In vitro fusion of tissue-derived endosomes and lysosomes

166 European Journal of Cell Biology 77, 166-174 (1998, November) . © Gustav Fischer Verlag· Jena

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In vitro fusion of tissue-derived endosomes and Iysosomes Johannes A. Schmid 1) a, h, Isabella Ellingera, Paul Kosma c a b C

Department of General and Experimental Pathology, University of Vienna, Vienna/Austria Department of Vascular Biology, University of Vienna, Vienna/Austria Institute of Chemistry, University of Agricultural Sciences, Vienna/Austria

Received March 5, 1998 Received in revised version July 27,1998 Accepted August 11, 1998

Endosome - lysosome - fusion - endocytosis - membrane 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.

1) Dr. Johannes A. Schmid, Department of Vascular Biology, University of Vienna, Brunnerstr. 59, A-1235 Vienna/Austria, e-mail: [email protected], Fax: ++ 186634623.

Introduction After internalisation by different mechanisms of endocytosis, macromolecules are first delivered to an early endocytic compartment in the periphery of the cell. This is the first site where different sorting processes take place, in order to separate molecules that have to be recycled to the cell surface, from those which are destined for degradation in lysosomes. In polarised cells, like hepatocytes, an additional sorting event takes place at the stage of this early endocytic compartment, the segregation of molecules that are transported to the opposite cell pole by transcytosis. Endocytic membrane traffic can be described by at least two different models. The first one, designated as vesicle-shuttle model of endocytosis, assumes that molecules destined for degradation in lysosomes are packaged into carrier vesicles which bud off from early endosomes and are subsequently transported along microtubules to the perinuclear area of the cell where they fuse with late endosomes. The second model, claims that early endosomes change their properties (after segregation of recycling or transcytotic molecules) in a maturation process to those of late endosomes while being transported themselves towards lysosomes [17, 18]. Obviously, fusion processes are an inherent part of the vesicle shuttle model, whereas they would be not essential for the transfer of internalised molecules along the lysosomal pathway, according to the maturation model. However, in the latter model, fusion events would be necessary for the transfer of newly synthesised lysosomal enzymes from the trans-Golgi network to late endosomes. For various cultured cells, like BHK or CHO cells, fusion events of endocytic compartments could be reconstituted in vitro using content mixing of selectively labelled endosomes. These studies allowed a more detailed insight into the endocytic membrane traffic of the investigated cell types [1, 7, 10, 16]. The results implied that early endosomes are able to fuse in vitro with other early endosomes, but not efficiently with late endosomes. Early carrier vesicles (ECV's) which pinch off from early endosomes, were capable of fusing with late endosomes, but just in the presence of polymerised microtubules

EJCB [1]. Similar observations were made for MDCK cells, which serve as a model for polarised cell types: Apically labelled early endosomes derived from these cells, did not fuse in vitro with early endosomes labelled from the basolateral side. Nevertheless, apically labelled carrier vesicles could mix their contents with carrier vesicles derived from the basolateral side in the presence of intact microtubules, probably via intermediate late endosomes [3]. Taken together, these data led to the assumption, that the ordered course of membrane traffic along the endocytic pathway is based on the specificity of the fusion processes involved (reviewed in [17]). However, more recent data suggest that apical and basolateral endosomes of MDCK cells exchange their contents rapidly in vivo, generating an interconnected endosomal element [24]. Thus, it seems possible that endosomal compartments are interacting with each other more dynamically in vivo than in vitro, and that the vectorial traffic of molecules along this pathway is rather regulated by sorting signals within these molecules than by signals within the vesicles carrying them. Moreover, it could be dependent on the cell type or the physiological status of the cell, whether endosome fusion is highly specific or not. Regardless from the question which model applies for the traffic from early to late endosomes, the question has to be raised, how the molecules are transferred from late endocytic compartments to lysosomes. The observation that lysosomes in contrast to late endosomes do not contain significant amounts of mannose-6-phosphate receptors [19] would support a vesicle shuttle model for this stage of the lysosomal pathway, as well. However, intermediate carrier vesicles, as described for the transport between early and late endosomes of BHK cells [1] were not reported between late endosomes and lysosomes, but it could be shown that these two compartments are able to fuse directly with each other in vitro [22,23]. The fusion was shown to be dependent on cytosol and ATP, and to be sensitive to NEM. This indicates the potential role of NEM-sensitive fusion factor (NSF), which is essential for various other membrane fusion events [8, 29], in late endosomelysosome fusion, as well. The observations that some components of late endosomes are not found in lysosomes, but that fusion processes do occur between these two compartments fit to a recently postulated model, where lysosomes fuse temporarily with endosomes ("kiss and run-model", [34]), thereby transferring lumenal molecules, as well as small vesicles inside of endosomes (multivesicular bodies) without major mixing of the surrounding membranes. However, the question of the specificity of this fusion event has not been exactly addressed. Moreover, it was not investigated in detail, whether endosomes derived from cells in the physiological environment of the organ, exhibit the same fusion specificity, as observed between endocytic compartments from cultured cells. Therefore, we aimed to investigate the fusion properties of endocytic compartments from rat liver, using a content-mixing assay with selectively labelled early or late endosomes and lysosomes. Our results demonstrate that tissue-derived early endosomes are able to interact in vitro with late endosomes in the absence of intact microtubules. In vitro, a content mixing can be observed also with lysosomes, likely via intermediate late endosomes. These observations indicate that the directional course of endocytic transport in rat liver cells is probably not regulated by a strict specificity of membrane fusions, as observed in BHK cells.

