Département de pathologie et biologie cellulaire, Université de Montréal, CP 6128, Succ. Centre ville, Montréal, QC, H3C 3J7, Canada
* Author for correspondence (e-mail: michel.desjardins{at}umontreal.ca)
Accepted 6 November 2002
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Summary |
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Key words: Endosomes, Lysosomes, Membrane fusion, Kiss and run
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Introduction |
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Lysosome and phagolysosome biogenesis are considered to occur by different
mechanisms. However, more and more evidences indicate that these two pathways
share common features. So far, most, if not all, of the proteins present on
endosomes have also been found on phagosomes
(Garin et al., 2001). Among
these markers, rab5, a member of the rab family of small GTPases present on
early endocytic and phagocytic structures has been particularly studied
(Chavrier et al., 1990
;
Gorvel et al., 1991
;
Bucci et al., 1992
;
Desjardins et al., 1994
). Its
apparent absence from late endosomes, where rab7 is observed, was instrumental
to the proposal that early and late endosomes are pre-existing organelles
displaying specific sets of proteins
(Griffiths and Gruenberg,
1991
). However, recent evidence clearly indicates that rab5 is
also associated to late endosomes in some cell types
(Jahraus et al., 1998
) and
that several rabs can be present on the same endosomes
(Sönnichsen et al.,
2000
). In a previous study, we used a RAW264.7 macrophage cell
line expressing a GTPase-defective rab5(Q79L) mutant
(Duclos et al., 2000
) to
demonstrate that phagolysosome biogenesis occurs through `kiss and run'
interactions (Desjardins,
1995
), regulated in part by rab5. In the present study, we used
the rab5 mutant cell line to determine to what extent the processes of
phagolysosome and lysosome biogenesis are related.
Our results demonstrate that endosome maturation occurs during the early stages of lysosome biogenesis. Rab5 regulates parts of this process by allowing transient interactions of a `kiss and run' nature between endosomes. Together, our results indicate that lysosome biogenesis occurs by mechanisms very similar to those involved in the maturation of phagosomes during phagolysosome biogenesis.
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Materials and Methods |
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Morphology of endosomes by electron microscopy
To determine the effect of rab5(Q79L) on endosome morphology, 10 mg/ml HRP
(Sigma) was internalized in transfected and control cells for 30 minutes.
Cells were then fixed in 1% glutaraldehyde. HRP was revealed by a DAB (Sigma,
St Louis, MO) reaction as described previously
(Desjardins et al., 1994).
Cells were post-fixed in 1% OsO4, dehydrated in alcohol, processed
for flat embedding in Epon 812 and observed at the Zeiss CEM 902 electron
microscope as described previously
(Desjardins et al., 1994
).
Time course of large endosomes formation
To determine the nature of the large endosomes, 1.5 mg/ml BSA-rhodamine
(kindly provided by Lucian Ghitescu, Université de Montréal,
Montréal) in DMEM was internalized for 2 minutes at 37°C. Cells
were then washed twice in cold PBS for 3 minutes with mild agitation.
BSA-rhodamine was chased for the indicated time points by incubating cells in
DMEM at 37°C. Cells were then rapidly observed at the epifluorescence
microscope without fixation.
To further analyze the nature of the large endosomes, 16 nm BSA-gold
particles were internalized for 30 minutes without a chase to form early and
late endosomes, or with a 16 hours-chase in order to empty early and late
endosomes, and fill lysosomes. Cells were then fixed and processed for
observation at the electron microscope as described
(Desjardins et al., 1994).
Effect of bafilomycin A1 on endosome morphology
To characterize the effect of bafilomycin A1 on various endosome
populations in wild-type RAW264.7 macrophages, 16 nm BSA-gold particles were
internalized in macrophages for 15 minutes, followed by 0 minutes, 60 minutes
or 16 hours of chase time in DMEM to fill endosomes at various stages of their
maturation. Cells were then treated with bafilomycin A1 (Kamiya Biomedical
Company, Tukwila, WA) in DMSO or with DMSO only for 30 minutes and prepared
for electron microscopy. To characterize the effect of bafilomycin A1 on
endosomes from rab5(Q79L)-expressing cells, late endosomes were formed by the
internalization of 35 nm BSA-gold particles for 15 minutes, followed by a 4
hour chase in DMEM. In the same cells, early endosomes were then formed by
internalization of 5 nm BSA-gold particles for 15 minutes. Cells were then
treated with bafilomycin A1 or with DMSO for 30 minutes, and prepared for
electron microscopy.
