Analysis of post-lysosomal compartments


Lysosomes are acidic intracellular compartments and are regarded as degradative and the end point, of the endocytic pathway. Here we provide evidence for the generation of acid hydrolase poor and non-acidic post-lysosomal compartments in NRK cells that have accumulated non-digestible macromolecules, Texas red-dextran (TR-Dex), within lysosomes. When TR-Dex was fed to the cells for 6 h, most of the internalized TR-Dex colocalized with a lysosomal enzyme, cathepsin D. With an increase in the chase period, however, the internalized TR-Dex gradually accumulated in cathepsin D-negative vesicles. These vesicles were positive for a lysosomal membrane protein, LGP85, and their formation was inhibited by treatment of the cells with U18666A, which impairs membrane transport out of late endosomal/lysosomal compartments, thereby suggesting that the vesicles are derived from lyso- somes. Interestingly, these compartments are non-acidic as judged for the DAMP staining. The results, therefore, suggest that the excess accumulation of non-digestible macromolecules within lysosomes induces the formation of acid hydrolase poor and non-acidic post-lysosomal compartments. The fact that treatment of the cells with lysosomotropic amines or a microtubule-de- polymerization agent resulted in extensive colocalization of TR-Dex with cathepsin D further indicates that the formation of the post-lysosomal compartments depends on the lysosomal acidification and microtubule organization. Furthermore, these results suggest bi-directional membrane transport between lysosomes and the post-lysosomal compartments, which implies that the latter are not resting compartments.

Keywords: Lysosomes; Post-lysosomes; Membrane traffic; Lysosomal storage disease

Lysosomes are acidic membrane-bound organelles which play a key role in the degradation of macromol- ecules internalized by endocytosis or sequestered by autophagy [1–4]. Small molecules produced by these processes are subsequently exported from lysosomes either through specific transporters present in the lim- iting membranes to the cytoplasm or by a vesicle-med- iated process to other intracellular compartments in order to maintain the homeostasis of the cell. Conse- quently, non-digestible materials and membranous whorls concentrate within lysosomes, which provide this organelle with its characteristic high density and heter- ogeneous morphology.Several models have been proposed for the biogenesis and maintenance of lysosomes [4,5]. They all suggest that lysosomes are not the end point of the endocytic pathway. Rather, lysosomes seem to be maintained by an extensive supply of membrane and lumenal compo- nents through direct fusion and fission with late en- dosomal compartments or the kiss-and-run process proposed by Storrie and Desjardins [6]. Therefore, it is proposed that lysosomes and late endosomes are in dynamic equilibrium [3]. Little is known, however, about whether less digestible molecules once accumu- lated within lysosomes are gradually degraded through fusion with late endosomes and/or lysosomes containing active lysosomal enzymes or merely continue to accu- mulate in lysosomes, which ultimately become resting lysosomes.

In the present study, we show that when non-digest- ible materials, such as Texas red-labeled dextran, were fed to NRK cells, they were excluded from acid hydrolase-enriched lysosomes and accumulated in non-acidic and less degradative compartments. The formation of these compartments was inhibited by a reagent that impairs membrane transport out of late endosomes and lysosomes, suggesting that these com- partments are produced through the post-lysosomal membrane trafficking. Our results further show that the formation of the post-lysosomal compartments seems to be regulated by an acidification of lysosomes and the microtubule organization.

Experimental procedures

Materials. Culture media and fetal bovine serum (FBS) were pur- chased from Gibco-BRL (Grand Island, NY). Filipin and nocodazole were obtained from Sigma Chemical (St. Louis, MO). Texas red- and Oregon green-conjugated dextran (Mr, 70,000 lysine fixable), and Alexa488-, Alexa594-, and Cascade blue-labeled secondary antibodies were purchased from Molecular Probes (Eugen, OR). U18666A was obtained from Biomol (Plymouth Meeting, PA).

