomponent complex formation on the droplet surface.     

Multiple-color fluorescence imaging of chromosomes and microtubules in living cells

Microscopic observation of fluorescently-stained intracellular molecules within a living cell provides a straightforward approach to understanding their temporal and spatial relationships. However, exposure to the excitation light used to visualize these fluorescently-stained molecules can be toxic to the cells. Here we describe several important considerations in microscope instrumentation and experimental conditions for avoiding the toxicity associated with observing living fluorescently-stained cells. Using a computer-controlled fluorescence microscope system designed for live observation, we recorded time-lapse, multi-color images of chromosomes and microtubules in living human and fission yeast cells. In HeLa cells, a human cell line, microtubules were stained with rhodamine-conjugated tubulin, and chromosomes were stained with a DNA-specific fluorescent dye, Hoechst33342, or with rhodamine-conjugated histone. In fission yeast cells, microtubules were stained with alpha-tubulin fused with the jellyfish green fluorescent protein (GFP), and chromosomes were stained with Hoechst33342.     

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.     

Cardiac fatty acid-binding proteins. Isolation and characterization of the mitochondrial fatty acid-binding protein and its structural relationship with the cytosolic isoforms

In the course of our studies on the structural diversity of the isoforms of cardiac fatty acid-binding proteins (cFABPs), a cardiac-type FABP from the matrix of bovine heart mitochondria was purified to homogeneity and obtained as a single 15-kDa protein with an isoelectric point of 4.9. The primary structures of this protein and of the two isoforms isolated from the cytosol (pI4.9-cFABP and pI 5.1-cFABP) were investigated by means of plasma desorption mass spectrometry and sequencing of peptides. All three proteins are amino-terminally blocked with an acetyl group and shown to be colinear with the sequence deduced from a cDNA clone for bovine heart fatty acid-binding protein (Billich, S., Wissel, T., Kratzin, H., Hahn, U., Hagenhoff, B., Lezius, A. G., and Spener, F. (1988) Eur. J. Biochem. 175, 549-556) except for the residue at position 98. This residue is demonstrated to be the molecular origin of bovine cFABP isoforms since pI 5.1-cFABP contains Asn98 in accordance with the sequence derived from the cDNA, whereas in pI 4.9-cFABP, this position is occupied by Asp98. Moreover, mitochondrial FABP is identical to pI 4.9-cFABP. Molecular masses of pI 4.9-cFABP (14,679 +/- 10 Da) and pI 5.1-cFABP (14,678 +/- 20 Da) determined by plasma desorption mass spectrometry coincide with that calculated from the cDNA (14,673 Da). Hence, residues linked to these proteins by posttranslational modification are not present, and the Asn-Asp exchange is the sole origin of heterogeneity of mitochondrial and cytosolic fatty acid-binding proteins from bovine heart.     

Fluorescence and multiphoton imaging resolve unique structural forms of sterol in membranes of living cells

Although cholesterol is an essential component of mammalian membranes, resolution of cholesterol organization in membranes and organelles (i.e. lysosomes) of living cells is hampered by the paucity of nondestructive, nonperturbing methods providing real time structural information. Advantage was taken of the fact that the emission maxima of a naturally occurring fluorescent sterol (dehydroergosterol) were resolvable into two structural forms, monomeric (356 and 375 nm) and crystalline (403 and 426 nm). Model membranes (sterol:phospholipid ratios in the physiological range, e.g. 0.5-1.0), subcellular membrane fractions (plasma membranes, lysosomal membranes, microsomes, and mitochondrial membranes), and lipid rafts/caveolae (plasma membrane cholesterol-rich microdomain purified by a nondetergent method) contained primarily monomeric sterol and only small quantities (i.e. 1-5%) of the crystalline form. In contrast, the majority of sterol in isolated lysosomes was crystalline. However, addition of sterol carrier protein-2 in vitro significantly reduced the proportion of crystalline dehydroergosterol in the isolated lysosomes. Multiphoton laser scanning microscopy (MPLSM) of living L-cell fibroblasts cultured with dehydroergosterol for the first time provided real time images showing the presence of monomeric sterol in plasma membranes, as well as other intracellular membrane structures of living cells. Furthermore, MPLSM confirmed that crystalline sterol colocalized in highest amounts with LysoTracker Green, a lysosomal marker dye. Although crystalline sterol was also detected in the cytoplasm, the extralysosomal crystalline sterol did not colocalize with BODIPY FL C(5)-ceramide, a Golgi marker, and crystals were not associated with the cell surface membrane. These noninvasive, nonperturbing methods demonstrated for the first time that multiple structural forms of sterol normally occurred within membranes, membrane microdomains (lipid rafts/caveolae), and intracellular organelles of living cells, both in vitro and visualized in real time by MPLSM.     

