3531
Journal of Cell Science 113, 3531-3541 (2000)
Printed in Great Britain © The Company of Biologists Limited 2000
JCS1535
Rab5 regulates the kiss and run fusion between phagosomes and endosomes
and the acquisition of phagosome leishmanicidal properties in RAW 264.7
macrophages
Sophie Duclos1, Roberto Diez1, Jérome Garin2, Barbara Papadopoulou3, Albert Descoteaux4,
Harald Stenmark5 and Michel Desjardins1,*
1Département
de pathologie et biologie cellulaire, Université de Montréal, C.P. 6128, Succ. Centre ville, Montréal, QC, Canada,
H3C 3J7
2Laboratoire de Chimie des protéines, CEA, 38054 Grenoble, France
3Centre de Recherche en Infectiologie, CHUQ, Pavillon CHUL, Ste-Foy, QC, Canada G1V 4G2
4INRS-Institut Armand-Frappier, Université du Québec, Laval, QC, Canada, H7V 1B7
5Department of Biochemistry, The Norwegian Radium Hospital, Montebello, N-0310 Oslo, Norway
*Author for correspondence (e-mail: michel.desjardins@umontreal.ca)
Accepted 20 July; published on WWW 13 September 2000
SUMMARY
Phagolysosome biogenesis is essential for the killing and
degradation of intracellular pathogens. It involves the
fusion of phagosomes with various endocytic organelles, a
process known to be regulated in part by Rab proteins. We
generated RAW 264.7 macrophages expressing an active
mutant of Rab5 (Rab5(Q79L)) to determine the role of
Rab5 in phagocytosis and phagolysosome biogenesis. Our
results indicate that Rab5 stimulates phagocytosis of latex
beads but not Fc or C3 receptor-mediated phagocytosis.
Rab5 also acts to restrict the complete fusion of
phagosomes with endosomes, a phenomenon allowing
exchange of solutes from the two compartments without
complete intermixing of their membrane (kiss and run). In
Rab5(Q79L)-expressing macrophages, uncontrolled fusion
events occurred, leading to the appearance of giant
phagosomes. These phagosomes could initiate their
maturation and acquire LAMP1, but failed to generate
the microbicidal conditions needed to kill intracellular
parasites. These results identify Rab5 as a key molecule
regulating phagosome-endosome fusion and as an essential
component in the innate ability of macrophages to restrict
the growth of intracellular parasites.
INTRODUCTION
(Kornfeld and Mellman, 1989; Claus et al., 1998). Thus, the
fusion of phagosomes with these organelles is fundamental for
phagocytic cells like macrophages to control the spread of
infection. Indeed, several microorganisms have evolved
mechanisms to inhibit phagosome-lysosome fusion and
survive within their host cells (for reviews see Finlay and
Falkow, 1997; Sinai and Joiner, 1997; Aderem and Underhill,
1999; Méresse et al., 1999).
Phagolysosome biogenesis was first believed to involve the
complete fusion of phagosomes with lysosomes (see
Rabinowitz et al., 1992). However, recent studies revealed that
phagosomes engage in a regulated series of fusion events, first
with early and late endosomes, which are necessary for the
subsequent fusion with lysosomes to proceed (Pitt et al., 1992;
Desjardins et al., 1994a, 1997; Jahraus et al., 1994; Via et al.,
1997). Despite this dynamic fusion activity, phagosomes
and endosomes maintain a relatively stable size over time
(Desjardins et al., 1994a). Based on some of these
observations, we proposed that phagosomes fuse with
endocytic organelles through repeated transient fusion events,
Intracellular pathogens internalized by phagocytosis are
sequestered in compartments originating from the plasma
membrane, the phagosomes. Newly formed phagosomes are
unable to kill and degrade their content and must therefore
engage in a complex process of maturation referred to as
phagolysosome biogenesis (Berón et al., 1995; Desjardins,
1995). This process involves the binding to and movement
along microtubules (Blocker et al., 1997, 1998) and the
multiple steps required for recognition and fusion with
endocytic organelles (Desjardins et al., 1997; Jahraus et al.,
1998). During these interactions, phagosomes acquire several
of the molecules required for their microbicidal activity. These
include proton pump ATPases involved in phagosome
acidification (Mellman, 1992) and a variety of hydrolases
responsible for the digestion of phagosome content (Claus et
al., 1998). Although hydrolases are present in various
populations of endocytic organelles, the cellular bulk of these
molecules are stored within late endosomes and lysosomes
Key words: Rab5, Phagosome, Kiss and run, Leishmania, Membrane
fusion
3532 S. Duclos and others
a process referred to as the ‘kiss and run’ hypothesis
(Desjardins, 1995; Storrie and Desjardins, 1996). This process
has several advantages. It allows organelles to exchange
contents without the complete mixing of their membranes,
limiting the need for large scale recycling processes. Limited
exchange of membrane molecules has the further potential to
allow the gradual transformation of organelles observed along
the endocytic and phagocytic pathways. Evidence for ‘kiss and
run’ exchanges has been obtained from various biological
systems including phagosome-endosome interaction (Wang
and Goren, 1987; Desjardins et al., 1994a, 1997), endosomeendosome interaction (Berthiaume et al., 1995), and exocytosis
of neurotransmitters (Alvarez de Toledo et al., 1993; Albillos
et al., 1997). In the later case, the fusion pore formed between
mast cell granules and the plasma membrane could stay open
for several seconds, allowing the complete transmitter release
without full fusion of the vesicle with the plasma membrane.
Transient fusions between Golgi tubules have also been
proposed to occur (Weidman, 1995). The molecular
mechanisms governing transient membrane interactions are
poorly understood.
Membrane fusion is the focus of intense investigation.