Antibodies. Rabbit polyclonal antibodies to rat LGP85 were raised against a purified LGP85 from isolated rat liver lysosomal membranes [7]. Mouse monoclonal antibodies to rat LGP85 were kindly provided by Kenji Akasaki (Fukuyama University, Japan). Rabbit polyclonal antibodies to rat cathepsin D have been previously described [8]. Mouse monoclonal antibodies to GM130 were purchased from Transduction Laboratories (Lexington, KY).

Cell culture. NRK cells were cultured in DMEM supplemented with 10% FBS, 2 mM glutamine, and 100 U penicillin/streptomycin/ml in a humidified atmosphere of 95% air and 5% CO2 at 37 °C. The cells were plated onto 13 mm coverslides, and after 24–48 h, were used for immunocytochemical experiments.

Immunofluorescence microscopy. Immunofluorescence microscopy was performed as described previously [9,10]. Briefly, cells cultured on coverslips were rinsed with phosphate-buffered saline (PBS), fixed immediately in 4% paraformaldehyde (PFA) in PBS, pH 7.4, for 30 min at room temperature, and permeabilized with 0.05% saponin in PBS for 15 min. Cells were quenched with 50 mM NH4Cl in PBS for 15 min and blocked with 1% bovine serum albumin (BSA) in PBS for 30 min. The cells were then incubated for 1 h with the primary antibody in blocking solution using the following dilutions: anti-LGP85 anti- body (1:300), anti-cathepsin D antibody (1:300), and anti-GM130 antibody (1:100). The cells were washed with blocking solution and incubated for 30 min with the secondary antibodies diluted in the blocking solution.

Coverslips were then washed three times with blocking solution, rinsed with water, and mounted onto glass slides. Visualization of the acidic compartments by 3-(2,4-dinitroanilino)-3′- amino-N-methyldipropylamine (DAMP) was carried out using the acidic granule kit (Oxford Biomedical Research, MI) according to the manufacturer’s instructions. The cells were analyzed by confocal laser scanning microscopy using a Radiance 2100 MP confocal microscope (Bio-Rad Laboratories, Richmond, CA) with an argon/krypton laser,Red Diode laser, and Blue Diode laser. Photographic images were acquired using a cooled CCD camera (MicroMAX; Princeton In- struments, Trenton, NJ), processed using IPlab software (Scanalytics, Fairfax, VA), and merged using Adobe Photoshop software (Adobe Systems, Mountain View, CA).

Dextran internalization. For dextran internalization, cells cultured on coverslips were incubated with Texas red- or Oregon green-labeled dextran (0.1 mg/ml) in serum-free DMEM with 1 mg/ml of BSA for 6 h at 37 °C and then cultured in normal medium for up to 12 h. After a chase, cells were washed with PBS, fixed in 4% PFA in PBS, per- meabilized with saponin in PBS, and immunostained for cathepsin D or LGP85.

Filipin staining. Cells cultured on coverslips were rinsed with PBS and fixed in 4% PFA in PBS, pH 7.4, for 30 min at room temperature. They were stained (and permeabilized) with 0.05% filipin in PBS for 1 h and then immunostained for cathepsin D or LGP85.

