* Department of Cell Biology, Department of Ophthalmology, Yale University School of Medicine, New Haven,
Connecticut 06520
Lysosomes are considered to be a terminal degradative compartment of the endocytic pathway, into which transport is mostly unidirectional. However, specialized secretory vesicles regulated by Ca2+, such as neutrophil azurophil granules, mast cell-specific granules, and cytotoxic lymphocyte lytic granules, share characteristics with lysosomes that may reflect a common biogenesis. In addition, the involvement of Ca2+ transients in the invasion mechanism of the parasite Trypanosoma cruzi, which occurs by fusion of lysosomes with the plasma membrane, suggested that lysosome exocytosis might be a generalized process present in most cell types.
Here we demonstrate that elevation in the intracellular free Ca2+ concentration of normal rat kidney
(NRK) fibroblasts induces fusion of lysosomes with the
plasma membrane. This was verified by measuring the
release of the lysosomal enzyme -hexosaminidase, the
appearance on the plasma membrane of the lysosomal
glycoprotein lgp120, the release of fluid-phase tracers
previously loaded into lysosomes, and the release of the
lysosomally processed form of cathepsin D. Exposure
to the Ca2+ ionophore ionomycin or addition of Ca2+containing buffers to streptolysin O-permeabilized
cells induced exocytosis of ~10% of the total lysosomes
of NRK cells. The process was also detected in other
cell types such as epithelial cells and myoblasts. Lysosomal exocytosis was found to require micromolar levels of Ca2+ and to be temperature and ATP dependent,
similar to Ca2+-regulated secretory mechanisms in specialized cells.
These findings highlight a novel role for lysosomes in cellular membrane traffic and suggest that fusion of lysosomes with the plasma membrane may be an ubiquitous form of Ca2+-regulated exocytosis.
Lysosomes are acidic organelles delimited by a single
membrane, containing a characteristic set of acid
hydrolases (Novikoff, 1961 Extracellular release of lysosomal contents, however,
has been described in several cell types, such as hepatocytes (LeSage et al., 1993 Despite these similarities, conventional lysosomes of
nonspecialized cells have not been generally described as
organelles that fuse with the plasma membrane upon stimulation. Recent studies of cell infection with the protozoan
parasite Trypanosoma cruzi have, however, provided evidence for the existence of a lysosomal exocytic pathway in
fibroblasts and epithelial cells. During T. cruzi invasion,
lysosomes are recruited to the parasite entry site and gradually fuse with the plasma membrane (Tardieux et al.,
1992 Taken together, the observations described above suggested that increases in [Ca2+]i might be sufficient to trigger lysosomal exocytosis in most cell types, in a similar fashion to the well-characterized process of Ca2+-regulated
exocytosis in specialized secretory cells. In this work we
provide evidence in favor of this hypothesis, demonstrating that conventional lysosomes can be induced to fuse
with the plasma membrane in a Ca2+, temperature- and
ATP-dependent fashion.
Materials
BSA, bombesin, and gold chloride were obtained from Sigma Chemical
Co. (St. Louis, MO); ionomycin and colchicine were from CalbiochemNovabiochem Corp. (La Jolla, CA). Lucifer yellow and FITC-transferrin
(human) were from Molecular Probes, Inc. (Eugene, OR). Reduced streptolysin O was from Murex Diagnostics (Dartford, UK), and hexokinase
was from Boehringer Mannheim Biochemicals (Indianapolis, IN). 3H-dextran (mol wt 70,000) was obtained from Amersham Corp. (Arlington Heights, IL). Human diferric 125I-transferrin was obtained from DuPont
(Wilmington, DE). Purified rabbit antibodies against cathepsin D were
from Biodesign Intl. (Kennebunk, ME). Trypanosome-soluble fraction
(TSF) was prepared from the infective stages of T. cruzi as described in
Rodríguez et al. (1995) Cell Culture
All cells were grown at 37°C with 5% CO2. Cultures of primary human fibroblasts (NIGMS; Coriell Institute for Medical Research, Camden, NJ),
NRK, J774, IMR-90, L6E9, and LLC-MK2 cell lines were grown in DME
containing 10% FBS. CHO cells were grown in Ionomycin Treatment
Confluent monolayers of NRK cells in 60-mm culture dishes were washed
with PBS and incubated with 0.5 ml of either PBS or 10 µM ionomycin
in PBS for the indicated times. The incubation buffer was collected and
centrifuged at 11,000 g for 5 min before performing N-Acetyl- For each sample, 350 µl of the incubation buffer was incubated for 15 min
at 37°C with 50 µl of 6 mM 4-methyl-umbellyferyl-N-acetyl- Lactate Dehydrogenase Activity Assay
For each point, 100 µl of the incubation buffer or 1:10 dilutions of NP-40
extracts prepared as described above were incubated with 900 µl of reaction buffer (0.23 mM NADH, 1 mM sodium pyruvate, 0.1% Triton X-100,
0.2 M Tris-HCl, pH 7.3). Decrease in absorbance at 340 nm was measured
in the spectrophotometer for either 2 min (permeabilized cells) or 5 min
(nonpermeabilized cells).
Lucifer Yellow and 3H-Dextran Loading of Lysosomes
and Detection
Cells were incubated for 4 h at 37°C in DME containing 10 mg/ml lucifer
yellow or for 1 h with 100 µCi/ml 3H-dextran, washed extensively with
PBS, and chased for another 2 h in DME 10% FCS before performing the
ionomycin or permeabilization treatments. The incubation buffer was collected and centrifuged for 5 min at 11,000 g. Lucifer yellow was detected
in each sample of supernatant by measuring the fluorescence at excitation
428 nm/emission 540 nm; 3H-dextran was measured in a scintillation
counter.
Staining for Surface lgp120
After the different treatments, NRK cells were incubated at 4°C for 30 min with culture supernatant from a mouse hybridoma line (Ly1C6) producing antibodies to rat lgp120 (kindly provided by I. Mellman, Yale University School of Medicine, New Haven, CT). Cells were then fixed with
2% paraformaldehyde for 15 min at 4°C, washed in PBS, and incubated
with rhodamine-conjugated anti-mouse IgG antibodies (Boehringer Mannheim Biochemicals) for 30 min at room temperature.
