Evidence for retrograde traffic between terminal lysosomes and the prelysosomal/late endosome compartment

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1 Journal of Cell Science 107, (1994) Printed in Great Britain The Company of Biologists Limited 1994 JCS Evidence for retrograde traffic between terminal lysosomes and the prelysosomal/late endosome compartment Andrea Jahraus 1, Brian Storrie 2, Gareth Griffiths 1 and Michel Desjardins 1, * 1 European Molecular Biology Laboratory, Cell Biology Programme, D Heidelberg, Germany 2 Department of Biochemistry and Nutrition, Virginia Polytechnic Institute and State University, Blacksburg, Virginia 24061, USA *Author for correspondence SUMMARY We have investigated the interactions occuring between the prelysosomal compartment, PLC/late endosome, and terminal lysosomes using an approach that allowed us to internalize and deliver specific tracers to these compartments, and look for evidence of their meeting. After internalization of sucrose, the lysosomes (sucrosomes), but not the PLC/late endosomes, became significantly swollen and visible in the light microscope. If invertase is then added to the medium it reaches the lysosomes where it cleaves sucrose into fructose and glucose. These sugars, unlike sucrose, can be transported into the cytosol, resulting in the disappearance of the sucrosomes. We previously showed that phagocytosed latex beads are delivered specifically to, and reside in, the PLC/late endosome, a stage earlier than the lysosomes in the endocytic pathway (Rabinowitz et al. (1992) J. Cell Biol. 116, ). In the present study, we demonstrate that invertase conjugated to the latex beads, and thus immobilized in late endosomes, has access to the sucrose present in the more distal lysosomes. Experiments using nocodazole indicate that this retrograde fusion event requires the presence of an intact microtubule network. The simplest interpretation of our results is that the two compartments fuse, allowing for a retrograde transport of sucrose from the lysosomes to the PLC/late endosomes. Key words: endocytosis, lysosome biogenesis, prelysosomal compartment INTRODUCTION Two distinct compartments have been identified in the terminal stages of the endocytic pathway in animal cells. The proximal prelysosomal compartment (PLC or late endosome) and the more distal lysosomes (Storrie, 1988; Kornfeld and Mellman, 1989; Griffiths and Gruenberg, 1991). These two compartments are distinct both structurally and functionally. In cultured cells the PLC/late endosome is usually a large pleomorphic structure that, after separation on Percoll gradients, has a light buoyant density. In contrast, lysosomes are simple secretion granule-like vesicles that are significantly more dense on Percoll gradients (Storrie, 1988). Although there is no record of any molecule being present in lysosomes that is absent in the PLC/late endosome, there are a number of proteins found in the latter, often in very high concentration, that are not detected in lysosomes. These include the cationindependent (CI) and cation-dependent (CD) mannose 6- phosphate receptors (MPR) (Griffiths et al., 1988; Geuze et al., 1988; Bleekemolen et al., 1988) as well as smaller amounts of recycling receptors such as the transferrin receptor (Killisch et al., 1992). Two proteins on the cytoplasmic side of the PLC/late endosome membrane are also not detected in lysosomes. First, the small GTP-binding protein rab7 (Chavrier et al., 1990; Rabinowitz et al., 1992). Second, the regulatory (RII) subunit of the cyclic AMP-dependent protein kinase, which is associated with the cytoplasmic surface of the PLC/late endosome (as well as with the plasma membrane, early endosomes and the TGN), but not with lysosomes (Griffiths et al., 1990b). Both the PLC/late endosomes and lysosomes have properties expected of functional degradative compartments, having a low luminal ph and being enriched in acid hydrolases as well as in a family of lysosomal membrane glycoproteins (lgps or lamps). Indeed, available data support the argument that some kind of dynamic equilibrium exists between the PLC/late endosomes and lysosomes. First, in NRK cells the immunogold density of labelling for lgp 120, an antigen prepared from membranes of dense lysosomes (Lewis et al., 1985), is the same in membranes of the PLC/late endosome and dense lysosomes (Griffiths et al., 1990a). If the PLC/late endosome was simply a station en route to the lysosomes one would expect the concentration of lgp to be considerably lower in that organelle relative to the terminal lysosomes. Second, when gold tracers taken up in many cell types by either fluid-phaseor receptor mediated-endocytosis are allowed to accumulate over periods longer than hours, these tracers are invariably found in high concentrations in both the PLC/late endosomes and lysosomes (Griffiths et al., 1988, 1990a; Parton et al., 1989; Griffiths and Gruenberg, 1991). This suggests that at steady state the tracers equilibrate between the two compartments. Third, when rat and mouse cells are fused together