Results and discussion

The dextran internalized in cells by a fluid-phase en- docytosis is delivered through endosomal compartments to lysosomes, where it accumulates due to its acid hy- drolase-resistant nature [11]. Therefore, we took ad- vantage of this to investigate the fate of lysosomes that had accumulated a non-digestible endocytic marker, TR-Dex. To this end, NRK cells that had internalized TR-Dex for 12 h were chased for up to 6 h. After the chase, the cells were fixed and permeabilized, and the intracellular distribution of the internalized TR-Dex was visualized in comparison with that of the lysosomal markers cathepsin D and LGP85. Most of the TR-Dex colocalized with the lysosomal enzyme cathepsin D (Figs. 1A–C), thereby indicating the accumulation of TR-Dex in lysosomes. After 3 h of chase, however, some TR-Dex began to appear in vesicles negative for ca- thepsin D immunostaining (Figs. 1D–F). This difference in the distribution of the internalized TR-Dex and ca- thepsin D continued to increase as the chase period in- creased. After 12 h of chase, most of the TR-Dex was detected in vesicles distinct from those containing ca- thepsin D (Figs. 1J–L). TR-Dex-positive, but cathepsin D-negative (TR-Dex+/CD—), compartments, however, were labeled with antibodies to a lysosomal membrane protein, LGP85 [7,12] (Figs. 1M–O), indicating a lyso- somal origin. On the other hand, even in normal cells that could not internalize TR-Dex, cathepsin D did not completely colocalize with LGP85 (data not shown). This seems to reflect a difference in their intracellular localization; cathepsin D is more restricted in its distri- bution in lysosomes than LGP85, part of which is also localized in late endosomes (data not shown). Such a difference in localization between cathepsin D and LGP85 apparently increased in cells that had internalized TR-Dex. In addition, an increased concentration of TR-Dex accelerated the formation of TR-Dex+/CD— compartments (data not shown). The appearance of TR- Dex+/CD— compartments, therefore, seems to depend on the excess accumulation of non-digestible macro- molecules within lysosomes.

To clarify whether TR-Dex+/CD— compartments in- deed originate from lysosomes, we used U18666A, a reagent that inhibits post-lysosomal membrane trans- port [13]. U18666A is known to accumulate free-cho- lesterol within late endosomes/lysosomes, which causes impairment of the membrane trafficking out of these compartments. Therefore, if TR-Dex+/CD— compart- ments were derived from lysosomes, U18666A treatment would lead to impairment of the formation of these compartments. As expected, in cells treated with U18666A, the internalized TR-Dex extensively colocal- ized with cathepsin D in large vesicles, where free cho- lesterol also accumulates, as evident from the staining of filipin, an antibiotic that can specifically bind with free cholesterol (Fig. 2). Even after a prolonged chase for up to 12 h in the presence of U18666A, TR-Dex remained colocalized with cathepsin D. These results apparently support the notion that TR-Dex+/CD— compartments are formed by membrane transport out of lysosomes.

Fig. 1. Internalized Texas red dextran was delivered to a compartment distinct from cathepsin D-containing lysosomes. NRK cells were fed with the medium containing TR-Dex (0.1 mg/ml) for 6 h and subsequently chased for the indicated periods of time with the normal medium. After the chase, cells were fixed, permeabilized, stained for cathepsin D, and visualized by confocal microscopy (A–L). In the lower columns, cells that had inter- nalized TR-Dex for 6 h and been chased for 12 h were stained for the lysosomal membrane protein LGP85 (M–O). The left columns (A, D, G, J, and M) show merged images of Texas red dextran and cathepsin D or LGP85. Bars, 20 lm.

To further examine whether TR-Dex+/CD— com- partments are acidic, a characteristic feature of lyso- somes, cells that had internalized TR-Dex for 12 h and been chased for 12 h were subsequently incubated with DAMP, which is a basic congener of dinitrophenol and accumulates in acidic organelles [14]. After 3 h of incu- bation with DAMP, cells were fixed and stained for DAMP using anti-dinitrophenol antibody. As shown in Figs. 3A–C, although some TR-Dex-containing vesicles were positive for DAMP staining, large numbers were significantly negative for DAMP. Similar results were obtained with LGP85-staining in the TR-Dex-internal- ized cells (Figs. 3D–F). On the other hand, most of the cathepsin D-containing compartments were positive for DAMP in the TR-Dex-internalized cells (Figs. 3G–I). Together with the results shown in Fig. 1, these results suggest that most, but not all, of the TR-Dex-containing vesicles were less acidic compartments than cathepsin D-positive lysosomes.

