2027
Journal of Cell Science 110, 2027-2040 (1997)
Printed in Great Britain © The Company of Biologists Limited 1997
JCS4416
Dense core lysosomes can fuse with late endosomes and are re-formed from
the resultant hybrid organelles
Nicholas A. Bright, Barbara J. Reaves, Barbara M. Mullock and J. Paul Luzio*
Department of Clinical Biochemistry, University of Cambridge, Addenbrooke’s Hospital, Hills Road, Cambridge CB2 2QR, UK
*Author for correspondence
SUMMARY
Electron microscopy was used to evaluate the function and
formation of dense core lysosomes. Lysosomes were preloaded with bovine serum albumin (BSA)-gold conjugates
by fluid phase endocytosis using a pulse-chase protocol.
The gold particles present in dense core lysosomes and late
endosomes were flocculated, consistent with proteolytic
degradation of the BSA. A second pulse of BSA-gold also
accumulated in the pre-loaded dense core lysosomes at
37°C, but accumulation was reversibly blocked by incubation at 20°C. Time course experiments indicated that
mixing of the two BSA-gold conjugates initially occurred
upon fusion of mannose 6-phosphate receptorpositive/lysosomal glycoprotein-positive late endosomes
with dense core lysosomes. Treatment for 5 hours with
wortmannin, a phosphatidyl inositide 3-kinase inhibitor,
caused a reduction in number of dense core lysosomes preloaded with BSA-gold and prevented a second pulse of
BSA-gold accumulating in them. After wortmannin
treatment the two BSA-gold conjugates were mixed in
swollen late endosomal structures. Incubation of NRK cells
with 0.03 M sucrose resulted in the formation of swollen
sucrosomes which were morphologically distinct from preloaded dense core lysosomes and were identified as late
endosomes and hybrid endosome-lysosome structures.
Subsequent endocytosis of invertase resulted in digestion of
the sucrose and re-formation of dense core lysosomes.
These observations suggest that dense core lysosomes are
biologically active storage granules of lysosomal proteases
which can fuse with late endosomes and be re-formed from
the resultant hybrid organelles prior to subsequent cycles
of fusion and re-formation.
INTRODUCTION
been described as multi-vesicular bodies (MVBs) when
observed by electron microscopy.
In recent years great strides have been made in understanding how newly synthesised lysosomal proteins are
delivered from the trans-Golgi network (TGN) to a prelysosomal compartment (PLC) with late endosomal properties (Griffiths et al., 1988; Ludwig et al., 1991). In particular,
the role of two mannose 6-phosphate receptors in transporting mannose 6-phosphate-tagged soluble proteins has been
well documented (Kornfeld, 1986; Kornfeld and Mellman,
1989). The function of cytoplasmic tail sequence motifs in
the direct delivery of lgps from the TGN to the PLC and
lysosomes, without trafficking via the cell surface, is also
well described (Rohrer et al., 1996; Honing et al., 1996;
Hunziker and Geuze, 1996). Similarly, there has been
progress in understanding, both descriptively and in terms of
detailed molecular mechanism, the passage of endocytosed
ligands and membrane proteins to late compartments of the
endocytic pathway. Much of the debate about the relative
importance of maturation (Stoorvogel et al., 1991; Murphy,
1991) or vesicular transport steps (Griffiths and Gruenberg,
1991) in delivery from the plasma membrane to late
endosomes has now been resolved (Gruenberg and Maxfield,
1995). However, very little is known about delivery of endocytosed ligands and membrane proteins to dense core
Contemporary definitions of lysosomes include the criteria that
they form the terminal compartment of endocytosis and contain
the bulk of acid hydrolases and lysosomal glycoproteins (lgps),
but no cation-independent mannose 6-phosphate receptor
(M6PR; Griffiths et al., 1988, 1990; Luzio, 1994; Hunziker and
Geuze, 1996; Rohrer et al., 1996). They can thus be distinguished from late endosomes which are M6PR positive,
although both compartments accumulate endocytosed tracers
(Geuze et al., 1988; Griffiths et al., 1988, 1990; Parton et al.,
1989). Since the discovery of lysosomes in the 1950s (De
Duve, 1963, 1983) they have been recognised as being heterogeneous in morphology, although in most mammalian cells
they have been observed as approximately spherical structures
(0.2-0.5 µm in diameter) with an amorphous electron-dense
matrix (for review see Holtzmann, 1989). In the present study
we refer to these organelles as dense core lysosomes and
provide further evidence that they can be clearly distinguished
from endosomes. Endosomes themselves are heterogeneous in
morphology and many contain intra-organelle vesicles (Trowbridge et al., 1994). Endosomal compartments (Hopkins et al.,
1990; Felder et al., 1990; Futter et al., 1996) and endocytic
carrier vesicles (ECVs; Gruenberg et al., 1989), which transfer
endocytosed ligands from early to late endosomes, have often
Key words: Endocytosis, Lysosome biogenesis, Electron Microscopy
2028 N. A. Bright and others
lysosomes nor indeed how these structures are formed and
whether they are re-used.
It has been shown previously that when normal rat kidney
(NRK) cells are incubated with BSA-gold conjugates, they take
them up by fluid phase endocytosis and accumulate them in
dense core lysosomes (Griffiths et al., 1988, 1990; Parton et
al., 1989). We have built on this observation to study the interaction between dense core lysosomes and late endosomes and
to identify the route by which endocytosed conjugates become
concentrated in these lysosomes. In the present study we have
demonstrated that dense core lysosomes which have received
an initial pulse of gold conjugate remain accessible to a subsequent pulse and that the initial site of mixing of the two
pulses is in hybrid structures formed by direct fusion of dense
core lysosomes with late endosomes. We have also studied the
effects, on accumulation of endocytosed conjugates in
lysosomes, of a temperature block and of the phosphatidyl
inositide 3-kinase (PI3-kinase) inhibitor wortmannin, which
causes dramatic morphological changes to late endosomes
(Reaves et al., 1996). Finally, we have formed sucrosomes in
the NRK cells by uptake of 0.03 M sucrose as previously
described (Cohn and Ehrenreich, 1969, DeCourcy and Storrie,
1991) and showed that, contrary to previous interpretations
(DeCourcy and Storrie, 1991; Montgomery et al., 1991;
Jahraus et al., 1994), these were swollen late endosomes which
could fuse with dense core lysosomes. Dense core lysosomes
could be re-formed from the resultant swollen, hybrid
lysosome-sucrosomes only after subsequent endocytosis of
invertase.