In vitro fusion of tissue-derived endosomes ond Iysosomes

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Materials and methods Materials

Biotin (NHS-LC-Biotin) was purchased from Pierce (Rockford, USA), peroxidase-conjugated anti-avidin antibodies were from Dako (Glostrup, Denmark) and XRITC-dextran was a gift from Prof. R. F. Murphy, Carnegie Mellon Univ., Pittsburgh, USA. Polymeric IgA was a gift from J. P. Vaerman. All other materials were purchased from Sigma (Munich, Germany). Galactosylated avidin was prepared at a molar ratio of 40: 1 as described [20,32]. Male Louvain rats were provided by the local animal breeding farm (Versuchstierzucht der Universitat Wien, Himberg, Austria) and had free access to food and water.

Labelling of endosomes and preparation of microsomal fractions Rat liver endocytic compartments were labelled in the isolated perfused rat liver essentially as described [15, 35]. Early endosomes were either labelled by short time labelling at 37°C or by recirculating perfusion for 2 h at 16°C under conditions where transport to late endosomes is blocked [21, 27, 35]. Pulse labelling for 1 min at 3rC was used for loading with FITC-dextran (IOmglml in KrebsHenseleith buffer, KHB: 118.4 mM NaCl, 4.75 mM KCl, 1.19 mM KH2 P0 4 , 1.185 mM MgS0 4 · 7H20, 2.57 mM CaCI 2 , 25 mM NaHC0 3 , 1 gil D-glucose' H 20). Labelling with avidin (1 mglml in KHB) was done for 2 h at 16°C with buffer containing 2 mg/ml BSA and 1 mglml mannan to prevent uptake by endothelial cells and Kupffer cells. The loading of early endosomes with BASOR was carried out by perfusion for 3 min at 3rC with 250-500 ~g of the marker [35]. Labelling of early endosomes with biotinylated polymeric IgA (B-pIgA) was achieved by intravenous injection of 200 ~g of the marker followed by ice-cold perfusion in situ 2 min after the injection. Late endosomes were labelled in the isolated perfused rat liver either with 1 mglml galactosylated avidin (gal-avidin; including 2 mglml mannan and 2 mgl ml BSA), or with 500~g BASOR for 3min at 37°C followed by marker-free perfusion for 10 min at 37°C. For the labelling of transcytotic vesicles, the same pulse-chase conditions were applied using 500 ~g B-IgA as marker. After differential labelling of early endosomes, late endosomes or transcytotic compartments, the livers were immediately cooled by perfusion with 200 ml ice-cold Hanks'-buffer containing 5 mM EGTA, to prevent further transport of the marker to subsequent compartments followed by perfusion with ice-cold 50 ml KAcSHM-buffer (100mM K-acetate, 85mM sucrose, IOmM Hepes, 1 mM MgCh, pH 7.4) containing 2~glmlleupeptin, 0.7~glml pepstatin and 50 ~glml PMSF as protease inhibitors. The tissue was homogenised in KAcSHM-buffer (3 ml/g liver) containing protease inhibitors using a Dounce homogenizer (6 strokes with a loose fitting and 2 strokes with a tight fitting pestle). The homogenate was centrifuged at 1500g for 10 min and the supernatant was subjected to centrifugation at 3400g for 10 min to obtain a post-nuclear supernatant (PNS, protein content: 30 mglml). Microsomes were prepared by centrifugation of PNS at 42000g for 70min. After removal of the supernatant the pellet was resuspended in KAcSHM buffer (in O.4ml/g liver) by 6 strokes of a tight fitting Dounce homogenizer. Aliquots of the resulting membrane preparations were frozen in liquid nitrogen and stored at -70°C. A decrease in fusion competence could not be observed within one year.

Preparation of endosome-enriched membrane fractions Endosome-enriched Golgi fractions were prepared by float-up centrifugation of microsomes. The microsomal pellet was suspended in 4 ml 1.15 M sucrose, transferred into a centrifugation tube and overlaid with 4 mil M sucrose and 4 ml 0.25 M sucrose. The step gradient was centrifuged at 200000g for 1.5 h in a swing-out rotor. Endosomes, which accumulate at the interface between 0.25 and 1 M sucrose, were collected with a syringe and suspended in 25 ml fusion buffer. The membranes were pelleted at 42000g for 70min, and resuspended in 1 ml fusion buffer.

168 J. A. Schmid, I. Ellinger, P. Kosmo Labelling of Iysosomes and preparation of lysosomal fractions 10 mg biotin-dextran or 20 mg XRITC-dextran were dissolved in 0.8 ml PBS and injected intravenously into rats. After 1.5-2 h the liver was removed and perfused with KHB for 1 h at 37 DC to chase the marker from endosomes into Iysosomes. Afterwards, the liver was cooled by perfusion as described, followed by perfusion with ice-cold KCISHM buffer (100 mM KCI, 85 mM sucrose, 10 mM Hepes, 1 mM MgCIz, pH 7.4) containing protease inhibitors. Homogenisation of the liver was carried out as described before. Nuclei and unbroken cells were removed by centrifugation at 500g for 10 min, followed by pelleting of Iysosomes at 27000g for 10 min. The pellet was resuspended in KCISHM buffer containing protease inhibitors (in 0.6ml/g liver) by 4 strokes in a loose fitting Dounce homogenizer. Aliquots were frozen in liquid nitrogen and stored at -70 DC. There was no significant loss of fusion competence detectable after storage.

