Effects of methylcyclodextrin on lysosomes.

The cholesterol complexing agent methyl-cyclodextrin (MCD) provides an efficient mean for the removal of cholesterol from biological membranes. In order to study the effects of this agent on the lysosomal membrane in situ, we treated HepG2 cells with MCD and studied the effects of this treatment on lysosomes in isolated fractions. We found that lysosomes prepared from treated cells are more sensitive to various membrane perturbing treatments such as: incubation of lysosomes in isotonic glucose, in hypotonic sucrose or in the presence of the lytic agent glycyl-L-phenylalanine 2-naphthylamide. The lysosomal membrane is also less resistant to increased hydrostatic pressure. Centrifugation methods were used to analyse the effect of MCD on lysosomes. Isopycnic centrifugation in sucrose density gradients demonstrates that the drug induces a reversible density increase of the lysosomes. Our study indicates that extracellularly added MCD can modify the properties of the lysosomal membrane in living cells. It suggests that MCD could be an effective tool to modulate the physical properties of lysosomes within intact cells and to monitor the cellular responses to such modifications.

Identification of HE1 as the second gene of Niemann-Pick C disease.

Niemann-Pick type C2 disease (NP-C2) is a fatal hereditary disorder of unknown etiology characterized by defective egress of cholesterol from lysosomes. Here we show that the disease is caused by a deficiency in HE1, a ubiquitously expressed lysosomal protein identified previously as a cholesterol-binding protein. HE1 was undetectable in fibroblasts from NP-C2 patients but present in fibroblasts from unaffected controls and NP-C1 patients. Mutations in the HE1 gene, which maps to chromosome 14q24.3, were found in NP-C2 patients but not in controls. Treatment of NP-C2 fibroblasts with exogenous recombinant HE1 protein ameliorated lysosomal accumulation of low density lipoprotein-derived cholesterol.

Uptake and intracellular fate of polyethylenimine in vivo.

Branched polyamines are extensively used as nonviral vectors for plasmid DNA in transfection experiments. Moreover, recently it has been shown that these compounds are able to eliminate prions from infected cells in cultures. It has been proposed that in both cases endosomes or lysosomes are the site of action. This raises the question of how these molecules are taken up by the cells and what is their intracellular fate. In the work presented here, the question has been addressed by investigating the uptake and the intracellular distribution of branched polyethyleneimine (25 kD) by centrifugation methods. The polyamine was labelled with (125)I-tyramine cellobiose and injected to the rat. The radioactive polymer is taken up after injection into the liver, kidney, spleen, and lungs and remains in these organs for many days. In the liver, it is found mainly in the hepatocytes. Intracellular distribution of radioactivity present in that organ was investigated by differential and isopycnic centrifugations. Early after injection, radioactivity exhibits a distribution pattern similar to that of alkaline phosphodiesterase, a plasma membrane marker. Later, the distribution pattern becomes similar to that of cathepsin C, a lysosomal enzyme. Radioactivity and hydrolase distributions in a sucrose gradient are similarly modified by a pretreatment of the rat with Triton-WR1339, a specific density perturbant of lysosomes. These results indicate that polyethyleneimine is endocytosed and reaches lysosomes. For many days it persists in these organelles probably due to its resistance to lysosomal hydrolases.

Endosomes, lysosomes: their implication in gene transfer.

Plasmid DNA, naked or bound to a non-viral vector, is taken up by endocytosis. As a result, it has to travel through the intracellular endocytic pathway involving endosomes and lysosomes. However, some DNA molecules must escape these organelles to reach the nucleus where transcription takes place. In this paper, we consider different factors that could affect the trafficking of plasmid DNA and influence transfection efficiency.

Subcellular localization of mannose 6-phosphate glycoproteins in rat brain.