2 146 A. Jahraus and others to form hybrid cells, the PLC/late endosomes fuse to form one functional compartment (Ferris et al., 1987; Deng et al., 1991). In contrast, the MPR-negative lysosomes maintain their discrete size and shape, and do not appear to fuse permanently to form a larger structure. Nevertheless, within a few hours after cell fusion both membrane antigens and luminal contents of the lysosomes from the two parental cell types are freely intermixed (Deng et al., 1991). We asked in the present study whether lysosomes can fuse in a retrograde manner with the PLC/late endosomes in vivo. Our approach is based on two observations. First, internalization by cells of sucrose, a non-degradable molecule, leads to the formation of large vesicles, referred to as sucrosomes. Further internalization of invertase (sucrase), added to the culture medium, causes a disappearance of all the preformed sucrosomes over a period of a few hours, indicating that these vesicles are still accessible to endocytic traffic (Cohn and Ehrenreich, 1969; Swanson et al., 1986; Ferris et al., 1987). We showed previously that these sucrosomes correspond to the lysosome compartment, since they are all reactive for lamps, but negative for the CI-MPR (DeCourcy and Storrie, 1991). The latter was, as expected, concentrated in structures in the perinuclear region whose shape was apparently unperturbed by sucrose. These data argue that within the endocytic pathway only the lysosomes display the property of swelling after accumulating sucrose. The second observation relevant to the present study comes from recent data on the phagocytic pathway in peritoneal macrophages. Latex beads taken up by phagocytosis accumulated with time in the PLC/late endosome compartment and were not found in lysosomes. The latter can, however, be labelled with internalized BSA/gold, which also accumulates in the PLC/late endosomes (Rabinowitz et al., 1992; Tassin et al., 1990). In these cells, the CI-MPR was restricted to one part of the PLC/late endosomes. The membrane of the PLC/late endosome compartment was enriched in lamp 1 and 2 as well as in the late endosomal GTP-binding protein rab7. In the present study we combine these two sets of observations using the following strategy. Sucrose was internalized in cells in order to swell the lysosomes, producing the morphologically distinct sucrosomes. Subsequently, the cells were allowed to take up latex beads to which invertase had been covalently bound. Thus, within the period of our experiments the invertase latex beads taken up by phagocytosis would effectively behave as resident markers of the PLC/late endosomes and the compartment-bound invertase would be free to act on any sucrose transported back from the sucrosomes to the PLC/late endosomes. The results suggest that lysosomes are indeed capable of fusing back with the PLC/late endosomes. MATERIALS AND METHODS Cell culture NRK-49F cells were cultured in Dulbecco s modifed Eagle s medium (DMEM) supplemented with 5% fetal calf serum, 100 i.u./ml penicillin and 100 µg/ml streptomycin, 1% non-essential amino acids (Gibco/BRL) and 4.5 g/l glucose. Cells were routinely grown in 100 mm Falcon tissue culture dishes in a 5% CO 2 atmosphere incubator at 37 C, and were passaged by trypsination 36 hours before use at a 1:10 dilution from 80% confluent dishes. Coupling of invertase to latex beads Invertase was coupled to carboxylated latex beads by modification of a protocol by Molday et al. (1975). Briefly, 3 ml of 10 mg/ml invertase (grade X, from Candida utilis, chromatographically purified; Sigma Chemical Company, St. Louis, Missouri) in 0.15 M NaCl, ph 7, was mixed on ice with µl of carboxylate-modified latex beads (10% suspension, particle diameter: 0.885± µm; Sigma). The ph was adjusted to and kept stable during the whole procedure. A 1.5 ml sample of a 30 mg/ml aqueous solution of EDAC ((1-ethyl-3-(3-dimethylaminopropyl) carbodiimide) hydrochloride; Sigma) was then added very slowly while stirring. Subsequently, the mixture was incubated for 4 hours with continuous stirring at 4 C and for 30 minutes at room temperature. The reaction was stopped by adding sodium acetate, ph 4.2, to a final concentration of 100 mm. The beads