Article |
Address correspondence to Satyajit Mayor, National Centre for Biological Sciences, Tata Institute for Fundamental Research (TIFR), UAS-GKVK Campus, Bellary Rd., Bangalore 560 065, India. Tel.: 91-80-3636420, ext. 4260 Fax: 91-80-3636662. E-mail: mayor{at}ncbs.res.in
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Abstract |
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Key Words: lysosome; endosome; maturation; cell culture; cathepsin L
* Abbreviations used in this paper: car, carnation; DAB, diaminobenzidine; dor, deep-orange; dSR, Drosophila scavenger receptor; F-Dex, FITC-dextran; Fl-mBSA, fluorescently conjugated mBSA; LR-Dex, lissamine rhodaminelabeled dextran; mBSA, maleylated BSA; MVB, multivesicular body; vps, vacuolar protein sorting; Ydor+, Dp(Y:1)1E.
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Introduction |
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This system of organelles is critical for a variety of processes in metazoans: cessation of mitogenic signaling, turnover of normal cellular proteins, disposal of abnormal proteins, antigen processing, and release of endocytosed nutrients in endolysosomes (Mullins and Bonifacino, 2001). Important clues to understanding mechanisms of development have come from analyses of animals carrying mutations in genes proposed to be involved in lysosomal delivery and degradation. Lysosomal function decides the range of action of morphogens during development by modulating the duration of intracellular signaling of ligand-bound signaling receptors (Babst et al., 2000; Dubois et al., 2001).
Genetic analysis has provided an understanding of the role of molecular players involved in secretory traffic and vacuole biogenesis in Saccharomyces cerevisiae (Horazdovsky et al., 1995; Wendland et al., 1998). Analyses of vacuolar protein sorting has led to the identification of over 50 genes involved in vacuolar biogenesis in yeast (Bryant and Stevens, 1998). These genes, referred to as vacuolar protein sorting (vps)* or vam genes (Wickner, 2002) are subdivided into six classes (AF) based on their vacuolar phenotypes (Banta et al., 1988; Robinson et al., 1988). Development of assays for vesicle fusion in vitro has also provided insight into the biochemical functions of these genes (Wickner, 2002). Since the yeast vacuole is proposed to be functionally analogous to lysosomes in metazoans (Lloyd et al., 1998; Odorizzi et al., 1998; Dell'Angelica et al., 2000), homologues of these genes are likely to be used in constructing endolysosomes in metazoans.
To analyze the role of genes involved in endolysosomal biogenesis in a metazoan, we have been able to reproducibly derive primary cultures of hemocytes from Drosophila melanogaster. We have shown (Guha et al., 2003) that larval hemocytes from animals mutant at the shibire locus exhibit a temperature-sensitive, reversible inhibition of receptor-mediated endocytosis, faithfully reproducing temperature-sensitive paralysis phenotype observed in shi flies (Krishnan et al., 1996).
Here we have examined the perturbation of late endosomal trafficking and degradation in two mutants, deep-orange (dor) and carnation (car). These genes are part of the "granule group" of eye color genes in Drosophila (Lloyd et al., 1998; Odorizzi et al., 1998; Spritz, 1999), which reduce both red and brown eye pigments and encode fly homologues of yeast genes, VPS18 and VPS33, respectively (Mullins and Bonifacino, 2001). 85 mutations affecting eye color have been isolated, and a subset is proposed to be involved in pigment granule biogenesis, an organelle whose biogenesis may resemble that of lysosomes (Lloyd et al., 1998; Dell'Angelica et al., 2000). The availability of a cell culture system provides an opportunity wherein mutations in genes involved in lysosomal biogenesis that give rise to phenotypes in the animal may be analyzed at the cellular level at high resolution.
VPS18 and VPS33 are part of class C vps genes, which also includes VPS11 and VPS16 and whose products assemble as a complex called the class C complex (Sato et al., 2000; Wurmser et al., 2000). The VPS18p homologue, Dor, associates with endosomal membranes in Drosophila cells (Sevrioukov et al., 1999). Clones of a null mutant, dor8 in the compound eye of Drosophila are pigmentation deficient, and consistent with an effect on lysosomal degradation, endocytosed HRP-Boss ligand accumulates intracellularly in aberrant multivesicular structures (Sevrioukov et al., 1999). The role of Dor in lysosomal function is further suggested by the lack of degradation of overexpressed Wingless-HRP in dor8 embryos (Dubois et al., 2001). However, the reason for this defect in degradation in terms of the pathways perturbed in a metazoan system is yet unknown. The VPS33p homologue, Car, associates with Dor in vitro (Sevrioukov et al., 1999); car genetically interacts with dor (Lindsley and Zimm, 1992). These observations implicate Dor and Car in biogenesis of endolysosomes in metazoa.