Our experimental data are consistent with dense core
lysosomes being re-usable storage granules, containing
proteases and other acid hydrolases, that fuse directly with late
endosomes to allow the commencement of degradation of
endocytosed material and which are re-formed from the hybrid,
fused organelle.
MATERIALS AND METHODS
Materials
Wortmannin (aliquoted and kept at −20°C as a 1 mM stock in DMSO),
BSA, invertase, gold chloride, tannic acid, tri-sodium citrate and
methyl cellulose were from Sigma Chemicals (Poole, Dorset, UK).
The rabbit polyclonal anti-rat lgp110 antiserum was previously
described (Reaves et al., 1996). The rabbit polyclonal anti-bovine
M6PR antibody was kindly provided by Dr Suzanne Pfeffer (Stanford
University, Stanford, CA; Pfeffer, 1987). The rabbit polyclonal antimouse cathepsin L antibody cross-reacts with rat fibroblast cathepsin
L (Punnonen et al., 1994) and was kindly provided by Dr Michael
Gottesman (National Cancer Institute, Bethesda, MD). Protein A conjugated to 10 nm or 15 nm colloidal gold was purchased from the
Department of Cell Biology, University of Utrecht.
Cell culture
NRK fibroblast cells were grown in Dulbecco’s modified Eagle’s
medium supplemented with 10% fetal calf serum (FCS), 100 i.u./ml
penicillin, 100 µg/ml streptomycin, 4.5 g/l glucose and 2 mM Lglutamine. Cells were grown in 25 or 75 cm2 tissue culture flasks in
a 5% CO2 incubator at 37°C.
Preparation of BSA-gold
5 nm, 10 nm and 15 nm colloidal gold was prepared by tannic acid/trisodium citrate reduction of gold chloride (Slot and Geuze, 1985). The
colloid was adjusted to pH 5.5 with NaOH and conjugated to sufficient BSA to afford protection from NaCl-induced flocculation. BSAgold was harvested using ultracentrifugation protocols which yielded
monodisperse preparations free of aggregates and unbound protein
(Slot and Geuze, 1981, 1984). The preparations were dialysed against
PBS and adjusted to an A520 of 1.4 with PBS.
Flocculation of gold colloids
To test if gold colloids were stabilised against electrolyte-induced
flocculation NaCl was added to try and destabilise the conjugates.
These were visualised by incubating with poly-L-lysine
coated/Formvar-carbon coated EM grids for 10 minutes, which were
then blotted, air dried and viewed in a Philips CM100 transmission
EM. The micrographs of stabilised BSA-10 nm gold and colloidal
gold devoid of adsorbed BSA and flocculated by addition of NaCl
(Slot and Geuze, 1981, 1984; De Mey, 1986) are shown in Fig. 1a,b.
Endocytosis of BSA-gold
1 ml of BSA-gold was added to 4 ml DMEM + 10% FCS and NRK
cells grown to ~80% confluence were incubated with the conjugatecontaining medium for 4 hours at 37°C followed by incubation in
conjugate-free medium for 20 hours as previously described (Reaves
et al., 1996). Cells were subsequently incubated with medium containing BSA-15 nm gold for 15 minutes to 4 hours with a 20 hour
chase, prior to fixation and processing for EM. In temperature block
experiments, cells which had been pre-loaded with BSA-5 nm gold
were incubated with BSA-15 nm gold at 20°C for 1 hour prior to
transfer to 37°C for 0, 30 or 60 minutes.
Treatment with wortmannin
After internalisation of BSA-5 nm gold for 4 hours and a 20 hour
chase the cells were incubated with 100 nM wortmannin in RPMI
medium (Sigma) + 1% FCS for 1 hour as previously described
(Reaves et al., 1996). The effects of wortmannin on the uptake and
trafficking of a subsequent pulse of BSA-15 nm gold were examined
after internalision of BSA-5 nm gold for 4 hours followed by a 20
hour chase, by incubating with 100 nM wortmannin for 1 hour
followed by endocytosis of medium containing BSA-15 nm gold in
the presence of 100 nM wortmannin for 4 hours (2× 2 hours; Reaves
et al., 1996).
Formation of sucrosomes
NRK cells which had internalised BSA-5 nm gold for 4 hours followed
by a 20 hour chase were subsequently incubated with medium containing 0.03 M sucrose for 4 to 24 hours to induce the formation of
sucrosomes (Cohn and Ehrenreich, 1969). The cells were then fixed
and processed for EM. In addition, cells were subsequently allowed to
endocytose BSA-15 nm gold for 4 hours in sucrose-free medium.
Sucrose was omitted from the medium in control experiments.
Cells in which the dense core lysosomes were pre-loaded with
BSA-5 nm gold followed by 0.03 M sucrose internalisation for 24
hours were allowed to internalise 0.5 mg/ml invertase for 30 minutes
to 8 hours. In control cells invertase was omitted from the medium.
Transmission electron microscopy
For routine EM, cells were removed from tissue culture flasks by
trypsinisation and pelleted in a bench-top microfuge at 500 g for 2
minutes. The cells were then fixed with 2.5% glutaraldehyde/2%
paraformaldehyde in 0.1 M Na cacodylate buffer, pH 7.2, for 3 hours
at room temperature, washed with 0.1 M Na cacodylate buffer, pH
7.2, and post-fixed in 1% osmium tetroxide in 0.1 M Na cacodylate
buffer, pH 7.2, for 1 hour. The cell pellet was then washed with 0.05
M Na maleate buffer, pH 5.2, and en bloc stained with 0.5% uranyl
acetate in 0.05 M Na maleate buffer, pH 5.2. The cell pellets were
then dehydrated in ethanol, exchanged into 1,2-epoxy propane and
embedded in Araldite CY212 epoxy resin (Agar Scientific, Stansted,
UK).