Lipid droplets formation in human endothelial cells in response to polyunsaturated fatty acids and 1‐methyl‐nicotinamide (MNA); confocal Raman imaging and fluorescence microscopy studies

In this work the formation of lipid droplets (LDs) in human endothelial cells culture in response to the uptake of polyunsaturated fatty acids (PUFAs) was studied. Additionally, an effect of 1‐methylnicotinamide (MNA) on the process of LDs formation was investigated. LDs have been previously described structurally and to some degree biochemically, however neither the precise function of LDs nor the factors responsible for LD induction have been clarified. Lipid droplets, sometimes referred in the literature as lipid bodies are organelles known to regulate neutrophil, eosinophil, or tumor cell functions but their presence and function in the endothelium is largely unexplored. 3D linear Raman spectroscopy was used to study LDs formation in vitro in a single endothelial cell. The method provides information about distribution and size of LDs as well as their composition. The incubation of endothelial cells with various PUFAs resulted in formation of LDs. As a complementary method for LDs identification a fluorescence microscopy was applied. Fluorescence measurements confirmed the Raman results suggesting endothelial cells uptake of PUFAs and subsequent LDs formation in the cytoplasm of the endothelium. Furthermore, MNA seem to potentiate intracellular uptake of PUFAs to the endothelium that may bear physiological and pharmacological significance.

Confocal Raman imaging of HAoEC cell with LDs.     

Specific localization of hepatic fatty acid-binding protein in the gastric brush cells of rats

Summary An immunocytochemical study by light- and electron microscopy using the antibody against rat hepatic fatty acid-binding protein (FABP) revealed the brush cells in the gastric epithelium of rats to be intensely immunoreactive. The immunoreactive cells were present in a group in the distal wall of the groove between forestomach and glandular stomach, as well as scattered singly in the surface and foveolar epithelia of the glandular stomach. Almost all immunoreactive brush cells had a thin basal process in contact with the basement membrane. No secretory granules with dense cores, similar to those of endocrine cells, were observed in the brush cells. The specific appearance of FABP-immunoreactivity in the brush cell indicates that this cell type is a distinct entity from other epithelial cells in the stomach and that FABP is a useful histochemical marker of the brush cells. FABP may be involved in the specific function(s) of this cell type related to fatty acid metabolism.     

Gender differences in the response of hepatic fatty acids and cytosolic fatty acid-binding capacity to alcohol consumption in rats

To investigate possible gender differences in the response of hepatic fatty acids and cytosolic fatty acid-binding capacity to ethanol consumption, both female and male rats (41 days of age) were pair fed liquid diets (with a littermate of the same sex) for 28 days. The diets contained 36% of energy either as ethanol or as additional carbohydrate. After ethanol feeding, the hepatic concentration of fatty acids increased 155% in females (P less than 0.01), whereas there was only a trend for an increase (22%) in males. This was associated with a much smaller increase of cytosolic fatty acid-binding capacity in females (58%) than in males (161%). Whereas the ethanol-induced increase in fatty acid-binding capacity provided an ample excess of binding sites for the fatty acids in males, the increase in females was barely sufficient for the binding of the large increase of fatty acids produced by ethanol in the females. The cytosolic protein responsible for this binding, the liver fatty acid-binding protein of the cytosol (L-FABPc), also promotes esterification of the fatty acids. In keeping with the postulated role of this protein, the ethanol-induced increases in hepatic triacylglycerols, phospholipids, and cholesterol esters were smaller in females than in males. The gender difference in cholesterol esters was associated with parallel changes in acyl-CoA transferase activity. A possible implication of the relatively small and most likely inadequate increase in liver fatty acid-binding capacity and fatty acid esterification during alcohol consumption in the females is that under these circumstances the risk for development of a potentially deleterious accumulation of fatty acids in the liver is increased, thereby contributing to the enhanced vulnerability of females to alcohol-induced hepatotoxicity.     

Watching proteins in the wild: fluorescence methods to study protein dynamics in living cells

The advent of GFP imaging has led to a revolution in the study of live cell protein dynamics. Ease of access to fluorescently tagged proteins has led to their widespread application and demonstrated the power of studying protein dynamics in living cells. This has spurred development of next generation approaches enabling not only the visualization of protein movements, but correlation of a protein's dynamics with its changing structural state or ligand binding. Such methods make use of fluorescence resonance energy transfer and dyes that report changes in their environment, and take advantage of new chemistries for site-specific protein labeling.     