Flow Cytometry
Confluent NRK cells were trypsinized and washed twice with PBS before
incubation with PBS or 10 µM ionomycin in PBS for 5 min at 37°C. 106
cells for each assay were centrifuged and resuspended in Ly1C6 supernatant (mAb to rat lgp120) or FITC-transferrin (0.5 mg/ml) at 4°C for 30 min. Cells were washed in PBS and fixed in 2% paraformaldehyde for 15 min. Anti-lgp-treated cells were further incubated with FITC-conjugated
anti-mouse antibodies (Boehringer Mannheim Biochemicals) for 30 min
at room temperature. Finally, cells were washed, resuspended in 0.5 ml
PBS, and analyzed on a FACS® Vantage Flow Cytometer (Becton Dickinson & Co., Mountain View, CA). Cells were illuminated at 488 nm and
emission was detected at 525 nm. Forward angle scatter, right angle scatter, and fluorescence intensity were recorded from 10,000 cells whose forward angle scatter fell above a threshold used to distinguish intact from damaged cells.
BSA-Gold Labeling of Lysosomes and EM
Lysosomes were labeled by the internalization of BSA adsorbed to 5 nm
gold particles, prepared according to published procedures (Slot and Geuze,
1985 Streptolysin O Permeabilization
Confluent monolayers of NRK cells in 60-mm culture dishes were washed
twice with 1 ml ice-cold buffer A (20 mM Hepes, 110 mM NaCl, 5.4 mM
KCl, 0.9 mM Na2HPO4, 10 mM MgCl2, 2 mM CaCl2, and 11 mM glucose,
pH 7.4) and incubated for 10 min at 4°C with streptolysin O (SLO) at 0.5 U/ml in buffer A. Cells were then washed once with 1 ml ice-cold buffer B
(20 mM Hepes, 100 mM K-glutamate, 40 mM KCl, and 5 mM EGTA, pH
7.2) containing 2 mM MgATP and 5 mM free Mg2+ (added as MgCl2). 0.5 ml
of buffer B with MgATP and Mg2+, or buffer B with MgATP, Mg2+, and
Ca2+/EGTA at the indicated concentrations, was added to the cells at
37°C for the indicated time periods. The concentrations of free Mg2+ and
Ca2+ were maintained using a Ca2+ or Mg2+/EGTA buffering system, calculated using the software developed by Foehr and Warchol (Ulm, Germany). Incubation buffer was collected for each sample and centrifuged
for 5 min at 11,000 g before performing enzyme assays. Total extracts were
obtained by incubation of culture dishes with 0.5 ml of PBS 1% NP-40,
followed by a 5-min centrifugation of the extract at 11,000 g. In the ATP
depletion experiment, cell permeabilization was performed in cells in suspension, which were previously trypsinized and washed before SLO permeabilization as described above. 106 cells were used for each assay. After
permeabilization, cells were incubated with hexokinase (150 U/ml) and
glucose 5 mM in buffer B with Mg2+ for 15 min at 37°C, before performing
the Ca2+-induced exocytosis assay.
Detection of Cathepsin D
Confluent IMR-90 cells in 150-mm culture dishes were either treated with
PBS or 10 µM ionomycin or permeabilized with SLO and incubated with
a 0 or 1 µM Ca2+ buffer as described above. The supernatants of these
cells (3 ml) were collected after 5 min and concentrated with a Centricon10 (Amicon Corp., Beverly, MA) to 50 µl (25 µg of total protein for PBS
and ionomycin samples and 80 µg for permeabilized cell samples). The total extract was obtained by addition of 1 ml of lysis buffer (150 mM NaCl,
50 mM Tris, pH 8.6, 1% NP-40) to the cells, and 15 µl (20 µg) was used for
detection. 4× concentrated SDS-PAGE loading buffer (62.5 mM Tris, pH
6.8, 10% glycerol, 2% SDS, and 5% Transferrin Release Assay
125I-transferrin release from SLO-permeabilized cells was performed essentially as described by Galli et al. (1994) Ionomycin Induces the Release of Lysosomal Contents
into the Extracellular Medium
To test if an increase in [Ca2+]i would stimulate exocytosis
of lysosomes, the calcium ionophore ionomycin was added
to NRK fibroblasts in a Ca2+-containing buffer (PBS with
1 mM CaCl2). Cells were incubated at 37°C with 10 µM
ionomycin, and release of the lysosomal enzyme
To verify if cell integrity was affected by the exposure to
ionomycin, activity of the cytoplasmic enzyme lactate dehydrogenase (LDH) was measured in the incubation buffer of treated and untreated cells. Fig. 1 b shows that LDH
levels in the incubation buffer were low in both conditions,
indicating that the observed release of Lysosomes can be specifically labeled by incubation of
cells with an endocytosis fluid-phase marker, followed by a
chase of several hours (Adams et al., 1982 Another fluid-phase tracer, BSA-gold, was used for
specific visualization of lysosomes by EM. NRK cell lysosomes were loaded with BSA-gold, and then treated with
10 µM ionomycin for different periods of time. In PBStreated control NRK cells, a population of vesicles containing BSA-gold and with the characteristic morphology and dimensions of lysosomes (Holtzman, 1989
To confirm that the BSA-gold-loaded vesicles consisted
of lysosomes, labeling with antibodies against the abundant lysosomal membrane glycoprotein lgp120 (Kornfeld
and Mellman, 1989
Ionomycin Induces the Appearance of lgp120 on the
Plasma Membrane
Additional evidence for Ca2+-dependent lysosomal fusion
with the plasma membrane was provided by exposure of the
luminal domain of lgp120 on the cell surface. After treating NRK cells with 10 µM ionomycin, surface staining was
performed in live cells at 4°C with an mAb recognizing the
luminal domain of lgp120. A gradual increase in extracellularly exposed lgp120 was detected over time in ionomycin-treated cells, but not in control PBS-treated cells (Fig.
3, a-c). As a control, surface staining of ionomycin-treated cells with an mAb for the cytosolic protein kinesin was
performed, showing no detectable fluorescence under the
same conditions (not shown).
To quantify the percentage of the ionomycin-treated
cell population that presented lgp120 on the plasma membrane, a similar experiment was performed with cells in
suspension. NRK fibroblasts were trypsinized, washed, and
incubated with 10 µM ionomycin for 5 min before incubation at 4°C with the anti-lgp120 mAb, followed by fixation.