Antibodies and immunofluorescence microscopy
To better confirm the nature of the large endosomes, immunofluorescence
analysis was performed with the following primary antibodies: the mAb 4F11
raised against a peptide from the C-terminal region of rab5 (a generous gift
of Angela Wandinger-Ness, National Center for Genome Resources, Santa Fe, New
Mexico); a rabbit polyclonal antibody directed against the C-terminal region
of rabaptin-5 (a kind gift of Marino Zerial, Max Planck Institute for
Molecular Cell Biology and Genetics)
(Stenmark et al., 1995); an
affinity-purified human autoantibody to EEA1
(Mu et al., 1995
); a rabbit
polyclonal antibody raised against rab7 (a kind gift of Stéphane
Méresse Centre d'Immunologie de Marseille-Luminy, Marseille);
monoclonal rat anti-LAMP1 and anti-LAMP2 (Developmental Studies Hybridoma
Bank, Department of Pharmacology and Molecular Sciences, Johns Hopkins
University School of Medicine, Baltimore, MD, and the Department of Biological
Sciences, University of Iowa, Iowa City, IA, under contract N01-HD-6-2915 from
the NICHD); a rabbit polyclonal antibody raised against flotillin1 (a kind
gift of Robert Parton, Centre for Microscopy and Microanalysis, Department of
Physiology and Pharmacology, and Institute of Molecular Bioscience, University
of Queensland, Brisbane). For all immunofluorescence experiments, cells were
grown on 18 mm round coverslips. Fixation was performed either in 4%
paraformaldehyde followed by 0.2% Triton X-100 permeabilization at room
temperature, or in 80% methanol/20% acetone for 20 minutes at -20°C. After
washes in PBS, cells were incubated in a blocking solution made of 2% BSA and
0.2% gelatin in PBS. Incubation with the primary antibodies was done for 1
hour at room temperature. After washes in PBS/1% BSA, cells were incubated
with the appropriate secondary antibodies [Texas-Red-conjugated anti-mouse
IgG, Texas-Red-conjugated anti-rabbit (BIO/CAN Scientific, Mississauga, ON,
Canada), ALEXA-conjugated anti-human IgG or ALEXA-conjugated anti-rat IgG
(Molecular Probes, Eugene, OR)] for 30 minutes in the dark, at room
temperature. Coverslips were mounted on Gelvatol (Air Products &
Chemicals, Allentown, PA) and observed with a Zeiss inverted epifluorescence
microscope, or with a Leica confocal fluorescence microscope.
Dextran segregation
To observe segregation of fluid phase tracers, macrophages on coverslips
were incubated for 30 minutes with mixtures of tetramethylrhodamine dextran,
average molecular weight 70,000 (TRDx70) plus fluorescein dextran, average
molecular weight 10,000 or 70,000 (FDx10 or FDx70), each at 5 mg/ml in DMEM.
After extensive washes in PBS, the tracers were chased for the indicated times
(0, 30, 120 or 240 minutes). Coverslips were mounted on Gelvatol and observed
at the Leica confocal fluorescence microscope.
Size-selective transfer of particles at the electron microscope
level
To further characterize the size-selective transfer of particles,
macrophages were incubated for either 15 or 30 minutes to allow the
endocytosis of a mixture of 100 nm BSA-coated latex beads and 16 nm BSA-gold
particles. After extensive washes in PBS, the tracers were chased for 0, 30,
or 120 minutes. Cells were then fixed and processed for observation at the
electron microscope as described
(Desjardins et al., 1994).
Coating of latex beads with BSA was done by incubating the beads with 5 mg/ml
BSA, as previously described (Duclos et
al., 2000
), with the difference that washes in this case were
performed in distilled water at 48,300 g.