were washed 3 times with PBS (phosphate buffered saline, ph 7.2) by centifugation in an SW 40 rotor for 12 minutes at 25,000 g at 4 C. To remove free invertase, a step sucrose gradient was used as previously described (Muller et al., 1980). The invertase beads, at the 10%-25% interface, were collected and dialysed extensively against PBS. The invertase activity was assayed as described by Ferris et al. (1987) and the colorimetric product was measured at 455 nm with a spectrophotometer. This assay can detect invertase amounts of SI (standard international) units (correspond to 1-2 ng invertase)/100 µl input. HRP was tagged to carboxylated beads by the same method. The enzyme-conjugated bead number was measured using a flow cytometer. The beads were then frozen in liquid nitrogen and stored in aliquots at 20 C. In all cases, the invertase-beads were stored frozen for no longer than one month and thawed only once. After this procedure, 86% of the beads were still monodisperse, and a loss of 10.8±8.2% s.d. of invertase activity was observed. Four parallel invertase-bead preparations made the same day were effectively identical with respect to the invertase conjugated: ± s.d. SI units invertase/bead. The SI units were referred to as soluble native invertase and hence should be considered to be measures of apparent enzyme activity. This corresponds on average to nearly 3650 invertase molecules/bead. Variation was observed for preparations made over a series of months, presumably related to differences in bead concentration, ph and temperature. Formation/disappearence of sucrosomes NRK cells grown on coverslips were incubated for 15 hours in culture medium containing 0.03 M sucrose to form sucrosomes. After 3 washes of 5 minutes in PBS and a 1 hour chase in sucrose-free medium to clear the early compartments of the endocytic pathway, a 2 hour pulse was made with invertase-beads in 4 ml sucrosefree medium ( SI units invertase per bead). The cells were washed 2 times with PBS and 3 times with culture medium and then chased for various times. After the internalization and after 1, 3 and 5 hours of chase in bead-free medium, the cells were observed for the presence of sucrosomes with a Zeiss Axiophot phase-contrast microscope. For photography, a 40 Planapo phase-contrast oil objective was used. The same procedure was performed with unconjugated carboxylated beads as a control. In another set of experiments to study the formation of sucrosomes, the cells were first incubated for 2 hours with invertase-beads, washed extensively, and the beads were then chased overnight. The cells were then incubated in 0.03 M sucrose in culture medium for 2 hours, 4 hours or 8 hours. The coverslips were then observed as described above. The same procedure was done using HRP-tagged beads as a control. Cryosections NRK cells were incubated for 2 hours with latex beads diluted 1:150

3 Interaction of lysosomes and late endosomes 147 in culture medium. The cells were then washed extensively with PBS and incubated for 43 hours in bead-free medium. During this chase time, the cells were incubated in 0.03 M sucrose culture medium for the last 20 hours. They were then detached by proteinase K treatment and fixed in 8% formaldehyde freshly made from paraformaldehyde as described previously (Griffiths et al., 1984). The fixative was supplemented with 0.03 M sucrose to maintain the sucrosome osmolarity. Ultrathin cryosections were prepared and double-labeled with antibodies and 6 nm or 9 nm Protein A-gold as described previously (Slot et al., 1991). lgp 120 (Lewis et al., 1985) was revealed with a rabbit polyclonal antibody (a kind gift from Dr I. Mellman), which is directed against the rat lysosomal integral glycoprotein. The 215 kda CI MPR (cation-independent mannose 6- phosphate receptor) (Griffiths et al., 1988) was revealed with a rabbit polyclonal antibody against the bovine receptor (a kind gift from Dr B. Hoflack). Determination of the stability of the invertase-bead conjugate Two dishes of NRK cells were incubated for 2 hours with invertase beads/dish ( SI units invertase per bead). After washing twice in ice-cold PBS and twice in ice-cold PBS-BSA, the beads were chased for 5 hours. Cells were washed again and scraped in ice-cold PBS. They were then centrifuged and the pellet was resuspended in 200 µl of 0.1% Triton X-100 in triple-distilled water and left on ice for 20 minutes. The beads were pelleted at 150,000 g, 150 µl of the supernatant was collected and the pellet was resuspended in 150 µl 0.1% Triton X-100 in triple-distilled water. The invertase assay was done as before with 100 µl of both supernatant and resuspended