Intracellular membrane trafficking requires Rab GTPases and
SNARE proteins which appear to act in conjunction to allow
vesicle movement, docking, fusion and fission (McBride et al.,
1999). Both Rab and SNARE proteins are present on
phagosomes. While the SNARE proteins syntaxins and
synaptobrevins have been identified on phagosomes at all
stages of their maturation (Hackham et al., 1996, 1998;
Desjardins et al., 1997), in some studies Rab5 and Rab7 have
been shown to associate transiently to maturing phagosomes
(Desjardins et al., 1994a,b; Via et al., 1997; Funato et al.,
1997). Rab5 and Rab7 have first been identified on early
endosomes and late endosomes, respectively (Chavrier et al.,
1990), and shown to be involved in endosome fusion (Gorvel
et al., 1991; Bucci et al., 1992; Feng et al., 1995; Méresse et
al., 1995; Vitelli et al., 1997). Seminal work has further allowed
us to characterize several of the Rab5 effectors, underlining the
complexity of membrane interaction among endovacuolar
organelles (see Novick and Zerial, 1997; Gonzalez and
Scheller, 1999). Despite the increasing body of data regarding
Rab5 interactions with its effectors, the role of Rab5 in the
interaction of phagocytic and endocytic organelles is not well
understood. Rab5 or some of its effectors have been shown
to regulate some aspects of phagosome-early endosome
interactions (Alvarez-Dominguez et al., 1996; Jahraus et al.,
1998; Alvarez-Dominguez and Stahl, 1999; Steele-Mortimer et
al., 1999). In the present study, we generated macrophage cell
lines expressing an active form of Rab5 (Rab5(Q79L)) to study
the role of this molecule in phagocytosis, phagosomeendosome interaction, and phagolysosome biogenesis. Our
results indicate that Rab5 activity controls the occurrence of
transient fusion events between phagosomes and endosomes.
The deregulation of Rab5 activity induces the formation of
latex beads- or Leishmania donovani-containing giant
phagosomes that are able to mature by acquiring hydrolases
and late endosome/lysosome membrane markers, but unable to
efficiently kill the intracellular parasite L. donovani. The
involvement of Rab5 in the acquisition of phagosomal
leishmanicidal properties identifies it as a key component in
the ability of macrophages to restrain the spread of infection.
MATERIALS AND METHODS
Cell culture and stable transfections
The murine macrophage cell line RAW 264.7 (American Type Culture
Collection, Rockville, MD) was cultured in DMEM, pH 7.4,
supplemented with 10% heat inactivated FBS (Life Technologies Inc.,
ON, Canada), 20 mM Hepes, pH 7.3-7.4, and antibiotics (100 U/ml
penicillin, 100 µg/ml streptomycin), at 37°C in 5% CO2. The myctagged Rab5a(Q79L) cDNA was cloned in the EcoRI site of the
expression vector pCIN4 (Rees et al., 1996). Stable transfections were
performed by electroporation essentially as described (Stacey et al.,
1993) except that 20 µg of linearized plasmid DNA were used for
the transfection. Clones were selected for their ability to grow in
500 µg/ml G418 (Calbiochem, San Diego, CA). Clones resistant to
the antibiotic were tested for the expression of myc Rab5(Q79L) by
RT-PCR using the following oligonucleotides primers: primer 1,
encoding the 5′ end of the c-myc epitope (5′GGAATTCGCCATGGAACAAAAACTC-3′) and primer 2, encoding the 3′ end of
Rab5 (5′GGAATTCTTAGTTACTACAACAC-3′), as well as by
immunofluorescence microscopy using the anti-myc epitope antibody
9E10 (Evan et al., 1985).
Parasites
Leishmania donovani promastigotes (Sudanese strain 1S) transfected
with the luciferase expression vector pGEM72f/anealuc (St-Denis et
al., 1999) were grown at 26°C in the presence of 50 µg/ml G418 in
RPMI 1640 with glutamine (Life Technologies Inc.) supplemented
with 20% heat inactivated FBS (Hyclone, Logan, UT), 10 mM
adenine, 0.0005% hemin in 50% triethanolamine, 1 µg/ml 6-biopterin,
0.0001% biotin in 95% ethanol, 20 mM MES, and antibiotics
(100 U/ml penicillin, 100 µg/ml streptomycin) at pH 5.5.
Promastigotes were grown to stationary or late stationary phases prior
to each experiment.
Antibodies and immunofluorescence microscopy
The primary antibodies used were: an affinity-purified rabbit antibody
to EEA1 (Simonsen et al., 1998), a monoclonal rat anti-LAMP1
(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), and a mouse monoclonal
antibody CA7AE directed against the repeating units of
lipophosphoglycan (LPG) (Tolson et al., 1989). 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, or in 80%
methanol/20% acetone for 20 minutes at –20°C. After washes in cold
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 IgM (BIO/CAN Scientific, Mississauga,
ON, Canada), ALEXA-conjugated anti-rabbit IgG or ALEXAconjugated anti-rat IgG (Molecular Probes, Eugene, OR)) for 30
minutes in the dark, at room temperature. Cells were mounted on
Gelvatol (Air Products & Chemicals, Allentown, PA) and observed at
the Zeiss inverted epifluorescence microscope.
Opsonization of latex beads and measurement of
phagocytic rates
The 0.8 µm latex beads (Sigma, St Louis, MO) were briefly sonicated
to disrupt aggregates, then diluted 1/10 in bidistilled water (ddH2O)
and washed by centrifugation 3 times 5 minutes at 12,000 g. For IgG
opsonization, beads were incubated for 2 hours at 37°C with 5 mg/ml
BSA in ddH2O under gentle agitation. They were then washed 3 times
Regulation of phagosome-endosome fusion by Rab5 3533
in BSS (124 mM NaCl, 5.8 mM KCl, 10 mM dextrose, 20 mM Hepes,
pH 7.4), and once in ddH2O. The beads were further incubated with
a mouse IgG antibody against BSA (Sigma) for 1 hour at 37°C,
followed by an overnight incubation at 4°C, with constant agitation.
Beads were then washed 3 times and resuspended in BSS.
Opsonization was controlled by incubation of the beads with an antimouse antibody coupled to the fluorochrome ALEXA (Molecular
Probes) for 5 minutes at room temperature and observation at the
Zeiss epifluorescence microscope. For complement coating, beads
were first washed in ddH2O. Mouse serum (Sigma) was centrifuged
in order to pellet contaminating cells. The serum was then diluted 1/5
in ddH2O and incubated 1:1 with the beads for 2 hours at 37°C,
followed by an overnight incubation at 4°C. Afterwards, beads were
washed several times and resuspended in BSS. Opsonization was
controlled with a mouse anti-C3 antibody coupled to the FITC
fluorochrome (ICN Biomedicals Inc., Aurora, OH).
To evaluate the level of phagocytosis of control and mutant cells,
0.8 µm naked, IgG-opsonized, or C3-opsonized latex beads diluted
1/100 in DMEM were internalized for 1 hour at 37°C, followed by a
chase of 1 hour. Naked latex beads of 3 µm were also internalized.
