Dense core lysosomes can fuse with late endosomes and are re-formed from the resultant hybrid organelles

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1 Journal of Cell Science 110, (1997) Printed in Great Britain The Company of Biologists Limited 1997 JCS Dense core lysosomes can fuse with late endosomes and are re-formed from the resultant hybrid organelles Nicholas A. Bright, Barbara J. Reaves, Barbara M. Mullock and J. Paul Luzio* Department of Clinical Biochemistry, University of Cambridge, Addenbrooke s Hospital, Hills Road, Cambridge CB2 2QR, UK *Author for correspondence SUMMARY Electron microscopy was used to evaluate the function and formation of dense core lysosomes. Lysosomes were preloaded with bovine serum albumin (BSA)-gold conjugates by fluid phase endocytosis using a pulse-chase protocol. The gold particles present in dense core lysosomes and late endosomes were flocculated, consistent with proteolytic degradation of the BSA. A second pulse of BSA-gold also accumulated in the pre-loaded dense core lysosomes at 37 C, but accumulation was reversibly blocked by incubation at 20 C. Time course experiments indicated that mixing of the two BSA-gold conjugates initially occurred upon fusion of mannose 6-phosphate receptorpositive/lysosomal glycoprotein-positive late endosomes with dense core lysosomes. Treatment for 5 hours with wortmannin, a phosphatidyl inositide 3-kinase inhibitor, caused a reduction in number of dense core lysosomes preloaded with BSA-gold and prevented a second pulse of BSA-gold accumulating in them. After wortmannin treatment the two BSA-gold conjugates were mixed in swollen late endosomal structures. Incubation of NRK cells with 0.03 M sucrose resulted in the formation of swollen sucrosomes which were morphologically distinct from preloaded dense core lysosomes and were identified as late endosomes and hybrid endosome-lysosome structures. Subsequent endocytosis of invertase resulted in digestion of the sucrose and re-formation of dense core lysosomes. These observations suggest that dense core lysosomes are biologically active storage granules of lysosomal proteases which can fuse with late endosomes and be re-formed from the resultant hybrid organelles prior to subsequent cycles of fusion and re-formation. Key words: Endocytosis, Lysosome biogenesis, Electron Microscopy INTRODUCTION Contemporary definitions of lysosomes include the criteria that they form the terminal compartment of endocytosis and contain the bulk of acid hydrolases and lysosomal glycoproteins (lgps), but no cation-independent mannose 6-phosphate receptor (M6PR; Griffiths et al., 1988, 1990; Luzio, 1994; Hunziker and Geuze, 1996; Rohrer et al., 1996). They can thus be distinguished from late endosomes which are M6PR positive, although both compartments accumulate endocytosed tracers (Geuze et al., 1988; Griffiths et al., 1988, 1990; Parton et al., 1989). Since the discovery of lysosomes in the 1950s (De Duve, 1963, 1983) they have been recognised as being heterogeneous in morphology, although in most mammalian cells they have been observed as approximately spherical structures ( µm in diameter) with an amorphous electron-dense matrix (for review see Holtzmann, 1989). In the present study we refer to these organelles as dense core lysosomes and provide further evidence that they can be clearly distinguished from endosomes. Endosomes themselves are heterogeneous in morphology and many contain intra-organelle vesicles (Trowbridge et al., 1994). Endosomal compartments (Hopkins et al., 1990; Felder et al., 1990; Futter et al., 1996) and endocytic carrier vesicles (ECVs; Gruenberg et al., 1989), which transfer endocytosed ligands from early to late endosomes, have often been described as multi-vesicular bodies (MVBs) when observed by electron microscopy. In recent years great strides have been made in understanding how newly synthesised lysosomal proteins are delivered from the trans-golgi network (TGN) to a prelysosomal compartment (PLC) with late endosomal properties (Griffiths et al., 1988; Ludwig et al., 1991). In particular, the role of two mannose 6-phosphate receptors in transporting mannose 6-phosphate-tagged soluble proteins has been well documented (Kornfeld, 1986; Kornfeld and Mellman, 1989). The function of cytoplasmic tail sequence motifs in the direct delivery of lgps from the TGN to the PLC and lysosomes, without trafficking via the cell surface, is also well described (Rohrer et al., 1996; Honing et al., 1996; Hunziker and Geuze, 1996). Similarly, there has been progress in understanding, both descriptively and in terms of detailed molecular mechanism, the passage of endocytosed ligands and membrane proteins to late compartments of the endocytic pathway. Much of the debate about the relative importance of maturation (Stoorvogel et al., 1991; Murphy, 1991) or vesicular transport steps (Griffiths and Gruenberg, 1991) in delivery from the plasma membrane to late endosomes has now been resolved (Gruenberg and Maxfield, 1995). However, very little is known about delivery of endocytosed ligands and membrane proteins to dense core