pellet. The beads were counted in the flow cytometer. Effect of nocodazole on the disappearance of sucrosomes NRK cells were plated on 5 mm coverslips and incubated with 10 µm nocodazole for 2.5 hours to assess the effect of nocodazole on microtubules in NRK cells. The coverslips were dipped into prewarmed PBS, and for 2 seconds into 5 different beakers containing 10 ml warm (37 C) extraction medium (0.08 M K-Pipes, ph 6.8, 5 mm EGTA, 1 mm MgSO 4, 0.5% Triton X-100). They were transferred directly for 4 minutes into 20 C methanol for fixation and then transferred into PBS. Standard methods for fluorescence immunolabeling were used as described previously (Deng et al., 1991) with a mouse monoclonal anti-tubulin antibody (a kind gift from Dr T. Kreis), which was visualized with a FITC-conjugated goat anti-mouse IgG. To address the effect of microtubule depolymerization by nocodazole on sucrosome disappearence, NRK cells were incubated in 0.03 M sucrose overnight to form sucrosomes. The cells were then incubated in medium without sucrose for 60 minutes and a 90 minute pulse was made with invertase-beads/dish ( SI units invertase per bead). Cells were washed extensively and chased for 30 minutes to recover. The cells were then incubated for 1 hour at 4 C in air medium (1 MEM, 4 mm NaHCO 3, 10 mm Hepes, 11 mg pyruvate/100 ml, 0.64% glucose, 1% 100 glutamine from Gibco/BRL) with or without 10 µm nocodazole and in culture medium for 2.5 hours with or without 10 µm nocodazole at 37 C. The cells were then observed and photographed as before. Morphometry To determine the cytoplasmic area occupied by sucrosomes, a point counting procedure with light micrographs (i.e. cell projection) was employed using a double-lattice system (Weibel, 1979). The number of large points over cell profiles and the number of small points over sucrosomes were counted (1 large point corresponded to 8 small points). The ratio of the number of small points over sucrosomes relative to the total small points over the cell profile gives the areal fraction of sucrosomes per cell projection. 100 cell profiles were scored at each time point. RESULTS Characterization of the sucrosomes and latex beadcontaining compartments Incubation of NRK cells in culture medium supplemented with 0.03 M sucrose effectively induced the formation of large vacuolated perinuclear structures, absent in normal cells (Fig. 1a), referred to as sucrosomes, which were easily visualized by light microscopy (Fig. 1b). Although sucrosomes started to appear within 30 to 60 minutes of incubation in sucrose-containing medium, the best results were obtained when cells were incubated in sucrose for 12 to 15 hours. At that time, the cells contain a large number of sucrosomes of maximal size. In agreement with earlier data (Cohn and Ehrenreich, 1969; Pesanti and Axline, 1975; Swanson et al., 1986; Ferris et al., 1987), subsequent internalization of free invertase resulted in the rapid disappearance of the sucrosomes (results not shown), demonstrating the accessibility of the sucrosomes for enzyme internalized from the medium. In the absence of invertase the sucrosomes were stable in size for more than 8-10 hours. Although not considered a professional phagocyte, NRK cells were able to internalize latex beads from the culture medium (Grinell and Geiger, 1986). The presence of sucrosomes in NRK cells did not seem to affect the ability of these cells to subsequently internalize latex beads. Indeed, internalization of 0.8 µm latex beads for 2 hours followed by various chase times resulted in the delivery of the beads to perinuclear structures that appeared distinct from the sucrosomes (Figs 2, 4 and 5). The average bead uptake per cell was 16±2 s.d. The ability to internalize beads was variable from cell to cell. A few cells failed to internalize any beads whereas a small number had taken up as many as 40 beads. For the experiments reported below it was crucial to establish that the sucrosomes indeed were the terminal lysosomes and that several hours after the uptake of the latex beads, these resided in a distinct compartment, the PLC/late endosomes. For this characterization we used CI MPR, which is present in PLC/late endosomes but not in lysosomes, and lgp 120, which is present in both compartments (Griffiths et al., 1988, 1990a; Geuze et al., 1988). Immunofluorescence microscopy clearly revealed the presence of lgp 120 on the membrane of the sucrosomes (Fig. 2a,b), while CI MPR labelling, punctate over the juxtanuclear cytoplasm, was not observed as a ring around the sucrosomes (Fig. 2c). This was confirmed by electron microscopy (Fig. 3). In contrast, the latex