Fusion of liposomes and rat brain microsomes examined by two assays (original) (raw)
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Low pH fusion of mouse liver nuclei with liposomes bearing covalently bound lysozyme
Biochimica et Biophysica Acta (BBA) - Biomembranes, 1987
Lysozyme covalently bound to liposomes induces the fusion of iiposomes with isolated mouse liver nuclei. The fusion behavior is very similar to the case of erythrocyte ghosts (Arvinte, T., Hildenhrand, K., Wahl, P. and Nicolau, C. (1986) Proc. Natl. Acad. Sci. USA 83, 962-966). Kinetic studies showed that membrane lipid mixing was completed within 15 min, as indicated from the resonance energy transfer (RET) measurements. For the resonance energy transfer kinetic measurements the iiposomes contained L-a-dipaimitoylphosphatidylethanolamine 0DPPE), labeled at the free amino group with the energy donor 7.nitrobenz-2-oxa-l,3-diazol-,l-yi (NBD) or with the energy acceptor tetramethylrhodamine. The lipid mixing at equilibrium was studied by the fluorescence recovery after photobleaching technique (FRAP). Liposomes (with/without lysozyme) containing Rh-labeled DPPE in their membranes were incubated with nuclei at 37°C, pH 5.2, for 30 min. After washing of nuclei by three centrifugations, 60-70% of the initial amount of labeled DPPE was associated with the nuclei in the ease of liposomes bearing lysozyme and only 7-10% in the case of iiposomes without lysozyme. For the nuclei incubated with liposomes having iysozyme, about 70% of the total Rh-labeled iipids present in the nuclei diffused in the nuclear membrane(s) (lateral diffusion constant of D = (1.4 + 0.5). 10 -9 cm2//s). By encapsulating fluorescein isothiocyanate-labeled dextran of 150 kDa molecular mass into the liposomes and using a microfiuorimetric method, it was shown that after the fusion a part of the liposome contents is found in the nuclei interior. In this lysozyme-induced fusion process between liposomes and nuclei or erythrocyte ghosts, the binding of lysozyme to the glycoconjugates contained in the biomemhranes at acidic pH seems to be the determining step which explains the high fnsogenic property of the iiposomes bearing iysozyme.
The Fusion of Liposomes to Rat Brain Microsomal Membranes Regulates Phosphatidylserine Synthesis
Journal of Neurochemistry, 1991
Rat brain microsomal membranes were fused to liposomes prepared with several pure lipids, namely, phosphatidylserine, phosphatidylinositol, phosphatidic acid, and mixtures of phosphatidic acid and phosphatidylcholine or phosphatidylethanolamine. The fusion between liposomes and microsomes was measured by the octadecyi rhodamine B chloride method. The extent and other properties of fusion largely depend on the lipid used to prepare liposomes; phosphatidic acid and phosphatidylinositol fuse more extensively than other lipid classes. The activity of serine base exchange is affected by the fusion between rat brain microsomes and lipids. It is strongly inhibited by phosphatidylserine, but it is activated by phosphatidic acid. The inhibition produced by phosphatidylserine on its own synthesis is proposed as a mechanism for controlling the formation of phosphatidylserine in rat brain microsomes. Key Words: Brain-Fusion-Microsome-Phosphatidylserine-Phospholipid.