2 2028 N. A. Bright and others lysosomes nor indeed how these structures are formed and whether they are re-used. It has been shown previously that when normal rat kidney (NRK) cells are incubated with BSA-gold conjugates, they take them up by fluid phase endocytosis and accumulate them in dense core lysosomes (Griffiths et al., 1988, 1990; Parton et al., 1989). We have built on this observation to study the interaction between dense core lysosomes and late endosomes and to identify the route by which endocytosed conjugates become concentrated in these lysosomes. In the present study we have demonstrated that dense core lysosomes which have received an initial pulse of gold conjugate remain accessible to a subsequent pulse and that the initial site of mixing of the two pulses is in hybrid structures formed by direct fusion of dense core lysosomes with late endosomes. We have also studied the effects, on accumulation of endocytosed conjugates in lysosomes, of a temperature block and of the phosphatidyl inositide 3-kinase (PI3-kinase) inhibitor wortmannin, which causes dramatic morphological changes to late endosomes (Reaves et al., 1996). Finally, we have formed sucrosomes in the NRK cells by uptake of 0.03 M sucrose as previously described (Cohn and Ehrenreich, 1969, DeCourcy and Storrie, 1991) and showed that, contrary to previous interpretations (DeCourcy and Storrie, 1991; Montgomery et al., 1991; Jahraus et al., 1994), these were swollen late endosomes which could fuse with dense core lysosomes. Dense core lysosomes could be re-formed from the resultant swollen, hybrid lysosome-sucrosomes only after subsequent endocytosis of invertase. Our experimental data are consistent with dense core lysosomes being re-usable storage granules, containing proteases and other acid hydrolases, that fuse directly with late endosomes to allow the commencement of degradation of endocytosed material and which are re-formed from the hybrid, fused organelle. MATERIALS AND METHODS Materials Wortmannin (aliquoted and kept at 20 C as a 1 mm stock in DMSO), BSA, invertase, gold chloride, tannic acid, tri-sodium citrate and methyl cellulose were from Sigma Chemicals (Poole, Dorset, UK). The rabbit polyclonal anti-rat lgp110 antiserum was previously described (Reaves et al., 1996). The rabbit polyclonal anti-bovine M6PR antibody was kindly provided by Dr Suzanne Pfeffer (Stanford University, Stanford, CA; Pfeffer, 1987). The rabbit polyclonal antimouse cathepsin L antibody cross-reacts with rat fibroblast cathepsin L (Punnonen et al., 1994) and was kindly provided by Dr Michael Gottesman (National Cancer Institute, Bethesda, MD). Protein A conjugated to 10 nm or 15 nm colloidal gold was purchased from the Department of Cell Biology, University of Utrecht. Cell culture NRK fibroblast cells were grown in Dulbecco s modified Eagle s medium supplemented with 10% fetal calf serum (FCS), 100 i.u./ml penicillin, 100 µg/ml streptomycin, 4.5 g/l glucose and 2 mm L- glutamine. Cells were grown in 25 or 75 cm 2 tissue culture flasks in a 5% CO 2 incubator at 37 C. Preparation of BSA-gold 5 nm, 10 nm and 15 nm colloidal gold was prepared by tannic acid/trisodium citrate reduction of gold chloride (Slot and Geuze, 1985). The colloid was adjusted to ph 5.5 with NaOH and conjugated to sufficient BSA to afford protection from NaCl-induced flocculation. BSAgold was harvested using ultracentrifugation protocols which yielded monodisperse preparations free of aggregates and unbound protein (Slot and Geuze, 1981, 1984). The preparations were dialysed against PBS and adjusted to an A 520 of 1.4 with PBS. Flocculation of gold colloids To test if gold colloids were stabilised against electrolyte-induced flocculation NaCl was added to try and destabilise the conjugates. These were visualised by incubating with poly-l-lysine coated/formvar-carbon coated EM grids for 10 minutes, which were then blotted, air dried and viewed in a Philips CM100 transmission EM. The micrographs of stabilised BSA-10 nm gold and colloidal gold devoid of adsorbed BSA and flocculated by addition of NaCl (Slot and Geuze, 1981, 1984; De Mey, 1986) are shown in Fig. 1a,b. Endocytosis of BSA-gold 1 ml of BSA-gold was added to 4 ml DMEM + 10% FCS and NRK cells grown to ~80% confluence were incubated with the conjugatecontaining medium for 4 hours at 37 C followed by incubation in conjugate-free medium for 20 hours as previously described (Reaves et al., 1996). Cells were subsequently incubated with medium containing BSA-15 nm gold for 15 minutes to 4 hours with a 20 hour chase, prior to fixation and processing for EM. In temperature block experiments, cells which had been pre-loaded with BSA-5 nm gold were incubated with BSA-15 nm gold at 20 C for 1 hour prior to transfer to 37 C for 0, 30 or 60 minutes. Treatment with wortmannin After internalisation of BSA-5 nm gold for 4 hours and a 20 hour chase the cells were incubated with 100 nm wortmannin in RPMI medium (Sigma) + 1% FCS for 1 hour as previously described (Reaves et al., 1996). The effects of wortmannin on the uptake and trafficking of a subsequent pulse of BSA-15 nm gold were examined after internalision of BSA-5 nm gold for 4 hours followed by a 20 hour chase, by incubating with 100 nm wortmannin for 1 hour followed by endocytosis of medium containing BSA-15 nm gold in the presence of 100 nm wortmannin for 4 hours (2 2 hours; Reaves et al., 1996). Formation of sucrosomes NRK cells which had internalised BSA-5 nm gold for 4 hours followed by a 20 hour chase were subsequently incubated with medium containing 0.03 M sucrose for 4 to 24 hours to induce the formation of sucrosomes (Cohn and Ehrenreich, 1969). The cells were then fixed and processed for EM. In addition, cells were subsequently allowed to endocytose BSA-15 nm gold for 4 hours in sucrose-free medium. Sucrose was omitted from the medium in control experiments. Cells in which the dense core lysosomes were pre-loaded with BSA-5 nm gold followed by 0.03 M sucrose internalisation for 24 hours were allowed to internalise 0.5 mg/ml invertase for 30 minutes to 8 hours. In control cells invertase was omitted from the medium. Transmission electron microscopy For routine EM, cells were removed from tissue culture flasks by trypsinisation and pelleted in a bench-top microfuge at 500 g for 2 minutes. The cells were then fixed with 2.5% glutaraldehyde/2% paraformaldehyde in 0.1 M Na cacodylate buffer, ph 7.2, for 3 hours at room temperature, washed with 0.1 M Na cacodylate buffer, ph 7.2, and post-fixed in 1% osmium tetroxide in 0.1 M Na cacodylate buffer, ph 7.2, for 1 hour. The cell pellet was then washed with 0.05 M Na maleate buffer, ph 5.2, and en bloc stained with 0.5% uranyl acetate in 0.05 M Na maleate buffer, ph 5.2. The cell pellets were then dehydrated in ethanol, exchanged into 1,2-epoxy propane and embedded in Araldite CY212 epoxy resin (Agar Scientific, Stansted, UK).

