Comparison of antigen contents in co-cultures by an in situ immunoradiometric assay.

A fast, sensitive and reproducible in situ immunoradiometric assay has been developed to compare relative contents of cellular markers in cultures. This assay is performed directly in the multi-well plate. After methanol fixation, antigens are identified by specific primary antibodies, followed by 125I-protein A. Cell-associated radioactivity is then measured in lysates using a gamma radiation counter and expressed with respect to protein content. By this method, differences in the level of any antigen retained by fixation can be easily quantified. The convenience, dynamic range of linearity and reproducibility of this technique compare favorably with Western blotting. Originally, the assay was designed to monitor the relative abundance of glial or neuronal cells in embryonic cerebral co-cultures upon various experimental conditions, by measuring related changes in glial fibrillary acidic protein (GFAP) or microtubule-associated protein 2 (MAP-2) content. It is proposed as a method of choice to quantify the effects of culture conditions or toxic agents on a specific cell type in mixed populations.

Retrieved constituents of large dense-cored vesicles and synaptic vesicles intermix in stimulation-induced early endosomes of noradrenergic neurons.

Two storage compartments in cultured noradrenergic neurons derived from the superior cervical ganglion from fetal pig have been defined using sucrose density gradient centrifugation and electron microscopy: (1) large dense-cored vesicles (LDV) contain noradrenaline and dopamine-beta-hydroxylase (DbetaH); (2) small electron-lucent vesicles contain acetylcholine and p38 and represent the noradrenergic small synaptic vesicles (SSV); no small dense-cored vesicles (SDV) could be detected. Our results demonstrate that internalized LDV membrane constituents are retrieved into early endosomes, as shown by the colocalization of retrieved DbetaH with the endosomal markers Rab5 and HRP in sucrose density gradients and on confocal microscopical images. Recycling of the SSV membranes via an endosomal intermediate is also confirmed in noradrenergic neurons. Finally, colocalization of retrieved DbetaH and retrieved p38 in stimulated neurons indicates that the two sets of constituents intermix. These data provide the first experimental evidence for a common early endosome in which SSV and LDV membrane constituents are internalized after exocytosis and imply that endosomal sorting is an important process for the generation of different secretory vesicles in the noradrenergic nerve terminal.

Three unrelated perturbations similarly uncouple fluid, bulk-membrane, and receptor endosomal flow in rat fetal fibroblasts.

We have compared the effects of three perturbations (treatment with 2 microM monensin, potassium depletion, and incubation in 0.35 M NaCl) on recycling of internalized fluid-phase, bulk-membrane, and receptor-mediated tracers in rat fetal fibroblasts. Monensin accelerated 2-fold the regurgitation of the fluid-phase tracer horseradish peroxidase (HRP), as previously described in these cells after potassium depletion or upon incubation in hypertonic medium (1), and all treatments severely inhibited transfer of HRP from endosomes to lysosomes. In comparison, recycling of (6-[N-(7-nitro-2,1,3-benzoxadiazol-4-yl)amino]hexanoyl glucosylsphingosine (C6-NBD-GlcCer), a fluorescent lipid used as a bulk-membrane probe, was not significantly affected while transferrin recycling was slowed down 2-fold. The striking similarities of these unrelated perturbations in their distinct effects on fluid, bulk-membrane, and receptor addressing point to common targets regulating these mechanisms.

Fractionation of human brain by differential and isopycnic equilibration techniques.

Fractionation of brain tissue by either differential or isopycnic centrifugation is a useful cytological and biochemical tool to study the intracellular localization of neuronal elements involved in neurotransmission. Several neuroreceptors and uptake sites were found to display a subcellular bimodal distribution in rat brain. However, in the human brain, little is known about the subcellular distribution of neurotransmitter receptors and amine uptake sites. Despite the inevitable post-mortem delay which seems to induce many more morphological changes than modifications of enzymatic or receptor distribution profile from the subcellular fractions, fractionation of human brain areas remains a valid procedure to explore the subcellular localization of neuronal constituents. This paper describes the methods used to separate human brain tissue. As we have previously demonstrated in rat and dog brains, our results indicate that differential and isopycnic fractionation techniques, used with a large number of markers such as enzymes, receptors and uptake sites, make it possible to separate tissue fractions enriched in nerve endings, dendrites, dendritic spines, plasma membranes or vesicles.

v-Src induces constitutive macropinocytosis in rat fibroblasts.