Chloroquine is known to inhibit the acidification of endosomal/lysosomal compartments [11]. In addition, chloroquine causes perturbation of the endocytic path- way as well as enlargement of endosomal/lysosomal compartments [15,16]. Since TR-Dex+/CD— compart- ments were less acidic, we assessed the effect of chloroquine on the morphological alteration of these com- partments. To this end, cells that had internalized TR- Dex for 12 h and been chased for 12 h were treated with 100 lM chloroquine for 3 h. As can be seen in Fig. 4, chloroquine caused the enlargement of not only cathep- sin D-positive, but also the less acidic TR-Dex-positive,compartments. Interestingly, chloroquine treatment resulted in extensive colocalization of TR-Dex with cathepsin D in the enlarged vacuoles (Fig. 4A(B)). In addition, NH4Cl, a lysosomotropic amine, displayed a similar effect to chloroquine (Fig. 4A(C)). These results, therefore, suggest that the enlargement of TR-Dex-positive compartments comes from increased fusion of lysosomes and post-lysosomal compartments and decreased fission from the fused compartments, and is not a consequence of the swelling due to impairment of acidification. This also implies the importance of the interior acidification of lysosomes in membrane transport between lysosomes and the post-lysosomal compartments.

Fig. 2. U18666A treatment impairs the formation of the post-lysosomal compartments. Cells were pretreated with U18666A (3 lg/ml) for 4 h and subsequently incubated for 24 h with TR-Dex in the presence of the drug. After that, cells were chased for the indicated periods of time in the presence of U18666A, fixed, stained for cathepsin D (C, G, K, and O; green) and cholesterol with filipin (D, H, L, and P; blue), and visualized by confocal microscopy. The left columns (A, E, I, and M) show merged images of TR-Dex, cathepsin D, and filipin. Bars, 20 lm.

Fig. 3. The post-lysosomal compartments are less acidic. Cells were internalized TR-Dex for 24 h and chased for 12 h. After chased, cells were incubated for 1 h with DAMP, fixed, stained for DAMP (B, E, and H; green) and LGP85 (F; red) or cathepsin D (I; red), and visualized by confocal microscopy. The left columns show the merged images of DAMP and TR-Dex (A), LGP85 (D), and cathepsin D (G). Bars, 20 lm.

Fig. 4. pH- and microtubule-dependent membrane traffic between lysosomes and post-lysosomes. (A) Cells internalized TR-Dex for 24 h and were chased for 12 h. Subsequently, they were incubated for 3 h with medium alone (A), 100 lM chloroquine (B), or 50 mM NH4Cl (C), fixed, and stained for cathepsin D (green). (B) Cells internalized TR-Dex for 6 h and were chased for 21 h. After that, they were treated with nocodazole (33 lM) for the indicated periods of time, fixed, stained for cathepsin D (A–D; green) or GM130 (E–H; green), and visualized by confocal microscopy. The left columns show merged images of TR-Dex and cathepsin D, and GM130. Bars, 20 lm.

It should be noted that chloroquine arrests the transport of LDL-derived cholesterol at lysosomes [17]. Therefore, one explanation for the extensive colocaliza- tion of cathepsin D and TR-Dex on chloroquine treat- ment may be an impairment of fission due to the accumulation of cholesterol in lysosomes. This may also imply that cathepsin D-positive lysosomes and the post- lysosomal compartments are maintained by a “kiss-and- run” process. Thus, membrane traffic between typical lysosomes and post-lysosomes seems to be bi-directional.