3 Endosome-lysosome fusion 2029 Ultrathin sections were cut using a diamond knife mounted on a Reichert Ultracut S ultramicrotome (Leica, Milton Keynes, UK), collected on EM grids and stained with uranyl acetate and Reynolds lead citrate (Reynolds, 1963). The sections were observed in a Philips CM 100 transmission electron microscope (Philips Electron Optics, Cambridge, UK) at an operating voltage of 80 kv. Immunoelectron microscopy Cells were prepared for immuno-em as described by Griffiths (1993). Cells were washed with PBS and fixed with 4% paraformaldehyde/0.1% glutaraldehyde in 250 mm Hepes, ph 7.2, at room temperature for 1 hour. The cells were scraped and pelleted at 1,000 g for 2 minutes in a microfuge. The cell pellets were subsequently embedded in 10% gelatin in PBS at 37 C, cooled on ice, trimmed and infused with 2.1 M sucrose in PBS overnight at 4 C prior to being frozen on aluminium stubs in liquid nitrogen. Frozen ultrathin sections were cut using a cryochamber attachment (Leica, Milton Keynes, UK), collected with 2.3 M sucrose in PBS and mounted on Formvar-carbon coated EM grids. Immunolabelling was performed using the Protein A-gold technique at room temperature (Slot and Geuze, 1983). Sections were incubated with 50 mm NH 4Cl in PBS for 10 minutes to quench unreacted aldehydes, transferred to 2% gelatin in PBS for 10 minutes and then 1% BSA in PBS for 10 minutes. Sections were incubated for 30 minutes with 5 µl of primary antibody diluted in PBS containing 5% FCS and 0.1% BSA. Primary antibodies utilised in these studies were rabbit anti-mannose-6-phosphate receptor (M6PR: diluted 1:50), rabbit anti-lgp110 (diluted 1:50) and rabbit anti-cathepsin L (diluted 1:100). The sections were washed with PBS/0.1% BSA (6 3 minutes) and incubated for 30 minutes with PBS/0.1% BSA containing Protein A conjugated to 10 or 15 nm colloidal gold. The sections were washed with PBS/0.1% BSA (2 5 minutes), PBS (4 5 minutes) and the complex stabilised using 1% glutaraldehyde in PBS (5 minutes). Finally the sections were rinsed with distilled water (5 3 minutes) and contrasted by embedding in 1.8% methyl cellulose/0.3% uranyl acetate (Tokuyasu, 1978). Sections were allowed to air dry prior to observation. Morphometry Quantitative analysis of the subcellular distribution of gold particles was performed on the EM at a magnification of 15,500. Individual random sections through cell pellets were systematically scanned and the presence of gold conjugates in endosomes, sucrosomes, lysosomes or organelles possessing intermediate morphology were scored and expressed as a percentage of the total gold-labelled organelles. Goldcontaining organelles were scored irrespective of the number of gold particles contained therein. Dense core lysosomes were defined as possessing an electron-dense lumenal content; endosomes and sucrosomes as possessing vacuolar morphology with an electronlucent lumenal content; organelles with intermediate morphology as possessing vacuolar morphology with a diffuse lumenal content of intermediate electron density. The data were tested for statistical significance with analysis of variance using Super Anova software (Abacus concepts Inc., version 1.11). Only P values 0.01 are shown. Unless otherwise stated results are expressed as a mean ± s.e.m, with the number of experiments (n) shown in parentheses. The number of cell profiles and gold-labelled organelles scored to generate the data are indicated in the figure legends. RESULTS Accumulation of BSA-gold in dense core lysosomes and late endosomes BSA-gold accumulated in dense core lysosomes and late endosomes of NRK cells after endocytosis of medium containing the conjugate for 4 hours at 37 C, followed by a chase period of 20 hours at 37 C in conjugate-free medium (Fig. 1c) as previously described (Griffiths et al., 1988, 1990; Parton et al., 1989; Reaves et al., 1996). Quantitation indicated that 83.8±2.3% (n=8) of the organelles that had accumulated gold were electron dense and therefore identified as dense core lysosomes. Immuno-EM indicated that the BSA-gold loaded dense core lysosomes could be immunolabelled with antibodies to lgp110 (Fig. 1d) and cathepsin L (Fig. 1e), but only rarely with antibodies to M6PR (not shown), thus providing verification that these structures were lysosomal. Of the organelles that had accumulated BSA-gold, 14.4±2.0% (n=8) possessed the morphological appearance of endosomes (Fig. 1f). Immuno-EM showed that they could be labelled with antibodies to lgp110 (Fig. 1g) and M6PR (Fig. 1h). Structures possessing intermediate or equivocal morphology accounted for 1.8±0.7% (n=8) of the BSA-gold loaded organelles. BSA-gold which had accumulated in the late endosomes and lysosomes had flocculated in a manner similar to NaCl-induced flocculation of gold colloids which had not been stabilised by addition of protein, as seen in Fig. 1b. In contrast, BSA-gold conjugates present in organelles of the early endocytic pathway had not flocculated (see below), but remained as discrete particles. Thus flocculation of gold conjugates was a feature of delivery to compartments containing active proteases. A second pulse of BSA-gold accumulated in dense core lysosomes pre-loaded with BSA-gold To test whether dense core lysosomes were biologically active after accumulating BSA-gold, NRK cells that had been preloaded with BSA-gold were allowed to endocytose a second pulse of BSA conjugated to colloidal gold of larger diameter for 4 hours followed by a 20 hour chase. After this treatment 85.9±0.5% (n=3) of the organelles containing BSA-15 nm gold were dense core lysosomes that also contained the smaller BSA-gold (Fig. 2a). These results