Cells were analyzed for surface staining by flow cytometry.
An increase in membrane staining for anti-lgp120 was observed in 95% of ionomycin-treated cells (Fig. 3 d).
Agonists That Mobilize Ca2+ from Intracellular Stores
Induce a Lower Level of Lysosomal Exocytosis
The results described above indicated that ionomycinmediated influx of Ca2+ from the extracellular medium induced fusion of lysosomes with the plasma membrane.
To verify if Ca2+ mobilized from intracellular stores by receptor-mediated agonists had the same effect, release of
The protozoan parasite Trypanosoma cruzi induces fusion of lysosomes with the plasma membrane at its invasion site (Tardieux et al., 1992 Ca2+ Concentrations between 1 and 5 µM Are
Sufficient for Optimal Exocytosis of Lysosomes in
Permeabilized Cells
To further characterize the process of Ca2+-dependent lysosome exocytosis, we next performed experiments in fibroblasts permeabilized with SLO (Miller and Moore, 1991 Initially, to determine the efficiency of the permeabilization procedure under our experimental conditions, SLOtreated NRK cells were stained with propidium iodide,
a fluorescent nuclear stain that does not penetrate intact
cells. Virtually all cells were found to be permeabilized, when
observed under a fluorescence microscope (not shown).
The release of
The optimal concentration of SLO for the permeabilization of NRK cells was determined for each batch of toxin
used and was found to vary between 0.2 and 0.5 U/ml, as
determined by titrations using the Ca2+-dependent Release of the cytosolic enzyme LDH from SLO-permeabilized cells was measured as a further control for permeabilization. Fig. 5 b shows that a significantly higher
level of the enzyme was released when the cells were permeabilized (Fig. 5 b). The total amount of LDH released
(5-7% of the total cellular content) reflects the relative
loss of cytosolic components occurring during the 5-min
period of the assay. Such minor cytosolic loss has been shown not to interfere significantly with secretory functions in previously characterized systems (Pimplikar et al.,
1994 To determine the optimal Ca+ concentration inducing
exocytosis of lysosomes in the permeabilized cell system,
we performed the
Elevated [Ca2+]i Induces Secretion of the Lysosomally
Processed Form of Cathepsin D
To confirm the lysosomal origin of the Ca2+-triggered exocytic products, we examined the lysosomal protease cathepsin D released into the supernatant of cells with elevated
[Ca2+]i. Cathepsin D is processed into a mature polypeptide of 31 kD only after being transported to lysosomes
(Gieselmann et al., 1983
Elevated [Ca2+]i Has a Minor
Stimulatory Effect on the Recycling of Transferrin
Receptor-containing Endosomes
To verify if the elevation in [Ca2+]i triggering secretion of
lysosomes also affected the exocytosis of other elements of
the endocytic pathway, we measured the release of previously internalized 125I-labeled transferrin using the same
SLO-permeabilized cell system. As described previously
(Galli et al., 1994
Characterization of Lysosome Exocytosis in
Permeabilized Cells
The presence of Ca2+-dependent lysosomal exocytosis was
tested in different cell types. Cells were permeabilized and
assayed for Table I.
Ca2+-dependent ). They receive newly
synthesized lysosomal enzymes from the biosynthetic pathway and are responsible for the degradation of internalized material from the endocytic and autophagic pathways. Therefore, lysosomes are generally considered to
be a terminal degradative compartment, the final destination of unidirectionally transported soluble macromolecules (DeDuve, 1963
; Kornfeld and Mellman, 1989
).
), activated platelets (Silverstein
and Febbraio, 1992
), pancreatic acinar cells (Hirano et al.,
1991
), macrophages (Tapper and Sundler, 1990
), and osteoclasts (Baron et al., 1990
). In neutrophils, fusion of lysosomes with incompletely formed phagosomes also results
in extracellular leakage of their contents (Pryzwansky et al.,
1979
). Based on the presence of hydrolytic enzymes and lysosomal membrane glycoproteins, a common biogenesis
has been proposed between lysosomes and specialized
Ca2+-regulated secretory vesicles, such as azurophil granules in neutrophils (Borregaard et al., 1993
), specific granules in mast cells (Jamur et al., 1986
), and lytic granules in
cytotoxic T lymphocytes (Burkhardt et al., 1990
; Peters et al.,
1991
).
; Rodríguez et al., 1996
). Intracellular free Ca2+ concentration ([Ca2+]i)1 transients, triggered in host cells by a
trypanosome-soluble factor (Burleigh and Andrews, 1995
),
cause reversible disassembly of the cortical actin cytoskeleton of normal rat kidney (NRK) fibroblasts (Rodríguez
et al., 1995
), similar to what has been described in regulated exocytosis. The T. cruzi-triggered [Ca2+]i transients
appear to be required for the lysosome-mediated invasion
process, since buffering or depletion of host cell intracellular Ca2+ blocks parasite entry (Tardieux et al., 1994
; Rodríguez et al., 1995
).
Materials and Methods
.
-MEM with 5% FBS.
Confluent monolayers containing 6 × 104 cells per cm2 were used for all
experiments.
-hexosaminidase,
3H-dextran, lucifer yellow, or cathepsin D detection assays. Total cell extracts were obtained by incubation of culture dishes with 0.5 ml of PBS 1%
NP-40 (NP-40), followed by a 5-min centrifugation of the extract at 11,000 g.
-D-Glucosaminidase (
-Hexosaminidase)
Activity Assay
-D-glucosaminide in sodium citrate-phosphate buffer, pH 4.5. The reaction was
stopped by addition of 100 µl of 2 M Na2CO3 and 1.1 M glycine, and the fluorescence was measured in an F-2000 spectrofluoremeter (Hitachi Instruments, Inc., Danbury, CT) at excitation 365 nm/emission 450 nm. To
determine the total cellular content of
-hexosaminidase, cell extracts
were diluted 1:10 and 350 µl was used for enzyme detection. In the TSF
experiment, background levels of
-hexosaminidase in TSF (<0.2% of the
total content of control NRK cells) were subtracted from each sample.