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Results |
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Giant endosomes display early and late endocytic features
To determine the nature of the giant endosomes present in
rab5(Q79L)-expressing macrophages, we characterized the kinetic of their
formation and some of their biochemical properties. First, we followed the
distribution of a fluorescent fluid tracer at various time points after its
internalization. This was done by internalizing BSA-rhodamine for 2 minutes
followed or not with chase incubations of increasing periods of time. The
rapid screening of these cells without fixation allowed us to observe that
normal macrophages display the rhodamine signal in small vesicles throughout
the cytoplasm at all the time points studied
(Fig. 2). In contrast, in
addition to the small vesicles, large endosomes filled with BSA-rhodamine were
present in rab5(Q79L)-expressing cells after the initial 2 minutes of
internalization (not shown), a time expected to fill early endosomes. Large
labeled vacuoles were still present after 10 minutes and even 30 minutes of
chase of the BSA-rhodamine marker (Fig.
2), a period of time largely sufficient to empty early endosomes
and load late endocytic organelles. However, at 60 minutes after
internalization, a chase period normally used to load lysosomes, the rhodamine
signal was now observed in smaller structures, possibly lysosomes
(Fig. 2), while large empty
vacuoles were visible in the rab5(Q79L)-expressing cells
(Fig. 2, inset). These results
indicate that the large endosomes formed in rab5(Q79L)-expressing cells
kinetically correspond to both early and late endosomes.
|
Giant endosomes are not lysosomes
In order to determine whether rab5(Q79L) also affects the morphology of
lysosomes, we internalized 16 nm BSA-gold particles and incubated cells for
long periods of time. After 30 minutes of internalization, the gold particles
were present in giant structures. BSA-gold was not observed in the numerous
small dense vesicles present in these cells
(Fig. 3A). In contrast, a 16
hour chase led to the accumulation of the gold particles in small dense
vesicles, most likely representing lysosomes, while the giant vacuoles still
present were devoid of the gold tracer
(Fig. 3B). These results
confirm that alteration of rab5 induces morphological changes to what can be
described as an early-endosomeslate-endosomes continuum, without
affecting the formation and morphology of lysosomes.
|
Giant endosomes gradually acquire resistance to bafilomycin
A1-induced fragmentation
Previous studies indicated that early endosomes could be fragmented by
treating cells with the proton pump inhibitor bafilomycin A1
(Clague et al., 1994;
D'Arrigo et al., 1997
). This
observation prompted us to determine whether the large endosomes in
rab5(Q79L)-expressing cells could be fragmented by this drug. By preloading
cells with BSA-gold for different periods of time to fill early endosomes,
late endosomes and lysosomes, we were able to show that, in control
macrophages, treatment with bafilomycin A1 resulted in the fragmentation of
early endosomes, but had no noticeable effect on the morphology of late
endosomes and lysosomes (Fig.
4). Based on these criteria, we performed similar experiments in
rab5(Q79L)-expressing cells preloaded with BSA-gold. Observation at the
electron microscope clearly showed that newly formed early endosomes could be
fragmented with bafilomycin A1 treatment while the late endosomes were
insensitive to the drug (Fig.
5). Indeed, in cells preloaded sequentially with 16 nm and 5 nm
BSA-gold to fill late endosomes with the large gold particles and early
endosomes with the small gold particles, only the early structures were
fragmented (Fig. 5). These
results indicate that while giant early endosomes can be fragmented by
bafilomycin A1, the enlarged structures are remodeled in a way that eventually
confers resistance to the fragmentation effect of this drug.