The intracellular transport of soluble lysosomal enzymes relies on the post-translational modification of N-linked oligosaccharides to generate mannose 6-phosphate (Man 6-P) residues. In most cell types the Man 6-P signal is rapidly removed after targeting of the precursor proteins from the Golgi to lysosomes via interactions with Man 6-phosphate receptors. However, in brain, the steady state proportion of lysosomal enzymes containing Man 6-P is considerably higher than in other tissues. As a first step toward understanding the mechanism and biological significance of this observation, we analyzed the subcellular localization of the rat brain Man 6-P glycoproteins by combining biochemical and morphological approaches. The brain Man 6-P glycoproteins are predominantly localized in neuronal lysosomes with no evidence for a steady state localization in nonlysosomal or prelysosomal compartments. This contrasts with the clear endosome-like localization of the low steady state proportion of mannose-6-phosphorylated lysosomal enzymes in liver. It therefore seems likely that the observed high percentage of phosphorylated species in brain is a consequence of the accumulation of lysosomal enzymes in a neuronal lysosome that does not fully dephosphorylate the Man 6-P moieties.

Uptake by rat liver and intracellular fate of plasmid DNA complexed with poly-L-lysine or poly-D-lysine.

Efficiency of transfection is probably dependent on the rate of intracellular degradation of plasmid DNA. When a non-viral vector is used, it is not known to what extent the plasmid DNA catabolism is subordinated to the catabolism of the vector. In the work reported here, the problem was approached by following the intracellular fate in rat liver, of plasmid [35S]DNA complexed with a cationic peptide poly-L-lysine that can be hydrolyzed by cellular peptidases or with its stereoisomer, poly-D-lysine, that cannot be split by these enzymes. Complexes of DNA with poly-L-lysine and poly-D-lysine are taken up to the same extent by the liver, mainly by Kupffer cells, but the intracellular degradation of nucleic acid molecules is markedly quicker when poly-L-lysine is injected. The association of DNA with the polycations inhibits DNA hydrolysis in vitro by purified lysosomes but similarly for poly-L-lysine and poly-D-lysine. The intracellular journey followed by [35S]DNA complexed with poly-L- or poly-D-lysine was investigated using differential and isopycnic centrifugation. Results indicate that [35S]DNA is transferred more slowly to lysosomes, the main site of intracellular degradation of endocytosed macromolecules, when it is given as a complex with poly-D-lysine than with poly-L-lysine. They suggest that the digestion of the vector in a prelysosomal compartment is required to allow endocytosed plasmid DNA to rapidly reach lysosomes. Such a phenomenon could explain why injected plasmid DNA is more stable in vivo when it is associated with poly-D-lysine.

Delta F508 CFTR localizes in the endoplasmic reticulum-Golgi intermediate compartment in cystic fibrosis cells.

We have studied the localization of mutant cystic fibrosis transmembrane regulator delta F508CFTR in pancreatic adenocarcinoma cells (CFPAC), which naturally express the mutant protein. Our goal was to investigate whether delta F508CFTR is strictly retained in the endoplasmic reticulum (ER) or alternatively whether it can be transported beyond the ER and reach the endoplasmic reticulum-Golgi intermediate compartment (ERGIC). This compartment, defined by the presence of the 53-kDa protein ERGIC-53, was identified by subcellular fractionation and by immunofluorescence. Part of the delta F508CFTR population and ERGIC-53 showed similar distributions in membrane fractions analyzed on Nycodenz density gradients. Both proteins were present in density fractions distinct from the ones containing the ER marker proteins calnexin and Sec61. Immunofluorescence microscopy of CFPAC cells revealed some colocalization of delta F508CFTR with ERGIC-53. Following incubation of CFPAC cells at 15 degrees C, a condition known to block ER to Golgi transport, both ERGIC-53 and delta F508CFTR subcellular localizations were altered. By contrast, this temperature shift had no effect on the localization of the ER marker Sec61. Our observations indicate that the abnormal protein delta F508CFTR can leak out of the ER and reach the ERGIC. These results support the idea that this intermediate compartment plays a role in the trafficking events leading to retention and finally degradation of the misfolded delta F508CFTR protein.