Endosome-lysosome fusion 2029
Ultrathin sections were cut using a diamond knife mounted on a
Reichert Ultracut S ultramicrotome (Leica, Milton Keynes, UK),
collected on EM grids and stained with uranyl acetate and Reynolds
lead citrate (Reynolds, 1963). The sections were observed in a Philips
CM 100 transmission electron microscope (Philips Electron Optics,
Cambridge, UK) at an operating voltage of 80 kV.
Immunoelectron microscopy
Cells were prepared for immuno-EM as described by Griffiths (1993).
Cells were washed with PBS and fixed with 4% paraformaldehyde/0.1% glutaraldehyde in 250 mM Hepes, pH 7.2, at room temperature for 1 hour. The cells were scraped and pelleted at 1,000 g for
2 minutes in a microfuge. The cell pellets were subsequently
embedded in 10% gelatin in PBS at 37°C, cooled on ice, trimmed and
infused with 2.1 M sucrose in PBS overnight at 4°C prior to being
frozen on aluminium stubs in liquid nitrogen. Frozen ultrathin
sections were cut using a cryochamber attachment (Leica, Milton
Keynes, UK), collected with 2.3 M sucrose in PBS and mounted on
Formvar-carbon coated EM grids.
Immunolabelling was performed using the Protein A-gold
technique at room temperature (Slot and Geuze, 1983). Sections were
incubated with 50 mM NH4Cl in PBS for 10 minutes to quench
unreacted aldehydes, transferred to 2% gelatin in PBS for 10 minutes
and then 1% BSA in PBS for 10 minutes. Sections were incubated for
30 minutes with 5 µl of primary antibody diluted in PBS containing
5% FCS and 0.1% BSA. Primary antibodies utilised in these studies
were rabbit anti-mannose-6-phosphate receptor (M6PR: diluted 1:50),
rabbit anti-lgp110 (diluted 1:50) and rabbit anti-cathepsin L (diluted
1:100). The sections were washed with PBS/0.1% BSA (6× 3 minutes)
and incubated for 30 minutes with PBS/0.1% BSA containing Protein
A conjugated to 10 or 15 nm colloidal gold. The sections were washed
with PBS/0.1% BSA (2× 5 minutes), PBS (4× 5 minutes) and the
complex stabilised using 1% glutaraldehyde in PBS (5 minutes).
Finally the sections were rinsed with distilled water (5× 3 minutes)
and contrasted by embedding in 1.8% methyl cellulose/0.3% uranyl
acetate (Tokuyasu, 1978). Sections were allowed to air dry prior to
observation.
Morphometry
Quantitative analysis of the subcellular distribution of gold particles
was performed on the EM at a magnification of 15,500. Individual
random sections through cell pellets were systematically scanned and
the presence of gold conjugates in endosomes, sucrosomes, lysosomes
or organelles possessing intermediate morphology were scored and
expressed as a percentage of the total gold-labelled organelles. Goldcontaining organelles were scored irrespective of the number of gold
particles contained therein. Dense core lysosomes were defined as
possessing an electron-dense lumenal content; endosomes and sucrosomes as possessing vacuolar morphology with an electronlucent
lumenal content; organelles with intermediate morphology as possessing vacuolar morphology with a diffuse lumenal content of intermediate electron density.
The data were tested for statistical significance with analysis of
variance using Super Anova software (Abacus concepts Inc., version
1.11). Only P values ≤0.01 are shown. Unless otherwise stated results
are expressed as a mean ± s.e.m, with the number of experiments (n)
shown in parentheses. The number of cell profiles and gold-labelled
organelles scored to generate the data are indicated in the figure
legends.
RESULTS
Accumulation of BSA-gold in dense core lysosomes
and late endosomes
BSA-gold accumulated in dense core lysosomes and late
endosomes of NRK cells after endocytosis of medium containing the conjugate for 4 hours at 37°C, followed by a chase
period of 20 hours at 37°C in conjugate-free medium (Fig. 1c)
as previously described (Griffiths et al., 1988, 1990; Parton et
al., 1989; Reaves et al., 1996). Quantitation indicated that
83.8±2.3% (n=8) of the organelles that had accumulated gold
were electron dense and therefore identified as dense core
lysosomes. Immuno-EM indicated that the BSA-gold loaded
dense core lysosomes could be immunolabelled with antibodies to lgp110 (Fig. 1d) and cathepsin L (Fig. 1e), but only
rarely with antibodies to M6PR (not shown), thus providing
verification that these structures were lysosomal. Of the
organelles that had accumulated BSA-gold, 14.4±2.0% (n=8)
possessed the morphological appearance of endosomes (Fig.
1f). Immuno-EM showed that they could be labelled with antibodies to lgp110 (Fig. 1g) and M6PR (Fig. 1h). Structures possessing intermediate or equivocal morphology accounted for
1.8±0.7% (n=8) of the BSA-gold loaded organelles.
BSA-gold which had accumulated in the late endosomes and
lysosomes had flocculated in a manner similar to NaCl-induced
flocculation of gold colloids which had not been stabilised by
addition of protein, as seen in Fig. 1b. In contrast, BSA-gold
conjugates present in organelles of the early endocytic pathway
had not flocculated (see below), but remained as discrete
particles. Thus flocculation of gold conjugates was a feature of
delivery to compartments containing active proteases.
A second pulse of BSA-gold accumulated in dense
core lysosomes pre-loaded with BSA-gold
To test whether dense core lysosomes were biologically active
after accumulating BSA-gold, NRK cells that had been preloaded with BSA-gold were allowed to endocytose a second
pulse of BSA conjugated to colloidal gold of larger diameter
for 4 hours followed by a 20 hour chase. After this treatment
85.9±0.5% (n=3) of the organelles containing BSA-15 nm gold
were dense core lysosomes that also contained the smaller
BSA-gold (Fig. 2a). These results indicate that dense core
lysosomes retained their ability to fuse with endocytic structures containing a subsequent pulse of BSA-15 nm gold despite
their lumenal content of pre-loaded gold. Flocculation of the
second pulse of gold suggested that the BSA had been
degraded, consistent with the hydrolytic enzymes in the
lysosome remaining biologically active.