Cells were then processed for electron microscopy. The number of
beads per cell profile was then counted on thin section at the electron
microscope. A minimum of 50 cells per sample was evaluated. Each
experiment was repeated at least 3 times. To allow comparison
between each experiment, the values obtained for control cells were
arbitrarily set at 1. The index for the mutant cells was subsequently
adjusted by using the same factor of conversion.
Morphology of phagosomes by electron microscopy
To determine the effect of Rab5(Q79L) on phagosome morphology,
cells were fed with latex beads or infected with Leishmania parasites
(3×107/ml in DMEM) at a cell/parasite ratio of 1 to 10 for 1 hour at
37°C, washed in cold PBS and chased in DMEM for 1 hour. To
observe the formation of early phagosomes, Leishmania parasites
were internalized for 20 minutes without a chase. In some cases,
16 nm gold particles coated with BSA were internalized using
standard procedures (Rabinowitz et al., 1992) for 30 minutes in order
to load endosomes, or internalized prior to infection for 30 minutes
followed by an overnight chase to load lysosomes. Cells were fixed
in 2% glutaraldehyde, 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., 1994a).
Size selective transfer experiment (‘kiss and run’)
To determine whether phagosomes and endosomes engage in
complete or transient fusion events, and if Rab5 is involved in this
process, we adopted the following strategy. We used endocytic tracers
of three different sizes and evaluated if they were all transferred to
phagosomes at once (complete fusion) or if there was a size-selective
transfer indicative of narrow transient openings between the
phagosome and the endosome membrane. Cells were infected with L.
donovani (3×107/ml in DMEM) for 1 hour at 37°C, washed in cold
PBS and chased for 1 hour in DMEM at 37°C. BSA-gold particles of
5 and 35 nm were then internalized together for 1 hour at 37°C, and
washed in cold PBS. This was followed by the internalization of
100 nm latex beads (Sigma) for 1 hour. Cells were then embedded in
Epon 812 and observed at the electron microscope. Quantitation was
performed on 50 phagosomes in 3 independent experiments.
2-D gel electrophoresis and protein identification
Phagosomes were formed by the internalization of 0.8 µm latex beads
for 60 minutes in culture medium. Cells were then incubated for 60
minutes in culture medium with or without 500 nM bafilomycin A1
(Kamiya Biomedical Company, Tukwila, WA). Latex bead-containing
phagosomes were isolated from control, bafilomycin-treated cells or
Rab5(Q79L) cells as described previously (Desjardins et al., 1994a,
1997). Proteins were separated according to their isoelectric point and
molecular mass using 18 cm immobilized pH gradient (IPG) strips,
pH 3-10 (Amersham), for the first dimension following the
manufacturer procedures. The second dimension was performed on
12% SDS acrylamide gels with 10% sucrose allowing separation of
proteins between 130 kDa and 15 kDa approximately. At the end of
the migration, gels were fixed and silver stained following standard
procedures. The gel polypeptide patterns were then analyzed using the
package software MELANIE II (Bio-Rad, Glattbrugg, Switzerland;
Appel et al., 1997). For mass spectrometry analysis, gels were stained
with zinc acetate without fixation. The protein spots were excised with
the acrylamide and analyzed by MALDI-TOF-MS (matrix-assisted
laser-desorption ionization time-of-flight mass spectrometry) as
described previously (Rabilloud et al., 1998).
Characterization of endosomal markers on
Leishmania containing-phagosomes
Cells were infected with L. donovani parasites (3×107/ml in DMEM)
for 30 minutes at 37°C. Cells were then washed and further incubated
or not for 120 minutes to form early phagosomes (30′/0) or late
phagosomes (30′/120′). The presence of the early (EEA1) and late
(LAMP1) endosomal markers was assessed in different experiments
using the corresponding antibodies, while parasites were labeled with
the anti-LPG monoclonal antibody CA7AE. Quantification of the
number of Leishmania-containing phagosomes positive for the
different markers was done as follow. Cells were first examined in the
red channel to localize parasites. The selected field was then observed
in the green channel to determine if the parasites were present or not
in EEA1 or LAMP1 positive phagosomes. Quantification was
performed on 100 phagosomes for each time point and each marker
in 3 different sets of experiments.
Survival of L. donovani in RAW 264.7 macrophages
To determine the survival rate of L. donovani in macrophages,
adherent cells were infected with luciferase-expressing parasites in
DMEM for one hour at 37°C, at a ratio of ten parasites per
macrophage. Uningested Leishmania were removed by three washes
with cold PBS. Macrophages were then incubated for one hour in
complete medium containing either 500 nM bafilomycin A1 (Kamiya
Biomedical Compary) in DMSO or only DMSO. After few washes in
cold PBS, cells were further incubated in complete medium and
survival rates were determined after 1, 6, 24, 48 and 72 hours postinfection by measuring luciferase activity in cell extracts.
Luciferase activity was measured in Leishmania-infected cells
extracts using the Promega Luciferase Assay system as recommended
by the manufacturer (Promega Corp, Madison, WI). Briefly, host cells
were lysed in 100 µl of 1× Cell Culture Lysis Reagent. Then, 20 µl
of cells extracts were mixed with 100 µl Luciferase Assay Reagent at
room temperature, and light emission was quantified in a luminometer
(Berthold, Nashua, NH). Since only living parasites express the
luciferase gene, the light emission produced by the reaction of
luciferase with its substrate is proportional to the number of living
Leishmania.
RESULTS
Rab5 is a small GTPase that plays important roles in membrane
interactions and fusion (see Mohrmann and van der Sluijs,
1999; Mills et al., 1999). This protein, first localized to early
endocytic organelles (Chavrier et al., 1990), is also present on
phagosomes (Desjardins et al., 1994a; Via et al., 1997). Its
function during phagocytosis, in phagosome properties and in
phagolysosome biogenesis is poorly understood. In the present
study, we transfected the mouse macrophage cell line RAW
3534 S. Duclos and others
5
Control
Rab5(Q79L)
Phagocytic index
LB3 PB1
4
3
2
1
0
3 µm 0.8 µm
"naked"
0.8 µm 0.8 µm
IgG
C3
Fig. 1. Rab5(Q79L) increases phagocytosis of serum proteinsopsonized latex beads but not C3- or IgG-opsonized beads.
Macrophages expressing Rab5(Q79L) or expressing the vector alone
were allowed to internalize 3 µm or 0.8 µm ‘naked’ latex beads
(opsonized by serum proteins), 0.8 µm IgG- or complementopsonized (C3) latex beads for 1 hour followed by a 1 hour-chase.