bead-containing structures were labelled for both lgp 120 and MPR as shown by immunofluorescence (Fig. 2d,e) and electron microscopic immunocytochemistry (Fig. 3). These results are consistent with previous observations in which the latex bead-containing structures were identified as the prelysosomal/late endosome compartments (Rabinowitz et al., 1992), and the sucrosomes as being terminal lysosomes (DeCourcy et al., 1991). Collectively, these results indicate that the latex bead-containing compartments and the sucrosomes are distinct organelles. In vivo determination of fusion between sucrosomes and late endocytic structures containing latex beads Our data argue that latex beads can serve as a marker of the PLC/late endosomes, while the sucrosomes can serve as a way

4 148 A. Jahraus and others to identify the terminal lysosomes. In the next series of experiments cells were incubated in sucrose-containing medium to form sucrosomes, followed by a subsequent internalization via phagocytosis of invertase covalently conjugated to latex beads. The strategy here was that, when coupled to the beads, the enzyme would be trapped for many hours in the PLC/late endosomes, and would not be delivered to the lysosomes/ sucrosomes Thus, only transfer between the two compartments would allow the meeting of sucrose and invertase, resulting in the degradation of sucrose to glucose and fructose, and in the disappearance of the sucrosomes. Conversely, if the terminal lysosomes are a dead end in the pathway such that their content never returns to earlier compartments, the sucrosomes should not be affected by the invertase beads. Internalization of these invertase-derivatized latex beads resulted in their appearance in perinuclear compartments, in a manner indistinguishable from that of unconjugated beads. To demonstrate that the beads were delivered to an endocytic compartment at a stage prior to that of the terminal lysosomes we tested whether internalization of invertase-derivated latex beads could prevent the subsequent formation of sucrosomes. For this, we first internalized invertase beads, allowed them to reach the late compartments in cells without sucrosomes, followed by a continuous incubation of the cells in sucrosecontaining culture medium. This approach effectively inhibited the formation of sucrosomes for a period of over 8 hours (Fig. 4b,d and f). Only a small number of sucrosomes were visible in the periphery of few cells. In contrast, sucrosomes had formed already minutes after sucrose internalization in cells preloaded with latex beads covalently coupled to horseradish peroxidase (Fig. 4a,c and e). Table 1 shows a quantitative analysis of the results, which confirm the qualitative observations shown in Fig. 4. Since sucrose is internalized through Table 1. Preloading of invertase-conjugated latex beads blocks the formation of sucrosomes Invertase-latex beads Control beads (HRP-tagged) Sucrose (areal density of sucrosomes (areal density of sucrosomes pulse (h) per cell projection*) per cell projection*) ± ± ± ± ± ±0.94 Estimation by point counting of areal density of sucrosomes in NRK cells that had first internalized either invertase-latex beads or HRP-latex beads followed by an incubation in sucrose-containing culture medium for different times. *Expressed as % of total cell area occupied by sucrosomes. fluid-phase endocytosis, these results strongly suggest that the beads are located in an intermediate compartment in the endocytic pathway of sucrose transport to lysosomes, which corresponds, according to our data and the work from Rabinowitz et al. (1992), to the PLC. In the next series of experiments, the sucrosomes were first allowed to form and the cells were subsequently fed with invertase-latex beads followed by various periods of chase. In this case, a progressive disappearance of the sucrosomes was already seen after 1 hour of chase (Fig. 5b). Indeed, sucrosomes were seldom observed in the cells after 3 to 5 hours of chase (Fig. 5d and f). The few sucrosomes observed tended to be localized at the cell periphery away from the perinuclear beads. In contrast, internalization of horseradish peroxidase (HRP)-latex beads had no effect on the sucrosomes for a period of over 7 hours (Fig. 5a,c and e). In this case, cells displayed numerous beads and sucrosomes in the perinuclear region at all experimental time points. We next quantified these results in two different ways. First, Fig. 1. Normal NRK cells (a) and cells containing sucrosomes (b). Internalization of 0.03 M sucrose for 6 hours leads to the formation of large vesicular structures, sucrosomes (S), around the nucleus (N). Bar, 10 µm.