). Cells were incubated with BSA-gold (OD 4.0 at 255 nm) for 3 h at
37°C, washed, and chased for 2 h in DME 10% FBS before addition of 10 µM
ionomycin for 2.5 and 10 min at 37°C. For resin embedding, cells were
fixed in 2.5% glutaraldehyde in 100 mM sodium cacodylate (pH 7.4), postfixed in 1% aqueous osmium tetroxide, en bloc stained in 1% uranyl acetate in 50 mM sodium maleate (pH 5.2), dehydrated in methanol, and embedded in Epon. Sections were contrasted with uranyl acetate and lead citrate. For immunocytochemistry, cells were fixed in 4% paraformaldehyde in 200 mM Hepes buffer, pH 7.2, scraped from the dish, pelleted, and
embedded in 10% gelatin. Embedded pellets were infiltrated with 2.1 M
sucrose in PBS and frozen by immersion in liquid nitrogen. Cryosectioning was performed using established methods, as described previously
(Webster et al., 1994
). Thawed sections were labeled with affinity-purified
rabbit anti-lgp120-tail antibodies (Rodríguez et al., 1996
) or mouse anti-
rat lysosomal membrane glycoprotein (lgp120) mAbs (Ly1C6). The mAb
was followed by application of a rabbit anti-mouse bridging antibody.
Binding of antibodies was visualized using protein A-gold probes (purchased
from Department of Cell Biology, University of Utrecht, The Netherlands).
-mercaptoethanol) was added to the
samples, which were heated to 95°C for 4 min before electrophoresis in a
10% SDS-polyacrylamide gel. Proteins were then transferred to Nytran
filters by semidry electroblotting (Schleicher & Schuell, Keene, NH). Blots
were probed with rabbit anti-cathepsin D antibodies (dilution 1:1,000 of a
12.5 mg/ml stock solution), followed by peroxidase-conjugated goat anti-
rabbit immunoglobulin G and enhanced chemiluminescence detection
(ECL; Amersham Intl., Buckinghamshire, UK).
. Confluent NRK cells plated at
2 × 104/cm2 24 h before were trypsinized and washed in buffer K (20 mM
Hepes, 128 mM NaCl, 3 mM KCl, 1 mM Na2HPO4, 1.2 mM MgSO4, 2.7 mM
CaCl2, and 11 mM glucose, pH 7.4, with NaOH) before incubation of 1.5 × 105 cells per µl in buffer K containing 20 µCi/ml human 125I-transferrin for
1 h at 37°C. Cells were washed once with ice-cold buffer K and twice with
buffer A, and permeabilization was performed as described above. Cells
were resuspended in buffer B and divided into 100-µl aliquots containing
106 cells each for determining transferrin release. Supernatants were collected after incubation at 37°C for 20 min or the indicated times, and the
amount of 125I-transferrin was determined in a scintillation counter. In each experiment, samples in triplicate were pelleted at the beginning of the
37°C incubation (defined as time 0), the amount of 125I-transferrin in the
supernatant was determined, and this value was subtracted from each sample. Cell extracts to quantify the total amount of internalized 125I-transferrin were obtained for each sample by incubation of pellets with 100 µl of
PBS 1% NP-40.
Results
-hexosaminidase was measured in the incubation buffer. This
enzyme is frequently used as a marker for lysosomes, since
90% of the total
-hexosaminidase in NRK cells is located
in this compartment (Griffiths et al., 1990
). A continuous
release of
-hexosaminidase, typically reaching ~10% of
the total enzyme content of the cells after 10 min, was specifically triggered by ionomycin (Fig. 1 a).
Fig. 1.
Ionomycin induces exocytosis of -hexosaminidase and
lysosomal fluid-phase tracers but not LDH from intact NRK fibroblasts. (a and b) NRK cells were incubated at 37°C with either
PBS (open circles) or 10 µM ionomycin in PBS (black circles). At
the indicated time points, the incubation buffer was collected and
assayed for
-hexosaminidase and LDH activity. The amount of
enzyme released at each point is expressed as a percentage of the
total content of enzyme in control cells. (c and d) Lysosomes of
NRK cells were loaded with lucifer yellow or 3H-dextran by fluidphase endocytosis followed by a 2-h chase, before treatment with
PBS or 10 µM ionomycin for 10 min. The incubation buffer was
collected, lucifer yellow was detected by reading the fluorescence, and 3H-dextran was detected by scintillation counting. The
amount of lucifer yellow or 3H-dextran released in each sample is
expressed as a percentage of the total amount of tracer present in
control cells. The data represent the average of triplicate determinations ±SD.
[View Larger Version of this Image (43K GIF file)]
-hexosaminidase
induced by the ionophore was not due to cell lysis.
). To confirm
that the
-hexosaminidase release described above was indeed a consequence of lysosome exocytosis, similar experiments were performed after loading lysosomes with the
soluble fluorescent tracer lucifer yellow or 3H-dextran. Release into the supernatant of ~10% of the total amount of
the tracers taken up by NRK cells was detected after 10min exposure to 10 µM ionomycin (Fig. 1, c and d). This
corresponds to an approximate increase of 6-10-fold over
the levels observed during the same period in PBS-treated
control cells.
) was observed, mostly clustered in the perinuclear area (Fig. 2, A
and B). In contrast, cells exposed to ionomycin showed a
more dispersed distribution of gold-loaded vesicles, with
accumulation in the proximity of the plasma membrane
(Fig. 2, C-F). Images that strongly suggested exocytic events,
with extracellular release of the gold complexes and other
membranous lysosomal contents, were observed at both 2.5 and 10 min after exposure to ionomycin (Fig. 2, D and
F). No similar images were detected in control cells (Fig. 2,
A and B).
Fig. 2.
Exocytosis of BSA-gold-loaded lysosomes is triggered by [Ca2+]i elevation. NRK cells loaded with 5 nm BSA-gold complexes
for 4 h followed by a chase of 2 h were incubated with PBS (A and B) or 10 µM ionomycin (C-H) for 2.5 (C and D) or 10 min (E-H). Transmission EM sections show gold-loaded vesicles (arrows), observed in close proximity to the plasma membrane in ionomycintreated cells (C, E, and F). Exocytosed BSA-gold was also detected after exposure to ionomycin (D and F). Labeling with antibodies
against lgp120 (10 nm gold) was detected on BSA-gold-containing vesicles (G and H). Small arrows indicate anti-lgp120 labeling on lysosomes; arrowheads indicate anti-lgp120 labeling on the plasma membrane after ionomycin treatment. Bars, 1 µM.