Apparent fusions of dense core lysosomes with endocytic
organelles could occasionally be observed (Fig. 2b).
Organelles possessing the morphological appearance of
endosomes accounted for 14.1±0.5% (n=3) of the total BSA15 nm gold-labelled structures. The BSA-15 nm gold, which
had also flocculated, frequently co-localised with the smaller
BSA-gold in these organelles (Fig. 2c).
Mixing of a second pulse of BSA-gold with preloaded BSA-gold occurred upon fusion of a late
endosome compartment with a dense core
lysosome
To clarify where mixing of a second pulse of BSA-gold with
pre-loaded BSA-gold occurred, a time-course was performed.
Cells were pre-loaded with BSA-5 nm gold and then incubated
with medium containing BSA-15 nm gold for 15 minutes to 4
hours. Mixing of the BSA-15 nm gold with the pre-loaded
BSA-5 nm gold occurred within 15 minutes, consistent with
2030 N. A. Bright and others
Fig. 1. Accumulation of BSAgold in lysosomes and late
endosomes of NRK cells.
BSA-10 nm gold conjugates
remained unaffected by the
addition of NaCl (a). However,
addition of NaCl to colloidal
gold devoid of adsorbed BSA
resulted in flocculation (b).
NRK cells were allowed to
endocytose BSA-10 nm gold
for 4 hours followed by a 20
hour chase in conjugate-free
medium. This resulted in the
accumulation of gold (small
arrowheads) in dense core
lysosomes (c). In frozen
sections these organelles could
be immunolabelled with
antibodies to lgp110 (d) and
cathepsin L (e) and visualised
with Protein A-15 nm gold
(large arrowheads). 14.4±2.0%
(n=8) of the BSA-10 nm goldlabelled organelles possessed
the morphology of endosomes
(f). These could be
immunolabelled with
antibodies to lgp110 (g) and
M6PR (h) and visualised with
Protein A-15 nm gold. Note
that the 10 nm gold has
flocculated in the lysosomes
and endosomes similar to
NaCl-induced flocculation of
the unconjugated gold colloid.
Bristle-like coats may
frequently be observed on
endosomes (arrow). Bar, 500
nm.
fusion between late endosomes containing BSA-15 nm gold
and dense core lysosomes containing BSA-5 nm gold. These
fusions resulted in hybrid organelles with the appearance of
endosomes containing diffuse lumenal contents and internal
membranes (Fig. 3a). Flocculation of the BSA-15 nm gold
occurred in these structures but conjugates that had not yet
encountered the pre-loaded gold remained particulate. The
hybrid organelles were immunoreactive with antibodies to
M6PR (Fig. 3b) and lgp110 (not shown). After 15 minutes
uptake the BSA-15 nm gold was not present in dense core
Endosome-lysosome fusion 2031
Fig. 2. Accumulation of BSA-15 nm gold in lysosomes and late
endosomes pre-loaded with BSA-10 nm gold. Lysosomes and late
endosomes of NRK cells were pre-loaded with BSA-10 nm gold
using a 4 hour pulse and 20 hour chase in conjugate-free medium.
BSA-15 nm gold was subsequently internalised using a 4 hour pulse
and 20 hour chase and accumulated in the pre-loaded lysosomes (a).
In b a dense core lysosome loaded with 10 nm and 15 nm gold has
apparently fused with an endocytic organelle (arrow). Organelles
possessing the morphology of endosomes frequently contained both
sizes of gold (c). These structures may arise from a fusion between a
non-gold labelled endosome and a lysosome containing both gold
conjugates or upon fusion of a BSA-10 nm gold-laden lysosome with
a 15 nm gold-laden endosome. Bar, 200 nm.
lysosomes (see Fig. 4a), but by 30 minutes, in addition to the
hybrid structures, a small population of dense core lysosomes
were labelled with both gold conjugates (Fig. 3c). After 1 hour
to 4 hours of uptake of the BSA-15 nm gold, an increasing
number of dense core lysosomes contained both sizes of gold
(Fig. 4a).
In another series of experiments cells pre-loaded with BSA5 nm gold were subsequently incubated with BSA-15 nm gold
at 20°C for 1 hour. The cells were either fixed and processed
for EM or returned to 37°C for 30 minutes or 1 hour prior to
fixation (Fig 4b; micrographs not shown). After 1 hour at 20°C,
97.9±1.2% (n=3) of the organelles labelled with BSA-15 nm
gold possessed endosomal morphology and contained no BSA5 nm gold. After returning the cells to 37°C for 1 hour, the
number of BSA-15 nm gold-labelled organelles with
endosomal morphology that were devoid of BSA-5 nm gold
had dropped significantly, and there was an increase in the
number of endosomes and dense core lysosomes containing
both sizes of gold (Fig. 4b).
Inhibition of accumulation of BSA-gold in dense
core lysosomes after treatment with wortmannin
NRK cells pre-loaded with BSA-5 nm gold were treated with
100 nM wortmannin for 1 hour at 37°C, resulting in the
formation of two populations of swollen late endosomes
(Reaves et al., 1996). Of the organelles that were labelled with
colloidal gold, 87.6±2.0% (n=6) were dense core lysosomes,
compared with 83.8±2.3% (n=8) in control cells. Thus,
lysosomes were unaffected by treatment with wortmannin for
1 hour. Swollen late endosomes accounted for 11.2±1.6%
(n=6) of the gold-labelled organelles, compared with
14.4±2.0% (n=8) normal late endosomes in control cells.
Organelles with intermediate morphology accounted for
1.2±0.5% (n=6) of the gold-containing structures (Fig. 5).