Cells were then prepared for electron microscopy and the number of
beads per cell profiles was counted at the electron microscope. To
allow comparison between each experiment, the values obtained for
control cells were arbitrarily set at 1 and the index for Rab5(Q79L)expressing cells adjusted using an appropriate conversion factor.
Rab5(Q79L)-expressing cells internalized 3 to 4 times more naked
latex beads than control cells. However, the internalization of beads
by either the FcR (IgG-coated beads) or the CR (C3-coated beads)
was similar in both cell types. These values represent the mean of the
analysis of 100 cell profiles in at least 3 independent experiments.
Error bars indicate the standard deviation.
264.7 with an active form of Rab5 (Q79L, with reduced
GTPase activity) to study the role of Rab5 in these processes.
Rab5(Q79L) stimulates non-specific phagocytosis
but not Fc or C3 receptor-mediated phagocytosis
The role of Rab5 during phagocytosis and on the subsequent
properties of phagosomes is poorly understood. To determine
whether Rab5 is involved in phagocytosis per se, we measured
the rate of internalization of latex beads in normal and in
Rab5(Q79L)-expressing cells. Three types of internalization
were analyzed: (a) non-specific internalization of ‘naked’ latex
beads through undefined receptors; (b) internalization of latex
beads opsonized with IgG through the Fc receptor (FcR); and
internalization of complement-opsonized latex beads through
the complement receptor (CR). In the case of so-called ‘naked’
beads, biochemical analysis using 2-D gel electrophoresis
has shown that these beads are in fact rapidly opsonized by
serum proteins present in the culture medium prior to their
internalization by macrophages, the main one being bovine
serum albumin (unpublished observation). Quantification at the
electron microscope of the number of beads present in cell
sections of the control and mutant cells indicated that three to
four times as many naked latex beads were internalized in
Rab5(Q79L)-expressing cells than in untransfected cells (Fig. 1).
Rab5(Q79L)-expressing cells were more efficient at
internalizing both 0.8 µm and 3 µm naked beads. In contrast, no
significant difference was observed in the internalization of
0.8 µm latex beads opsonized with IgG or complement. These
results indicate that Rab5 is unlikely to play a role in FcR or C3
receptor-mediated phagocytosis. The rise in internalization of
naked latex beads in mutant cells is possibly linked with the
stimulation of internalization processes more related to fluid
phase endocytosis or macropinocytosis (Araki et al., 1996) as
expression of Rab5(Q79L) was shown to stimulate fluid phase
endocytosis in other cell types (Stenmark et al., 1994).
Interestingly, overexpression of Rab5 in HeLa cells has also been
shown to stimulate receptor-mediated adenovirus endocytosis,
by as yet unknown mechanisms (Rauma et al., 1999).
Rab5(Q79L) induces the formation of giant
phagosomes
Next, we determined whether the Rab5(Q79L) mutation had
any effect on the morphology of phagosomes containing either
latex beads or the intracellular protozoan parasite Leishmania
donovani. Cells were fed latex beads or infected with L.
donovani promastigotes for 60 minutes followed by a chase
period of 60 minutes, and prepared for electron microscopy.
In normal cells, latex beads or Leishmania were observed
individually in phagosomes with the membrane tightly
surrounding the particle (not shown) or the parasite (Fig. 2A).
The presence of two parasites in the same phagosome was
never observed. In Rab5(Q79L)-expressing cells, latex beads
or Leishmania were observed in very large lucent vacuoles
with a loose membrane. In many cases (over 25% of the
phagosomes observed), these vacuoles contained two or more
parasites (Fig. 2B). These results indicate that Rab5 can also
modulate the fusion properties of phagosomes, including
phagosome-phagosome fusion, the only way by which two L.
donovani can be in the same vacuole (Fig. 3).
To further analyze the formation of giant phagosomes in
Rab5(Q79L)-expressing macrophages, we asked whether
these large vacuoles were formed at the entry step, or were the
result of subsequent interactions with intracellular organelles.
For this experiment, we internalized Leishmania donovani
parasites for 20 minutes without a chase in order to form very
early phagosomes. At the electron microscope, the parasites
were observed in phagosomes of normal size (not shown),
indicating that the giant structures were not formed during
the internalization step, but were rather generated from
phagosome interaction with an internal pool of membranes,
after their formation.
Electron microscopy clearly shows that large endosomes
fuse with phagosomes. However, the possibility that
lysosomes, which are able to fuse with phagosomes in certain
conditions, also constitute a source of membrane deserves
some consideration. To determine which compartments
contribute membranes to generate the large phagosomes, we
first infected cells with Leishmania for 60 minutes followed by
a 60 minutes-incubation in culture medium to get rid of the
non-internalized parasites. BSA-gold particles were then
internalized for 30 minutes, which is sufficient to fill early and
late endosomes but too short to significantly fill lysosomes. In
these conditions, gold particles were observed in large
endosomes and in 98% of the phagosomes indicating that
fusion between these two organelles occurred. In a second set
of experiments, BSA-gold particles were internalized first for
Regulation of phagosome-endosome fusion by Rab5 3535
phagosomes indicating that fusion with lysosomes is unlikely
to contribute significantly to the enlargement of phagosomes
in Rab5(Q79L)-transfected cells (not shown).
Rab5 regulates the ‘kiss and run’ fusion of
phagosomes with endosomes
The results obtained so far strongly suggest that Rab5 is
involved in the regulation of fusion between phagosomes and
endosomes. We proposed a few years ago that Rab proteins
could regulate the nature of the interaction occurring between
these organelles by limiting the complete fusion and mixing of
endosomes with phagosomes, a phenomenon referred to as ‘kiss
and run’ (Desjardins, 1995; Storrie and Desjardins, 1996).