Physical association between the adipocyte fatty acid-binding protein and hormone-sensitive lipase: a fluorescence resonance energy transfer analysis

Previous in vitro studies have established that hormone sensitive lipase (HSL) and adipocyte fatty acid-binding protein (AFABP) form a physical complex that presumably positions the FABP to accept a product fatty acid generated during catalysis. To assess AFABP-HSL interaction within a cellular context, we have used lipocytes derived from 293 cells (C8PA cells) and examined physical association using fluorescence resonance energy transfer. Transfection of C8PA cells with cyan fluorescent protein (CFP)-HSL, yellow fluorescent protein (YFP)-adipocyte FABP, or YFP-liver FABP revealed that under basal conditions each protein was cytoplasmic. In the presence of 20 microm forskolin, CFP-HSL translocated to the triacylglycerol droplet, coincident with BODIPY-FA labeled depots. Fluorescence resonance energy transfer analysis demonstrated that CFP-HSL associated with YFP-adipocyte FABP in both basal and forskolin-treated cells. In contrast, little if any fluorescence resonance energy transfer could be detected between CFP-HSL and YFP-liver FABP. These results suggest that a pre-lipolysis complex containing at least AFABP and HSL exists and that the complex translocates to the surface of the lipid droplet.     

Nonresonant confocal Raman imaging of DNA and protein distribution in apoptotic cells

Nonresonant confocal Raman imaging has been used to map the DNA and the protein distributions in individual single human cells. The images are obtained on an improved homebuilt confocal Raman microscope. After statistical analysis, using singular value decomposition, the Raman images are reconstructed from the spectra covering the fingerprint region. The data are obtained at a step interval of approximately 250 nm and cover a field from 8- to 15- micro m square in size. Dwell times at each pixel are between 0.5 and 2 s, depending on the nature and the state of the cell under investigation. High quality nonresonant Raman images can only be obtained under these conditions using continuous wave high laser powers between 60 and 120 mW. We will present evidence that these laser powers can still safely be used to recover the chemical distributions in fixed cells. The developed Raman imaging method is used to image directly, i.e., without prior labeling, the nucleotide condensation and the protein distribution in the so-called nuclear fragments of apoptotic HeLa cells. In the control (nonapoptotic) HeLa cells, we show, for the first time by Raman microspectroscopy, the presence of the RNA in a cell nucleus.     

Human T cell epitope mapping of the Schistosoma mansoni 14-kDa fatty acid-binding protein using cells from patients living in areas endemic for schistosomiasis

The development of a defined anti-schistosomiasis vaccine would contribute to the current control strategy mainly because immunization provides long-lasting immunity to the disease. Sm14, one of the six Schistosoma mansoni antigens selected by WHO as a candidate to compose a subunit vaccine against schistosomiasis, has been associated with resistance to S. mansoni infection in human beings and is able to induce protection in the murine model. To identify human T cell epitopes in Sm14, we used the TEPITOPE algorithm to select peptides that would most likely bind to several HLA-DR molecules. In this study, three Sm14 epitopes were selected and produced as synthetic peptides. Human T cell responses from schistosomiasis patients living in endemic areas in Brazil were determined by proliferation assay and IL-5 and IFN-gamma measurements. Differential peptide recognition and cytokine production in response to Sm14 epitopes were observed in individuals resistant to S. mansoni infection versus susceptible individuals. Sm14(32-48) and Sm14(53-69) peptides were preferentially recognized by peripheral blood mononuclear cells (PBMCs) of S. mansoni-resistant individuals, and Sm14(53-69) induced significant production of IFN-gamma. Additionally, Sm14(32-48) and Sm14(53-69) were "promiscuous" peptides, since they were able to induce cellular immune responses in individuals carrying 10 and 8, respectively, of the 11 HLA-DR molecules expressed in the studied population. Among Sm14 synthetic peptides tested in this study, we identified Sm14(32-48) and Sm14(53-69) as promising candidates to compose an anti-schistosomiasis vaccine, since they seem to be related to resistance to human schistosomiasis.     

Localization of brain-type fatty acid-binding protein in Kupffer cells of mice and its transient decrease in response to lipopolysaccharide

Brain-type fatty acid-binding protein (B-FABP) was localized in Kupffer cells of liver of postnatal day 10 (P10) and older mice in immunolight and electron microscopy as well as by in situ hybridization histochemistry. The immunoreaction products were localized in the cytoplasmic matrix but not within the nucleus. After peritoneal injection of lipopolysaccharide (LPS), the immunoreaction for B-FABP decreased markedly in Kupffer cells at 1 h postinjection and thereafter gradually recovered to the preinjection level by 24 h postinjection, although no decrease in the mRNA expression was detected in Northern blotting throughout the course after the injection. The specific localization of B-FABP, but not the other FABPs, in Kupffer cells, and its rapid decrease after LPS injection suggest the intimate involvement of B-FABP in Kupffer cells in the inflammatory reaction, probably through mediation of n-3 polyunsaturated fatty acids, which are strong binders of B-FABP.