When cells pre-loaded with BSA-5 nm gold were treated
with 100 nM wortmannin for 5 hours at 37°C, under conditions
in which swollen late endosomal structures were maintained,
an effect on dense core lysosomes was observed. In contrast to
the lack of effect after treatment for 1 hour, 56.2±0.2% (n=3)
of the BSA-5 nm gold-labelled organelles were dense core
lysosomes, 26.4±1.7% (n=3) were swollen endosomal structures and 17.4±2.2% (n=3) possessed intermediate morphology (Fig. 5). These data were consistent with fusion of dense
core lysosomes with late endosomes, and inhibition of the reformation of dense core lysosomes from the resultant hybrid
organelles. To test this hypothesis and determine the effects of
wortmannin upon the accumulation of BSA-gold in dense core
2032 N. A. Bright and others
Fig. 3. Time-course of delivery of BSA-15 nm gold into NRK cells pre-loaded with
BSA-5 nm gold. Lysosomes and late endosomes were pre-loaded with BSA-5 nm
gold using a 4 hour pulse and 20 hour chase. BSA-15 nm gold was then
internalised for 15 minutes (a-b) or 30 minutes (c). Mixing of the gold conjugates
could first be observed in organelles possessing the morphology of endosomes after
15 minutes of BSA-15 nm gold internalisation (a). These could be immunolabelled
with antibodies to M6PR and visualised using Protein A-10 nm gold (medium
arrowhead) on frozen sections (b). Note that both gold conjugates have flocculated.
After 30 minutes (c) of BSA-15 nm gold uptake both sizes of gold could be
observed in the same dense core lysosomes. Note that BSA-15 nm gold has not
flocculated in endosomes which do not possess 5 nm gold. Bar, 200 nm.
a
% of total BSA-15 nm gold-labelled organelles.
100
75
Endosome + BSA15.
50
Endosome
+ BSA15 & BSA5.
Dense core lysosome
+ BSA15 & BSA5.
25
sucrose 24h
then 240
240
+ 20h chase
240
120
60
30
15
0
Duration of BSA-15 nm Au internalisation (mins).
b
% of total BSA-15 nm gold-labelled organelles.
100
75
50
25
60
30
0
0
Time at 37oC (mins).
Fig. 4. Quantitation of the time course of distribution of BSA-15 nm gold in
NRK cells, (a) after uptake at 37°C, or (b) after 1 hour at 20°C followed by
uptake at 37°C. Cells were pre-loaded with BSA-5 nm gold for 4 hours followed
by a 20 hour chase. Results are presented as a mean of triplicate experiments ±
s.e.m. (a) No. of cells/section = 81±6; no. of organelles/section 344±33. (b) No.
of cells/section = 66±9; no. of organelles/section = 173±23. The final set of
columns on the right hand side of (a) indicates the distribution of BSA-15 nm
gold after 4 hours internalisation into NRK cells previously allowed to
accumulate 0.03 M sucrose for 24 hours.
Endosome-lysosome fusion 2033
100
intermediate structures
% of total gold-labelled organelles.
endosomes
75
dense core lysosomes
**
50
*
25
**
0
0
1
5
Duration of wortmannin treatment (h).
Fig. 5. Quantitation of the distribution of pre-loaded BSA-5 nm gold
after a 4 hour pulse and 20 hour chase followed by incubation with
100 nM wortmannin. Results are presented as a mean (0 hours: n=8;
1 hour: n=6; 5 hours: n=3) ± s.e.m. *P≤0.01; **P≤0.0001, when
compared with control cells or cells treated for 1 hour with 100 nM
wortmannin. No. of cells/section = 55±5; no. of organelles/section =
94±19.
lysosomes, cells pre-loaded with BSA-5 nm gold were treated
with 100 nM wortmannin for 1 hour and subsequently
incubated in medium containing BSA-15 nm gold and 100 nM
wortmannin for 4 hours. The BSA-15 nm gold accumulated in
swollen late endosomes but did not appear in dense core
lysosomes pre-loaded with BSA-5 nm gold (Fig. 6). These
swollen endosomes could be immunolabelled for M6PR and
Fig. 6. The effect of wortmannin on
endocytosis of BSA-15 nm gold.
Lysosomes and late endosomes were preloaded with BSA-5 nm gold using a 4
hour pulse and 20 hour chase. The cells
were then incubated with 100 nM
wortmannin for 1 hour and additionally
for 4 hours with BSA-15 nm gold in the
presence of 100 nM wortmannin. BSA-15
nm gold accumulated in the swollen
endocytic compartments but was not
found in association with dense core
lysosomes (stars) pre-loaded with BSA-5
nm gold. Bar, 200 nm.
lgp110 (data not shown). Many of the swollen organelles
contained both sizes of gold conjugate and those containing
BSA-5 nm gold could be immunolabelled for cathepsin L (data
not shown), consistent with them resulting from the fusion of
lysosomes with late endosomes. In control experiments, in
which wortmannin was omitted, the second pulse of BSA-gold
accumulated in dense core lysosomes as in previous experiments.
Sucrose accumulated in swollen late endosomes
(sucrosomes) distinct from dense core lysosomes
Previous investigators have shown that uptake of sucrose into
cells which cannot digest it causes swelling of late endocytic
compartments (Cohn and Ehrenreich, 1969; DeCourcy and
Storrie, 1991; Montgomery et al., 1991; Jahraus et al., 1994).
To investigate the effects of this treatment on dense core
lysosomes, NRK cells were pre-loaded with BSA-5 nm gold.
The cells were then incubated with medium containing 0.03 M
sucrose for 8 hours at 37°C, fixed and processed for EM.
Swollen structures (sucrosomes) appeared after this treatment
(Fig. 7). The number of dense core lysosomes loaded with the
BSA-gold decreased after this treatment (Fig. 8) but those that
remained were morphologically unaffected (Fig. 7a). Fine
filaments could occasionally be observed attaching lysosomes
to the sucrosomes (Fig. 7b). Immuno-EM confirmed the
identity of the swollen sucrosomes as late endosomes, or
endosome-lysosome hybrids, by virtue of their M6PR (Fig. 7c)
and lgp110 (Fig. 7d) immunoreactivity. In sucrosomes containing BSA-gold, cathepsin L immunoreactivity was also seen
(Fig. 7e), consistent with these being endosome-lysosome
hybrids.