indicate that dense core lysosomes retained their ability to fuse with endocytic structures containing a subsequent pulse of BSA-15 nm gold despite their lumenal content of pre-loaded gold. Flocculation of the second pulse of gold suggested that the BSA had been degraded, consistent with the hydrolytic enzymes in the lysosome remaining biologically active. Apparent fusions of dense core lysosomes with endocytic organelles could occasionally be observed (Fig. 2b). Organelles possessing the morphological appearance of endosomes accounted for 14.1±0.5% (n=3) of the total BSA- 15 nm gold-labelled structures. The BSA-15 nm gold, which had also flocculated, frequently co-localised with the smaller BSA-gold in these organelles (Fig. 2c). Mixing of a second pulse of BSA-gold with preloaded BSA-gold occurred upon fusion of a late endosome compartment with a dense core lysosome To clarify where mixing of a second pulse of BSA-gold with pre-loaded BSA-gold occurred, a time-course was performed. Cells were pre-loaded with BSA-5 nm gold and then incubated with medium containing BSA-15 nm gold for 15 minutes to 4 hours. Mixing of the BSA-15 nm gold with the pre-loaded BSA-5 nm gold occurred within 15 minutes, consistent with

4 2030 N. A. Bright and others Fig. 1. Accumulation of BSAgold in lysosomes and late endosomes of NRK cells. BSA-10 nm gold conjugates remained unaffected by the addition of NaCl (a). However, addition of NaCl to colloidal gold devoid of adsorbed BSA resulted in flocculation (b). NRK cells were allowed to endocytose BSA-10 nm gold for 4 hours followed by a 20 hour chase in conjugate-free medium. This resulted in the accumulation of gold (small arrowheads) in dense core lysosomes (c). In frozen sections these organelles could be immunolabelled with antibodies to lgp110 (d) and cathepsin L (e) and visualised with Protein A-15 nm gold (large arrowheads). 14.4±2.0% (n=8) of the BSA-10 nm goldlabelled organelles possessed the morphology of endosomes (f). These could be immunolabelled with antibodies to lgp110 (g) and M6PR (h) and visualised with Protein A-15 nm gold. Note that the 10 nm gold has flocculated in the lysosomes and endosomes similar to NaCl-induced flocculation of the unconjugated gold colloid. Bristle-like coats may frequently be observed on endosomes (arrow). Bar, 500 nm. fusion between late endosomes containing BSA-15 nm gold and dense core lysosomes containing BSA-5 nm gold. These fusions resulted in hybrid organelles with the appearance of endosomes containing diffuse lumenal contents and internal membranes (Fig. 3a). Flocculation of the BSA-15 nm gold occurred in these structures but conjugates that had not yet encountered the pre-loaded gold remained particulate. The hybrid organelles were immunoreactive with antibodies to M6PR (Fig. 3b) and lgp110 (not shown). After 15 minutes uptake the BSA-15 nm gold was not present in dense core

5 Endosome-lysosome fusion 2031 Fig. 2. Accumulation of BSA-15 nm gold in lysosomes and late endosomes pre-loaded with BSA-10 nm gold. Lysosomes and late endosomes of NRK cells were pre-loaded with BSA-10 nm gold using a 4 hour pulse and 20 hour chase in conjugate-free medium. BSA-15 nm gold was subsequently internalised using a 4 hour pulse and 20 hour chase and accumulated in the pre-loaded lysosomes (a). In b a dense core lysosome loaded with 10 nm and 15 nm gold has apparently fused with an endocytic organelle (arrow). Organelles possessing the morphology of endosomes frequently contained both sizes of gold (c). These structures may arise from a fusion between a non-gold labelled endosome and a lysosome containing both gold conjugates or upon fusion of a BSA-10 nm gold-laden lysosome with a 15 nm gold-laden endosome. Bar, 200 nm. lysosomes (see Fig. 4a), but by 30 minutes, in addition to the hybrid structures, a small population of dense core lysosomes were labelled with both gold conjugates (Fig. 3c). After 1 hour to 4 hours of uptake of the BSA-15 nm gold, an increasing number of dense core lysosomes contained both sizes of gold (Fig. 4a). In another series of experiments cells pre-loaded with BSA- 5 nm gold were subsequently incubated with BSA-15 nm gold at 20 C for 1 hour. The cells were either fixed and processed for EM or returned to 37 C for 30 minutes or 1 hour prior to fixation (Fig 4b; micrographs not shown). After 1 hour at 20 C, 97.9±1.2% (n=3) of the organelles labelled with BSA-15 nm gold possessed endosomal morphology and contained no BSA- 5 nm gold. After returning the cells to 37 C for 1 hour, the number of BSA-15 nm gold-labelled organelles with endosomal morphology that were devoid of BSA-5 nm gold had dropped significantly, and there was an increase in the number of endosomes and dense core lysosomes containing both sizes of gold (Fig. 4b). Inhibition of accumulation of BSA-gold in dense core lysosomes after treatment with wortmannin NRK cells pre-loaded with BSA-5 nm gold were treated with 100 nm wortmannin for 1 hour at 37 C, resulting in the formation of two populations of swollen late endosomes (Reaves et al., 1996). Of the organelles that were labelled with colloidal gold, 87.6±2.0% (n=6) were dense core lysosomes, compared with 83.8±2.3% (n=8) in control cells. Thus, lysosomes were unaffected by treatment with wortmannin for 1 hour. Swollen late endosomes accounted for 11.2±1.6% (n=6) of the gold-labelled organelles, compared with 14.4±2.0% (n=8) normal late endosomes in control cells. Organelles with intermediate morphology accounted for 1.2±0.5% (n=6) of the gold-containing structures (Fig. 5). When cells pre-loaded with BSA-5 nm gold were treated with 100 nm wortmannin for 5 hours at 37 C, under conditions in which swollen late endosomal structures were maintained, an effect on dense core lysosomes was observed. In contrast to the lack of effect after treatment for 1 hour, 56.2±0.2% (n=3) of the BSA-5 nm gold-labelled organelles were dense core lysosomes, 26.4±1.7% (n=3) were swollen endosomal structures and 17.4±2.2% (n=3) possessed intermediate morphology (Fig. 5). These data were consistent with fusion of dense core lysosomes with late endosomes, and inhibition of the reformation of dense core lysosomes from the resultant hybrid organelles. To test this hypothesis and determine the effects of wortmannin upon the accumulation of BSA-gold in dense core