We noticed that most of the TR-Dex+/CD— vesicles tend to localize more in the cell periphery than cathepsin D-positive, but TR-Dex-negative vesicles, which were relatively concentrated in the perinuclear region.Endosomal/lysosomal compartments are distributed in the perinuclear region dependent on microtubules. Re- cent studies have further demonstrated several mole- cules involved in the spatial localization of late endosomes and lysosomes to the perinuclear region. These molecules include Rab7 interacting lysosomal protein (RILP) [18], human Vam6p [19], and Rab34 [20]. Notably, it has been shown that RILP interacts directly with Rab34, which can regulate the spatial dis- tribution of lysosomes by modulating the activity of dynein–dynactin [20]. In order to examine whether the distribution of TR-Dex+/CD— vesicles is regulated by microtubules, cells that had internalized TR-Dex for 12 h and been chased for 12 h were further treated for up to 6 h with 33 lM nocodazole, a microtubule-depo- lymerization agent, which allows the perinuclear lo- calization of late endosomes/lysosomes to disperse throughout the cytoplasm [21]. The disruption of the microtubule organization by nocodazole treatment was confirmed by the scattered cytoplasmic punctate distribution of the cis-Golgi marker GM130 [22], which is normally distributed in the juxtanuclear region as reticular structures, even after treatment for 1 h. Nocodazole treatment allowed the cathepsin D-positive vesicles to move throughout the cytoplasm. Furthermore, after treatment for 6 h, cathepsin D-posi- tive vesicles began to cluster. On the other hand, al- though after treatment with nocodazole for 1 h there was no significant change in the distribution of TR-Dex- positive vesicles, the rate of colocalization with ca- thepsin D increased. After 6 h, most of the TR-Dex colocalized with cathepsin D in clustering lysosomes. These results together suggest that the formation of post-lysosomes is regulated by intact microtubules. It is conceivable, therefore, that extension of the docking/ tethering time of post-lysosomes with lysosomes induced by the microtubule-depolymerization causes the equili- bration of constituents between these compartments, and as a result, may lead to inhibition of the formation of post-lysosomes. Presumably, after docking/fusing with the post-lysosomes and subsequent fission, lysosomes rapidly migrate toward the minus-end of microtubules in the juxtanuclear region, while the post- lysosomes tend to remain in, or move to, the cell periphery. In contrast to the minus-end motor protein dynein-dependent spatial distribution of lysosomes [18], the possibility that the migration to and positioning within the cell periphery of the post-lysosomes is de- pendent on the plus-end motor protein kinesin [23,24], therefore, cannot be completely ruled out.
The early onset or late-onset age-related diseases cause an abnormal accumulation of proteins and lipids within lysosomes and lead to the dysfunction of this organelle (reviewed in [25]). They include lysosomal storage disorders that are caused by a lysosomal en- zyme deficiency and neurodegenerative disorders such as Alzheimer’s disease, Huntington’s disease, and Par- kinson’s disease. In some cases, the accumulation of aggregated proteins is the inherent toxicity, a hallmark of Alzheimer’s disease. It is of particular interest that in
Alzheimer’s disease which involves an abnormal accu- mulation of b-amyloid (Ab) within lysosomes and leads to the dysfunction of this organelle [26], Ab1–42 inter- nalized and accumulated as protease-resistance insolu- ble aggregates in lysosomes can disrupt the lysosomal proton gradient by generating free radicals [27,28]. In this case, Ab1–42 aggregates appear to mediate intra- lysosomal membrane oxidation dependent upon the presence of hydrogen peroxide and free iron. Such an oxidative reaction in lysosomes also results in the for- mation of indigestible macromolecules, such as lipo- fuscin, through aldehyde-mediated cross-linking with free amino groups within proteins, leading to a pro- gressive accumulation and cell death [29]. A similar mechanism, therefore, might operate in the cells that accumulated TR-Dex described in this study. The fact that the post lysosomal compartments were not enriched with the lysosomal enzyme cathepsin D, however, suggests that non-digestible molecules accu- mulated within lysosomes are gradually excluded by a process which involves selective sorting from the resi- dent content and membrane proteins and/or digestible molecules. Further experiments are necessary to eluci- date the mechanism involved in the formation of the post-lysosomal compartments by accumulation of non- digestible molecules within lysosomes.