To determine the effects of prolonged sucrose endocytosis,
NRK cells were pre-loaded with BSA-gold and the effects of
2034 N. A. Bright and others
Fig. 7. The effect of 0.03 M sucrose internalisation upon lysosomes pre-loaded with BSA-5 nm gold in NRK cells. Cells were pre-loaded with
BSA-5 nm gold using a 4 hour pulse and 20 hour chase, and subsequently incubated with 0.03 M sucrose for 8 hours to induce the formation of
sucrosomes. The organelles which accumulated sucrose were morphologically distinct from the gold-laden dense core lysosomes (a).
Lysosomes and endosomes could occasionally be observed to be attached via fine filaments (arrows) as seen at a greater magnification (b).
However, after this duration of sucrose internalisation 87.6±2.0% (n=6) of the pre-loaded BSA-5 nm gold was present in swollen sucrosomes
(c-e). These structures could be immunolabelled with antibodies to M6PR (c), lgp110 (d) and cathepsin L (e) and visualised using Protein A-10
nm gold in frozen sections. Bars: 500 nm (a,c-e); 200 nm (b).
sucrose internalisation for 4 to 24 hours were examined (Fig.
8a). Prolonged uptake of sucrose resulted in the depletion of
labelled dense core lysosomes and concomitant accumulation
of gold in swollen sucrosomes. This appeared to be an all-ornothing effect, as evidenced by the paucity of organelles with
intermediate morphology. In control cells incubated in medium
without sucrose the steady state distribution of BSA-gold was
unaffected.
Dense core lysosomes were re-formed from
sucrosome-lysosome hybrids upon internalisation
of invertase
It has been shown previously that endocytosis of invertase
into cells containing pre-formed sucrosomes results in the
efficient collapse and disappearance of these structures (Cohn
and Ehrenreich, 1969). In the present experiments internalisation of medium containing invertase, after the formation of
sucrosomes over a 24 hour period and concomitant depletion
of lysosomes pre-loaded with BSA-gold, resulted in the
regeneration of dense core lysosomes containing BSA-gold
(Fig. 8b).
A time-course of invertase uptake showed a decrease in the
number of labelled sucrosomes, a transient rise of organelles
possessing intermediate morphology and a gradual rise in the
number of re-formed dense core lysosomes. These results
indicate that as the content of sucrosomes was digested the
organelles transiently possessed intermediate morphology
prior to reforming morphologically identifiable dense core
Endosome-lysosome fusion 2035
b
100
90
90
80
70
dense core lysosomes.
60
50
sucrosomes.
40
intermediate structures.
30
20
10
% of total gold-labelled organelles.
% of total gold-labelled organelles.
a
100
80
70
60
dense core lysosomes.
50
sucrosomes.
40
intermediate structures.
30
20
10
0
0
0
4
8
12
16
20
24
Duration of 0.03 M sucrose uptake (h).
0
1
2
3
4
5
6
7
8
Duration of 0.5 mg/ml invertase uptake (h).
Fig. 8. Quantitation of the distribution of pre-loaded BSA-gold after internalisation of 0.03 M sucrose (a), or after 24 hours sucrose
accumulation followed by uptake of 0.5 mg/ml invertase (b). Results are presented as a mean of triplicate experiments ± s.e.m (a), or duplicate
experiments ± range (b). (a) No. of cells/section = 84±9; no. of organelles/section = 83±6. (b) No. cells/section = 75±5; No. organelles/section
= 15±2.
lysosomes. In cells to which invertase was not added the gold
remained in swollen sucrosomes (Fig. 9).
The presence of sucrosomes prevented
accumulation of a second pulse of BSA-gold in
dense core lysosomes
We examined the effect of the presence of sucrosomes on internalisation and subsequent trafficking of a second pulse of
BSA-gold. Cells were pre-loaded with BSA-5 nm gold and
then incubated in the presence of 0.03 M sucrose for 24 hours.
The cells were then allowed to internalise BSA-15 nm gold for
4 hours. In contrast to the observation in control cells (Fig. 4a),
internalised BSA-15nm gold accumulated in structures with
the characteristic morphology of ECVs (Fig. 10a; Gruenberg
et al., 1989). These did not contain BSA-5nm gold and were
clearly distinct from the swollen sucrosomes (Fig. 10b). Only
a small percentage of the sucrosomes contained both sizes of
gold and any residual dense core lysosomes contained only the
first pulse of BSA-5 nm gold (for quantitation see Fig. 4a).
These observations imply that fusions between sucrosomes and
Fig. 9. The effect of invertase internalisation on sucrosomes. Cells were pre-loaded with BSA-5 nm gold using a 4 hour pulse and 20 hour
chase, and subsequently allowed to internalise 0.03 M sucrose for 24 hours resulting in the formation of sucrosomes and depletion of dense
core lysosomes. Cells were then incubated in medium without invertase (a) or + 0.5 mg/ml invertase for 8 hours (b). Endocytosis of invertase
resulted in digestion of the sucrose and re-formation of dense core lysosomes labelled with BSA-gold (b). Bar, 500 nm.
2036 N. A. Bright and others
Endosome-lysosome fusion 2037
Fig. 10. The effect of the presence of sucrosomes on endocytosis and
subsequent trafficking of BSA-gold. Cells were pre-loaded with
BSA-5 nm gold using a 4 hour pulse and 20 hour chase, and then
allowed to internalise 0.03 M sucrose for 24 hours, prior to
incubation with medium containing BSA-15 nm gold for 4 hours.
This resulted in the accumulation of ECVs in which the BSA-15 nm
gold had not flocculated (a). These structures were clearly distinct
from the sucrosomes (b). Frozen sections were immunolabelled with
antibodies to M6PR (c), lgp110 (d) and cathepsin L (e) and
visualised using Protein A-10 nm gold. Residual dense core
lysosomes (stars) contain only the first pulse of BSA-5 nm gold and
not the second pulse of BSA-15 nm gold. ECVs containing
particulate BSA-15 nm gold (white arrowheads) were not
immunoreactive with antibodies to M6PR, lgp110 or cathepsin L.