Fusion of cationic liposomes with mammalian cells occurs after endocytosis
Biochimica et Biophysica Acta (BBA) - Biomembranes, 1995
The interaction of cationic liposomes prepared using either dioleoyltrimethylammonium propane (DOTAP) or 3~8-(N-(N',N'-dimethylaminoethane)carbamoyl)cholesterol (DC-CHOL) with model membranes and with cultured mammalian cells was examined using an assay developed for monitoring virus-cell fusion (Stegmann et al. (1993) Biochemistry 32, 11330-11337). Lipid mixing between cationic liposomes and liposomes composed of DOPE/dioleoylphosphatidyiglycerol (DOPG) or dioleoylphosphatidylcholine (DOPC)/DOPG was insensitive to pH in the range of pH 4.5-7.0 and was not affected by sodium chloride concentration in the range of 0-150 mM. Lipid mixing was dependent on dioleoylphosphatidylethanolamine (DOPE), since cationic liposomes prepared using dioleoylphosphatidylcholine (DOPC) were incapable of lipid mixing with DOPC/DOPG liposomes. The interaction of cationic liposomes with Hep G-2 and CHO D-cells was also studied. For both cell types, liposome-cell lipid mixing was rapid at 37 ° C, beginning within minutes and continuing for up to 1 hour after uptake. The extent of lipid mixing was decreased at 15 ° C, especially at later (> 20 min) time points. This suggests that at least part of the observed lipid mixing occurred after reaching cellular lysosomes. No lipid mixing was seen at 4 ° C. Monensin inhibited lipid mixing between cationic liposomes and the cells, despite having no effect on liposome uptake. Inhibition of endocytic uptake of liposomes, either by incubation in hypertonic media or by depletion of cellular ATP with sodium azide and 2-deoxyglucose abolished liposome-cell fusion in both cell types. These data demonstrate that binding to the cell surface is insufficient for cationic liposome-cell fusion and that uptake into the endocytic pathway is required for fusion to occur.
Induction of fusion in aggregated and nonaggregated liposomes bearing cationic detergents
Biochimica et Biophysica Acta (BBA) - Biomembranes, 1989
The addition of polyanlnnic polymers such as poly(aspartic acid) (PASP), DNA or dextran sulfate to liposomes composed of phosphatidyleholine (PC) and cholesterol (chol), and bearing the quaternary ammonium detergent ll[(i,l,3,3.tetramethylbutyl)crw,-~soxylethoxyIethytidlmethylbenzylammonium hydroxide (DEBDA[OH]) ~ulted in tipflsome .lg~egatlon mtd fusion. Lipo~m~llposome fusi~ w~ studied by ~ing fl~e~ently labeled liposomes and fluore~cance-dequenehi~tg (DQ) methods. Addition of monoanions, such as aspartate or acetate, to liposomes bearing DEBDAIOH] caused neither their a88regation nor liposome-Iip~me htsion. A~regation of liposomes bearing DEBDAIOH] by the binding pair avidin-biotin did not result in their fusion. Fusion in such agg~gated liposomes was observed by the addition of cha0n~c anions, such as niirate or thiocyanate, or by PASP. A variety of other quaternary ammonium detergents hehaved simgarly to DEBDAIOH] in their ability to confer f~sogenie properties upon PC/ehol llposomes. The relevance of these findings to the mech~ism of liposome-liposome fusion is discussed.
In vitro fusion of tissue-derived endosomes and lysosomes
European Journal of Cell Biology, 1998
Endosomelysosomefusionendocytosismembrane traffic We investigated the in vitro fusion of different endocytic compartments derived from perfused rat liver, where the cells are assumed to be in their physiological state. Specifically labelled early, late and transcytotic endosomes, as well as Iysosomes were tested for their fusion properties. In addition to the expected ATP-dependent fusion between early endosomes, we observed fusion between early and late endosomes with similar efficiency, kinetics and cytosol dependence. Fusion between early endosomes and transcytotic vesicles could not be detected. Prolonged incubation of complementary labelled early endosomes under fusion-supporting conditions followed by Percoll gradient centrifugation revealed the occurrence of fusion product at a dense position, indicating fusion events between light and dense compartments. Incubation of membrane preparations containing avidinlabelled endosomes and biotin-dextran-Ioaded Iysosomes resulted in the formation of avidin-biotin complexes, indicating that fusion between early and late endosomes is followed by fusion with Iysosomes. This was verified by colocalization of f1uorescently labelled endosomes and Iysosomes, as assessed by laser scanning microscopy. Endosome fusion, as well as content mixing between endosomes and Iysosomes, were dependent on temperature and ATP, and could be inhibited by N-ethylmaleimide (NEM). The NEM-sensitivity was localised on endosomes and in the cytosol, but not on Iysosomes. These observations indicate that early and late endosomes of rat liver exhibit a high fusion competence in vitro, promoting not only homotypic, but also heterotypic fusion. Abbreviations: BASOR Biotinylated asialo-orosomucoid.-BHK Baby hamster kidney cells.-CHO Chinese hamster ovary cells.-NEM N-ethylmaleimide. NSF NEM-sensitive fusion factor.-PNS post-nuclear supernatant.