The role of v-Src as regulator of fluid-phase pinocytosis was investigated in Rat-1 cells expressing a stable (Rat-1/BB16) or a thermosensitive (Rat-1/tsLA29) v-Src protein. In the second cell line, this protein is inactive when cells are cultured at 40 degrees C but recovers its tyrosine kinase activity upon transfer to 34 degrees C, resulting into a transformed phenotype. The rate of fluid-phase pinocytosis of the tracer horseradish peroxidase was 2-fold higher in v-Src-transformed fibroblasts (Rat-1/BB16, Rat-1/tsLA29 cultured at 34 degrees C) as compared to non-transformed cells (Rat-1, Rat-1/tsLA29 kept at 40 degrees C). In contrast, receptor-mediated endocytosis of transferrin was poorly affected, suggesting that structures distinct from clathrin-coated pits are involved in pinocytosis stimulation. By light and electron microscopy, transformed cells frequently contained large peroxidase-labeled pinocytic vesicles located near to membrane ruffles, demonstrating that stimulation of pinocytosis corresponds to induction of constitutive macropinocytosis. Stimulation of pinocytosis occurred more than 8 hours after transfer to the permissive temperature, whereas transfer to the non-permissive temperature partially reversed the stimulation within 2 hours. Protein synthesis inhibition for 6 hours abrogated pinocytosis stimulation in transformed cells, indicating that constitutive macropinocytosis induced by v-Src depends on continuous synthesis of a short-lived regulatory machinery.

Quantitative analysis of clustering on biological membranes: methodology and application to ligand-induced asialoglycoprotein receptor redistribution on rat hepatocytes.

Ligand-induced receptor clustering is the first step in receptor mediated endocytosis of asialoglycoproteins by rat hepatocytes. This well-characterized receptor was used as a model system to set up a general method for the quantitative analysis and the visualization of molecular clustering on surface replicas, using a two-step approach. In the first step, aiming at the quantitation of clustering, gold-labeled asialoglycoprotein receptors on the cell surface were assumed to reflect two populations, one of clustered and one of not-clustered receptors. The experimental distribution of nearest neighbor distances of labeled receptors was adjusted by least square fitting to the sum of two functions, each corresponding to the theoretical nearest neighbor distance distribution of randomly distributed points, corresponding to non-clustered and clustered particle concentrations, respectively. The resolution of the experimental nearest neighbor distribution into these two components yielded an objective estimate of the number of labeled receptors in clusters and of the total surface of clusters. The second step, aiming at the visualization of clusters, rested on the fact that the distance of a point to its neighbors is shorter in a cluster than outside the cluster. Accordingly, each particle was ordered according to the sum of the distances to its nearest neighbors. Clustered particles with the lowest cumulative distances were extracted, in proportion of the extent of clustering determined in the first step, and displayed on the computer screen. This translated the two components into the two corresponding populations of identified particles. This method demonstrated that, if rat hepatocytes were incubated at 4 degrees C with either asialofetuin-gold complexes before fixation, or with asialofetuin prior to fixation and immunogold labeling of the ASGP-R, up to 65% of the receptors became clustered on the dorsal cell surface in areas where receptors could be concentrated up to 20-fold, as compared with randomly distributed molecules. In ultrathin sections, clusters essentially corresponded to clathrin-coated pits. In principle, this method is generic and can be applied to other biological systems, especially when statistical analysis is needed for studying the dynamics of the clustering process.

Conversion of angiotensin II into active fragments by an endosomal pathway in bovine adrenal medullary cells in primary culture.