To study biogenesis of endolysosomes and role(s) of Dor and Car in this process, we have followed the endocytic fate of molecules internalized by endogenously expressed anionic ligand binding receptors resembling Drosophila scavenger receptors (dSRs; unpublished data) using fluorescently labeled protein ligands for dSR together with probes for fluid phase endocytosis (e.g., fluorescently labeled dextrans) in hemocytes. We show that in Rab7-positive endosomal system, multivesicular late endosomes contain both Dor and Car and mature into small dense Dor-negative but Car-positive endosomes. These endosomes eventually fuse with tubular lysosomes. We have then addressed the role of Dor and Car in biogenesis of these different types of Rab7-positive endosomal compartments using a combination of high resolution fluorescence microscopy and EM. We provide evidence that Dor is required for fusion of Golgi-derived vesicles rich in hydrolytic enzymes with large multivesicular endocytic compartments. On the other hand, Car appears to function in removal of Dor from maturation-competent Rab7-positive late endosomes. It is also involved in fusion of small dense Dor-negative, Car-positive organelles with tubular lysosomes, providing evidence for a function independent of Dor.
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Results |
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After a 5-min pulse, increasing chase times (Fig. 1, DF) result in major morphological changes in endosomal compartments accessed by Fl-mBSA and LR-Dex, clearly distinguishing them from MVBs observed at 5 min. After a 15-min chase period, endosomes appear smaller (0.51.0 µm) and show complete colocalization of Fl-mBSA and fluid tracer within the endosome (Fig. 1 D, arrows and inset). EM analysis confirms the formation of small HRP-filled electron-dense structures (Fig. 1 G iii). After a 1-h chase, endosomal morphology is tubular-vesicular (Fig. 1 E), and this persists for 2 (Fig. 1 F) to 4 h (data not depicted). This tubular morphology is sensitive to osmotic stress (unpublished data), analogous to tubular lysosomes observed in mammalian bone marrowderived macrophages labeled with the fluid tracer lucifer yellow (Knapp and Swanson, 1990) and conventional fixing protocols. Tubules are not observed by fluorescence (Fig. 3 C) and EM analyses (Fig. 1 G iv) of fixed samples. Comparison of the morphology of endosomes as a function of chase times in live cells versus those obtained in fixed cells is shown in Fig. 2.
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Multivesicular large sized endosomes in wild-type cells mature into fusion-inaccessible small dense organelles
We next probed the process by which endosomal cargo is trafficked between large sized endosomes and small dense endosomes formed in wild-type cells. There are mainly two ways this trafficking can happen: (1) via transformation of the large sized endosomes into the small dense endosomes (maturation process) or (2) vesicle budding from large sized endosomes, fusing with small dense endosomes (vesicle shuttle) (Fig. 4 B). These processes have distinct predictions for the mixing of endosomal contents between two temporally separated endocytic probe pulses (Fig. 4 A). Measuring the ratio of the amount of the two probes in colocalized endosomal compartments at different chase times provides a method of distinguishing between the vesicle shuttle and maturation models of endosomal trafficking between two types of organelles (Stoorvogel et al., 1991; Dunn and Maxfield, 1992).
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The mere loss of fusion accessibility does not discriminate between the two models. If a vesicle shuttle mechanism is involved in traffic between large endosomes and small compartments (or if small compartments represent vesicles in transit between two stages of the endocytic pathway), with increasing chase times (Fig. 4 B) the endosomes accessible to the second pulse will gradually lose the first pulse to the next set of compartments. However, in a maturation process the endosomes become the next set of compartments, thus those endosomes accessible to the second pulse would always contain similar levels of the first pulse during the entire chase period. These processes may be distinguished by measuring the ratio of the amount of first pulse to second pulse in colocalized compartments with increasing chase times (Dunn and Maxfield, 1992).