5 Interaction of lysosomes and late endosomes 149 Fig. 2. Immunofluorescence characterization of the latex bead-containing compartments and sucrosomes. Sucrosomes (S) were formed by incubating the cells in 0.03 M sucrose for 15 hours. Latex beads were internalized for 2 hours and chased for 20 hours. (a,b) Immunolocalization of lgp 120. The labelling appears as a ring around the large perinuclear sucrosomes. (c) Immunolocalization of the CI MPR. The labelling appears as a punctate pattern in the juxtanuclear cytoplasm, while no distinct ring-like labelling (as seen for the lgp 120) is observed over the sucrosomes. This is especially evident in the peripheral region. (d,e) Colocalization of lgp 120 (d) and latex beads (e) in cells without sucrosomes. The blue-dyed beads are fluorescent in the rhodamine channel. Arrows point to bead-containing structures positive for lgp 120 in the periphery of the cell. Bars, 10 µm.

6 150 A. Jahraus and others Fig. 3. Immunoelectron microscopic localization of lgp 120 and the CI MPR in a double-labeling analysis. Invertase-latex beads (ILB) were internalized for 2 hours and chased for 43 hours, including a sucrose pulse of 20 hours at the end of the chase time. (a,b) The membrane surrounding the beads, which appear here in a tangential section, are labeled with both lgp 120 (large gold, arrowheads) and CI MPR (small gold, arrows) similar to the neighboring late endosomal compartment (LE). (c,d) Only lgp is associated with the membrane of the sucrosomes (S). Arrows point to two CI MPR hits over the sucrosome lumen. Nu, nucleus. Bars, 0.2 µm.

7 Interaction of lysosomes and late endosomes 151 we estimated the relative proportion of cells that had at least one visible sucrosome (sucrosome-positive; Table 2). These data clearly show the effect of invertase beads on the disappearance of sucrosomes, in contrast to control HRP-beads, which had no significant effect on the sucrosomes. Furthermore, Table 3 shows the results of the morphometric analysis in which the proportion of the area of cells in the light micrographs occupied by sucrosomes was estimated by point counting. These data also show the effective disappearance of sucrosomes in the presence of invertase beads relative to the control beads. Together, these results argue that the contents of sucrosomes and the bead-containing organelles must mix, presumably by fusion of the organelles, in order to allow the degradation of the sucrose by the invertase-tagged beads. We then investigated whether the degradation of sucrose and the resulting disappearance of the sucrosomes was caused by the possible dissociation of invertase from the beads followed by transport of free enzyme molecules to the lysosomes. Cells were allowed to internalize invertase-latex beads for 2 hours followed by a chase of 5 hours, after which cells were lysed

8 152 A. Jahraus and others Fig. 4. Kinetic analysis of sucrosome formation in cells containing latex beads (controls, a,c and e) or invertase-latex beads (b,d and f). In all cases the cells first internalized beads for 2 hours, followed by an overnight chase in medium free of beads. Subsequently, sucrose was added to the medium and internalized for 2 hours (a and b), 4 hours (c and d) and 8 hours (e and f). In the experiments using latex beads (LB), sucrosomes (S) were formed and persisted throughout the experiments. With invertase-latex beads (ILB), however, the formation of sucrosomes was inhibited at all time points. In this case, some sucrosomes are observed at the periphery of a few cells (arrowheads). Bars, 10 µm.

9 Interaction of lysosomes and late endosomes 153 Fig. 5. Kinetic analysis of sucrosome disappearance after internalization of latex beads (control: a,c and e) or invertase-latex beads (b,d and f). In all cases the sucrosomes were formed by continuous internalization of sucrose for 15 hours followed by a chase of 1 hour in sucrose-free medium. The beads were then internalized for 2 hours and chased for 1 hour (a and b), 3 hours (c and d) and 5 hours (e and f). In the controls that have internalized unconjugated latex beads (LB), sucrosomes (S) are observed throughout the experiment, while the internalization and chase of invertase-latex beads (ILB) results in the progressive disappearance of the sucrosomes. Bars, 10 µm.