However, BSA-15 nm gold which had been delivered to sucrosomes
had clearly flocculated (d, e). Bar, 500 nm.
ECVs were impaired by the accumulation of the indigestible
sucrose. The second pulse of BSA-15 nm gold had not flocculated in the ECVs, whereas gold present in sucrosomes containing the first pulse of BSA-5 nm gold had flocculated (Fig.
10e). Immuno-EM of the ECVs containing the second pulse of
gold revealed that they were not immunoreactive for M6PR
(Fig. 10c), lgp110 (Fig. 10d) or cathepsin L (Fig. 10e), consistent with them being an earlier endocytic compartment than
late endosomes.
DISCUSSION
We have studied the interaction and fusion of dense core
lysosomes with late endosomes in cultured cells. Using fused
hybrid cell systems, others have shown that lysosomes and
endosomes are dynamic structures that interchange content and
membrane markers (Deng and Storrie, 1988; Deng et al.,
1991). In cell-free experiments we have previously shown that
content mixing occurs when lysosomes and late endosomes
derived from rat liver hepatocytes are incubated in the presence
of cytosol and an energy source (Mullock et al., 1989, 1994).
However, in none of these systems was it possible to distinguish between direct fusion of late endosomes and lysosomes,
vesicular transport or even ‘kiss and run’ (for various models
to explain content mixing see Berg et al., 1995, and Storrie and
Desjardins, 1996). Futter et al. (1996) demonstrated in HEp-2
cells, a cell type in which MVBs are the dominating endocytic
organelle (van Deurs et al., 1993, 1995), that attachment and
fusion of endocytic MVBs occurred with non-electron dense,
M6PR negative MVBs which were pre-loaded with horseradish peroxidase. Our present study extends this observation to
the fusion of late endosomes with dense core lysosomes and
allows us to propose a model for the accumulation of endocytosed ligands in dense core lysosomes which involves fusion
of pre-existing lysosomes with late endosomes followed by reformation of dense core lysosomes from the resultant hybrid
structures.
We have shown that not only is it possible for dense core
lysosomes to accumulate BSA-gold, as previously described
by others (Griffiths et al., 1988, 1990; Parton et al., 1989), but
that these lysosomes can acquire a second pulse of BSA-gold
showing that they are re-usable. Intermixing of the two
separate pulses of BSA-gold was consistent with fusion of
lysosomes pre-loaded with the first pulse and M6PR
positive/lgp positive late endosomes containing the second
pulse. The resultant hybrid compartments were the site of flocculation of BSA-gold indicating that they contained proteases
(van Deurs et al., 1995). Direct fusion events between late
endosomes and lysosomes were indicated by the observation
of apparent fusion profiles, consistent with delivery of the
entire content of proteolytic enzymes of a dense core lysosome
to the newly formed hybrid structure. The high lumenal protein
concentration of a dense core lysosome is probably an
unfavourable environment for proteolytic activity. After fusion
with a late endosome, diffusion of lysosomal content into the
lumen of the hybrid organelle may well provide a more suitable
aqueous and acidic milieu for protease action.
A temperature block of 20°C inhibited the appearance of
BSA-gold in lysosomes. When cells containing pre-loaded
lysosomes accumulated a second pulse of BSA-gold at 20°C
and were then transferred to 37°C, mixing of gold occurred,
with flocculation of the second pulse after 30 minutes and the
presence of both sizes of gold in dense core lysosomes after
60 minutes. These data are compatible with results of previous
work using temperature blocks to prevent delivery of endocytosed markers to lysosomes (Dunn et al., 1980; Miller et al.,
1986; Marsh et al., 1983; Felder et al., 1990; Futter et al.,
1996).
We have extended our previous observations on the effect of
the PI3-kinase inhibitor, wortmannin, on late endocytic compartments (Reaves et al., 1996). By quantitating the effect on
pre-loaded lysosomes we have confirmed that treating NRK
cells with 100 nM wortmannin for 1 hour has no measurable
effect on the morphology of dense core lysosomes, yet causes
the appearance of swollen late endosomal compartments. It is
not known which of several wortmannin-sensitive PI3-kinases
may be involved in controlling the morphology and function
of these compartments (Reaves et al., 1996). During subsequent incubation of the cells for 5 hours in the presence of
wortmannin, the swollen compartments received delivery of
flocculated BSA-5nm gold from pre-loaded dense core
lysosomes, consistent with direct fusion between swollen
endosomes and dense core lysosomes. A new finding was that
re-formation of dense core lysosomes, from the swollen hybrid
compartments formed after these fusions, was prevented by
wortmannin. Thus a further membrane traffic step which
requires, or is modulated by, PI3-kinase activity, but which is
not recycling of M6PR to the TGN (Nakajima and Pfeffer,
1997), may be added to previous lists (Shepherd et al., 1996).
Contrary to the interpretation of data in some previous
reports (Cohn and Ehrenreich, 1969; Deng and Storrie, 1988;
DeCourcy and Storrie, 1991; Montgomery et al., 1991; Jahraus
et al., 1994), we have determined that sucrosomes, induced by
accumulation of indigestible sucrose, are late endosomes and
hybrid endosome-lysosome structures, as determined by
M6PR immunoreactivity, but not terminal lysosomes. After
prolonged sucrose uptake, fusions between the sucrosomes and
dense core lysosomes resulted in the appearance of pre-loaded
BSA-gold in sucrosome-lysosome hybrids and the depletion of
dense core lysosomes. Direct contact and apparent fusions
were occasionally seen in the electron microscope. In previous
experiments, Deng and Storrie (1988) demonstrated that
species-specific lysosomal membrane proteins from one cell
type could be transferred to sucrosomes of another in a fused,
2038 N. A. Bright and others
heterotypic, hybrid cell system. Although sucrosomes were
considered to be mature lysosomes in this system, these data
nevertheless provide further evidence for membrane traffic
between lysosomes and sucrosomes.