Expression of Lamp-1 and Lamp-2 and their interactions with galectin-3 in human tumor cells.

Lysosomal-membrane-associated glycoproteins (Lamps) 1 and 2 are rarely found on the plasma membranes of normal cells. There is evidence suggesting an increase in their cell-surface expression in tumor cells, with some data indicating that the adhesion of some cancer cells to the extracellular matrix is partly mediated by interactions between Lamps and E-selectin and between Lamps and galectins (endogenous-galactoside-binding lectins). The present study examined the expression of Lamp-1 and Lamp-2 and their interactions with galectin-3 in different human tumor cell lines. Indirect immunofluorescence staining revealed accumulation of Lamp molecules at the edges of A2058 human metastasizing melanoma cells suggesting that these glycoproteins could participate in cell adhesion. Flow cytometry showed the presence of cell-surface Lamps in A2058, HT1080 (human fibrosarcoma) and CaCo-2 (human colon-adenocarcinoma) cells. Treatment with 2 mM sodium butyrate for 24 and 48 hr resulted in a significant increase in Lamps surface expression. A strong binding of A2058 to recombinant galectin-3 was detected by FACS. The application of 2 and 5 mM butyrate for the same incubation period enhanced galectin-3 binding to Lamps-expressing cells. Our results support the idea that Lamps may be considered a new family of adhesive glycoproteins participating in the complex process of tumor invasion and metastasis.

Cationic lipids destabilize lysosomal membrane in vitro.

Addition of cationic lipids to plasmid DNA considerably increases the efficiency of transfection. The mechanism has not yet been elucidated. A possibility is that these compounds destabilize biological membranes (plasma, endosomal, lysosomal), facilitating the transfer of nucleic molecules through these membranes. We have investigated the problem by determining if a cationic lipid N-(1-(2,3-dioleoxy)propyl)-N,N,N,-trimethylammonium methyl-sulfate (DOTAP, Boehringer, Mannheim, Germany) affects the integrity of rat liver lysosomal membrane. We have measured the latency of beta-galactosidase, a lysosomal enzyme, and found that incubation of lysosomes with low concentrations of DOTAP causes a striking increase in free activity of the hydrolase and even a release of the enzyme into the medium. This indicates that lysosomal membrane is deeply destabilized by the lipid. The phenomenon depends on pH, it is less pronounced at pH 5 than at pH 7.4. Anionic compounds, particularly anionic amphipathic lipids, can to some extent prevent this phenomenon. It can be observed with various cationic lipids. A possible explanation is that cationic liposomes interact with anionic lipids of lysosomal membrane, allowing a fusion between the lipid bilayers which results in a destabilization of the organelle membrane.

Supramolecular assemblies from lysosomal matrix proteins and complex lipids.

Most lysosomal hydrolases are soluble enzymes. Lamp-II (lysosome-associated membrane protein-II) is a major constituent of the lysosomal membrane. We studied the aggregation of a series of lysosomal molecules. The aggregation-sensitive lysosomal marker enzymes were optimally aggregated at intralysosomal pH. A similar pH dependence was recorded for aggregation of Lamp-II. The pH-dependent loss of solubility of isolated Lamp-II required components of the lysosome extract. Conditions of mild acid pH promoting aggregation triggered the formation of complexes with lipids of lysosomal origin. We fractionated a membrane-free lysosome extract by gel-filtration chromatography and could reconstitute assemblies in vitro from separated fractions. We found some selectivity in the lysosomal proteins binding to complex lipids, phosphatidylcholine, sphingomyelin, and phosphatidylethanolamine being most effective. We propose that the formation at pH 5.0 of such supramolecular assemblies between lysosomal proteins and lipids occurs within the intralysosomal environment. Some possible consequences of such an intralysosomal matrix formation on organelle function are discussed.

Cationic lipids delay the transfer of plasmid DNA to lysosomes.