After a 45-min chase period, although most of the endosomes labeled by the first pulse are fusion inaccessible (Fig. 4, C and E), there are a few endosomal structures where the first pulse colocalizes with the second pulse. The ratio of the amount of the first pulse in endosomes marked by the second pulse remains constant throughout the chase period (Fig. 4 F), consistent with a maturation process (Fig. 4 B). This demonstrates that Rab7-positive late endosomes of large multivesicular morphology transform into small dense organelles via a maturation process. After maturation into fusion-inaccessible endosomes, the small sized endosomes finally mix their contents in tubular lysosomes (for example, if cells treated as in Fig. 4 D are further chased for 1 h; data not depicted). Note that these endosomal-morphological transformations are not necessary for the maturation process (Fig. S3, available at http://www.jcb.org/cgi/content/full/jcb.20010166/DC1).
Endosomal degradation in hemocytes from wild-type and eye color mutant alleles of dor and car
To determine the extent of degradation of endocytosed protein ligands in the endolysosomal system in wild-type (Canton-S strain) cells, Cy3-mBSA was pulsed into cells for 5 min (Fig. 5 A) and chased for different times. Quantification of total cell (Fig. 5 B) and endosome-associated (unpublished data) fluorescence at the end of each chase time showed a dramatic reduction (8085%) between 1 and 2 h. At this time, the endocytosed probes are in tubular, Rab7-positive endosomes (Figs. 13), indicating this compartment as a major site for endosomal degradation. The reduction in fluorescence is inhibited by a protease-inhibitor cocktail (Fig. 5 C), confirming that loss in fluorescence is a measure of protein degradation in these endosomal compartments.
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Cells from the only available allele of car, car1 showed a small but significant (P < 0.0001) impairment in degradation of Cy3-mBSA after a 2-h chase (Fig. 5 C), suggesting a role for Car in degradation. Although Car associates with Dor in the same protein complex (Sevrioukov et al., 1999), its role in endosomal function is yet to be determined. To test whether car is involved with dor in endosomal degradation, we used a double mutant of dor and car (dor1car1), which does not survive beyond the prepupal stage as reported previously (Lindsley and Zimm, 1992). The cell culture system that we developed afforded us an opportunity to analyze endosomal trafficking in this mutant combination. Cells from dor1car1 showed the most severe defect in endosomal degradation (Fig. 5 A, bottom, and C). There is a small but significant (P < 0.05) difference between the degradation defect in dor1car1 and dor1, consistent with a role for both Car and Dor in endosomal degradation.
The impairment of degradation in cells from mutant animals may be due to an acidification defect in mutant cells, causing a perturbation of lysosomal degradation. This possibility is ruled out, since the extent of endosomal acidification in compartments labeled by a 5-min pulse or a subsequent 2-h chase in wild-type and mutant cells are comparable (Fig. 5 D). Another possibility may be due to differences in the extent of internalization of Cy3-mBSA. However, endosomal fluorescence in hemocytes from wild-type and dor1car1 is comparable after the 5-min pulse (Fig. 5 A, compare left panels). Thus, the degradation defects observed in the mutant cells are likely to be due to alterations in biogenesis of late endosomes or lysosomes.
Mutant alleles of dor and car show blockage in morphological transformation at distinct stages of endolysosomal biogenesis but mature with normal kinetics
We next examined the morphology of endosomal compartments accessed by dSR ligands and fluid phase in live hemocytes from dor4, dor1, car1, and dor1car1. Following a similar pulsechase protocol outlined earlier (Fig. 1), we find that the net internalization of probes was not affected in any of the mutants studied (Fig. 5). The probes are delivered to Rab7-positive (unpublished data) large endosomes in all alleles (Fig. 6). Distinct from cells from wild-type (Fig. 1 D) and car1 animals (Fig. 6 E), the large endosomes in mutant dor alleles appear blocked in progression to later stages (Fig. 6, A and B, dor1; C, dor4; H, dor1car1). This defect is completely rescued by overexpression of Dor; in cells from dor4/Ydor+ endosomal progression is similar to wild type (Fig. 6 I). Ultrastructure analysis using HRP as a fluid phase probe showed no difference in the morphology and formation of the MVB and the small dense organelle between car1 and Canton-S cells (unpublished data). In the cells from the synthetic lethal mutant dor1car1, after a 2-h chase, many endosomes showed an aberrant distribution of endocytosed probes wherein the dSR ligand is often distributed on the endosomal membrane and the fluid tracer is present in the lumen of exaggerated large sized endosomes (34 µm; Fig. 6 H, inset). Cells from car1 animals exhibited a block in transition of small dense vesicles to tubular-vesicular compartments; even after a chase of 2 h (Fig. 6 G compared with Fig. 1 F) or 4 h (data not depicted) only small vesicular structures are observed.