6 2032 N. A. Bright and others Fig. 3. Time-course of delivery of BSA-15 nm gold into NRK cells pre-loaded with BSA-5 nm gold. Lysosomes and late endosomes were pre-loaded with BSA-5 nm gold using a 4 hour pulse and 20 hour chase. BSA-15 nm gold was then internalised for 15 minutes (a-b) or 30 minutes (c). Mixing of the gold conjugates could first be observed in organelles possessing the morphology of endosomes after 15 minutes of BSA-15 nm gold internalisation (a). These could be immunolabelled with antibodies to M6PR and visualised using Protein A-10 nm gold (medium arrowhead) on frozen sections (b). Note that both gold conjugates have flocculated. After 30 minutes (c) of BSA-15 nm gold uptake both sizes of gold could be observed in the same dense core lysosomes. Note that BSA-15 nm gold has not flocculated in endosomes which do not possess 5 nm gold. Bar, 200 nm. 100 a Endosome + BSA15. Endosome + BSA15 & BSA5. Dense core lysosome + BSA15 & BSA h chase sucrose 24h then 240 % of total BSA-15 nm gold-labelled organelles. Duration of BSA-15 nm Au internalisation (mins). b % of total BSA-15 nm gold-labelled organelles. 60 Time at 37 o C (mins). Fig. 4. Quantitation of the time course of distribution of BSA-15 nm gold in NRK cells, (a) after uptake at 37 C, or (b) after 1 hour at 20 C followed by uptake at 37 C. Cells were pre-loaded with BSA-5 nm gold for 4 hours followed by a 20 hour chase. Results are presented as a mean of triplicate experiments ± s.e.m. (a) No. of cells/section = 81±6; no. of organelles/section 344±33. (b) No. of cells/section = 66±9; no. of organelles/section = 173±23. The final set of columns on the right hand side of (a) indicates the distribution of BSA-15 nm gold after 4 hours internalisation into NRK cells previously allowed to accumulate 0.03 M sucrose for 24 hours.

7 Endosome-lysosome fusion 2033 % of total gold-labelled organelles Duration of wortmannin treatment (h). intermediate structures endosomes dense core lysosomes Fig. 5. Quantitation of the distribution of pre-loaded BSA-5 nm gold after a 4 hour pulse and 20 hour chase followed by incubation with 100 nm wortmannin. Results are presented as a mean (0 hours: n=8; 1 hour: n=6; 5 hours: n=3) ± s.e.m. *P 0.01; **P , when compared with control cells or cells treated for 1 hour with 100 nm wortmannin. No. of cells/section = 55±5; no. of organelles/section = 94±19. lysosomes, cells pre-loaded with BSA-5 nm gold were treated with 100 nm wortmannin for 1 hour and subsequently incubated in medium containing BSA-15 nm gold and 100 nm wortmannin for 4 hours. The BSA-15 nm gold accumulated in swollen late endosomes but did not appear in dense core lysosomes pre-loaded with BSA-5 nm gold (Fig. 6). These swollen endosomes could be immunolabelled for M6PR and ** * ** lgp110 (data not shown). Many of the swollen organelles contained both sizes of gold conjugate and those containing BSA-5 nm gold could be immunolabelled for cathepsin L (data not shown), consistent with them resulting from the fusion of lysosomes with late endosomes. In control experiments, in which wortmannin was omitted, the second pulse of BSA-gold accumulated in dense core lysosomes as in previous experiments. Sucrose accumulated in swollen late endosomes (sucrosomes) distinct from dense core lysosomes Previous investigators have shown that uptake of sucrose into cells which cannot digest it causes swelling of late endocytic compartments (Cohn and Ehrenreich, 1969; DeCourcy and Storrie, 1991; Montgomery et al., 1991; Jahraus et al., 1994). To investigate the effects of this treatment on dense core lysosomes, NRK cells were pre-loaded with BSA-5 nm gold. The cells were then incubated with medium containing 0.03 M sucrose for 8 hours at 37 C, fixed and processed for EM. Swollen structures (sucrosomes) appeared after this treatment (Fig. 7). The number of dense core lysosomes loaded with the BSA-gold decreased after this treatment (Fig. 8) but those that remained were morphologically unaffected (Fig. 7a). Fine filaments could occasionally be observed attaching lysosomes to the sucrosomes (Fig. 7b). Immuno-EM confirmed the identity of the swollen sucrosomes as late endosomes, or endosome-lysosome hybrids, by virtue of their M6PR (Fig. 7c) and lgp110 (Fig. 7d) immunoreactivity. In sucrosomes containing BSA-gold, cathepsin L immunoreactivity was also seen (Fig. 7e), consistent with these being endosome-lysosome hybrids. To determine the effects of prolonged sucrose endocytosis, NRK cells were pre-loaded with BSA-gold and the effects of Fig. 6. The effect of wortmannin on endocytosis of BSA-15 nm gold. Lysosomes and late endosomes were preloaded with BSA-5 nm gold using a 4 hour pulse and 20 hour chase. The cells were then incubated with 100 nm wortmannin for 1 hour and additionally for 4 hours with BSA-15 nm gold in the presence of 100 nm wortmannin. BSA-15 nm gold accumulated in the swollen endocytic compartments but was not found in association with dense core lysosomes (stars) pre-loaded with BSA-5 nm gold. Bar, 200 nm.