According to this model, the transient fusion between
phagosomes and endosomes would be characterized by the
formation of a fusion complex that would limit the size of the
lumenal molecules exchanged between these organelles. To
determine if Rab5 plays a role in the kiss and run fusion, we
infected cells with Leishmania and then internalized by fluid
phase endocytosis a mixture of solute markers of various sizes
including BSA-gold particles of 5 and 35 nm and latex particles
of 100 nm. At the electron microscope, we observed that all the
control cells had internalized the three types of particles in
endosomes in the close vicinity of phagosomes. However, in
these cells, although phagosomes housing Leishmania
contained preferentially the small gold particles of 5 and 35 nm,
most of them did not contain 100 nm latex particles (Fig. 4). In
contrast, in Rab5(Q79L)-expressing cells, the vast majority of
phagosomes contained small and large gold particles as well as
100 nm latex particles. Quantitative analysis confirmed that the
three size markers were in more than 85% of the phagosomes
in mutant cells. In contrast, a constant decrease was measured
in control cells. While about 65% of the phagosomes contained
the 5 nm gold particles and about 55% contained the 35 nm
gold particles, the 100 nm latex beads were found in only 15%
of the phagosomes (Fig. 4). Moreover, 300 nm latex beads were
also internalized in Leishmania infected cells. These beads were
never observed in Leishmania-containing phagosomes in
control cells, while several Leishmania-containing phagosomes
also contained the 300 nm latex beads in Rab5(Q79L)expressing cells (not shown). In Rab5(Q79L)-expressing cells,
large fusion necks were frequently observed between
endosomes and phagosomes, suggesting that complete fusion
between these organelles is occurring (see Fig. 3A).
Fig. 2. Rab5(Q79L) induces the formation of giant phagosomes. In
control RAW 264.7 cells (A), after 60 minutes of internalization
followed by a 60 minutes-chase, Leishmania promastigotes (L) are
found individually in phagosomes (P) with the membrane tightly
surrounding the parasite. In Rab5(Q79L)-expressing RAW 264.7
cells (B), parasites are observed in large vacuoles with loose
membrane, that can contain several parasites. Bars, 1 µm.
30 minutes followed by an overnight chase to load lysosomes.
After this procedure, gold particles were observed in small
dense vesicles while the large endosomes were mostly empty
(not shown). When cells were then infected with Leishmania
parasites and incubated long enough to allow extensive
interactions between phagosomes and endocytic organelles,
gold particles were observed in less than 28% of the large
Giant phagosomes engage in a maturation process
Next, we asked whether the biogenesis of phagolysosomes was
altered in the transfected cells. Based on several studies, it is now
well established that phagosome maturation is accompanied by
the loss of early endocytic markers and the acquisition of
molecules of late endocytic nature (Pitt et al., 1992; Desjardins
et al., 1994a,b; Via et al., 1997; Scianimanico et al., 1999). In
the present study, the maturation of phagosomes in control and
Rab5(Q79L)-transfected cells was first evaluated by comparing
the nature of their soluble proteins, mostly made of hydrolases.
This was done by isolating non opsonized latex bead-containing
phagosomes and comparing their protein content by 2-D gel
electrophoresis. Phagosomes from control and Rab5(Q79L)transfected cells displayed protein patterns of overall similar
complexity (Fig. 5A). In contrast, phagosomes isolated from
bafilomycin-treated cells, a drug that inhibits endosome
3536 S. Duclos and others
maturation (or endosomal transport; Clague et al., 1994; van
Weert et al., 1995; van Deurs et al., 1996) and phagosome
maturation (C. Rondeau and M. Desjardins, unpublished
observations), displayed simplified 2-D patterns where several
major protein spots were present in much lower quantity (Fig.
5A). Some of these spots excised from the 2-D gels were
analyzed and shown to be the 46 kDa
form of cathepsin D and the 31 kDa
cleaved form of this protein. These
results indicate that inhibition of
phagosome maturation, in our case by
bafilomycin A1, is accompanied by the
failure to acquire high levels of
cathepsin D and to process this
protein. There was also inhibition of
accumulation of other hydrolases (J.
Garin and M. Desjardins, unpublished
results). Accordingly, the overall
similarity
observed
between
phagosomes isolated from control and
Rab5(Q79L) cells, as well as their
ability to process cathepsin D clearly
indicates that phagosomes from
Rab5(Q79L)-expressing cells mature
like those of control cells. Among the
maturation markers shared by both cell
types were the A and B subunits of the
vacuolar (H+)-ATPase (V-ATPase)
responsible for the acidification of
phagosomes, a key process for the
biogenesis of phagolysosomes.
The
ability
of
Leishmaniacontaining phagosomes from control
and Rab5(Q79L)-expressing cells to
mature was assessed by monitoring the
loss of the early marker EEA1 with
time and the acquisition of the
late
marker
LAMP1.
By
immunofluorescence microscopy, we
found that, in both control and mutant
cells, phagosomes were able to recycle
EEA1 and acquire LAMP1. The
quantitative analysis of the acquisition
of these markers is shown in Fig. 5B.
Next, we determined whether
phagosomes in control and mutant
macrophages
can
acquire
the
microbicidal properties needed to kill
the intracellular parasite Leishmania.
Rab5(Q79L) alters the
leishmanicidal properties of
phagosomes
To determine if Rab5 is involved in
the acquisition of the microbicidal
properties of phagosomes, we measured
the survival rates of Leishmania
donovani promastigotes expressing
luciferase in control and Rab5(Q79L)expressing cells. The results obtained
indicated that normal cells were able to
generate conditions inside phagosomes that efficiently killed a
great proportion of the Leishmania parasites within 72 hours
(Fig. 6). In contrast, Rab5(Q79L)-expressing macrophages were
5 to 10 times less efficient at killing Leishmania in 4 distinct
experiments. By comparison, the maturation-deficient
bafilomycin A1-treated macrophages were unable to kill
Fig. 3. Rab5(Q79L) stimulates phagosome-phagosome fusion. (A) Multiple fusion events
between endosomes (E) loaded with 16 nm gold particles (small black dots) and Leishmaniacontaining phagosomes (P). Arrowheads indicate all the fusion necks formed between the
different compartments. (B) An example of fusion between two Leishmania-containing
phagosomes. Bars, 1 µm.
Regulation of phagosome-endosome fusion by Rab5 3537
A
B
D
E
C
F
Fig. 4. Rab5 is involved in the regulation of size-selective kiss and run fusion events between endosomes and phagosomes. In a fusion assay
between endosomes and phagosomes at the electron microscope, cells were infected with L. donovani to form phagosomes. Cells were then
allowed to internalize particles of three different sizes (5, 35 and 100 nm) by endocytosis, and further incubated to allow interaction between
phagosomes and endosomes. L. donovani-containing phagosomes (L) in control cells (A) received mainly 5 nm-gold particles from endosomes,
even if endosomes (E) in the close vicinity contained particles of all sizes (thin arrows indicate 100 nm-latex particles). Thick arrows point at
the phagosome membrane, tightly surrounding the parasite. Bar, 0.5 µm. (B) A higher magnification where 5 nm-gold particles (arrowheads)
can be clearly observed in the lumen of the phagosome. Bar, 0.1 µm. In Rab5(Q79L)-expressing cells (D-F), complete fusion between
phagosomes and endosomes occur leading to the formation of giant phagosomes (P) where the three types of particles can be observed.