Percoll gradient centrifugation After in vitro fusion for various periods of time, aliquots were removed, diluted in 5 ml of ice-cold gradient buffer (53 mM NaCI, 53 mM KCI, 132 mM sucrose, 1.1 mM EDTA, 5.3 mM Hepes, pH 6.8) and mixed with an equal volume of 66 % Percoll (Pharmacia) in 0.25 M sucrose. The suspension was subjected to centrifugation at 4 DC in a vertical rotor (Beckman VTi 50) for 60min at 25000g with slow acceleration and deceleration resulting in 1.18 x 1010 rad2/sec. The gradient was fractionated and membranes were lysed by addition of stopbuffer. The amount of avidin-BASOR fusion product in the fractions was quantified by ELISA as described.

Statistics The given values represent the mean of at least 3 independent measurements. Error bars indicate the standard deviation.

Cytosol preparation An isolated rat liver was perfused for lOmin at 37 DC with KHB, followed by perfusion with ice-cold KHB for 7 min and perfusion with 50ml of ice-cold KAcSHM. The liver was homogenised in KAcSHM as described (2.5 ml/g liver). The homogenate was centrifuged at 12000g for lOmin followed by centrifugation at 100000g for 60min. The fatty layer on top of the supernatant was removed, and the clear supernatant (cytosol) was stored in aliquots at -70 DC (protein concentration: 35 mg/ml). This primary cytosol was desalted using a Sephadex G25 column equilibrated with KAcSHM to prepare gel-filtered cytosol. Depletion of NEM-sensitive fusion factor (NSF) was carried out by incubation of gel-filtered cytosol at 37"C for 15 min [2].

Preparation of peripheral membrane proteins

Rat liver microsomes were washed with hypotonic buffer (20 mM Hepes-KOH, pH 7.0; 0.5 mM EGTA), pelleted for 15 min at 200000g (in a Beckman Airfuge), suspended in high salt buffer (0.5 M KCI in hypotonic buffer) and incubated for 30 min on ice to release peripheral membrane proteins. The suspension was again centrifuged for 15 min at 200000g and the supernatant was spin desalted on a Sephadex G25 column equilibrated with fusion buffer. The preparation contained approximately 3 mg/ml of peripheral membrane proteins and was stored at -70 DC.

In vitro fusion assays In vitro fusion was carried out in KAcSHM or KCISHM buffer, containing 0.2 % BSA, 2 mM MgCIz, 1 mM DTI, 20 !-1M EGTA and 50!-lg/ ml biocytin. Cytosol was usually added at a final concentration of 20 mg/ml. Either an ATP-regenerating system (final concentration: 2.5mM K-ATP, pH 7.4; 8mM phosphocreatine and 11 ulml phosphocreatine kinase) or an ATP-depleting system (5 u/ml hexokinase, 10 mM deoxy-glucose) was added. All reagents were mixed on ice, followed by addition of 10 times concentrated fusion buffer and distilled water to give an isotonic solution before avidin-labelled endosomes were added. The suspension was carefully mixed and incubated on ice for 5 min to allow the blocking of extravesicular avidin by biocytin. Biotin-dextran labelled Iysosomes were added and the reaction was started by warming to 37 DC. After incubation for the time indicated in the figure legends, the fusion reaction was stopped by lysis of the membranes with stop buffer (VIO of the assay volume: 10 % Triton X-lOO, 1 % SDS and 0.5 mg/ml biocytin). The avidin-biotin-dextran complex that had formed during the fusion reaction was measured by ELISA as described [26]. Avidin-BASOR complex was quantified in the same way, using anti-ai-acid glycoprotein 1: 1000 for coating of plates. Treatment of membranes or cytosol with NEM was carried out for 30 min on ice (final concentration: 2mM), followed by a quenching of excess NEM with 3 mM DTI (for 10 min on ice) before mixing of the individual components and starting the fusion reaction. Confocal laser scanning microscopy was carried out with a MRC600 model (BioRad, Munich, Germany) mounted on a Zeiss Axiovert microscope, using a 63 x oil immersion objective with a numerical aperture of 1.40 and filter sets for separation of fluorescein and rhodamine fluorescence.