[View Larger Version of this Image (147K GIF file)]
) was performed on cryosections. Specific lgp120 label was detected on the membrane of vesicles accumulated next to the plasma membrane in ionomycin-treated cells (Fig. 2, G and H). Anti-lgp120 labeling was also detected on the plasma membrane of ionomycintreated cells (Fig. 2 H), an observation that was confirmed
by immunofluorescence in live cells, as described below
(Fig. 3, a-d).
Fig. 3.
Ionomycin induces appearance of lgp120 on the plasma
membrane. NRK cells, either attached to coverslips (a-c) or in
suspension (d), were incubated with PBS or 10 µM ionomycin in
PBS at 37°C, followed by immunofluorescent surface labeling
with an mAb to a luminal domain of lgp 120. (a) lgp120 surface
staining 10 min after exposure to PBS; (b) lgp120 surface staining
3 min after exposure to ionomycin; (c) lgp120 surface staining 10 min after exposure to ionomycin. (d) FACS® analysis of NRK
cells in suspension treated for 5 min with PBS (black line) or 10 µM ionomycin in PBS (gray line). Cells were incubated with antilgp120 mAbs for 30 min at 4°C before fixation. Bar, 5 µm.
[View Larger Version of this Image (39K GIF file)]
-hexosaminidase was measured in the supernatant of
cells treated with several concentrations of the signaling
peptide bombesin. As shown in Fig. 4 a, bombesin also induced lysosomal exocytosis in a dose-dependent way, but to a much lesser extent than ionomycin (a 1.5-fold maximum increase). When tested for induction of translocation
to the plasma membrane of the lysosomal protein lgp120,
bombesin was found to induce levels detectable by immunofluorescence (Fig. 4, c and d).
Fig. 4.
Ca2+ agonists induce a low level of -hexosaminidase
release and appearance of lgp120 in the plasma membrane. (a)
NRK cells were incubated with different concentrations of bombesin for 5 min, and the incubation buffer was assayed for
-hexosaminidase activity. The amount of enzyme released at each
point is expressed as a percentage of the total content of enzyme
in control cells. The data represent the average of triplicate determinations ± SD. (b) Same as a, except that cells were incubated
for 5 min with PBS, TSF, or heat-inactivated TSF. (c) lgp120 surface staining 5 min after exposure to PBS; (d) lgp120 surface
staining of NRK cells 5 min after exposure to 10 µM bombesin.
Bar, 5 µM.
[View Larger Version of this Image (86K GIF file)]
; Rodríguez et al., 1996
). It
also triggers IP3 formation and release of Ca2+ from intracellular stores in host cells, through a signaling factor
present in soluble extracts of the parasites (Burleigh and
Andrews, 1995
; Burleigh et al., 1997
). To verify if the trypanosome-soluble factor induced lysosome exocytosis, release of
-hexosaminidase was measured in the supernatant of cells treated with native or heat-inactivated TSF.
As shown in Fig. 4 b, a low level of stimulated
-hexosaminidase exocytosis, of ~1.2-fold over the background levels, was induced in NRK cells by native TSF in a 5-min
assay. Heat inactivation of TSF (95°C, 5 min) resulted in
the loss of Ca2+-agonist activity (not shown) and of the
ability to induce lysosomal exocytosis (Fig. 4 b).
),
a system that allows precise control of the intracellular
Ca2+ concentration.
-hexosaminidase was then measured in
permeabilized and nonpermeabilized cells, in the absence or presence of 1 and 5 µM Ca2+. As shown in Fig. 5 a, in a
5-min assay, an approximate fivefold increase in the release of
-hexosaminidase from permeabilized cells was
triggered by addition of 1 or 5 µM Ca2+.
Fig. 5.
Release of -hexosaminidase and LDH from intact and
SLO-permeabilized NRK cells. Intact cells (black columns) and
SLO-permeabilized cells (white columns), were incubated in permeabilization buffer containing either 0, 1, or 5 µM Ca2+ for 5 min at 37°C. Supernatants were assayed for (a)
-hexosaminidase and (b) LDH activity. The amount of enzyme released in each
sample is expressed as a percentage of the total enzyme content
of control cells. The data represent the average of triplicate determinations ±SD.
[View Larger Version of this Image (18K GIF file)]
-hexosaminidase release assay described above. Concentrations of SLO lower or higher than the optimal one resulted
in decreased Ca2+-induced release of
-hexosaminidase
(not shown).
).
-hexosaminidase, lucifer yellow, and
3H-dextran release assays in buffers with Ca2+ concentrations ranging from 0-5 µM. Optimal release of lysosomal markers, reaching 7-10-fold above the levels observed in
the absence of Ca2+, was found to require 1-5 µM Ca2+,
although exocytosis was already detectable at 0.1 µM Ca2+
(Fig. 6, a-c).
Fig. 6.
Ca2+-dependent
release of -hexosaminidase,
lucifer yellow, and 3H-dextran from permeabilized
NRK cells. SLO-permeabilized cells were incubated for
5 min at 37°C in permeabilization buffer containing different Ca2+ concentrations.
(a)
-hexosaminidase activity released. (b) Lucifer yellow released from previously
loaded cells. (c) 3H-Dextran
released from previously loaded cells. Values are expressed as a percentage of the total content of either
-hexosaminidase, lucifer yellow, or 3H-dextran in control cells. The data represent the average of triplicate determinations ±SD.
[View Larger Version of this Image (13K GIF file)]
). Elevation of [Ca2+]i in the human fibroblast cell line IMR-90, by treatment with ionomycin or by addition of 1 µM Ca2+ buffer to SLO-permeabilized cells, induced the appearance in the supernatant
of the 31-kD mature form of cathepsin D (Fig. 7, lanes 3 and 5). A band of identical migration was also detected in
whole cell extracts (Fig. 7, lane 1). The intermediate (47 kD)
and precursor (53 kD) forms of the protein (Gieselmann
et al., 1983
) were detected in the supernatant at similar
levels in each condition, probably reflecting the constitutive extracellular release of unprocessed cathepsin D that
can occur before reuptake and targeting to lysosomes (von
Figura and Hasilik, 1986; Kornfeld and Mellman, 1989
).
The higher molecular mass bands also appearing in Fig. 7
are due to nonspecific cross-reaction of the antibodies.