Plasmid 35S DNA, naked or associated with different cationic lipid preparations was injected to rats. Subcellular distribution of radioactivity in the liver one hour after injection, was established by centrifugation methods. Results show that at that time, 35S DNA has reached lysosomes. On the contrary, when 35S DNA was complexed with lipids, radioactivity remains located in organelles whose distribution after differential and isopycnic centrifugation, is clearly distinct from that of arylsulfatase, lysosome marker enzyme. Injection of Triton WR 1339, a specific density perturbant of lysosomes, four days before 35S DNA injection causes a density decrease of radioactivity bearing structures, apparent one hour after naked 35S DNA injection but visible only after more than five hours, when 35S DNA associated with a cationic lipid is injected. These observations show that cationic lipids delay the transfer to lysosomes, of plasmid DNA taken up by the liver.

Soluble form of Lamp II in purified rat liver lysosomes.

Lamp II (for lysosomal associated membrane protein II) is an integral type I glycoprotein. It consists of a very large and heavily glycosylated luminal domain, a single transmembrane segment, and a short cytoplasmic tail. We show that in highly purified lysosomes from rat liver, Lamp II, immunodetected with a monoclonal antibody on Western blots, it surprisingly distributed between a membrane bound form and a "soluble" form. The partition of the protein between the membrane and the content of lysosomes is strongly pH dependent. The soluble Lamp II population is sensitive to pH dependent aggregation as it is for many lysosomal content enzymes.

Phagocytosis by rat liver: relationships between phagosomes and lysosomes.

To study the transfer of phagocytosed components from phagosomes to lysosomes, we have investigated phagocytosis by rat liver of killed Staphylococcus aureus labelled with (125)I tyramine cellobiose. Lysosomes were identified by injecting the animals with Triton WR1339, a non ionic detergent that is endocytosed by the liver and accumulates in lysosomes, causing a marked decrease of their density; that allows these organelles to be well separated from other particles in a density gradient. Bacteria were quickly taken up by the liver; their uptake is followed by a slow degradation as ascertained by the increase of acid-soluble radioactivity in the homogenates with time. Triton WR1339 injection does not affect the uptake and the degradation of the particles. Differential centrifugation of homogenates shows that at any time after injection, most of the radioactivity is recovered in the mitochondrial fractions. Distributions of acid precipitable and acid soluble radioactivities amongst subcellular structures present in mitochondrial fractions were studied by isopycnic centrifugation in sucrose gradients, at increasing times after bacteria injection. Results show that: 1) acid-precipitable radioactivity is quasi-exclusively present in gradient fractions of high density, well separated from the fractions where there are recovered lysosomes; 2) with time, acid-soluble radioactivity is more and more associated with lysosomes, however, a significant proportion can be detected for many hours after injection, in gradient fractions where acid-precipitable radioactivity is located. The most plausible explanation of our observations is that phagocytosed particles are degraded in phagosomes and that the degradation products are delivered to lysosomes, probably by a vesicular process.

Deleterious effects of xanthine oxidase on rat liver endothelial cells after ischemia/reperfusion.

Previous studies have demonstrated that reactive oxygen species are involved in ischemic injury. The present work was undertaken to determine in vivo the role of xanthine oxidase in the oxygen free radical production during rat liver ischemia and to examine the activity of antioxidant enzymes (superoxide dismutase, catalase and glutathione peroxidase) during the same period. Our results indicate a 4-fold increase in xanthine oxidase activity between 2 and 3 hours of normothermic ischemia, in parallel with a decrease in cell viability. Moderate hypothermia delays both events. Under the same conditions, the activity of oxygen radical scavenging enzymes remains unchanged. Moreover, we have compared in vitro the susceptibility of isolated liver cells to an oxidative stress induced by O2.-, H2O2 and .OH. Our results reveal that endothelial cells are much more susceptible to reactive oxygen species than hepatocytes, probably because they lack H2O2-detoxifying enzymes. These findings suggest that xanthine oxidase might play a major role in the ischemic injury mainly at the level of the sinusoidal space where most endothelial cells are located.

Uptake of exogenous DNA by rat liver: effect of cationic lipids.