8 2034 N. A. Bright and others Fig. 7. The effect of 0.03 M sucrose internalisation upon lysosomes pre-loaded with BSA-5 nm gold in NRK cells. Cells were pre-loaded with BSA-5 nm gold using a 4 hour pulse and 20 hour chase, and subsequently incubated with 0.03 M sucrose for 8 hours to induce the formation of sucrosomes. The organelles which accumulated sucrose were morphologically distinct from the gold-laden dense core lysosomes (a). Lysosomes and endosomes could occasionally be observed to be attached via fine filaments (arrows) as seen at a greater magnification (b). However, after this duration of sucrose internalisation 87.6±2.0% (n=6) of the pre-loaded BSA-5 nm gold was present in swollen sucrosomes (c-e). These structures could be immunolabelled with antibodies to M6PR (c), lgp110 (d) and cathepsin L (e) and visualised using Protein A-10 nm gold in frozen sections. Bars: 500 nm (a,c-e); 200 nm (b). sucrose internalisation for 4 to 24 hours were examined (Fig. 8a). Prolonged uptake of sucrose resulted in the depletion of labelled dense core lysosomes and concomitant accumulation of gold in swollen sucrosomes. This appeared to be an all-ornothing effect, as evidenced by the paucity of organelles with intermediate morphology. In control cells incubated in medium without sucrose the steady state distribution of BSA-gold was unaffected. Dense core lysosomes were re-formed from sucrosome-lysosome hybrids upon internalisation of invertase It has been shown previously that endocytosis of invertase into cells containing pre-formed sucrosomes results in the efficient collapse and disappearance of these structures (Cohn and Ehrenreich, 1969). In the present experiments internalisation of medium containing invertase, after the formation of sucrosomes over a 24 hour period and concomitant depletion of lysosomes pre-loaded with BSA-gold, resulted in the regeneration of dense core lysosomes containing BSA-gold (Fig. 8b). A time-course of invertase uptake showed a decrease in the number of labelled sucrosomes, a transient rise of organelles possessing intermediate morphology and a gradual rise in the number of re-formed dense core lysosomes. These results indicate that as the content of sucrosomes was digested the organelles transiently possessed intermediate morphology prior to reforming morphologically identifiable dense core

9 Endosome-lysosome fusion a 100 b % of total gold-labelled organelles dense core lysosomes. sucrosomes. intermediate structures. % of total gold-labelled organelles dense core lysosomes. sucrosomes. intermediate structures Duration of 0.03 M sucrose uptake (h). Duration of 0.5 mg/ml invertase uptake (h). Fig. 8. Quantitation of the distribution of pre-loaded BSA-gold after internalisation of 0.03 M sucrose (a), or after 24 hours sucrose accumulation followed by uptake of 0.5 mg/ml invertase (b). Results are presented as a mean of triplicate experiments ± s.e.m (a), or duplicate experiments ± range (b). (a) No. of cells/section = 84±9; no. of organelles/section = 83±6. (b) No. cells/section = 75±5; No. organelles/section = 15±2. lysosomes. In cells to which invertase was not added the gold remained in swollen sucrosomes (Fig. 9). The presence of sucrosomes prevented accumulation of a second pulse of BSA-gold in dense core lysosomes We examined the effect of the presence of sucrosomes on internalisation and subsequent trafficking of a second pulse of BSA-gold. Cells were pre-loaded with BSA-5 nm gold and then incubated in the presence of 0.03 M sucrose for 24 hours. The cells were then allowed to internalise BSA-15 nm gold for 4 hours. In contrast to the observation in control cells (Fig. 4a), internalised BSA-15nm gold accumulated in structures with the characteristic morphology of ECVs (Fig. 10a; Gruenberg et al., 1989). These did not contain BSA-5nm gold and were clearly distinct from the swollen sucrosomes (Fig. 10b). Only a small percentage of the sucrosomes contained both sizes of gold and any residual dense core lysosomes contained only the first pulse of BSA-5 nm gold (for quantitation see Fig. 4a). These observations imply that fusions between sucrosomes and Fig. 9. The effect of invertase internalisation on sucrosomes. Cells were pre-loaded with BSA-5 nm gold using a 4 hour pulse and 20 hour chase, and subsequently allowed to internalise 0.03 M sucrose for 24 hours resulting in the formation of sucrosomes and depletion of dense core lysosomes. Cells were then incubated in medium without invertase (a) or mg/ml invertase for 8 hours (b). Endocytosis of invertase resulted in digestion of the sucrose and re-formation of dense core lysosomes labelled with BSA-gold (b). Bar, 500 nm.