We previously demonstrated that angiotensin II (AII) is internalized in primary cultures of bovine adrenal medullary cells by receptor-mediated endocytosis. In the present work, we followed internalized AII in these cells by means of confocal laser scanning microscopy combined with analytical subcellular fractionation techniques and compared its fate with that of transferrin and horseradish peroxidase. The integrity of [125I]AII was investigated by chromatography. With pulse-chase experiments, internalized AII could only be detected in endosomes using either fluorescence microscopy or fractionation studies. With chase, most of the radioactivity initially associated with the cells was rapidly released into the medium, as converted fragments (> 60%), essentially as AIV (80% of the fragments). Fragments efficiently bound to bovine adrenal medullary and cortical cells, a binding that was specifically displaced by AIV > AIII = AII. These results indicate that AII is taken up in bovine adrenal medullary cells and can be rapidly converted in the endosomal pathway into fragments that bind specifically to putative angiotensin receptors. These fragments are presumably biologically active and could act on either the chromaffin cell itself (autocrine) or the cortical cells (paracrine).

Subcellular distribution of receptor sites in human brain: differentiation between heavy and light structures of high and low density.

Studies of the subcellular localization of neuroreceptors in the rat brain have shown that most of them are associated with light and low density subcellular fractions. In two human brain areas, quite different subcellular distributions were observed. After fractionation by differential centrifugation of frontal cortex homogenates, benzodiazepine and serotonin 5-HT2 receptors were mainly found in the heavy mitochondrial (M) fraction, whereas mu-opiate and muscarinic cholinergic receptors were mainly concentrated in the microsomal (P) fraction. In human putamen, the presynaptic markers of dopaminergic nerve terminals (neurotensin receptors, dopamine uptake sites and amine vesicular transporter-binding sites), benzodiazepine receptors and serotonin uptake sites were recovered both in the high and low density fractions, whereas the muscarinic, opiate and, to a lesser extent, dopamine D2 receptors were mostly concentrated in the microsomal fraction. In the cerebral cortex, after isopycnic centrifugation in sucrose gradients, neuroreceptors were found in the high density fractions where the peaks of cytochrome oxidase and that of nerve endings, as identified by amine uptake and by means of electron microscopy were also found. A single peak of benzodiazepine receptors was observed in high density (1.15-1.17 g/ml) fractions suggesting that these receptors are much more concentrated in the nerve terminals or dendrites rather than in the dendritic spines or vesicles. The fact that muscarinic and opiate receptors were recovered in the P fraction with plasma membrane constituents and also in M and L fractions, which is confirmed by a bimodal distribution in sucrose gradient, suggests that they are localized in both the nerve terminals or dendrites and in the small vesicles or dendritic spines. In the putamen, much of the specific binding to uptake sites for dopamine and serotonin was recovered in the high density fractions, but the existence of another peak at a lower density indicates the presence of microsomal uptake sites. The results indicate that differential and isopycnic fractionation methods performed on human brain samples, make it possible to separate tissue fractions enriched in nerve endings, dendrites, dendritic spines, plasma membranes or vesicles.

Clathrin polymerization is not required for bulk-phase endocytosis in rat fetal fibroblasts.