|
|
Giant endosomes display both early and late endocytic markers
The nature of the large endosomes was further studied by localizing a
series of well-known endocytic markers by immunofluorescence microscopy. These
included: the early endosome-associated molecules EEA1 and rabaptin-5; the
late endosome marker rab7, as well as flotillin1, a protein recently shown to
accumulate on subdomains of maturing phagosomes
(Dermine et al., 2001); and the
late endosome/lysosome markers LAMP1 and LAMP2
(Fig. 6). In control cells,
where large vacuoles are not usually observed, all these markers were detected
on small vesicles distributed throughout the cytoplasm (not shown). In
rab5(Q79L)-expressing cells, in addition to small vesicles, the large
endosomes were also labeled for all of these markers
(Fig. 6), confirming the
kinetic data (Fig. 2) showing
that the enlarged structures correspond to both early and late endosomes. This
point was also demonstrated by the observation of EGFP-rab5(Q79L) fusion
proteins on large endosomes displaying the late endocytic marker LAMP1
(Fig. 6). Of further interest,
while the labeling for LAMP1 and LAMP2 was evenly distributed on the membrane
of the giant organelles, all the other markers, including flotillin1,
displayed punctate patterns of labeling, which suggests that they are
associated with subdomains of the endosome membrane.
|
Rab5 regulates the `kiss and run' fusion between endosomes
In macrophages, the GTPase activity of rab5 regulates the transient fusion
events (`kiss and run') between phagosomes and endosomes required for
phagolysosome biogenesis (Duclos et al.,
2000). This was demonstrated by the loss of size-selective
transfer of particles between these compartments upon the expression of a
GTPase-deficient form of rab5. To investigate whether rab5-regulated transient
fusion events also occur along the endocytic pathway during lysosome
biogenesis, we followed the fate of fluorescent dextran molecules of different
sizes [average mol wt of 10,000 (green) and 70,000 (red)] after their
co-internalization in macrophages. In control cells, the two dextran
populations were found in the same endocytic organelles (labeled in yellow)
after 30 minutes of internalization (Fig.
7). These molecules were rapidly sorted, within 30 minutes of
chase, to distinct endosomes displaying red or green labeling, and then
observed in segregated compartments for the entire duration of the experiment
(up to 240 minutes of chase) (Fig.
7). In contrast, the small and large dextran molecules remained
co-localized for as long as 150 minutes after endocytosis in
rab5(Q79L)-expressing cells, their separation to distinct endocytic organelles
only starting after a 240 minute chase
(Fig. 7). In control cells at
240 minutes, the size segregation of the two dextran molecules did not appear
as extensive as for the 30'/120' time point, which indicates that
some remixing between late endocytic structures could occur, as suggested
previously (Luzio et al.,
2000
). Together, these results indicate that the GTPase activity
of rab5 regulates, to some extent, the transient nature (`kiss and run') of
the fusion events occurring between endosomes in the early part of the process
of lysosome biogenesis.
|
These results were further confirmed at the electron microscope by internalizing 16 nm BSA-gold particles and 100 nm BSA-coated latex beads. At the earliest time point observed (15 minutes of internalization), 63% and 61% of endosomes contained particles of both sizes in control and rab5(Q79L)-expressing cells, respectively (Fig. 8). In control cells, the particles started to segregate into different endosomal compartments 60 minutes after their internalization (Fig. 8), and were still observed in separated endosomes 150 minutes after endocytosis (Fig. 8). In contrast, as described for the dextran molecules by fluorescence, the two sizes of particles remained co-localized for as long as 150 minutes after endocytosis in rab5(Q79L)-expressing cells (Fig. 8), which demonstrates the size-selective nature of the fusion events occurring along the endocytic pathway and is expected from `kiss and run'-type interactions.
|
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Discussion |
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Do endosomes mature?