10 2036 N. A. Bright and others

11 Endosome-lysosome fusion 2037 Fig. 10. The effect of the presence of sucrosomes on endocytosis and subsequent trafficking of BSA-gold. Cells were pre-loaded with BSA-5 nm gold using a 4 hour pulse and 20 hour chase, and then allowed to internalise 0.03 M sucrose for 24 hours, prior to incubation with medium containing BSA-15 nm gold for 4 hours. This resulted in the accumulation of ECVs in which the BSA-15 nm gold had not flocculated (a). These structures were clearly distinct from the sucrosomes (b). Frozen sections were immunolabelled with antibodies to M6PR (c), lgp110 (d) and cathepsin L (e) and visualised using Protein A-10 nm gold. Residual dense core lysosomes (stars) contain only the first pulse of BSA-5 nm gold and not the second pulse of BSA-15 nm gold. ECVs containing particulate BSA-15 nm gold (white arrowheads) were not immunoreactive with antibodies to M6PR, lgp110 or cathepsin L. However, BSA-15 nm gold which had been delivered to sucrosomes had clearly flocculated (d, e). Bar, 500 nm. ECVs were impaired by the accumulation of the indigestible sucrose. The second pulse of BSA-15 nm gold had not flocculated in the ECVs, whereas gold present in sucrosomes containing the first pulse of BSA-5 nm gold had flocculated (Fig. 10e). Immuno-EM of the ECVs containing the second pulse of gold revealed that they were not immunoreactive for M6PR (Fig. 10c), lgp110 (Fig. 10d) or cathepsin L (Fig. 10e), consistent with them being an earlier endocytic compartment than late endosomes. DISCUSSION We have studied the interaction and fusion of dense core lysosomes with late endosomes in cultured cells. Using fused hybrid cell systems, others have shown that lysosomes and endosomes are dynamic structures that interchange content and membrane markers (Deng and Storrie, 1988; Deng et al., 1991). In cell-free experiments we have previously shown that content mixing occurs when lysosomes and late endosomes derived from rat liver hepatocytes are incubated in the presence of cytosol and an energy source (Mullock et al., 1989, 1994). However, in none of these systems was it possible to distinguish between direct fusion of late endosomes and lysosomes, vesicular transport or even kiss and run (for various models to explain content mixing see Berg et al., 1995, and Storrie and Desjardins, 1996). Futter et al. (1996) demonstrated in HEp-2 cells, a cell type in which MVBs are the dominating endocytic organelle (van Deurs et al., 1993, 1995), that attachment and fusion of endocytic MVBs occurred with non-electron dense, M6PR negative MVBs which were pre-loaded with horseradish peroxidase. Our present study extends this observation to the fusion of late endosomes with dense core lysosomes and allows us to propose a model for the accumulation of endocytosed ligands in dense core lysosomes which involves fusion of pre-existing lysosomes with late endosomes followed by reformation of dense core lysosomes from the resultant hybrid structures. We have shown that not only is it possible for dense core lysosomes to accumulate BSA-gold, as previously described by others (Griffiths et al., 1988, 1990; Parton et al., 1989), but that these lysosomes can acquire a second pulse of BSA-gold showing that they are re-usable. Intermixing of the two separate pulses of BSA-gold was consistent with fusion of lysosomes pre-loaded with the first pulse and M6PR positive/lgp positive late endosomes containing the second pulse. The resultant hybrid compartments were the site of flocculation of BSA-gold indicating that they contained proteases (van Deurs et al., 1995). Direct fusion events between late endosomes and lysosomes were indicated by the observation of apparent fusion profiles, consistent with delivery of the entire content of proteolytic enzymes of a dense core lysosome to the newly formed hybrid structure. The high lumenal protein concentration of a dense core lysosome is probably an unfavourable environment for proteolytic activity. After fusion with a late endosome, diffusion of lysosomal content into the lumen of the hybrid organelle may well provide a more suitable aqueous and acidic milieu for protease action. A temperature block of 20 C inhibited the appearance of BSA-gold in lysosomes. When cells containing pre-loaded lysosomes accumulated a second pulse of BSA-gold at 20 C and were then transferred to 37 C, mixing of gold occurred, with flocculation of the second pulse after 30 minutes and the presence of both sizes of gold in dense core lysosomes after 60 minutes. These data are compatible with results of previous work using temperature blocks to prevent delivery of endocytosed markers to lysosomes (Dunn et al., 1980; Miller et al., 1986; Marsh et al., 1983; Felder et al., 1990; Futter et al., 1996). We have extended our previous observations on the effect of the PI3-kinase inhibitor, wortmannin, on late endocytic compartments (Reaves et al., 1996). By quantitating the effect on pre-loaded lysosomes we have confirmed that treating NRK cells with 100 nm wortmannin for 1 hour has no measurable effect on the morphology of dense core lysosomes, yet causes the appearance of swollen late endosomal compartments. It is not known which of several wortmannin-sensitive PI3-kinases may be involved in controlling the morphology and function of these compartments (Reaves et al., 1996). During subsequent incubation of the cells for 5 hours in the presence of wortmannin, the swollen compartments received delivery of flocculated BSA-5nm gold from pre-loaded dense core lysosomes, consistent with direct fusion between swollen endosomes and dense core lysosomes. A new finding was that re-formation of dense core lysosomes, from the swollen hybrid compartments formed after these fusions, was prevented by wortmannin. Thus a further membrane traffic step which requires, or is modulated by, PI3-kinase activity, but which is not recycling of M6PR to the TGN (Nakajima and Pfeffer, 1997), may be added to previous lists (Shepherd et al., 1996). Contrary to the interpretation of data in some previous reports (Cohn and Ehrenreich, 1969; Deng and Storrie, 1988; DeCourcy and Storrie, 1991; Montgomery et al., 1991; Jahraus et al., 1994), we have determined that sucrosomes, induced by accumulation of indigestible sucrose, are late endosomes and hybrid endosome-lysosome structures, as determined by M6PR immunoreactivity, but not terminal lysosomes. After prolonged sucrose uptake, fusions between the sucrosomes and dense core lysosomes resulted in the appearance of pre-loaded BSA-gold in sucrosome-lysosome hybrids and the depletion of dense core lysosomes. Direct contact and apparent fusions were occasionally seen in the electron microscope. In previous experiments, Deng and Storrie (1988) demonstrated that species-specific lysosomal membrane proteins from one cell type could be transferred to sucrosomes of another in a fused,