Journal of Cell Biology, 2000
We have investigated the requirement for Ca2+ in the fusion and content mixing of rat hepatocyte late endosomes and lysosomes in a cell-free system. Fusion to form hybrid organelles was inhibited by 1,2-bis(2-aminophenoxy) ethane-N,N,N′,N′-tetraacetic acid (BAPTA), but not by EGTA, and this inhibition was reversed by adding additional Ca2+. Fusion was also inhibited by methyl ester of EGTA (EGTA-AM), a membrane permeable, hydrolyzable ester of EGTA, and pretreatment of organelles with EGTA-AM showed that the chelation of lumenal Ca2+ reduced the amount of fusion. The requirement for Ca2+ for fusion was a later event than the requirement for a rab protein since the system became resistant to inhibition by GDP dissociation inhibitor at earlier times than it became resistant to BAPTA. We have developed a cell-free assay to study the reformation of lysosomes from late endosome–lysosome hybrid organelles that were isolated from the rat liver. The recovery of electron dense lysosomes was ...
The Journal of Membrane Biology, 1994
We investigated the effect of several parameters, such as temperature, pH and proteins, on the fusion between synaptosomes, freshly isolated from rat brain cortex, and large unilamellar phosphatidylserine liposomes. These studies were carried out in both peroxidized and nonperoxidized synaptosomes. Mixing of membrane lipids was monitored using a fluorescence resonance energy transfer assay. Ascorbate (0.8 mM)/ Fe 2+ (2.5 gM)-induced peroxidation of synaptosomes enhanced the fusion process (twofold) which may reflect an increase in synaptosomal protein hydrophobicity and hence a facilitation of intermembrane aggregation. The fusion process was shown to be temperature sensitive, a reduction in the extent being observed (twofold) as the temperature was lowered from 37 to 25~ This effect may be due to changes in membrane fluidity. The fusion process is pH dependent, an increase in both kinetics and extent being observed when the pH was lowered from 7.4 to 5.5. A significant inhibition (92% at pH 7.4; 35% at pH 5.5) of the interaction between synaptosomes and liposomes by trypsin pretreatment of synaptosomes was found, thus indicating that the fusion reaction is a protein-mediated process. The inhibitory effect of trypsin at pH 5.5 is not so strong as that at physiological pH. These results suggest that, in addition to the involvement of proteins, nonspecific interactions between the synaptosomal and liposomal membranes under acidic conditions may also play a role in the fusion process. The investigation of binding of synaptosomes to liposomes under several experimental conditions provided evidence for the participation of proteins in membrane aggrega-Correspondence to: M.C. Pedroso de Lima tion, as well as for the role of electrostatic forces in this process, at mild acidic pH.
Biochemistry, 1988
Addition of the quaternary ammonium detergent [ [ [ (1,1,3,3-tetramethylbutyl)cresoxy]ethoxy]ethyl]dimethylbenzylammonium hydroxide (DEBDA [OH]) and the fluorescent probes N-(7-nitro-2,1,3-benzoxadiazol-4-y1)phosphatidylethanolamine and N-(lissamine rhodamine B sulfony1)phosphatidylethanolamine (N-NBD-PE and N-Rh-PE, respectively) to liposomes composed of phosphatidylcholine (PC) and cholesterol (chol) resulted in the formation of fluorescently labeled liposomes bearing DEBDA[OH]. Incubation of the anionic polymer poly(aspartic acid) (PASP) with such liposomes resulted in strong agglutination, indicating an association between the negatively charged PASP and the positively charged liposome-associated DEBDA[OH]. Addition of PASP to a mixture of fluorescently labeled and nonlabeled liposomes, both carrying DEBDA[OH], resulted in a significant increase in the extent of fluorescence, namely, fluorescence dequenching. The degree of the fluorescence dequenching was dependent upon the ratio between the nonfluorescent and the fluorescent liposomes, upon the temperature of incubation, and upon the amount * Author to whom correspondence should be addressed.