Rab5 was originally described as an early endosome marker
(Chavrier et al., 1990). Thus,
it was not surprising that mutations affecting that molecule would alter the
properties of early endosomes. For example, the expression of a
GTPase-defective mutant of rab5 in epithelial cells was shown to induce the
formation of giant endocytic structures identified as early endosomes
(Stenmark et al., 1994
). Late
endosomes and lysosomes were not altered in these cells, supporting the
concept of pre-existing organelles (mutation to a protein residing in early
endosomes would affect mainly, if not only, this compartment). However, in our
study, expression of a GTPase-deficient mutant of rab5 in RAW264.7 macrophages
brought an interesting observation. Our results clearly indicate that the
giant endocytic structures present in these cells display features of both
early and late endosomes. This was demonstrated by kinetic studies showing
that a tracer internalized by fluid phase endocytosis remained in giant
endosomes for long periods of time, largely sufficient to load late endocytic
organelles. In addition, the giant endosomes were shown to display archetypal
markers of early endosomes such as rab5 and its effectors rabaptin5 and EEA1
(Chavrier et al., 1990
;
Stenmark et al., 1995
;
Mu et al., 1995
), as well as
several of the markers known to associate with late endosomes. These include
the well-studied rab7 (Chavrier et al.,
1990
; Zerial and Stenmark,
1993
; Méresse et al.,
1995
), LAMP1 and LAMP2
(Griffiths et al., 1988
;
Kornfeld and Mellman, 1989
;
Rabinowitz et al., 1992
), and
flotillin1, a membrane-associated molecule recently shown to accumulate on
maturing phagosomes (Dermine et al.,
2001
). Moreover, we showed that several of the large endosomes
display early (rab5) and late (LAMP1) endocytic markers simultaneously, in
agreement with a recent report (Rosenfeld
et al., 2001
). Partial co-localization of these markers was also
observed in control cells. However, because of the small size of the endocytic
structures in these cells, it was difficult to determine whether both markers
were effectively present on the same endosomes. Thus, our results suggest that
rather than being composed of distinct early and late endosomes, macrophages
possess a heterogeneous population of endocytic organelles displaying mixed
features. This supports previous results showing that, in J774 macrophages, in
addition to its association to early endosomes, rab5 is present on late
endocytic organelles, a feature proposed to be unique to this cell type
(Jahraus et al., 1998
).
Further observation indicated that the enlarged endocytic structures
present in macrophages expressing the Q79L mutant of rab5 sequentially
displayed characteristics of both early and late endosomes. Indeed, we showed
that treatment of cells with bafilomycin A1, a vacuolar proton pump inhibitor,
induced the fragmentation of early endosomes in normal macrophages, as
previously described in other cell lines
(Clague et al., 1994;
D'Arrigo et al., 1997
), with no
noticeable effect on the size or overall morphology of late endosomes and
lysosomes. By internalizing BSA-gold of two different sizes for various
periods of time, we were able to fill the early and late endosomes of a given
cell with different tracers. In Q79L mutant cells treated with bafilomycin,
the enlarged endosomes containing the early endocytic tracer were fragmented,
whereas the morphology of enlarged endosomes containing the late endocytic
tracer was not altered. These results confirmed that the enlarged endocytic
structures induced by the Q79L mutation are dynamically modified and acquire
with time the property of late endocytic structures to resist the fragmenting
effect of bafilomycin. Together, these results indicate that endosomes are
gradually modified during a process allowing their acquisition of late
endocytic markers and features (resistance to bafilomycin fragmentation).
These series of events are reminiscent of the maturation process taking place
during phagolysosome biogenesis, where a sequential acquisition of rab GTPases
has been observed (Desjardins et al.,
1994
), and where `kiss and run' fusion events regulated by rab5
have been shown to occur (Duclos et al.,
2000
).
As shown both at the light and electron microscope, albeit that the
expression of rab5Q79L had a profound effect on the morphology of early-late
endosomes, the mutation did not affect the transfer of the endocytic tracers
to small dense lysosomes. These results suggest that although rab5 is an
important regulator of fusion along the early part of lysosome biogenesis, it
is unlikely to be involved in the late endosomes to lysosomes traffic. At this
point, it is still unclear how the terminal formation of the lysosomal
compartment occurs. It could happen by fragmentation of the late endosomes or,
as proposed by Luzio et al. (Luzio et al.,
2000), by the formation of a hybrid organelle between late
endosomes and lysosomes, followed by condensation of the content of this
organelle as well as selective recycling of membrane constituents.
The membrane of endosomes displays microdomains
The enlarged endosomes formed in rab5(Q79L)-expressing cells increased our
ability to observe the pattern of distribution of endocytic markers at the
surface of endosomes. We found that several of these markers, including
flotillin1, displayed a punctate pattern on endosomes. Interestingly,
flotillin 1, a marker also displaying a punctate distribution on phagosomes,
has recently been shown to accumulate in lipid rafts domains on the membrane
of these organelles (Dermine et al.,
2001). There is now increasing evidence suggesting the existence
of specialized microdomains on the membrane of endocytic organelles (for
reviews, see Gruenberg, 2001
;
Miaczynska and Zerial, 2002
).