To assess the role of clathrin in the bulk endocytic flow of rat foetal fibroblasts, the rate of internalization of fluid-phase and membrane-lipid tracers were compared, under control conditions and after inhibition of endocytic clathrin-coated pit formation. After intracellular potassium depletion or upon cell transfer into 0.35 M NaCl, the rate of internalization of receptor-bound transferrin and the residual membrane area of plasmalemmal clathrin-coated pits and vesicles were similarly decreased by approximately 90%. In contrast, the initial rate (< 5 min) of intracellular accumulation of the fluid-phase tracer HRP was not affected. Both in control and treated cells, the rate of HRP accumulation declined after approximately 5 min, and was twofold lower in treated cells, due to enhanced regurgitation. After correction for regurgitation, the endocytic rate constant was similar to measurements at shorter intervals and identical in control and treated cells. Similarly, the rate of internalization and the steady-state level of intracellular accumulation of two fluorescent lipid derivatives, 6-[N-(7-nitro-2,1,3-benzoxadiazol-4-yl)amino]hexanoylglucosylsp hingosine (C6-NBD-GlcCer) and 1-[4-(trimethylamino)phenyl]-6-phenylhexa-1,3,5-triene (TMA-DPH), were not affected by potassium depletion, indicating that the endocytic membrane traffic was equally preserved. Finally, the size distribution of primary endocytic particles that were accessible to HRP within 15 s before glutaraldehyde fixation was also indistinguishable in control and potassium-depleted cells. The simplest explanation is that clathrin polymerization is necessary to concentrate receptor-bound ligands in primary endocytic vesicles, but superfluous to the basic endocytic machinery in rat foetal fibroblasts.

Identification of a specific epitope on the extracellular domain of the LDL-receptor of Trypanosoma brucei brucei.

We have previously shown that an antiserum raised against the 86-kDa fragment of the low-density lipoprotein-receptor (LDL-receptor) of bloodstream Trypanosoma brucei brucei shows extensive cross-reactivity with the mammalian LDL-receptor. Here we report on the production and characterisation of 30 monoclonal antibodies (mAbs) raised against the same 86-kDa fragment of the T. b. brucei LDL-receptor. Of these, only 8 mAbs recognise in an ELISA test the purified (presumably intact) 145-kDa LDL-receptor. Seven of them also recognise the LDL-receptors isolated from rat and rabbit, whereas one mAb (1A9) is specific for the trypanosome LDL-receptor. A pool of several mAbs inhibits by 90% the specific binding of 125I-LDL to trypanosomes at 4 degrees C, but does not interfere with binding of 125I-LDL to rat fibroblasts. 125I-mAb 1A9 is efficiently taken up by T. b. brucei at 30 degrees C and its uptake is inhibited by an excess of unlabelled LDL particles, indicating that mAb 1A9 follows the LDL-receptor pathway. Uptake of 125I-mAb 1A9 by rat fibroblasts is less efficient and is not significantly reduced by an excess of unlabelled LDL. MAb 1A9 as well as other pooled mAbs activate rabbit complement, leading to lysis of trypanosomes in vitro. We conclude that the T. b. brucei LDL-receptor contains at least one specific epitope that is accessible on live cells to antibodies and which can activate the complement system.

Role of acidic compartments in Trypanosoma brucei, with special reference to low-density lipoprotein processing.

In the bloodstream form of Trypanosoma brucei, specific receptors mediate the endocytosis of host low-density lipoprotein particles. We have explored the fate of ligand and receptor with a biochemical approach, using inhibitors of the endocytotic apparatus. The weak base chloroquine rapidly accumulates in trypanosomes, its uptake is prevented by the proton ionophore monensin, and it induces the swelling of intracellular vacuoles, indicating that their content is acidic. Cell-associated LDL is rapidly degraded into intermediately sized fragments and TCA-soluble material that can be recovered in cell extracts and extracellular medium. Chloroquine, leupeptin and E64, but not PMSF, efficiently prevent LDL proteolysis, suggesting that degradation occurs in those acidic compartment(s) and is mediated by thiol-protease(s). Both monensin and chloroquine decrease the number of LDL receptors exposed at the cell surface, a phenomenon amplified in the presence of LDL. This provides indirect evidence that internalised LDL receptors are recycled at a rate which is slowed down by receptor occupancy and by agents that impair acidification of the endocytic organelles. Finally, chloroquine decreases by half the growth rate of procyclic trypanosomes in vitro at 5 micrograms ml-1. At 40 mg kg-1 per day, it also slows down the increase in parasitaemia and prolongs the survival time of infected mice by up to 2 days.

Ligand-induced clustering of asialoglycoprotein receptors on rat hepatocytes at 4 degrees C.