(E,F) Higher magnifications of regions of the phagosome from Rab5(Q79L)-expressing cells, where 100 nm-latex particles (thin arrows) can be
observed. Bars: 0.5 µm (D); 0.1 µm (E,F). (C) Histogram of the quantitative analysis of the size-selective fusion experiments clearly indicates
that a high proportion of phagosomes from Rab5(Q79L)-expressing cells contain the three markers. In contrast, less than 15% of the
phagosomes from control cells contain the 100 nm particles. These results represent the mean of three independent experiments, in which 50
phagosomes per cell section were analyzed. Error bars indicate the standard deviation.
Leishmania, demonstrating that inhibition of phagolysosome
biogenesis results in the inability to generate microbicidal
conditions within phagosomes. These results indicate that
despite the apparent maturation of phagosomes in Rab5(Q79L)transfected cells, the proper function of Rab5 is required for the
acquisition of leishmanicidal properties by macrophages.
DISCUSSION
Phagolysosome biogenesis is a regulated process that involves
the sequential interaction of newly formed phagosomes with
early endosomes, late endosomes and lysosomes (Pitt et al.,
1992; Desjardins et al., 1994a, 1997; Jarhaus et al., 1998). In
the present study, we provide evidence that Rab5 regulates the
kiss and run fusion occurring between phagosomes and
endosomes, a process essential for phagolysosome biogenesis.
Rab5 and its effectors have been shown to regulate the fusion
properties of early endocytic structures (see Novick and Zerial,
1997). Although Rab5 was also shown to be involved in
phagosome-endosome fusion in vitro (Alvarez-Dominguez et
al., 1996; Jahraus et al., 1998), its roles in this process are still
3538 S. Duclos and others
Fig. 5. (A) 2-D gel analysis of latex bead-containing phagosomes
formed in control and Rab5(Q79L)-expressing cells. Protein patterns
of latex bead-containing phagosomes isolated from control and
Rab5(Q79L)-expressing macrophages display a similar degree of
complexity. In contrast, phagosomes from bafilomycin-treated cells
exhibit a simpler pattern expected from immature phagosomes.
Among the proteins notably reduced in bafilomycin-treated
phagosomes is the 46 kDa form (white arrows) and the 31 kDa
cleaved form of cathepsin D (black arrows). Actin (asterisks) is
present in relatively equal amount in each gel. These results indicate
that the expression of Rab5(Q79L) does not interfere with the
maturation-associated acquisition of hydrolases by phagosomes.
Insets 1 and 2 illustrate that phagosomes from control and
Rab5(Q79L)-expressing cells acquire comparable levels of the A and
B subunits of the V-ATPase, respectively. (B) L. donovani
containing-phagosomes mature by losing early membrane markers
and acquiring late membrane markers in control and Rab5(Q79L)expressing macrophages. Cells were infected for 30 minutes with L.
donovani parasites in order to form phagosomes. These phagosomes
were chased or not for 120 minutes, and labeled with antibodies
against the early marker EEA1 or the late marker LAMP1 to assess
the acquisition and loss of these markers with time. The histogram
shows that the two cell types present the same pattern of maturation
in regard to the acquisition and loss of these particular markers. The
results represent the mean of the observation of 100 phagosomes in
three independent experiments. Error bars represent the standard
deviation.
poorly understood. The finding that alteration of Rab5 GTPase
activity leads to the formation of giant phagosomes indicates
that Rab5 regulates the nature of the fusion events occurring
between phagosomes and endocytic organelles. In normal cells,
endovacuolar organelles are dynamic structures exchanging
molecules and displaying intense fusion activities. Despite
these interactions, endosomes maintain their integrity and do
not all fuse to produce one big endocytic organelle. Indeed,
regulated fusion and fission or budding events equilibrate each
other and contribute to the maintenance of endosomes of
relatively stable size. During phagolysosome biogenesis, we
have proposed that repeated transient interactions between
phagosomes and endosomes would limit the membrane mixing
of these compartments, while allowing exchange of their
lumenal content (Desjardins, 1995; Desjardins et al., 1997). We
further proposed that the GTPase activity of Rab5 might
regulate the ‘kiss and run’ fusion occurring between
phagosomes and endosomes. This idea is supported by the
finding that Rab5 can act as a timer for endocytic membrane
fusion (Rybin et al., 1996), a process possibly linked to the
recruitment of the Rab5 effectors needed for fusion (Stenmark
et al., 1995; Simonsen et al., 1998; McBride et al., 1999). In
the latter study, it was shown that EEA1, a Rab5 effector,
directly interacts with Syntaxin 13, a member of the SNARE
machinery. Together with other Rab5 effectors (Rabex-5 and
Rabaptin-5), as well as NSF, these proteins assemble into large
oligomers, which could form fusion pores reminiscent of viral
fusion pores (McBride et al., 1999). This is in accordance with
the recent results showing the selective recruitment of Rab5 on
‘hot spots’ at the site of fusion on endosome membranes
(Roberts et al., 1999). Furthermore, different Rab proteins,
including Rab5, were shown to be present within distinct
domains on the endosome membrane (Sonnichsen et al., 2000).
Altogether, these studies suggest that Rab5 and its effectors
could perform their functions in focal area of the phagosome
membrane where fusion pore formation or bridges between
endocytic organelles occur.
In the present study, we provide further evidence that Rab5
is indeed involved in restricting the complete fusion of
phagosomes and endosomes. In normal cells, around half of
the Leishmania-containing phagosomes (cell profiles at the
Regulation of phagosome-endosome fusion by Rab5 3539
Control
Rab5(Q79L)
Control + Baf
Rab5(Q79L) + Baf
Luciferase (RLU/s)
107
106
105
104
0
24
48
72
Time post-infection (h)
Fig. 6. Survival rate of Leishmania donovani parasites in control and
Rab5(Q79L)-expressing macrophages. Control (squares) and
Rab5(Q79L)-expressing (circles) cells were infected for 60 minutes
with Luciferase-expressing L. donovani. Cells were then incubated
for 1 hour with 500 nM bafilomycin A1 in DMSO (filled symbols) or
DMSO only (open symbols), and chased in DMEM for the indicated
time points. After cell lysis, the luciferase activity (corresponding to
living parasites) was quantitated in a luminometer. Phagosomes of
control cells (open squares) gradually kill their content, as seen by
the constant decrease of luciferase activity over time. In contrast,
phagosomes from Rab5(Q79L)-expressing cells (open circles) can be
as much as ten times less efficient at killing parasites over a 72-hour
period. As a control, phagosomes from bafilomycin A1-treated cells
(which do not mature) do not acquire the microbicidal properties
needed to kill parasites in control (filled squares) and Rab5(Q79L)transfected (filled circles) macrophages. These results are
representative of three independent experiments made in triplicate.