For example, early endosomes and recycling endosomes have been shown to be
enriched in lipid rafts (Mukherjee et al.,
1998
; Gagescu et al.,
2000
). Several reports have indicated the presence of members of
the docking and fusion machinery, such as SNAREs, on specialized lipid
microdomains (Schnitzer et al.,
1995
; Galli et al.,
1996
; Lafont et al.,
1999
). Others have reported the co-localization of rab5 and one of
its effectors (EEA1) on microdomains at the surface of endosomes, although not
formally identified as lipid rafts
(McBride et al., 1999
). The
punctate distribution of EEA1 has also been observed on the membrane of
phagosomes (S.D. and M.D., unpublished). In living cells, rab5 was shown to
accumulate on microdomains representing `hot spots' for fusion on endosomes
(Roberts et al., 1999
).
Furthermore, rab4, rab5 and rab11 have recently been shown to define distinct
membrane domains on endosomes in the recycling pathway
(Sönnichsen et al.,
2000
). Unraveling the exact function of these microdomains on
endocytic organelles will provide a better understanding of lysosome
biogenesis.
`Kiss and run' and lysosome biogenesis
We proposed that because most of the molecules found in endosomes are also
present in phagosomes, the two pathways share common mechanistic features
(Storrie and Desjardins,
1996). For example, all the small GTPases of the rab family
identified on endocytic structures have been found on phagosomes
(Garin et al., 2001
). One of
these molecules, rab5, was shown to promote `kiss and run' interactions
between endosomes and phagosomes during phagolysosome biogenesis, through its
GTPase activity (Duclos et al.,
2000
). We show here that rab5 plays a similar role by allowing
transient fusion events between endocytic organelles during the early parts of
lysosome biogenesis. As a consequence, endocytic tracers of various sizes,
co-internalized in macrophages, are rapidly segregated in distinct populations
of endosomes. This size-selective transfer most probably reflects the
occurrence of fusion events between endosomes containing the small and large
tracers with endosomes devoid of tracer, as proposed previously
(Berthiaume et al., 1995
).
Indeed, in rab5 Q79L-expressing cells, the ability to promote `kiss and run'
fusion is lost and endosomes fuse in ways allowing the constant mixing of
tracers. With time, endosomes are remodeled to a point where rab5 is either no
longer present or functional, allowing `kiss and run' fusion events to resume
and the efficient sorting of tracers to lysosomes to occur.
Based on our results, and by analogy with phagolysosome biogenesis, we
propose a model in which endocytic organelles are part of a continuum of
vesicles dynamically remodeled. This remodeling, regulated by protein
complexes such as rab5 and its effectors, as well as members of the SNARE
family of proteins, involves series of transient interactions of a `kiss and
run' nature. These interactions could take place at specialized membrane
domains, as suggested by the punctate distribution of a number of proteins
involved in membrane fusion at the surface of endocytic organelles. In this
model, the GTPase activity of rab5 would act as a timer
(Rybin et al., 1996),
regulating the fusion/fission process between endosomes. Rab5-driven transient
interactions would allow the maintenance of the size and integrity of
endocytic organelles in the early parts of the pathway. Small GTPases other
than rab5 could be involved in the subsequent steps of lysosome biogenesis
(Mullock et al., 1998
). A
potential candidate being rab7, a protein present on late endosomes
(Feng et al., 1995
;
Méresse et al., 1995
;
Papini et al., 1997
;
Vitelli et al., 1997
) recently
shown to be essential for the maintenance of the perinuclear lysosome
compartment (Bucci et al.,
2000
). In the near future, proteomics and lipidomics studies of
isolated endosomes and phagosomes will probably contribute to uncover all the
molecules and their interactions involved in the pathways of lysosome and
phagolysosome biogenesis.
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Acknowledgments |
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