10 154 A. Jahraus and others Table 2. Disappearence of sucrosomes in cells fed with invertase-conjugated latex beads Chase Invertase-latex beads Control beads (HRP-tagged) of beads (sucrosome-positive (sucrosome-positive (h) cells (%)*) cells (%)*) NRK cells were first incubated in sucrose-containing culture medium to form sucrosomes and then invertase-latex beads or HRP-latex beads were internalized for 2 h followed by various chase times. *Expressed as % of cells displaying at least one sucrosome. Table 3. Disappearence of sucrosomes in cells fed with invertase-conjugated latex beads Chase Invertase-latex beads Control beads (HRP-tagged) of beads (areal density of sucrosomes (areal density of sucrosomes (h) per cell projection*) per cell projection*) ± ± ± ± ND 3.64±0.82 Areal density of sucrosomes in NRK cells incubated in sucrosomecontaining culture medium first, to form sucrosomes, and then with invertaselatex beads or HRP-latex beads for 2 h, followed by different chase times. *% of cell area. ND, not determined. with 0.1% Triton X-100 in water. The lysate was then centrifuged at 150,000 g for 20 minutes, and invertase activity was assayed on the pellet (bead-associated invertase) and the supernatant (free invertase). The results showed that minimal invertase activity was detectable in the supernatant (A 455 =0.003±0.002 s.d., an amount about equal to the limit of detection of the assay) and that the vast majority of invertase activity was in the pellet together with the latex beads (A 455 =0.072±0.006 s.d.). Assuming the invertase activity in the supernatant is above the noise level of the assay, this corresponds to 4% of total invertase delivered to the cells. To examine if this small amount of free active invertase could have the same dramatic effect on sucrosomes as the internalized invertase beads, NRK cells that had formed sucrosomes overnight were incubated in 5 µg/ml invertase for 2 hours and then chased for 5 hours in invertase-free medium. This concentration of invertase was calculated to give a delivery of invertase from the extracellular medium to the lysosomes equivalent to that which might have been released from the beads (Steinman et al., 1974, 1976; Adams et al., 1982, for basis of calculations). The sucrosome positive and negative cells were then quantitated as before (see Table 2). This control showed that this minimal level of invertase was not able to remove sucrosomes in the same manner as invertase-latex beads did: only 12% of cells fed with invertase-latex are sucrosome positive whereas 78% of cells incubated in medium with free invertase still have sucrosomes. Indeed, even a fold higher free invertase concentration in the medium was insufficient to produce the full invertase-bead effect (29% versus 12% sucrosome positive cells). Together these results strongly indicate that the effect observed with invertase-latex beads was due to delivery of sucrose from lysosomes to the PLC/late endosomes rather than the delivery of released enzyme from the PLC/late endosomes to the lysosomes. Nocodazole experiments Previous cell fusion experiments using the sucrosome approach demonstrated that mixing between invertase-containing late endocytic structures from one cell population and sucrosomes of a second cell population was dependent upon the preservation of a well-organized microtubule network (Deng and Storrie, 1988; Deng et al., 1991). In the next experiments we tested whether meeting of the lysosomes and the PLC/late endosomes was dependent on microtubules. For this, cells were allowed to form sucrosomes and subsequently to internalize invertase-derivatized latex beads to late compartments as before. Nocodazole was then added in order to depolymerize microtubules and the cells were examined. The effect of the drug was confirmed by tubulin immunofluorescence labelling (Fig. 6a,b). In nocodazole-treated cells, the presence of invertase-latex beads caused little disappearance of sucrosomes after 3 hours (Fig. 6d). In contrast, in control cells not treated with nocodazole the invertase-latex beads caused the disappearance of the sucrosomes (Fig. 6c). The point counting analysis shows that 1.1±0.6% s.d. of the total cell area in the micrographs was occupied by sucrosomes in cells not treated with nocodazole. In contrast, the value was three time higher in cells treated with nocodazole (3.3±1.0% s.d.). DISCUSSION According to the classical concepts of endocytosis, the flow of internalized material destined for degradation in the endocytic pathway follows a unidirectional movement towards the terminal lysosomes (de Duve, 1983; Kornfeld and Mellman, 1989; Murphy, 1991). In the present study we investigated whether retrograde traffic between the terminal lysosomes and the preceding PLC/late endosomes occurs. To do so, we used sucrose to swell and reveal the lysosomes, and invertase-latex beads as a marker of the PLC/late endosomes. Our data clearly show that internalization of invertase-latex beads in cells