Results We aimed at investigating the in vitro fusion properties of endosome fractions derived from cells in the physiological environment of their tissue. For that purpose we used the model of isolated perfused rat liver. Since it is difficult to obtain purified preparations of early and late endosomes, respectively, we relied on the possibility of differential labelling that can be achieved in that system. We applied the welldefined labelling conditions of Wall and Hubbard [35] to load early, sinusoidal endosomes or late, perinuclear endosomes with biotinylated asialo-orosomucoid (BASOR), which is internalised by the asialoglycoprotein receptor. In addition we used biotinylated polymeric IgA (B-pIgA) for the labelling of early or transcytotic endosomes. The differentially labelled endocytic compartments were characterised in our group by various techniques to prove the selectivity of the labelling. First, morphological analysis revealed a clearly distinct distribution, showing labelled early endosomes at the periphery of hepatocytes whereas late endosomes were located in the pericanalicular area (data not shown). Moreover, early endosomes could be clearly distinguished from late endosomes and transcytotic vesicles by free-flow electrophoresis of endosomeenriched Golgi fractions [9, 30]. Although, a complete separation of early and late endosomes can not be achieved by that technique, the tracers occurred as single peaks with a Gaussian-like distribution arguing against a significant colabelling of both compartments. Finally, membrane preparations containing differentially labelled early or late endosomes were unequivocally different in their characteristics of in vitro acidification, showing a higher lumenal acidity for late endosomes ([12] and additional data not shown). For in vitro fusion assays, post-nuclear supernatants containing BASOR-Iabelled early or late endosomes, respectively, were incubated at 37°C in the presence of cytosol with microsomes containing avidin-labelled early endosomes. We observed an ATP-dependent fusion signal not only between early endosomes, but also between early and late endosomes with comparable efficiency (Fig.la). Similarly, homotypic fusion between late endosomes could be detected (data not shown). In addition, we set up a fusion assay with early endosomes and transcytotic vesicles containing biotinylated polymeric IgA (B-pIgA). In contrast to the fusion between early endosomes, no significant fusion was detected between avidinloaded early endosomes and B-pIgA labelled transcytotic vesicles (Fig. Ib). Thus, the segregation of endocytic and transcy-

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Fig. 1. Early endosomes fuse in vitro with complementary labelled early endosomes or late endosomes, but not with transcytotic compartments. (a) In vitro fusion between early endosomes (EE/EE) or between early and late endosomes (EEILE) in the presence of an ATPregenerating system (ATP-regen.) or an ATP-depleting system (ATPdepl.). Avidin-labelled early endosomes were suspended at DoC in fusion buffer containing biocytin as scavenger for extravesicular avidin. Cytosol was present at a concentration of 25 mglml. BASORloaded early or late endosomes were added and the fusion reaction was started by warming to 37°C. After 30 min the reaction was stopped by mixing with lysis buffer containing biocytin. The full signal was determined by mixing of avidin- and BASOR-loaded membrane fractions with detergent in the absence of biocytin. The amount of avidinBASOR complexes was measured by ELISA. (b) Avidin-labelled early endosomes (EE) were incubated with membrane preparations containing either early endosomes loaded with biotinylated pIgA (E-IgA) or transcytotic compartments loaded with biotinylated pIgA (T-IgA). The incubation was for 30 min at 37°C in the presence of an ATPregenerating system (ATP-regen.) or an ATP-depleting system (ATPdepl.).

totic pathways that is observed in vivo, is apparently reflected by the fusion specificity in vitro. In vitro fusion between avidin-loaded early endosomes and BASOR-labelled early or late endosomes was not only observed with crude membrane preparations, but also with endosome-enriched membrane fractions. In that case, the signal related to vesicular protein was about 45 times higher than with microsomal fractions, proving that the process was endosome-specific. However, we noticed that the fusion signal related to the amount of marker in the sample was slightly lower with endosome-enriched membrane fractions. This could be an indication for a loss of fusion factors or a partial damage of the endosomal integrity caused by the purification process. Due to this observation and in order to keep the in vitro system close to the physiological conditions, we decided to use post-nuclear supernatants or microsomal membrane preparations for the fusion assays. For quantification of the fusion efficiency, we related the fusion signal to the maximum amount of avidin-BASOR complexes that could be obtained with the applied membrane

169

preparations after addition of detergent to the samples in the absence of biocytin. At a cytosol concentration of 25 mg/ml, the fusion signal (in the presence of biocytin as scavenger for extravesicular marker) reached about 48 % of the maximum signal. Since the part of marker which is outside of the vesicles contributes to the maximum signal, but not to the fusion signal in the presence of biocytin, the fusion efficiency has to be calculated considering the latency of the limiting marker. For PNS, the latency of the marker (BASOR) was in the range of 70 %. Thus, a fusion efficiency of about 68 % was obtained for both early/early and earlyflate endosome fusion. The high fusion efficiency was apparently reached in the absence of intact microtubules, since the preparation of cytosol and membrane fractions at 4 ee disrupts microtubules [14]. Moreover, neither homotypic fusion between early endosomes nor heterotypic fusion between early and late endosomes were affected by incubation in the presence of 20 ftM taxol which induces the polymerisation of endogenous tubulin [3], indicating that both fusion processes are rather independent from intact microtubules. The formation of microtubules in the presence of taxol was verified by confocal microscopy using anti-tubulin antibodies (data not shown). Despite the differences between early and late endosomes in free-flow electrophoresis, in vitro acidification and intracellular localization, we observed striking similarities between early/early and earlyflate endosome fusion not only for the efficiency of the fusion and the independence from intact microtubules, but also concerning the kinetics and cytosoldependence of the fusion process. The fusion could be detected without any particular lag phase with a rapid increase of the signal within the first 5 min after warming to 37 ee, reaching a plateau after 5 to 10 min which persisted for at least 30min, indicating that the avidin-BASOR complexes are not degraded within that time (Fig.2a). Furthermore, we found very similar requirements for cytosolic factors. For both early/ early and early/late endosome fusion, the cytosol dependence was remarkably high with an increase of the fusion signal up to the highest protein concentration that we could reach in our system (about 25 mg/ml; Fig. 2b). The fast kinetics of the reaction without any detectable lag phase implies that the fusion partners might interact with each other already at O°c. This was supported by experiments on the NEM-sensitivity of the fusion. When the BASORcontaining membrane preparation was mixed at ooe with microsomes containing avidin-labelled endosomes before addition of NEM (2 mM), only a minor reduction of the fusion signal was observed. In contrast, separate treatment of BASOR- and avidin-labelled endosomes with NEM at ooe, followed by mixing of the components and incubation at 37 ee, abolished the fusion signal nearly completely (Fig.3a). The simplest explanation for this observation is that the complementary labelled endosomes interact with each other already at 0 ee and form a pre-fusion complex which is insensitive to a subsequent treatment with NEM. The interaction at 0 ee is apparently inhibited by prior treatment of the separated membranes. The strong inhibition of endosome fusion in that case (without NEM-treatment of the cytosol), indicates that NEMsensitive factors which are essential for the fusion, are associated with endosomal membranes. Interestingly, preincubation of one endosome subset under fusion-supporting conditions eliminated its capacity to fuse with the complementary labelled endosome population in a