12 2038 N. A. Bright and others heterotypic, hybrid cell system. Although sucrosomes were considered to be mature lysosomes in this system, these data nevertheless provide further evidence for membrane traffic between lysosomes and sucrosomes. Uptake of sucrose, interposed between two pulses of BSAgold, prevents the accumulation of the second pulse in dense core lysosomes, presumably by preventing membrane retrieval and associated regeneration of dense core lysosomes. Cohn and Ehrenreich (1969) and Swanson et al. (1986) demonstrated that sucrosomes collapse down to phase-dense organelles after treatment with invertase. In the present study we have confirmed these observations and shown that sucrosomes which had acquired the pre-loaded gold content of dense core lysosomes over an extended period were capable of re-forming dense core lysosomes after endocytosis of invertase. Jahraus et al. (1994) concluded that there was retrograde traffic from lysosomes to the late endosome. They demonstrated the efficient disappearance of pre-formed sucrosomes after endocytosis and immobilisation of invertase-conjugated latex beads in late endosomes. However, it is likely that late endosome-late endosome fusions account for the disappearance of sucrosomes in their system. In the present study we observed numerous apparent fusion events between sucrosomes (see also Cohn and Ehrenreich, 1969). Further evidence for homotypic late endosome-late endosome fusions has been provided by Deng et al. (1991) in fused heterotypic, hybrid cells. Our data are consistent with the observations of Swanson et al. (1986) and Montgomery et al. (1991) who demonstrated that sucrosomes exhibit reduced fusions with earlier endocytic organelles. After sucrose uptake we observed the accumulation of a subsequent pulse of BSA-gold in ECVs, the carrier vesicles responsible for transfer from early to late endosomes (Gruenberg et al., 1989; Aniento et al., 1993). Our data suggest that proteolytic digestion in NRK cells commences in the endocytic pathway after fusion of a late endosome with a dense core lysosome. Some of the products of digestion will be transported across the membrane of the hybrid organelle into the cytosol. Membrane and contents will be selectively retrieved and re-cycled from the hybrid organelle to sites such as the TGN (eg. for M6PR) and possibly earlier endocytic compartments resulting in the re-formation of the dense core lysosome. This process would account for the diverse range of morphology and immunoreactivity encountered in these organelles. Membrane retrieval and re-formation of lysosomes from the hybrid organelles would require adaptor molecules and/or coat proteins to select and pinch off the membrane destined for re-cycling. There is accumulating evidence that such coat proteins do exist on endosomes (Whitney et al., 1995; Aniento et al., 1996; Stoorvogel et al., 1996), and lysosomes (Traub et al., 1996). The plasma membrane adaptor molecule α-adaptin has also been demonstrated on endosomes in the presence of GTPγS (Seaman et al., 1993), and on lysosomes after a purified fraction has been incubated with cytosol in the presence of ATP (Traub et al., 1996). A model outlining the fusion of dense core lysosomes with late endosomes and showing the re-formation of re-usable dense core lysosomes from the resultant hybrid organelles is shown in Fig. 11. The concept of direct fusion of lysosomes with other organelles, particularly during autophagy, is not new (see for example De Duve, 1963; Lawrence and Brown, 1992). Fusion Membrane retrieval ECV M6PR-, lgp-, protease- Late endosome M6PR+, lgp+, protease- Fusion Endosome / lysosome hybrid M6PR+, lgp+, protease+ Re-formation Dense core lysosome M6PR-, lgp+, protease+ Re-usable Blocked by 100 nm wortmannin and sucrose accumulation Fig. 11. Fusion of dense core lysosomes with late endosomes and subsequent re-formation. The data presented in this study support a model whereby ECVs formed from early endosomes fuse with, and deliver their content to, pre-existing late endosomes. Acid hydrolaserich electron-dense lysosomes fuse with late endosomes to generate hybrid organelles in which digestion of internalised material occurs. Dense core lysosomes are re-formed after selective recovery of membrane. The lysosome may then be re-used in subsequent cycles of fusion, digestion and re-formation. + indicates presence; indicates depletion but not necessarily exclusion. Recently there has been much interest in the concept of the secretory lysosome in haemopoietic cells, where secretory granules with many of the properties classically associated with lysosomes undergo a triggered fusion with the plasma membrane (G. M. Griffiths, 1996). Considering the dense core lysosome as a re-formable, re-usable storage granule that constitutively fuses with and secretes into a late endosome is thus not without precedent (see for example De Duve and Wattiaux, 1966; Hales, 1978). The suggestion that lysosomes may be storage organelles for acid hydrolases was also made by Tjelle et al. (1996), who demonstrated that the main proteolytic degradation of endocytosed proteins in a macrophage cell line

13 Endosome-lysosome fusion 2039 took place in late endosomes despite the observation that lysosomes contained the bulk of lysosomal enzymes. Our data are consistent with the proposal by G. Griffiths (1996) that lysosomes are mostly storage vesicles for mature lysosomal enzymes which are injected by fusion into late endosomal compartments which may be regarded as collectively representing the cell stomach. The observation of direct fusion between dense core lysosomes and late endosomes begs the questions of the molecular mechanisms of recognition and fusion. It is possible that the recognition and docking process involves the filamentous structures observed between endosomes and lysosomes in the present study (Fig. 7b) and previously observed between MVBs in HEp-2 cells (Futter et al., 1996). 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