Fig. 7.
Elevation in [Ca2+]i induces secretion of the 31-kD lysosomally processed form of cathepsin D. Rabbit anti-cathepsin
D antibodies were used to probe a Western blot containing a lysate and concentrated supernatants of IMR-90 human fibroblasts. (Lane 1) total lysate; (lane 2) concentrated supernatant of
cells treated with PBS for 5 min; (lane 3) concentrated supernatant of cells treated with 10 µM ionomycin for 5 min; (lanes 4 and
5) concentrated supernatants of SLO-permeabilized cells incubated for 5 min in buffers containing 0 or 1 µM Ca2+, respectively. Different ECL exposures were performed for each treatment to allow visualization of the secreted mature 31-kD cathepsin D band.
[View Larger Version of this Image (41K GIF file)]
), permeabilized cells released 125I-transferrin linearly for ~20 min after permeabilization (Fig. 8 a).
Confirming previous reports that describe a small stimulation in the exocytosis of transferrin receptor-containing
vesicles upon treatment with ionophores or Ca2+-mobilizing agonists (Buys et al., 1984
; Wiley and Kaplan, 1984
), there was a minor increase (1.5-fold) in the extracellular
release of transferrin at increasing Ca2+ concentrations
(Fig. 8 b). The release of apotransferrin into the medium
after recycling to the cell surface allows the use of externally added, labeled transferrin to estimate the number of receptors on the surface at a given time. Using this
method, we found that the number of receptors available
for binding of FITC-transferrin on the surface of NRK
cells was also slightly increased after exposure to 10 µM
ionomycin (Fig. 8 c).
Fig. 8.
Effect of elevated
[Ca2+]i on exocytosis of
transferrin receptor-containing endosomes. (a) Kinetics
of release of preinternalized 125I-transferrin from SLOpermeabilized NRK cells in
the absence of Ca2+. (b) Release of preinternalized 125Itransferrin from SLO-permeabilized NRK cells at different Ca2+ concentrations. Values are expressed as a
percentage of the total 125Itransferrin present in each sample (cell associated plus released). The data represent the average of triplicate determinations ±SD. (c)
FACS® analysis of NRK cells in suspension treated for 5 min with PBS (black line) or 10 mM ionomycin in PBS (gray line). Cells were incubated with FITC-transferrin for 30 min at 4°C before fixation.
[View Larger Version of this Image (14K GIF file)]
-hexosaminidase release for 5 min at 37°C.
To control for permeabilization efficiency in each cell type,
LDH release was measured during the 5-min assay time.
Levels of LDH release ranging from 10-20% confirmed
that cells were efficiently permeabilized (not shown). In
LLC-MK2 and L6E9 cells, a preincubation of 3 min at 37°C
in the absence of Ca2+ was necessary to allow pore formation before addition of the 1 µM Ca2+ buffer. As shown in
Table I, Ca2+-dependent lysosomal exocytosis was detected in various cell types, including a primary culture of
human fibroblasts as well as epithelial and myoblast cell
lines. Enhancement in
-hexosaminidase release over background levels varied from 2-12-fold among the various cell
types. The lowest Ca2+-induced lysosome exocytosis levels
in this group were observed with the macrophage cell line
J774. Interestingly, the fusion of lysosomes with phagosomes in J774 and monocyte-derived macrophages was recently reported not to be regulated by Ca2+ (Zimmerli et al.,
1996
).
-Hexosaminidase Secretion in
Different Cell Types
The kinetics of Ca2+-induced lysosomal secretion in
NRK cells, as measured by release of -hexosaminidase,
showed a rapid increase in the first 5 min that did not significantly change with longer incubation times (Fig. 9 a).
Conversely, there was a continuous increase in the extracellular accumulation of LDH, due to cytoplasmic leakage
from the permeabilized cells. As mentioned before, however, the degree of cytosol loss that occurred during the
first 5 min after permeabilization is not sufficient to account for the sharp decrease in
-hexosaminidase secretion; as shown in Fig. 8 a, transferrin recycling, a process
known to depend on cytosolic factors (Podbilewicz and
Mellman., 1990; Galli et al., 1994
), proceeds in a linear
fashion for at least 20 min, under the same conditions.
To investigate the role of microtubules in the Ca2+-regulated lysosome exocytosis, NRK cells were treated with 10 µM colchicine for 1 h in the absence of serum. This is a condition that induces extensive microtubule depolymerization, as visualized by immunofluorescence with antitubulin antibodies (not shown). A small reduction of ~15% in lysosomal exocytosis, reproducible in several experiments, was observed (Fig. 9 b).
We next investigated whether -hexosaminidase release
from SLO-permeabilized NRK cells exhibited the properties expected for an exocytic process. The release was
found to be temperature dependent; when cells previously
shifted for 5 min to 37°C to allow SLO pore formation
were returned to ice, Ca2+-dependent
-hexosaminidase
release was almost totally blocked (Fig. 9 c).
-Hexosaminidase release was also found to be dependent on the
presence of MgATP. Cells were first permeabilized and
then exposed or not to an ATP-depleting system (hexokinase 150 U/ml, glucose 5 mM) for 15 min at 37°C. A significant inhibition of Ca2+-triggered
-hexosaminidase secretion was observed in ATP-depleted cells (Fig. 9 d).
In this work we demonstrate that increase in [Ca2+]i triggers exocytosis of lysosomes, in NRK fibroblasts and other cell types not considered "professional" Ca2+-regulated secretory cells. To confirm the lysosomal nature of the secretory vesicles in our experiments, we used four different approaches, taking advantage of molecular markers and functional characteristics of lysosomes.
First, we measured the release into the extracellular medium of the lysosomal enzyme -hexosaminidase. About
90% of the total
-hexosaminidase of NRK cells is located
in dense lysosomes (Griffiths et al., 1990
). Therefore, the
release of ~10% of the total cellular content of this enzyme into the medium upon increase in [Ca2+]i strongly
suggested the occurrence of lysosomal exocytosis. This
was observed in both ionomycin-treated and in SLO-permeabilized cells, in the presence of Ca2+-containing buffers.