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It has been reported that late endosomes and lysosomes from rat liver are able to fuse in vitro [23]. Therefore, our findings of fusion between early and late endosomes imply the possibility of further content mixing with lysosomes. To test that, we established an assay to detect the fusion between endosomes and lysosomes derived from rat liver. For this purpose, we used lysosomes that were loaded with the non-degradable marker biotin-dextran. Early endosomes labelled with avidin, or late endosomes labelled with galactosylated avidin, were used as potential fusion partners. In the latter case, the marker was taken up by the asialoglycoprotein receptor, which could be confirmed by competition with asialo-orosomucoid (data not shown). To obtain a selective labelling of lysosomes, we applied the marker in situ for 1.5-2 hours, followed by perfusion of the liver for another hour, to chase residual endosomal marker into lysosomes. As shown in Fig. 5, incubation of post-nuclear supernatants containing early or late avidin-loaded endosomes, respectively, with biotin-dextran-labelled lysosomes for 30 min under the conditions used for in vitro fusion of endosomes, led to an ATP-dependent content mixing in both cases. Since early endosomes were able to fuse with late endosomes in our sys-

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Fig. 2. Kinetics and cytosol dependence of early/early (EE/EE) and earlynate (EEILE) endosome fusion. (a) Kinetics of endosome fusion. Aliquots of fusion samples incubated in the presence of an ATPregenerating system ( + ATP) or an ATP-depleting system (-ATP) were taken at the indicated time points after warming to 37°C and the reaction was stopped by addition of lysis buffer including biocytin. (b) Cytosol dependence of endosome fusion. Different amounts of cytosol were added to the fusion buffer prior to the addition of membrane fractions. Incubation was for 30 min at 37°C in the presence of an ATPregenerating system.

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subsequent fusion assay, indicating that essential fusion factors are depleted during the preincubation. Most strikingly, the fusion signal could be restored after the preincubation by addition of peripheral membrane proteins, but not by addition of cytosol (Fig. 3b). The fusion-restoring potential of peripheral membrane proteins was abolished by treatment with NEM, supporting the notion that NEM-sensitive fusion factors that are associated with endosomal membranes, are depleted during the preincubation. Since early and late endocytic compartments of rat liver differ in their density [22], fusion between these compartments would probably induce an increase in density of the early endosomes that are involved. To test this possibility, we incubated membrane preparations under fusion conditions for prolonged time and subjected the suspension to Percoll gradient centrifugation, followed by quantification of the avidinBASOR fusion product in the fractions. Interestingly, the fusion complex could be found at a quite dense position of the gradient close to that of lysosomes. This was even the case when both markers were in early endocytic compartments, indicating that fusion between early, light endosomes can be followed by subsequent fusion with compartments of higher density, thus implying multiple fusion events (Fig. 4). The occurrence of the fusion product at a denser position seemed to be a fusion-specific process, since the bulk of membranes was not shifted and a significant part of endosomal marker remained at a light position of the gradient, arguing against an unspecific aggregation of membranes.

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Fig. 3. Membrane-association of NEM-sensitive fusion factors. (a) Membrane fractions containing BASOR-Iabelled endosomes (donor) or avidin-labelled endosomes (acceptor) were either left untreated (-) or incubated with 2 mM NEM ( +). The incubation of membranes with NEM was either carried out separated from each other or after mixing at O°C (combined). The cytosolic fraction was left untreated. (b) Avidin-labelled early endosomes were preincubated for 0 min or 30 min at 37°C in the presence of cytosol and an ATP-regenerating system. After the preincubation cytosol, peripheral membrane proteins (p.m.p.) or NEM-treated peripheral membrane proteins (p.m.p.l NEM) were added as indicated, followed by addition of BASORlabelled early endosomes and subsequent incubation at 37°C for 30min.