The platinum-carbon replica technique was applied to rat hepatocyte monolayers to visualize the distribution of the receptor responsible for the clearance of asialoglycoproteins. In a first series of experiments, hepatocytes were mildly fixed, either immediately after transfer to 4 degrees C, or after up to 24 h of incubation at 4 degrees C in the presence or absence of soluble asialofetuin (ASF). The asialoglycoprotein receptors were then immunolabeled by a rabbit antiserum followed by protein A-gold complexes. Average labeling was 41 +/- 2 particles x microns -2 (mean +/- SEM, n = 50), corresponding to an efficiency of about 25% as compared to radioligand binding data. Nearest neighbor analysis of gold particles showed an almost random distribution on cells incubated without asialofetuin, but significant clustering after exposure to the ligand. In a second series of experiments, hepatocytes were incubated at 4 degrees C for 2 or 24 h with asialofetuin adsorbed onto 15-nm gold particles (2 x 10(12) particles x ml-1, 32 molecules x particle-1), then fixed. On average, 20 +/- 2 (n = 14) and 38 +/- 6 (n = 19) particles x microns -2 were observed after 2 and 24 h, respectively. By comparison to control preparations, clustering of particles was found at both time intervals, although no significant difference could be detected between 2 and 24 h using the Kolmogorov-Smirnov test when the abundance of particles was taken into consideration. Summarizing, (1) in absence of ligand, the asialoglycoprotein receptor is almost randomly distributed on rat hepatocytes, (2) both soluble ASF and ASF-gold complexes induce receptor clustering, (3) even at 4 degrees C, the plasma membrane remains sufficiently fluid for receptor clustering.

Trypanosoma brucei brucei: antigenic stability of its LDL receptor and immunological cross-reactivity with the LDL receptor of the mammalian host.

The rapid growth of Trypanosoma brucei brucei in the blood and tissue fluids of vertebrates requires the receptor-mediated endocytosis of LDL from the host (Coppens et al. 1987; Gillett and Owen 1987) and is slowed by a monospecific rabbit antiserum against the purified LDL receptor of the parasite. We have used this antiserum in combination with several well-characterized antigenic variants (originating from stock 427: MITat 1.1a, 1.3a, 1.4a, 1.5a, 1.5d, 1.8b) to examine whether the LDL receptor of T. b. brucei is a stable surface antigen, common to all parasite variants despite antigenic variation of the major surface glycoprotein, and whether it is immunologically distinct from the LDL receptor of the host. At low concentrations, binding at 4 degrees C of rat LDL to several variants of T. b. brucei and to isolated rat hepatocytes was inhibited to a similar extent by the antiserum. In double immunodiffusion, a single precipitation line was observed, showing continuity between the extracts of all variants as well as between that of trypanosomes and of mammalian tissues. In Western blots of trypanosome extracts, the LDL receptor was strongly labeled as a single band of Mr 145,000, whereas with a rat liver extract, a single band of similar electrophoretic mobility was weakly labeled at a high concentration of the antiserum. In conclusion, the LDL receptor occurred in all variants of T. b. brucei, was a stable surface antigen despite variation of the major surface glycoprotein, and displayed biochemical and immunological similarities with the LDL receptor of the rat host.

A rapid method purifies a glycoprotein of Mr 145,000 as the LDL receptor of Trypanosoma brucei brucei.

The trypanosome LDL receptor has been isolated from bloodstream form and cultured insect-stage trypanosomes as a protein of Mr 145,000, using a rapid purification procedure in the presence of a cocktail of protease inhibitors, whereas previously a polypeptide of Mr 86,000 was purified as the LDL receptor. Both the 145,000 and the 86,000 polypeptides are glycosylated and recognized by a monospecific antibody raised against the 86,000 species. This antibody inhibits LDL binding to the intact trypanosomes, to the isolated 145,000 receptor and to the 86,000 species. Hence, the previously isolated 86,000 polypeptide is a degradation product probably representing the cleaved-off ectodomain of the trypanosome LDL receptor.