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Staining of intracellular organelles with an acidophillic dye in live cells from wild-type and mutant animals is consistent with this interpretation. LysotrackerTM labels all endocytically accessible compartments in wild-type cells, whereas in addition to endocytically accessible organelles, large sized endocytically inaccessible structures are also labeled in dor1car1. Most of the endocytically inaccessible LysotrackerTM-labeled structures in this mutant correspond to phase-lucent vesicles that accumulate inside cells (unpublished data). This suggests that endosomes in mutant cells mature into fusion-inaccessible acidic endosomes, incapable of lateral fusion with each other.
Deep-orange is involved in the delivery of Golgi-derived enzymes to late endosomal compartments
The effects of mutant alleles of dor characterized thus far do not provide an explanation for reduced endosomal degradation observed in cells from mutant animals. To determine whether defects in endosomal degradation observed in cells from dor alleles are due to alterations in specific trafficking steps, we monitored the delivery of Golgi-derived hydrolases to the endolysosomal system. Cysteine proteinases like cathepsin L are important constituents of the lytic system in lysosomes (Turk et al., 2001; Zwad et al., 2002) and are delivered directly from the Golgi to MVBs and lysosomes in the pro form where they undergo cleavage to yield the mature proteinase (Turk et al., 2001). To detect the delivery of Golgi cargo we have used antiserum generated against Sacrophaga peregrina procathepsin L that cross-reacts with Drosophila cathepsin Llike enzyme encoded by cp1 (Tryselius and Hultmark, 1997) and monitored the time course of intersection of endocytosed F-Dex with this immunoreactivity. At the earliest time monitored (5 min), a fraction (20%) of large sized F-Dexcontaining endosomes is labeled by antiserum against procathepsin L in cells from wild-type (Fig. 7 A, open arrowhead) and car1 animals (unpublished data). This suggests that at least some of Golgi-derived procathepsin L is delivered to the 5 min, Rab7-positive large sized endosomes. At this time, endosomes in cells from dor alleles and dor1car1 cells do not show any detectable procathepsin L staining (unpublished data). When a 15-min pulse of F-Dex is chased for longer times in cells from wild-type and car1 animals an increasing fraction of F-Dexcontaining endosomes shows staining for procathepsin L as seen qualitatively in Fig. 7, B and C, and quantitatively in Fig. 7 F. However, in none of the dor alleles, is significant procathepsin L staining detected even at late times (Fig. 7, DF; dor4, unpublished data). This defect is completely rescued in cells overexpressing Dor (dor4/Ydor+; Fig. 7 F and Fig. S4, available at http://www.jcb.org/cgi/content/full/jcb.20010166/DC1).
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Eye color mutants affect the removal of Dor and Car from Rab7-positive endosomes
To obtain a mechanistic understanding of Dor and Car in biogenesis of endolysosomes, we examined the localization of Dor and Car on different Rab7-positive endosomal compartments in wild-type cells. F-Dex was pulsed and chased into morphologically distinct stages of the endosomal system as before (Fig. 2), and Dor and Car immunoreactivity associated with corresponding compartments was analyzed. Dor is associated with compartments labeled by a 15-min pulse of F-Dex (Fig. 8 A, inset). However, endosomal compartments accessed by a 15-min pulse followed by a 1-h chase are devoid of Dor immunoreactivity (Fig. 8 B, arrowheads). This is similar to observations made in garland cells wherein dextran beads are first seen in Dor-positive compartments and subsequently in Dor-negative compartments (Sevrioukov et al., 1999). Quantitative analysis shows that immediately after a 15-min pulse of F-Dex, 90% of all F-Dexcontaining endosomes are -Dor positive, whereas almost all endosomes accessed after a 1-h chase are devoid of detectable Dor immunoreactivity (Fig. 8 F). In a more detailed temporal analysis, after a pulse of 5 min wherein a majority of endosomes are large sized and Dor positive, a chase of 45-min results in <20% of F-Dexcontaining endosomes retaining Dor immunoreactivity on exclusively small sized endosomes observed at this time (Fig. 8 F, inset).