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Fig. 4. Density after in vitro fusion of endosomes. Microsomes containing BASOR-Iabelled early endosomes were incubated with avidinlabelled early endosomes in the presence of cytosol and an ATPregenerating system for 90 min at 37°C. The fusion was stopped by dilution with ice-cold fusion buffer and the suspension was mixed with isotonic Percoll-solution followed by density gradient centrifugation as described in Materials and methods. The gradient was fractionated from the bottom and the membranes were lysed by addition of detergent solution. The amount of avidin-BASOR-complex was quantified in the fractions by ELISA as described and compared to the activity of the lysosomal enzyme ~-N-acetylhexosaminidase. The normallocalisation of early endosomes, late endosomes and Iysosomes on the gradient is indicated by arrows.

tern, we assume that the content mixing between early endosomes and Iysosomes occurs via an intermediate earlyllate endosome fusion. Although early endosomes, labelled at 16°C for prolonged time, cannot be distinguished by morphology or free-flow electrophoresis from early endosomes loaded by short-time labelling at 37°C, they could be somehow primed for late endosomal characteristics, thereby influencing their interaction with late endosomes and subsequently with Iysosomes. To test this possibility and to obtain additional evidence for an interaction in vitro between early endosomes and Iysosomes, we investigated whether early endosomes pulselabelled for 1 min at 37°C with FITC-dextran show mixing with XRITC-dextran-Iabelled lysosomes upon incubation of post-nuclear supernatants under fusion-supporting conditions. Morphological analysis by confocal laser scanning microscopy revealed a partial colocalization of both markers after the incubation (Fig. 6). Although this method can not distinguish between aggregation and fusion, the formation of avidinbiotin-dextran complexes under the same incubation conditions indicates that content mixing can occur between kinetically early endocytic compartments and Iysosomes in vitro, most likely via intermediate fusion with unlabelled late endosomes. As observed for the in vitro fusion of endosomes, treatment of the membrane components with NEM was able to suppress the content mixing. For a further differentiation of the effect of NEM on the fusion reaction, we used normal or NSFdepleted cytosol, either untreated or treated with NEM. In addition, we incubated either just one or both membrane preparations with NEM, in order to find out whether NEMsensitive fusion factors are localised on both endosomes and Iysosomes. Treatment of normal, gel-filtered cytosol with NEM resulted in 70 % reduction of the fusion signal (Fig. 7). NEM treatment of the lysosomal fraction only, did not inhibit the content mixing between early endosomes and lysosomes,

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Fig. 5. Content mixing in vitro of early and late endosomes with Iysosomes. Microsomal membrane preparations with avidin-labelled early endosomes (EE) or gal-avidin labelled late endosomes (LE) were incubated in the presence of cytosol (20 mg/ml) and biocytin with biotin-dextran-Iabelled lysosomes (Lys). The reaction was carried out either under ATP-regenerating or ATP-depleting conditions and the signal was compared to the maximum signal after lysis of the vesicles in the absence of biocytin. suggesting that Iysosomes do not contain NEM-sensitive fusion factors. In contrast, NEM treatment of endosomal membranes inhibited fusion by 80 %, indicating that NEMsensitive factors that are associated with the endosomal membrane, are important for the fusion process. Incubation of the cytosol for 15 min at 37°C which was reported to completely inactivate NSF [2], reduced the fusion signal only by 20 % of controls, whereas NEM-treatment of NSF-depleted cytosol caused a reduction by 70 %, implying that additional NEM-sensitive factors different from NSF are present in the cytosol. These unknown cytosolic factors are apparently not able to compensate for the NEM-sensitive factors on endosomal membranes, since the combination of NSF-

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Fig. 6. Confocal laser scanning microscopy after incubation of early endosomes with lysosomes. Early endosomes were labelled by perfusion of rat liver for 1 min at 37°C with FITC-dextran, Iysosomes were labelled in situ with XRITC-dextran, followed by marker-free perfusion of the liver to chase marker from endosomes to Iysosomes. The differently labelled membrane fractions were incubated for 30 min in vitro under fusion supporting conditions in the presence of an ATPregenerating system. The membrane suspension was mounted on a glass slide and investigated by confocal laser scanning microscopy. Filter sets for the separation of fluorescein and rhodamine fluorescence were used. (A) Fluorescence of the FITC-channel, (B) fluorescence of the rhodamine channel. Two compartments are visible with a clear colocalisation of markers. Some other compartments display only FITC-dextran fluorescence, thereby showing that no spill-over of the different fluorescence signals occurs.

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Fig. 7. Effect of NEM on content mixing between early endosomes and Iysosomes. Cytosol was filtered through a Sephadex G25 column equilibrated with fusion buffer (gel-filtered cytosol) or filtered and depleted of NSF by incubation at 37°C for 15 min (NSF-depleted cytosol). Cytosol (Cyt), Iysosomes (Lys) or both Iysosomes and endosomes, separated from each other (Lys/EE) were treated with 2mM NEM at O°C for 30 min followed by quenching of excess NEM with 3 mM DTI before starting the fusion reaction by warming to 37°C. The fusion signal is expressed in percent of the control fusion.

depleted cytosol and NEM-treated endosomes abrogated the fusion signal completely. In contrast, NEM-treated endosomes combined with cytosol containing NSF exhibited about 20 % residual fusion activity indicating that the cytosolic NSF is capable of taking the role of the membrane-associated NEM-sensitive factors.