Second, we examined the extracellular release of tracers
previously loaded into lysosomes by fluid-phase endocytosis. A chase of 2 h after the uptake period was used to ensure delivery of the endocytic tracer to lysosomes (Adams
et al., 1982; Rodríguez et al., 1996
). The release into the
medium of lucifer yellow or 3H-dextran previously loaded
into lysosomes was measured after elevating the [Ca2+]i with
ionomycin or by exposing permeabilized cells to Ca2+-containing buffers. The results were essentially identical to those obtained for
-hexosaminidase secretion, with maximum release values for fluid-phase tracers reaching between 10 and 20% of the total cellular content. Another
fluid-phase tracer, BSA-gold, was used to visualize the
distribution of lysosomes by EM after elevation in [Ca2+]i.
In contrast to normal cells, after [Ca2+]i elevation, a large
number of BSA-gold-containing vesicles, with the size and
morphological characteristics of lysosomes, was observed
closely associated with the plasma membrane. In several instances, lysosomal contents were detected in direct contact with the extracellular medium, strongly suggesting the
occurrence of exocytosis.
Third, we monitored the appearance on the plasma
membrane of a luminal epitope from the lysosomal integral membrane glycoprotein lgp120. This protein is highly
enriched in lysosomes and is not detectable on the surface
of NRK cells under normal conditions (Harter and Mellman, 1992). The appearance of lgp120 on the surface of
the large majority of ionomycin-treated NRK cells, as demonstrated by flow cytometry, indicates that lysosomal exocytosis occurred homogeneously in the majority of cells of
the population.
Fourth, we studied the extracellular release of the protease cathepsin D, frequently used as a marker for lysosomes. This enzyme is synthesized in the ER as a 53-kD
precursor, which is subsequently cleaved into a 47-kD intermediate form, and then to an active 31-kD mature
polypeptide. The last processing event that generates the
31-kD mature form occurs after transport to dense lysosomes (Gieselmann et al., 1983). We observed that after elevation of [Ca2+]i, in both ionomycin-treated and SLOpermeabilized cells, a 31-kD band specifically recognized by
anti-cathepsin D antibodies appeared in the extracellular
medium.
The results obtained with these four different approaches, combining the use of ionomycin and of SLOpermeabilized cells, indicate that elevation in [Ca2+]i triggers exocytosis of lysosomes in fibroblasts. We cannot, however, rule out the participation of late endosomes,
since in some cell types these organelles, in addition to
containing hydrolases and lgp120 (Griffiths et al., 1988),
appear to retain soluble tracers even after long chase periods (Rabinowitz et al., 1992
).
Parallel experiments measuring the recycling of transferrin receptor-containing endosomes to the plasma membrane showed that lysosomes are the main vesicle population that undergoes exocytosis upon [Ca2+]i elevation. A
small stimulation in transferrin release and in the number
of transferrin receptors on the cell surface was observed upon elevation of [Ca2+]i, confirming previous findings
(Buys et al., 1984; Wiley and Kaplan, 1984
). However, the
increase in exocytosis triggered by Ca2+ in NRK cells was
several fold higher for lysosomes than for early endosomes, under the same conditions. Furthermore, transferrin receptor-containing vesicles fused efficiently with the
plasma membrane in the absence of Ca2+, while lysosomes
did not. Constitutive transport from the TGN to the
plasma membrane is also known to occur at similar rates in
the absence or presence of Ca2+ up to 1 µM (Miller and
Moore, 1991
).
Increases in [Ca2+]i regulate many secretory events, such
as neurotransmitter release (Smith and Augustine, 1988)
and exocytosis of secretory granules (Holz et al., 1991
). Intracellular constitutive fusion mechanisms, such as ER to
Golgi transport (Beckers and Balch, 1989
) or fusion of nuclear membrane vesicles (Sullivan et al., 1993
), require
basal levels of Ca2+ (~100 nM); regulated exocytosis fusion events in specialized secretory cells (Holz et al., 1991
)
and lysosome-phagosome fusion in neutrophils (Jaconi et al.,
1990
) require higher [Ca2+]i, ~0.5-5 µM in average. Significantly higher Ca2+ concentrations, reaching hundreds
of micromolar in microseconds, are involved in triggering
synaptic vesicle fusion at the neuron terminal (Heidelberger et al., 1994
). Using the SLO-permeabilized system,
which allows the equilibration of intracellular and extracellular Ca2+ concentrations (Ahnert-Hilger et al., 1989
),
we were able to define the optimal [Ca2+]i required to trigger lysosomal exocytosis in NRK cells as ~1 µM.
The similar [Ca2+]i levels necessary to trigger lysosome
exocytosis in fibroblasts and regulated secretion in specialized cells add evidence in support of a common biogenesis
between lysosomes and Ca2+-regulated secretory granules
(Borregaard et al., 1993; Jamur et al., 1986
; Burkhardt et al.,
1990
; Peters et al., 1991
). The temperature and MgATP
dependence we observed for lysosomal exocytosis is also
consistent with what is known for other regulated secretory systems. Kinetic studies of exocytosis in neuroendocrine cells revealed the existence of multiple Ca2+-activated
steps and have shown that secretion can be dissociated into MgATP-dependent and -independent stages, requiring
distinct cytosolic factors (Bittner and Holz, 1992
; Neher
and Zucker, 1993
; Hay and Martin, 1992
; Chamberlain et al.,
1995
). Involvement of components of the NSF-SNAPSNARE putative fusion machinery (Sollner et al., 1993
) in
several Ca2+-dependent exocytic processes has been reported, but the precise events in which they participate are
presently unclear (DeBello et al., 1995
; Chamberlain et al.,
1995
; Bi et al., 1995
).
The kinetics of Ca2+-dependent lysosome exocytosis in
SLO-permeabilized NRK cells showed no significant increase after the first 5 min. This does not appear to be due
to depletion of essential cytosolic components, since recycling of transferrin receptor-containing vesicles, a process
known to require cytosolic factors, proceeds in permeabilized cells at increasing rates for at least 20 min (Fig. 8 a,
compare with Fig. 9 a). Furthermore, after a significantly longer period of permeabilization (15 min), the Ca2+induced -hexosaminidase release measured in a 5-min assay was only reduced in ~40% (Fig. 9 d, compare with 9 b).