We have investigated by using centrifugation methods, the uptake and the intracellular fate of 35S DNA by rat liver and the effect on these processes of N-(1-(2,3-dioleoxyloxy)propyl)-N,N,N,-trimethylammonium-methyl-sul fate(DOTAP, Boehringer, Mannheim, Germany), an artificial cationic lipid frequently used in transfection experiments. Labeled DNA molecules are quickly taken up by the liver but a progressive degradation takes place with time. Subcellular distribution of the radioactivity was established after differential and isopycnic centrifugation. Results indicate that 35S DNA enters liver cells by endocytosis and reaches lysosomes. The uptake of 35S DNA is not modified if the molecule is associated with DOTAP but marked differences are observed after internalization of the macromolecule. When DOTAP is used, radioactive products remain for a long time in low density organelles distinct from lysosomes indicating that the transfer of internalized DNA to these organelles is delayed by the cationic lipid. These results suggest that cationic lipids could favor transfection by preventing the delivery of DNA to lysosomes, allowing these molecules to be kept intact and available for transfer from endosomes to cytosol for a long time.

Altered intracellular processing and enhanced secretion of procathepsin D in a highly deviated rat hepatoma.

Both freshly-isolated rat hepatocytes and Morris hepatoma 7777 cells synthesized cathepsin D as a precursor that was either processed intracellular to smaller mature forms or secreted into the medium. The pattern of mature enzyme forms was different in the 2 cell types. In addition, the relative amount of precursor secreted was much higher for hepatoma cells. Monensin strongly enhanced the secretion and also impaired the intracellular transport-linked maturation of procathepsin D in hepatocytes, while it markedly inhibited intracellular maturation and only slightly increased secretion of the pro-enzyme in hepatoma cells. Ammonium chloride influenced the intralysosomal segregation and maturation of procathepsin D in hepatocytes but not in hepatoma cells. Our observations indicate that (i) the lysosomal segregation of cathepsin D was less efficient and its fractional secretion higher in hepatoma cells than in hepatocytes; (ii) in the 2 cell types, delivery to lysosomes and processing of procathepsin D were differently sensitive to increases in the vacuolar pH.

Uptake of dopamine by rat hepatocytes in vitro.

The present results showed that uptake of dopamine (DA) by rat isolated hepatocytes was mediated, in addition to simple diffusion, mainly by a transporter-involved process, with Km of 66.8 mumol and Vmax of 52.3 pmol.min-1/10(5) cells. The process was pH- and temperature-dependent and required an activation energy of 4.12 kcal.mol-1 (Q10 = 1.25) in the range of 2.0-12.7 C and 13.0 kcal.mol-1 (Q10 = 2.0) in the range of 12.7-39.0 C. Cysteine residue having free thiol group was unrelated to the activity of the transporter. Catecholamines, serotonin, and cocaine inhibited the DA transport, but tyramine (TA) and tryptamine, as well as benztropine and imipramine (which are potent inhibitors for hepatic TA transporter and neuronal DA transporter), had no inhibitory effect on the transport of DA in these cells. These results indicated that DA was taken up into hepatocytes by a distinct carrier. NaF and mastoparan influenced the transport activity in these cells further, suggesting that signal transducing G-proteins may be involved in the regulation of DA transporter in rat hepatocytes.

Uptake by rat liver of bovine growth hormone free or bound to a monoclonal antibody.

In the work reported here, we have compared the elimination from the blood, the uptake by the liver and the intracellular distribution of bovine growth hormone, free(Gh) or bound to a monoclonal antibody (GhAb). Results show that: a) the elimination from the blood is more rapid for Gh than for GhAb; b) both molecules are quickly taken up by the liver; c) probably after travelling through endosomes, Gh and GhAb get to lysosomes where they are degraded. However, Gh mostly ends in hepatocyte lysosomes while GhAb is recovered to a large extent in sinusoidal cell lysosomes; and d) binding by isolated hepatocytes is markedly less efficient for GhAb than for Gh.