Error bars represent the standard deviation.
electron microscope) interacted and fused with early endocytic
organelles, as shown previously (Desjardins and Descoteaux,
1997; Scianimanico et al., 1999). These interactions are,
however, regulated in such a way that only partial fusions
occur. This is obvious from the observation that tracers of three
different sizes (5, 35 and 100 nm) present in the same
endosomes are not transferred to phagosomes in bulk, as
expected from complete fusion. Instead, a preferential transfer
of the 5 nm and the 35 nm particles occurs, but not the 100 nm,
as expected from transient fusion of the two compartments.
Narrow membrane bridges connecting endosomes and
phagosomes containing either latex beads, Leishmania
donovani or the intracellular pathogen Brucella abortus have
been documented (Desjardins et al., 1997; Desjardins and
Descoteaux, 1997; Pizarro-Cerdà et al., 1997). Complex
selective fusion of phagosomes with endosomes containing
only small gold particles was ruled out since most of the
endosomes contained more than one tracer (see Fig. 4 and
Desjardins et al., 1997). A role in the limitation of membrane
fusion has been suggested for other Rab proteins. In neurons,
Rab3 might be involved in modulating the levels of
neurotransmitter release (Geppert and Sudhof, 1998).
Interestingly, ‘kiss and run’ type of interactions between
neurotransmitter-containing vesicles and the plasma membrane
have been shown to occur in chromaffin cells (Alés et al.,
1999). Indeed, patch amperometry experiments have shown
that high concentrations of calcium induce the release of
catecholamines to the cell medium without full fusion of the
vesicle with the plasma membrane. Further studies will be
required to fully understand the molecular mechanisms
underlying these types of membrane interactions.
Rab5(Q79L) also induced the formation of giant
phagosomes containing several parasites, a phenomenon not
normally observed in Leishmania donovani-infected cells. This
further indicates that inhibition of Rab5 GTPase activity also
influences phagosome-phagosome fusion, as suggested by
images of fusion intermediates seen at the electron microscope
(see Fig. 3). Normally, the division of L. donovani in infected
cell is accompanied by the fission of the phagosome resulting
in the separation of the two daughter parasites in distinct tight
membrane organelles. Interestingly, some species of
Leishmania such as L. amazonensis, which are internalized in
tight phagosomes are eventually observed in giant
phagosomes, referred to as parasitophorous vacuoles, few
hours after infection (Veras et al., 1992, 1994). The molecular
mechanisms involved at the membrane level to allow the
sudden formation of these giant structures is not understood.
Inhibition of the Rab5 GTPase activity has important
consequences in the ability of phagosomes to kill intracellular
pathogens. Despite our observations that phagosomes from
both control and Rab5(Q79L)-expressing cells engage in a
similar maturation process, intracellular survival of
Leishmania donovani promastigotes was increased by 5- to 10fold in the latter. The reduced leishmanicidal activity of
Rab5(Q79L)-expressing macrophages may be related to the
increased phagosome size, which might dilute microbicidal
molecules, such as hydrolases, to concentrations below their
effective level. Enlargement of endosomes in Rab5(Q79L)expressing cells has been shown to decrease their ability to
degrade ricin (D’Arrigo et al., 1997). Indeed, the impaired
function either to initiate fission after the transient fusion or to
induce budding after a ‘complete’ fusion would have direct
consequence on the capacity of endocytic organelles to
concentrate their lumenal molecules. A very similar hypothesis
has been put forward to explain the persistence of Salmonella
typhimurium within spacious phagosomes after infection of
macrophages (Alpuche-Aranda et al., 1994). At that time, it
was speculated that the macrophage microbicidal activity
requires a close-fitting phagosome in order to efficiently
concentrate toxic compounds. This idea is further supported by
the recent finding that the increased ability of macrophages to
kill Listeria monocytogenes following Rab5 overexpression is
linked to a decrease of the size of phagosomes (AlvarezDominguez and Stahl, 1999). In this case, the overexpressed
native Rab5 protein still displays its GTPase activity and its
ability to stimulate both fusion and fission. Interestingly, a
process of cargo protein concentration by membrane retrieval
is also proposed in the biosynthetic pathway (Warren and
Mellman, 1999).
Other conditions that could reduce the overall microbicidal
activity of vacuoles include alteration of the acidification
process. It was shown for example that phagosomes containing
3540 S. Duclos and others
Mycobacteria were unable to acidify properly, a condition
linked to the absence of the proton pump ATPases responsible
for endovacuolar organelle acidification (Sturgill-Koszycki et
al., 1994). In the present study, we were able to show that
phagosomes from Rab5(Q79L)-expressing cells acquire the A
and B subunits of the V-ATPase to levels similar to those of
control cells, indicating that acidification is likely to occur
normally in both cell types. This goes along previous
observations showing that acidification in Rab5 GTPexpressing cells occurs normally (D’Arrigo et al., 1997).
Furthermore, the presence of cleaved forms of cathepsin D, as
well as an accumulation of LAMP molecules, in phagosomes
from control and Rab5(Q79L)-expressing cells indicates that
maturation of these organelles is not altered. In contrast, the
group of Russell (Ullrich et al., 1999) has shown recently that
Mycobacteria-containing phagosomes accumulate the
unprocessed 51 kDa proform of cathepsin D.
Finally, another interesting finding of our study is that Rab5
was shown to stimulate phagocytosis of serum-opsonized latex
beads, but not phagocytosis mediated through the FcR or CR.
Phagocytic uptake by these receptors has been shown to be
regulated in part by small GTPases of the Rho family. Indeed,
Caron and Hall (1998) have demonstrated recently that Fcγinduced phagocytosis is mediated by Cdc42 and Rac, while
complement-induced phagocytosis depends on the activity of
Rho. Accordingly, our results suggest that Rab5 might act in a
similar way, albeit with receptors others than the FcR or CR,
that have not been identified at this point. Further studies will
be required to firmly establish the role of Rab5 in this process.