Discussion Our results strongly support the view that early endosomes of rat liver are able to fuse in vitro not only with each other, but also with late endosomes with similar high efficiency, but not with transcytotic vesicles. Since late endosomes of rat liver are able to fuse in vitro with lysosomes [23], a fusion between early and late endosomes could subsequently result in content mixing with lysosomes. However, for cultured cells, studied so far, like CHO or BHK cells, it was shown that early endosomes do not fuse efficiently with late endosomes in vitro [1, 4, 10]. For these cell types, a clear difference in the protein composition of early and late endosomes was reported [1, 10, 31]. Moreover, it could be shown for BHK cells, that endocytosis in the presence of the microtubule-depolymerising agent nocodazol, leads to the labelling of carrier vesicles between early and late endocytic compartments, which are no longer capable of fusing in vitro with early endosomes, but show content mixing with late endosomes in the presence of intact microtubules [1, 10]. In contrast to the distinct protein composition of different endocytic compartments in CHO or BHK cells, it was reported that early and late endosomes from rat liver exhibit a nearly identical protein pattern, which was taken as an argument for a potential maturation process from early to late stages of endocytosis in hepatocytes [5, 11]. At the other hand, this similarity would be a possible explanation for the findings that early and late endosomes of rat liver are able to fuse with each other in vitro. In any case, the fact that early and late endosomes of different cell lines or tissues, can either be very similar or clearly different in their composition leads to the suggestion that they could also be different in their

fusion abilities with other compartments along the endocytic pathway. In our model system of tissue derived endosomes, we found that a fusion process does occur between early and late endocytic compartments, and in addition, that it proceeds with a remarkably high efficiency. We do not think that a contamination of our late endosome membrane fraction with labelled early endosomes, could cause the observed fusion signal between early and late endosomes, since at least 75 % of the marker are localised in late endosomes after the applied labelling conditions [35]. Moreover, the observed fusion efficiency of early/late endosome fusion was very similar to fusion of early endosomes with each other, rendering it unlikely, that the fusion signal can be explained simply by the presence of some labelled early endosomes in the late endosome preparation. A further support for this notion is the observation of a content mixing in vitro between labelled early endosomes and lysosomes upon incubation of post-nuclear supernatants. An interaction could be observed even with very specifically labelled compartments, using early endosomes labelled for only one minute with FITC-dextran and lysosomes labelled for prolonged time in vivo followed by 1 h chase. Since late endosome/lysosome fusion has been shown already for rat liver [23], the observed mixing of early endosomal and lysosomal tracers might be explained by fusion between early and late endosomes. The fact that we did not observe fusion between early endosomes and transcytotic vesicles implies that fusion selectivity might be important for the segregation of lysosomal and transcytotic pathways in hepatocytes, while probably other mechanisms than fusion specificity might regulate the vectorial course of endocytic transport to lysosomes. The observed difference between some cultured cells and rat liver-derived cells concerning the specificity of endosome fusion indicates that the interactions of endosomes are probably not regulated by mechanisms that are common for all cell types. This is supported by our findings that also the characteristics of fusion differs from that reported for different cultured cells. The kinetics of endosome fusion, for instance, were faster than many other comparable fusion processes [4, 7,16]. Interestingly, similar kinetics were reported for the in vitro fusion of endosomes derived from the human hepatoma cell line HepG2 [25]. In addition, the cytosol dependence is similar for HepG2 and rat liver endosome fusion, but is clearly different from that of CHO cells [31] or macrophages [6], thus further implying that the characteristics of endosomal membrane fusion is dependent on the cell type. The fast kinetics combined with the observation that no lag phase exists for rat liver endosome fusion suggests that the fusion partners might interact already at O°C with each other, thus leading to an immediate fusion signal after warming to 37°C. This is supported by the findings that treatment of both membrane preparations together with NEM after mixing at O°C could not significantly inhibit the fusion, whereas separate incubation of the membranes with NEM at O°C prevents a subsequent fusion nearly completely. Since a strong inhibition of the fusion, was achieved with treatment of the membrane preparations without any treatment of the cytosol, it is evident that an essential part of the NEM-sensitivity is localised on endosomal membranes. This is in agreement with a recent report, showing that NSF is already associated with membranes of clathrin-coated vesicles [33]. However, in many other studies of endosome fusion, NEMsensitive factor(s) were found mainly in the cytosol fraction [4, 25], or just in part associated with membranes [7]. Neverthe-