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In cells from dor mutants (including dor1car1), levels of Dor immunostaining are lower than those observed in wild-type hemocytes (unpublished data; Sevrioukov et al., 1999). However, Dor (Fig. 9, AC and G) and Car (Fig. 9 H) immunoreactivity are persistent on the Rab7-positive large sized endosomes compared with wild-type cells. This defect is completely rescued in cells from dor4/Ydor+ animals (Fig. 9 F). These results strongly suggest that mutations in dor prevent normal release of Dor and Car proteins from endosomal membranes.
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Discussion |
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In contrast to dor, only a single mutant allele of another eye color gene, the Drosophila homologue of VPS33, car1 (Lindsley and Zimm, 1992), has been characterized. Cells from this mutant background have a small but significant endosomal degradation defect. In this respect, car1 behaves like a "weak" allele of a mutation in the class C complex genes. Distinct from dor4 cells that exhibit only a similar defect in endosomal degradation but clearly mislocalize procathepsin L into aberrant structures, procathepsin L is delivered normally to endocytic organelles in cells from car1. Normal endosomal delivery of procathepsin L in car1 mutant suggests that there may be a redundant role for Car in this process. In conjunction with genes of the class C complex, VPS11, VPS16, and VPS18, recent studies (Webb et al., 1997; Nichols et al., 1998; Gerrard et al., 2000) have implicated VPS45 as another sec1 gene involved in Golgi to endosome traffic in S. cerevisiae. It is likely that similar to yeast, in addition to car, a VPS45 homologue may participate in this step (Littleton, 2000). Likely candidates for this gene exist at 85D-E (Lloyd et al., 2000). The reason for a degradation defect may be due to the failure of endosomal contents in car1 to be well mixed in a large stable pool of degradative enzymes normally found in tubular lysosomes of wild-type cells.
A suggestion for a role for Car in endosomal delivery of Golgi-derived cargo comes from analyses of the endocytic phenotype of cells from the synthetic lethal double mutant dor1car1. A small but significant enhancement of the dor1 endosomal degradation defect and an exaggeration of the aberration of endosomal morphology (compared with the defects observed in dor1 alone) implicate car in the same step; endosomal degradation may be a more sensitive read out of the impairment of delivery of cargo from the Golgi.
Large sized endosomes that accumulate in mutant dor alleles and dor1car1 mature with normal kinetics into fusion-inaccessible organelles but fail to lose Dor and Car. This suggests that, unlike morphological progression, persistent localization of Dor and Car does not impair maturation. Both the mutations in the dor alleles map to the COOH-terminal cysteine-rich RING domain of the protein; dor4 has a frame-shift mutation, which adds 30 new amino acids, whereas dor1 has a similar lesion as the VPS18tsf allele in terms of a mutation in a RING finger cysteine (Rieder and Emr, 1997). The inability of dor mutants to lose Dor immunoreactivity from endosomes may be a consequence of the specific molecular lesion in the RING domain in mutant alleles of dor. This is likely to affect the localization dynamics of Car. Car remains persistently localized on the membrane (Fig. 9 H), consistent with a possible physical interaction between Dor and Car (Sevrioukov et al., 1999).
These experiments clearly indicate sequential use of Dor (possibly with other players of the class C complex), first in forming large sized endosomes (fusion between Rab7-positive endosomes and Golgi-derived vesicles) and later in progression to small sized Dor-negative structures. Removal of Dor is implicated in this progression; Dor is retained on the Rab7-positive large endosomes in all dor mutants. At the same time, in wild-type and car1 cells morphological progression of the large sized to small sized endosomes precedes the loss of Dor from the small sized endosomes. This may be explained by a mechanism where Dor function is necessary for this progression and is then followed by inactivation of the membrane-associated Dor leading to its subsequent removal.
Our results suggest that Car is responsible for the inactivation of Dor. This is because in car1 cells there is delayed endosomal dissociation of Dor from the small sized endosomes, a phenotype that is greatly enhanced by overexpression of Dor (car1/Ydor+ cells; Fig. 10). This overexpression also results in blockage of the morphological transition of the Rab7-positive endosomes (but not its maturation). These observations together suggest that Dor must undergo a Car-dependent modulation necessary for morphological progression to the small sized endosomes, before its removal from the membrane.