Lysosomal enzymes in the human endometrium: a biochemical study in untreated and levonorgestrel-treated women.

The activities of four lysosomal enzymes, i.e. N-acetyl-beta-hexosaminidase, acid phosphatase, alpha-D-mannosidase and alpha-L-fucosidase have been measured in extracts of endometrial biopsies from untreated and levonorgestrel-treated women of fertile age. Values were compared with protein and DNA content, as well as with lactate dehydrogenase activity, used as reference constituents. In parallel, organ cultures were established from the same endometrial specimens and the release of lysosomal enzymes into the medium was followed. The human endometrium possesses a rich lysosomal equipment, comparable to that found in the human liver. In the untreated cycles, the activities of lysosomal enzymes show a coordinate response to the hormonal changes, decreasing by about 40% from the proliferative to the mid-late secretory phase. Long-term levonorgestrel treatment causes a marked cytoplasmic atrophy, as shown by decreased protein content and lactate dehydrogenase activity, whereas DNA content remains unchanged. In contrast, N-acetyl-beta-hexosaminidase, one of the most active lysosomal enzymes studied, shows a higher specific activity upon levonorgestrel. In both untreated and treated endometria, the organ cultures provide biochemical evidence for a higher release of N-acetyl-beta-hexosaminidase than of lactate dehydrogenase, indicating active secretion of the lysosomal enzyme. During levonorgestrel treatment, there was no correlation between clinically recognized spotting-bleeding patterns and lysosomal enzyme content in, or release from, the endometrium.

Marker enzymes in rat liver vesicles involved in transcellular transport.

In order to label the vesicles involved in transcellular transfer (transcytosis) through hepatocytes, polymeric IgA (pIgA) was conjugated to horseradish peroxidase (HRP) and injected into rats. The endosomes containing this ligand at 10 or 20 min after injection were isolated by the diaminobenzidine-induced density-shift procedure and their content in various marker enzymes was measured. The endosomes carrying pIgA-HRP 10 min after injection contained only traces of 5'-nucleotidase and low amounts of alkaline phosphodiesterase I. The estimated marker enzyme content is similar to that observed for the particles containing galactosylated bovine serum albumin conjugated to HRP, a ligand degraded in lysosomes. However, 20 min after injection, the transcytotic endosomes showed a marked enrichment in 5'-nucleotidase and especially in alkaline phosphodiesterase I. The results confirm the heterogeneity of rat liver endosomes and substantiate the concept of distinct endosomal compartments.

A quantitative model of traffic between plasma membrane and secondary lysosomes: evaluation of inflow, lateral diffusion, and degradation.

We present here a mathematical model that accounts for the various proportions of plasma membrane constituents occurring in the lysosomal membrane of rat fibroblasts (Draye, J.-P., J. Quintart, P. J. Courtoy, and P. Baudhuin. 1987. Eur. J. Biochem. 170: 395-403; Draye, J.-P., P. J. Courtoy, J. Quintart, and P. Baudhuin. 1987. Eur. J. Biochem. 170:405-411). It is based on contents of plasma membrane markers in purified lysosomal preparations, evaluations of their half-life in lysosomes and measurements of areas of lysosomal and plasma membranes by morphometry. In rat fibroblasts, structures labeled by a 2-h uptake of horseradish peroxidase followed by a 16-h chase (i.e., lysosomes) occupy 3% of the cellular volume and their total membrane area corresponds to 30% of the pericellular membrane area. Based on the latter values, the model predicts the rate of inflow and outflow of plasma membrane constituents into lysosomal membrane, provided their rate of degradation is known. Of the bulk of polypeptides iodinated at the cell surface, only 4% reach the lysosomes every hour, where the major part (integral of 83%) is degraded with a half-life in lysosomes of integral to 0.8 h. For specific plasma membrane constituents, this model can further account for differences in the association to the lysosomal membrane by variations in the rate either of lysosomal degradation, of inflow along the pathway from the pericellular membrane to the lysosomes, or of lateral diffusion.