filled with sucrosomes effectively led to a progressive disappearance of the sucrosomes, as expected if meeting occurs between the enzyme invertase and its substrate sucrose. In contrast, the sucrosomes remained stable when control beads lacking invertase were used. We could demonstrate that the disappearance of the sucrosomes resulted from their interactions with the PLC/late endosomes containing the invertase-beads, and not from transport to the lysosomes of free invertase following its dissociation from the beads. Indeed, internalization of invertase-beads followed by a chase of several hours did not decrease the invertase activity associated with the latex beads, and no significant free invertase activity was detected in the supernatant of lysed cells. Under these conditions, essentially all the invertase activity was pelletable together with the latex beads. Additional observations suggest that direct contact, and presumably fusion, between the PLC/late endosomes and the sucrosomes, rather than transport of free invertase, is needed for the sucrose to be degraded. In some cases, cells that had effectively internalized invertase beads to the perinuclear region appeared to contain some sucrosomes in peripheral areas where beads were absent. Under these conditions, the peripheral sucrosomes were stable over a long period. In addition, in the few cells that had not internalized invertase-latex beads, sucrosomes remained stable

11 Interaction of lysosomes and late endosomes 155 Fig. 6. Effect of nocodazole on the kinetics of disappearance of sucrosomes in cells that have internalized invertase-latex beads. (a,b) Immunofluorescent pattern of microtubules using anti-tubulin antibodies in control cells (a) and in cell treated with 10 µm nocodazole for 2.5 hours (b). (c,d) Sucrosomes (S) were formed as before and invertase-latex beads (ILB) were internalized for 90 minutes. The cells were then washed at 4 C for 1 hour without nocodazole (c) or with 10 µm nocodazole (d). The beads were then chased for 2.5 hours in medium without nocodazole (c) or with 10 µm nocodazole (d). Although the sucrosomes (S) disappeared in the absence of nocodazole (c), they are still clearly present in cells treated with nocodazole (d). Bar, 10 µm.

12 156 A. Jahraus and others for a long period, demonstrating the lack of free invertase in the medium. The need for close contact between sucrosomes and the bead-containing PLC/late endosomes to allow degradation of sucrose is also supported by the results obtained when we disrupted the microtubule organization with nocodazole. In this case, we observed an inhibition of the disappearance of the sucrosomes, under conditions where both the invertase-beads and the sucrose had reached their respective compartments before nocodazole treatment. Indeed, these results suggest that the interactions between PLC/late endosomes and lysosomes are dependent on the presence of a well-preserved microtubule organization. Previous studies using colchicine suggested that microtubules were not involved in the process of sucrosome disappearance after internalization of free invertase by endocytosis (Pesanti and Axline, 1975). However, recent studies using a cell fusion approach clearly showed the involvement of microtubules in the meeting and fusion between late endocytic structures and the sucrosomes (Deng and Storrie, 1988; Deng et al., 1991). In our case, since nocodazole by itself had no effect on the invertase activity in vitro, these results further suggest that the beads and the sucrose were in distinct compartments when the nocodazole treatment was started. In an earlier publication (Rabinowitz et al., 1992) we argued that the latex bead-containing compartments (PLC/late endosomes) are proximal to the lysosomes. This notion is supported in the present study by the experiment showing that when invertase-beads were internalized first, subsequent incubation of the cells in sucrose-containing medium did not lead to the formation of sucrosomes. This suggests that sucrose, which is internalized by fluid-phase endocytosis, is degraded in the invertase-bead-containing compartment before reaching the lysosomes. Several kinetic studies have shown that fluid-phase tracers, accumulated for hours in late endocytic organelles (referred to as lysosomes), could reappear in the culture medium (Besterman et al., 1981; Buktenica et al., 1987; Blomhoff et al., 1989). Although a retrograde transport of these tracers through the endocytic apparatus could be linked to these observations, the conclusions from these reports favored the existence of a regurgitation process analogous to exocytosis. Moreover, other reports showed no back flow of internalized HRP to the medium (Steinman et al., 1974, 1976; Storrie et al., 1984). The present study is the first to assess directly the possibility of retrograde traffic between well identified and characterized organelles along the endocytic pathway. 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