Our results implicate Car in a step that is likely to be independent of Dor. First, in wild-type cells, although Dor and Car associate with Rab7-positive MVBs, only Car remains associated with small dense Rab7-positive organelles before fusion with the tubular-vesicular compartments. Second, in the car1 mutant, Dor-negative and Car-positive small dense endosomal structures formed by maturation of large MVBs that fail to fuse with tubular lysosomes accumulate. This shows that Car remains functionally associated with the endosomal system in the absence of Dor. Consistent with this hypothesis, although Car has been shown to physically interact with Dor, not all membrane-bound Car is in a complex with Dor (Sevrioukov et al., 1999). Furthermore, in yeast Vps33p is obtained in a membrane-associated complex with Vps16p in a deletion strain of VPS18 (Sato et al., 2000).
Data presented here strongly suggest that although Dor may function similar to VPS18 in its role in Golgi to endosome traffic, the Sec1p homologue Car may be involved in the Dor-dependent and -independent steps, potentially modulating interaction with endosome stage-specific SNAREs. One of the two point mutations in car1 (Val to Gly at position 249) is located in the region conserved in all Vps33 family members (Sevrioukov et al., 1999), implicating this region in the regulation of SNARE function. Analyses of the endosomal phenotype of syntaxin 7 mutant fly cells will be necessary to address this question. Finally, the role of Rab7 in the context of this late endosomal pathway needs to be elucidated, since different from yeast, Dor and Car dissect the Rab7-positive endosomal compartments into subsets based on their fusion with either Golgi-derived compartments or with the lysosome.
In conclusion, these studies provide evidence for interplay of Dor and Car in ordering the sequence of endosomal biogenesis. Functional analyses of genes involved in this pathway in a metazoan cell is extremely relevant because these and other studies suggest that the endolysosomal system in higher eukaryotes is likely to be more complex than that present in yeast; some genes involved in cellular functions are present only in higher eukaryotes (Dell'Angelica et al., 2000).
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Materials and methods |
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Fly stocks
All Drosophila stocks obtained from the Bloomington Stock Center were grown at 20°C in cornmeal agar bottles. dor1car1 was provided by Mani Ramaswami (University of Arizona, Tucson, AZ). Squash-GAL4 was provided by Dan Kiehart (Duke University, Durham, NC).
Cell culture
Cells from larvae were obtained as described previously (Braun et al., 1998). Briefly, third instar larvae were surface sterilized, and hemolymph was collected by puncturing the integument using dissection forceps into 150 µl of complete medium (CM; Schneider's insect medium supplemented with 10% nonheat inactivated FBS, 1 µg/ml bovine pancreatic insulin, 150 µg/ml penicillin, 250 µg/ml streptomycin, 750 µg/ml glutamine) in 35-mm coverslip-bottom dishes (Mayor et al., 1998). Labeling incubations were performed on adherent hemocytes 2 h postdissection.
Cell labeling
Adherent hemocytes were washed (three times with medium 1 [Mayor et al., 1998]) before addition of endocytic probes. Fl-mBSA and dextran were used at 800 ng/ml and 1 mg/ml, respectively, in labeling medium (LM; Schneider's insect medium supplemented with 1.5 mg/ml BSA). Hemocytes were incubated with the endocytic probes for the indicated times (pulse period) and washed extensively with medium 1 before further incubation of cells for different intervals of time (chase periods) in CM. Fluorescently labeled mBSA probes were completely competed by incubation of cells with unlabeled mBSA (0.8 mg/ml). Cells were imaged live in imaging medium (IM; medium 1 supplemented with 1 mg/ml BSA and 2 mg/ml D-glucose), or fixed in 2.5% PFA in medium 1 for 20 min before imaging.
Immunofluorescence microscopy
Labeled and fixed cells were permeabilized using 0.4% Igepal for 13 min and incubated in blocking solution (BS; medium 1 with 2 mg/ml BSA), before incubation with primary antiserum. The primary antisera were obtained and used at the indicated dilutions in BS as described in Table I. Cells were incubated with labeled secondary antibodies diluted in BS for 45 min.
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Ratiometric pH estimation
To measure endosomal acidification, we used the pH sensitivity of F-Dex (Ohkuma and Poole, 1978). Cells were incubated with F-Dex for 5 min and either imaged immediately or after a 2-h chase. Two images were collected for each field, one before and the other after endosomal pH was neutralized using 10 µM nigericin (Sabharanjak et al., 2002). FITC fluorescence associated with the endosomes per cell before and after addition of nigericin was quantified using MetamorphTM software (Universal Imaging Corp.) and corrected for photobleaching during consecutive exposures. The extent of photobleaching was determined by exposing cells for exactly the same time without any treatment. Ratio of FITC fluorescence associated with the endosomes before addition of nigericin to that after its addition is a measure of the extent of endosomal acidification. Each data point was obtained from >10 cells per allele.