Uptake of sucrose, interposed between two pulses of BSAgold, prevents the accumulation of the second pulse in dense
core lysosomes, presumably by preventing membrane retrieval
and associated regeneration of dense core lysosomes. Cohn and
Ehrenreich (1969) and Swanson et al. (1986) demonstrated that
sucrosomes collapse down to phase-dense organelles after
treatment with invertase. In the present study we have
confirmed these observations and shown that sucrosomes
which had acquired the pre-loaded gold content of dense core
lysosomes over an extended period were capable of re-forming
dense core lysosomes after endocytosis of invertase. Jahraus et
al. (1994) concluded that there was retrograde traffic from
lysosomes to the late endosome. They demonstrated the
efficient disappearance of pre-formed sucrosomes after endocytosis and immobilisation of invertase-conjugated latex beads
in late endosomes. However, it is likely that late endosome-late
endosome fusions account for the disappearance of sucrosomes
in their system. In the present study we observed numerous
apparent fusion events between sucrosomes (see also Cohn and
Ehrenreich, 1969). Further evidence for homotypic late
endosome-late endosome fusions has been provided by Deng
et al. (1991) in fused heterotypic, hybrid cells. Our data are
consistent with the observations of Swanson et al. (1986) and
Montgomery et al. (1991) who demonstrated that sucrosomes
exhibit reduced fusions with earlier endocytic organelles. After
sucrose uptake we observed the accumulation of a subsequent
pulse of BSA-gold in ECVs, the carrier vesicles responsible
for transfer from early to late endosomes (Gruenberg et al.,
1989; Aniento et al., 1993).
Our data suggest that proteolytic digestion in NRK cells
commences in the endocytic pathway after fusion of a late
endosome with a dense core lysosome. Some of the products
of digestion will be transported across the membrane of the
hybrid organelle into the cytosol. Membrane and contents will
be selectively retrieved and re-cycled from the hybrid organelle
to sites such as the TGN (eg. for M6PR) and possibly earlier
endocytic compartments resulting in the re-formation of the
dense core lysosome. This process would account for the
diverse range of morphology and immunoreactivity encountered in these organelles. Membrane retrieval and re-formation
of lysosomes from the hybrid organelles would require adaptor
molecules and/or coat proteins to select and pinch off the
membrane destined for re-cycling. There is accumulating
evidence that such coat proteins do exist on endosomes
(Whitney et al., 1995; Aniento et al., 1996; Stoorvogel et al.,
1996), and lysosomes (Traub et al., 1996). The plasma
membrane adaptor molecule α-adaptin has also been demonstrated on endosomes in the presence of GTPγS (Seaman et al.,
1993), and on lysosomes after a purified fraction has been
incubated with cytosol in the presence of ATP (Traub et al.,
1996).
A model outlining the fusion of dense core lysosomes with
late endosomes and showing the re-formation of re-usable
dense core lysosomes from the resultant hybrid organelles is
shown in Fig. 11. The concept of direct fusion of lysosomes
with other organelles, particularly during autophagy, is not new
(see for example De Duve, 1963; Lawrence and Brown, 1992).
ECV
M6PR-, lgp-,
protease-
Fusion
Late endosome
M6PR+, lgp+,
protease-
Fusion
Re-usable
Endosome / lysosome
hybrid
M6PR+, lgp+,
protease+
Membrane retrieval
Blocked by 100 nM
wortmannin and
sucrose accumulation
Re-formation
Dense core lysosome
M6PR-, lgp+,
protease+
Fig. 11. Fusion of dense core lysosomes with late endosomes and
subsequent re-formation. The data presented in this study support a
model whereby ECVs formed from early endosomes fuse with, and
deliver their content to, pre-existing late endosomes. Acid hydrolaserich electron-dense lysosomes fuse with late endosomes to generate
hybrid organelles in which digestion of internalised material occurs.
Dense core lysosomes are re-formed after selective recovery of
membrane. The lysosome may then be re-used in subsequent cycles
of fusion, digestion and re-formation. + indicates presence; −
indicates depletion but not necessarily exclusion.
Recently there has been much interest in the concept of the
secretory lysosome in haemopoietic cells, where secretory
granules with many of the properties classically associated
with lysosomes undergo a triggered fusion with the plasma
membrane (G. M. Griffiths, 1996). Considering the dense core
lysosome as a re-formable, re-usable storage granule that constitutively fuses with and secretes into a late endosome is thus
not without precedent (see for example De Duve and Wattiaux,
1966; Hales, 1978). The suggestion that lysosomes may be
storage organelles for acid hydrolases was also made by Tjelle
et al. (1996), who demonstrated that the main proteolytic
degradation of endocytosed proteins in a macrophage cell line
Endosome-lysosome fusion 2039
took place in late endosomes despite the observation that
lysosomes contained the bulk of lysosomal enzymes. Our data
are consistent with the proposal by G. Griffiths (1996) that
lysosomes are mostly storage vesicles for mature lysosomal
enzymes which are injected by fusion into late endosomal compartments which may be regarded as collectively representing
the ‘cell stomach’. The observation of direct fusion between
dense core lysosomes and late endosomes begs the questions
of the molecular mechanisms of recognition and fusion. It is
possible that the recognition and docking process involves the
filamentous structures observed between endosomes and
lysosomes in the present study (Fig. 7b) and previously
observed between MVBs in HEp-2 cells (Futter et al., 1996).
We are currently investigating the role of cytosolic factors in
direct fusion between dense core lysosomes and late
endosomes in the previously published cell-free system derived
from rat liver hepatocytes (Mullock et al., 1994). Our unpublished data suggests that such fusion requires NSF and αSNAP,
as is the case for many other fusion events found on membrane
traffic pathways (Rothman, 1994).
This work was funded by the Medical Research Council. We thank
Howard Davidson, Rainer Duden, Nick Hales, John Hutton and
Margaret Robinson for much valuable discussion.
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(Received 14 February 1997 - Accepted 23 June 1997)