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less, the latter study implied already an equilibrium between membrane-bound and unbound forms, where the relation could be dependent on the cell type or the physiological status of the cell. In addition to the well-defined NSF, which is essential in a majority of membrane fusion events, other NEMsensitive factors have been shown to playa role in endosome fusion of different cultured cells [28, 36]. Similarly, our results of the rat liver model indicate that NEM-sensitive factors different from NSF contribute to the process of endosomelysosome fusion. The observation that NSF-depletion of the cytosol caused only a reduction of the fusion signal by 20 % , whereas NEM-treatment of gel-filtered cytosol decreased the signal by 70 %, leads to the suggestion that an additional NEM-sensitive factor is involved in the process. This factor is apparently not able to substitute for the NEM-sensitive factors on the endosomal membrane, since the combination of NEM-treated endosomes and Iysosomes with NSF-depleted cytosol, eliminated the fusion signal completely. A tentative model {Fig. 8) would predict that about 50 % of the NEMsensitivity are attributed to NSF, with about 20 % localised in the cytosol and 30 % associated with endosomal membranes, and that another 50 % of the NEM-sensitivity are different from NSF and are localised in the cytosol. According to this model, NSF is assumed to be essential for the fusion process, with the ability to shuttle between a membrane-associated and a cytosolic status. The other NEM-sensitive factor apparently stimulates the reaction, but is not capable of taking the role of NSF. Clearly, further investigations are necessary to characterise this factor and to elucidate its role in endocytic membrane traffic of rat liver. In conclusion, our results imply that fusion events between early and late endocytic compartments of rat liver do occur in vitro. A possible explanation for this observation would be that the factors regulating a fusion selectivity in vivo are not active in our in vitro assay. However,. the given similarity in protein composition of early and late endosomes in hepatocytes [5, 11), implies that they are structurally and functionally not as different as observed for BHK or CHO cells. This would be in agreement with a recent report [13], describing that endosomes of HEp-2 cells containing EGFEGF receptor complexes constitute functionally a single compartment which follows a maturation process before fusion with Iysosomes. Nevertheless, endocytic membrane traffic of all cells investigated so far, is vectorial and specific. In principle, this vectorial nature can be maintained by a specificity of the fusion events involved, as assumed for BHK cells, but it

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could be also maintained by other mechanisms like the spatial organisation of the compartments in the cell and sorting of certain components at defined stages of the pathway. In this case, fission processes instead of fusion events would regulate the endocytic traffic. It could be dependent on the cell type or the physiological status of the cell whether fusion or fission of membranes is actually regulating the ordered course of endocytosis. However, additional studies are required to elucidate the processes of endosome fusion, as well as endosomelysosome fusion, especially for cells that are close to their normal physiological status within the tissue. Acknowledgments. We gratefully acknowledge the contributions of Renate Fuchs and Sandra L. Schmid to both the practical and theoretical basis of this project, as well as to stimulating discussions. We also thank Robert E Murphy for providing us with XRITC-dextran, and Peter Wyskowsky for excellent technical assistance. This work was supported by the Austrian Science Foundation (Project: 8435-Med).

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c.,

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50% Fig. 8. Theoretical model of NEM-sensitive factors in endosomelysosome fusion as explained in the main text.

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174 J. A. Schmid, I. Ellinger, P. Kosmo [14] Goltz, J. S., A. W. Wolkoff, P. M. Novikoff, R. J. Stockert, P. Satir: A role for microtubules in sorting endocytic vesicles in rat hepatocytes. Proc. Natl. Acad. Sci. USA 89,7026-7030 (1992). [15] Graf, J., M. Peterlik: Ouabain-mediated sodium uptake and bile formation by isolated perfused rat liver. Am. J. Physiol. 230, 87lHl85 (1976). [16] Gruenberg, J., G. Griffiths, K. E. Howell: Characterization of the early endosome and putative endocytic carrier vesicles in vivo and with an assay of vesicle fusion in vitro. J. Cell BioI. 108, 1301-1316 (1989). [17] Gruenberg, J., F. R. Maxfield: Membrane transport in the endocytic pathway. Curro Opin. Cell BioI. 7, 552-563 (1995). [18] Hubbard, A. L.: Endocytosis. Curro Opin. Cell BioI. 1, 675---{)83 (1989). [19] Kornfeld, S., I. Mellman: The biogenesis of lysosomes. Annu. Rev. Cell BioI. 5,483-525 (1989). [20] Lee, R. T., Y. C. Lee: Synthesis of 3-(2-aminoethylthio)propyl glycosides. Carbohydr. Res. 37, 193-201 (1974). [21] Mueller, S. C., A. L. Hubbard: Receptor-mediated endocytosis of asialoglycoproteins by rat hepatocytes: receptor-positive and receptor-negative endosomes. J. Cell BioI. 102, 932-942 (1986). [22] Mullock, B. M., W. J. Branch, M. van Schaik, L. K. Gilbert, J. P. Luzio: Reconstitution of an endosome-lysosome interaction in a cell-free system. J. Cell BioI. 108,2093-2099 (1989). [23] Mullock, B. M., J. H. Perez, T. Kuwana, S. R. Gray, J. P. Luzio: Lysosomes can fuse with a late endosomal compartment in a cellfree system from rat liver. J. Cell BioI. 126, 1173-1182 (1994). [24] Odorizzi, G., A. Pearse, D. Domingo, I. S. Trowbridge, C. R. Hopkins: Apical and basolateral endosomes of MDCK cells are interconnected and contain a polarized sorting mechanism. J. Cell BioI. 135, 139-152 (1996). [25] Pitt, A., A. L. Schwartz: Reconstitution of human hepatoma endosome-endosome fusion in vitro: potential roles for an endoprotease and a phosphoprotein phosphatase. Em. J. Cell BioI. 55, 328-335 (1991).

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