Imaging and image processing
Confocal and wide field imaging was performed exactly as described (Sabharanjak et al., 2002). All images were processed for output purposes using Adobe Photoshop® software.
Quantitative analyses of colocalization
Quantification of colocalization for measuring maturation kinetics was performed as described (Dunn and Maxfield, 1992; Sabharanjak et al., 2002). All processing including determination of colocalization was performed using similar parameters regardless of the type of endocytic tracer used. Colocalization index was calculated as the ratio of the colocalized intensity to the total intensity of the probe in endosomes in each cell. Maximum extent of colocalization obtained by this method is 94% for the colocalization of cointernalized F-Dex and Cy3-mBSA in the same cell. Ratios of fluorophore intensities in individual endosomes were obtained by determining the individual fluorophore intensities in endosome that exhibited colocalization. For the quantification of endocytic probe-containing endosomes colocalized with immunodetected proteins, the total number of endosomes labeled by the endocytic tracer was identified and counted manually using MetamorphTM software (Universal Imaging Corp.). Endosomes positive for the immunodetected protein were recorded and expressed as the percentage of the total number of endosomes. Each experiment consisted of two dishes with >15 cells per dish.
HRP quenching assay
To ascertain lumenal connectivity (Mayor et al., 1998) of optically colocalized Cy3-mBSA and F-Dex in endosomes, HRP was cointernalized as a fluid phase probe along with F-Dex at 1 mg/ml in the presence of 0.5 mg/ml mannan. To prevent the internalization of HRP by mannose receptor expressed on hemocytes, cells were also preincubated with Mannan. To estimate the extent of fusion of two discrete pulses of endocytosed probes, hemocytes were incubated with Cy3-mBSA for 5 min in LM and chased for either 5 or 45 min in CM before a second pulse of 5 min of F-Dex and HRP. HRP-mediated quenching was performed at 4°C for 45 min (Mayor et al., 1998). Fluorescence of Cy3-mBSA and F-Dex was quantified using the MetamorphTM software (Universal Imaging Corp.). The extent of HRP-mediated quenching was expressed as the percentage of Cy3-mBSA fluorescence that was quenched by exposure to H2O2. Efficacy of HRP quenching was independently confirmed by ensuring that cointernalized F-Dex fluorescence was completely quenched for each condition.
EM
Hemocytes incubated with 1.5 mg/ml HRP in LM were fixed using 2.5% PFA for 3 min, and HRP enzymatic activity was developed with DAB and 0.003% H2O2 diluted in medium 1 at 4°C for 45 min. The cells were washed and postfixed using a mixture of 1.5% gluteraldehyde and 2.5% PFA for 1 h, treated with osmium tetroxide, dehydrated, and embedded in araldite (TAAB). Sections (50150 nm) were viewed using a Jeol CXII 100 transmission electron microscope. Images were captured on photographic emulsion and scanned at 1,200 dpi for output purposes.
Online supplemental material
Figs. S1S6 are available at http://www.jcb.org/cgi/content/full/jcb.20010166/DC1. Fig. S1 provides information regarding the specificity of the Rab7 antisera used and the nature of the compartments containg this marker. Fig. S2 shows that Hrs, Deep-orange, Carnation, and Hook are colocalized in the late endolysosomal system in larval hemocytes. Fig. S3 shows that mutant alleles of dor and car do not affect the maturation kinetics of large sized endosomes. Fig. S4 shows that the block in endosomal delivery of Golgi-derived hydrolase is completely rescued in hemocytes from dor4/Ydor+. Fig. S5 provides evidence that Golgi morphology is not affected in hemocytes from the eye color mutants. Fig. S6 shows that both Deep-orange and Carnation label GFP-Rab7positive late endosomes accessed by 5-min pulse of Fl-mBSA.
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Acknowledgments |
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This work was supported by a grant from the Department of Biotechnology, India, and from intramural funds from the National Centre for Biological Sciences. V. Sriram is supported by a Kanwal Rekhi fellowship from the Tata Institute of Fundamental Research endowment fund and Appam. S. Mayor is a Senior Research fellow of the Wellcome Trust.
Submitted: 29 October 2002
Revised: 31 March 2003
Accepted: 2 April 2003
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