The fact that only 10-20% of the lysosome population (as
defined by the total content of
-hexosaminidase or fluidphase tracers) appears to be capable of Ca2+-triggered exocytosis might reflect heterogeneity in spatial distribution and/or functional characteristics. Similar results have been
described for chromaffin cells, where the Ca2+-triggered
release of catecholamines is limited to 20-30% of the total
cellular content (Dunn and Holz, 1983
). Consistent with this interpretation, we observed only a very minor inhibition in lysosome secretion after microtubule depolymerization, suggesting that most of the lysosomes that undergo exocytosis are not transported for long distances,
but are already located close to the plasma membrane before the Ca2+ stimulation. Microtubule-based movement
was actually shown not to be required for exocytosis in
several systems, being only critical for delivery of newly
synthesized material to specific locations (Kelly, 1990
).
A recent report, based on capacitance measurements,
showed that elevation in [Ca2+]i triggers massive exocytosis in CHO cells and 3T3 fibroblasts, resulting in a surface
area increase of 20-30% in 4 min (Coorsen et al., 1996).
The Ca2+ concentration range described in that study to
trigger exocytosis is the same we determined for lysosome
exocytosis in NRK cells, from 0.1 to 4 µM. Interestingly,
the rate of exocytosis observed by those authors was found
to increase strongly with [Ca2+]i, while the amount did not.
This finding is consistent with our observation that only
10-20% of the lysosome population is capable of Ca2+triggered exocytosis, with no significant increase after 5 min. Our present study reinforces the suggestion that
Ca2+-regulated exocytosis is not restricted to specialized
secretory cells, and it strongly suggests that lysosomes are
the source of the large amount of exocytosed membrane
detected in that previous study (Coorsen et al., 1996
).
Another earlier observation consistent with our present
results is the Ca2+-dependent quantal release of acetylcholine loaded by endocytosis in CHO cells (Morimoto et al.,
1995). Although the loading and chase periods used in that
study were short (~15 min), it is conceivable that endocytosed acetylcholine reached lysosomes before exocytosis was triggered by Ca2+. Recently, Ca2+-regulated secretion
of glycosaminoglycan chains was detected in CHO fibroblasts (Chavez et al., 1996
). Although the Ca2+ concentration range for maximal secretion (1-5 µM) is the same as
the one required for lysosomal exocytosis, the glycosaminoglycan-containing organelles described in that study were
found to be less dense than lysosomes, and did not colocalize with lgp on immunofluorescence assays.
Our data indicate that optimal secretion of lysosomes in
NRK cells occurs at Ca2+ concentrations of ~1 µM. Therefore, a more significant level of exocytosis was initially expected in our experiments with the agonists bombesin and
TSF. These agents were previously shown to induce [Ca2+]i
transients reaching low micromolar levels in NRK cells
(Burleigh and Andrews, 1995). However, there is increasing evidence that IP3-mediated mobilization of Ca2+ from
intracellular stores can occur in a highly localized fashion, giving rise to a heterogeneous pattern of [Ca2+]i elevation
in cells (Hahn et al., 1992
; Paradiso et al., 1995
). It is conceivable that in our agonist treatment experiments, only lysosomes in the immediate vicinity of areas with elevated Ca2+
were able to fuse with the plasma membrane. It is interesting to note, in this context, that the mobilization of lysosomes to the plasma membrane by the parasite T. cruzi
was recently shown to involve only lysosomes located at a
distance of ~12 µm from the parasite invasion site (Rodríguez et al., 1996
).
Regulated secretion of lysosomal components has been
described previously mostly as part of specialized processes. In osteoclasts, Ca2+ regulation of lysosomal enzyme release was suggested (Davidson et al., 1994), and, in
neutrophils, both secretion of the lysosome-related azurophil granules (Sengelov et al., 1993
) and lysosome-phagosome fusion (Jaconi et al., 1990
) are Ca2+-dependent processes. Release of lysosomal contents from platelets and
pancreatic acinar cells is triggered by specific agonists, suggesting a possible Ca2+-regulated pathway (Febbraio and
Silverstein, 1990
; Hirano et al., 1991
; Grondin and Beaudoin, 1996
). Certain carcinoma cells present lgps in their
surface (Saitoh et al., 1992
; Garrigues et al., 1994
) and secrete lysosomal enzymes (Sloane et al., 1986
). A model of
secretory lysosomes fusing directly with the plasma membrane was proposed for mediating this process in colon
carcinoma cells (Saitoh et al., 1992
). Therefore, it is conceivable that the Ca2+-regulated lysosomal exocytosis we
describe here corresponds to a ubiquitous mechanism that
might be upregulated in certain pathological conditions.
A physiological function for a ubiquitous, Ca2+-dependent exocytosis of lysosomes is unclear. One intriguing
possibility, strongly suggested by our data, is that lysosomes
are the intracellular organelles recently proposed to be responsible for membrane resealing in wounded cells (Bi et
al., 1995; Miyake and McNeil, 1995
). Membrane resealing
was initially shown to require Ca2+-regulated exocytosis,
in 3T3 fibroblasts and sea urchin embryos (Steinhardt et
al., 1994
). More recently, the exocytic vesicles observed to
fuse with the plasma membrane at sites of injury, in 3T3 fibroblasts and bovine endothelial cells, were proposed to
belong to the endosomal/lysosomal compartment, based
on their labeling with lipophilic fluorescent dyes (Miyake
and McNeil, 1995
). In light of our present results, it is conceivable that a localized [Ca2+]i elevation, caused by Ca2+
influx through damaged membranes, triggers the fusion of
nearby lysosomes with the plasma membrane. Lysosome
exocytosis may therefore be at least one of the mechanisms underlying the essential process of plasma membrane repair in all cells.
Received for publication 4 September 1996 and in revised form 4 February 1997.
Please address all correspondence to Norma W. Andrews, Department of Cell Biology, Yale University School of Medicine, 333 Cedar Street, New Haven, CT 06520. Tel.: (203) 785-4314. Fax: (203) 785-7226.We thank H. Tan and B. Burleigh for photography, B. Burleigh and A. Sinai for helpful discussions and comments on the manuscript, R. Carbone for FACS® analysis, and A. Ma for excellent technical assistance.
This work was supported by the National Institutes of Health grant RO1AI34867 to N.W. Andrews.
[Ca2+]i, intracellular free Ca2+ concentration; LDH, lactate dehydrogenase; lgp, lysosomal membrane glycoprotein; NRK, normal rat kidney; SLO, streptolysin O; TSF, trypanosomesoluble fraction.