Altogether, our results indicate that Rab5 is involved in
regulating the duration and/or frequency of fusion between
phagosomes and endosomes in macrophages, allowing these
organelles to engage in ‘kiss and run’ interactions. This enables
the transfer of the solute content of endocytic organelles to
phagosomes without significant increase of phagosome size.
This process, required for phagolysosome biogenesis and the
acquisition of phagosome leishmanicidal properties, identifies
Rab5 as a key regulator of macrophage lytic activity.
The authors thank Christiane Rondeau, Sylvie Kieffer and Anik StDenis for technical support and Jean Léveillé for photographic work.
This work was supported by grants from the Medical Research
Council (MRC) of Canada to A.D. (MT-12933) and M.D. (MT-12951)
and from FCAR Equipe to A.D. and M.D.; S.D. is the recipient of a
studentship from the MRC, A.D. is an MRC Scholar and M.D. is a
Scholar from FRSQ.
REFERENCES
Aderem, A. and Underhill, D. M. (1999). Mechanisms of phagocytosis in
macrophages. Annu. Rev. Immunol. 17, 593-623.
Albillos, A., Dernick, G., Horstmann, H., Almers, W., Alvarez de Toledo,
G. and Lindau, M. (1997). The exocytotic event in chromaffin cells
revealed by patch amperometry. Nature 389, 509-512.
Alés, E., Tabares, L., Poyato, J. M., Valero, V., Lindau, M. and Alvarez de
Toledo, G. (1999). High calcium concentrations shift the mode of exocytosis
to the kiss-and-run mechanism. Nature Cell Biol. 1, 40-44.
Alpuche-Aranda, C., Racoosin, E. L., Swanson, J. A. and Miller, S. I.
(1994). Salmonella stimulate macrophage macropinocytosis and persist
within spacious phagosomes. J. Exp. Med. 179, 601-608.
Alvarez de Toledo, G., Fernandez-Chacon, R. and Fernandez, J. M. (1993).
Release of secretory products during transient vesicle fusion. Nature 363,
554-558.
Alvarez-Dominguez, C., Barbieri, A. M., Berón, W., Wandinger-Ness, A.
and Stahl, P. D. (1996). Phagocytosed live Listeria monocytogenes
influences rab5-regulated in vitro phagosome-endosome fusion. J. Biol.
Chem. 271, 13834-13843.
Alvarez-Dominguez, C. and Stahl, P. D. (1999). Increased expression of
Rab5a correlates directly with accelerated maturation of Listeria
monocytogenes phagosomes. J. Biol. Chem. 274, 11459-11462.
Appel, R. D., Palagi, P. M., Walther, D., Vargas, J. R., Sanchez, J. C.,
Ravier, F., Pasquali, C. and Hochstrasser, D. F. (1997). Melanie II–a thirdgeneration software package for analysis of two-dimensional
electrophoresis images: I. Features and user interface. Electrophoresis 18,
2724-2734.
Araki, N., Johnson, M. T. and Swanson, J. A. (1996). A role for
phosphoinositide 3-kinase in the completion of macropinocytosis and
phagocytosis by macrophages. J. Cell Biol. 135, 1249-1260.
Berón, W., Alvarez-Dominguez, C., Mayorga, L. and Stahl, P. D. (1995).
Membrane trafficking along the phagocytic pathway. Trends Cell Biol. 5,
100-104.
Berthiaume, E. P., Medina, C. and Swanson, J. A. (1995). Molecular sizefractionation during endocytosis in macrophages. J. Cell Biol. 129, 989-998.
Blocker, A., Severin, F. F., Burkhardt, J. K., Bingham, J. B., Yu, H., Olivo,
J. C., Schroer, T. A., Hyman, A. A. and Griffiths, G. (1997). Molecular
requirements for bi-directional movement of phagosomes along
microtubules. J. Cell Biol. 137, 113-129.
Blocker, A., Griffiths, G., Olivo, J. C., Hyman, A. A. and Severin, F. F.
(1998). A role for microtubule dynamics in phagosome movement. J. Cell
Sci. 111, 303-312.
Bucci, C., Parton, R. G., Mather, I. H., Stunnenberg, H., Simons, K.,
Hofflack, B. and Zerial, M. (1992). The small GTPase rab 5 functions as
a regulatory factor in the early endocytic pathway. Cell 70, 715-728.
Caron, E. and Hall, A. (1998). Identification of two distinct mechanisms of
phagocytosis controlled by different Rho GTPases. Science 282, 1717-1721.
Chavrier, P., Parton, R. G., Hauri, H. P., Simons, K. and Zerial, M. (1990).
Localization of low molecular weight GTP binding proteins to exocytic and
endocytic compartments. Cell 62, 317-329.
Clague, M. J., Urbe, S., Aniento, F. and Gruenberg, J. (1994). Vacuolar
ATPase activity is required for endosomal carrier vesicle formation. J. Biol.
Chem. 269, 21-24.
Claus, V., Jahraus, A., Tjelle, T., Berg, T., Kirschke, H., Faulstich, H. and
Griffiths, G. (1998). Lysosomal enzyme trafficking between phagosomes,
endosomes, and lysosomes in J774 macrophages. Enrichment of cathepsin
H in early endosomes. J. Biol. Chem. 273, 9842-9851.
D’Arrigo, A., Bucci, C., Toh, B. H. and Stenmark, H. (1997). Microtubules
are involved in bafilomycin A1-induced tubulation and Rab5-dependent
vacuolation of early endosomes. Eur. J. Cell Biol. 72, 95-103.
Desjardins, M., Huber, L. A., Parton, R. G. and Griffiths, G. (1994a).
Biogenesis of phagolysosomes proceeds through a sequential series of
interactions with the endocytic apparatus. J. Cell Biol. 124, 677-688.
Desjardins, M., Celis, J. E., van Meer, G., Dieplinger, H., Jahraus, A.,
Griffiths, G. and Huber, L. A. (1994b). Molecular characterization of
phagosomes. J. Biol. Chem. 269, 32194-32200.
Desjardins, M. (1995). Biogenesis of phagolysosomes: the ‘kiss and run’
hypothesis. Trends Cell Biol. 5, 183-186.
Desjardins, M. and Descoteaux, A. (1997). Inhibition of phagolysosomal
biogenesis by the Leishmania lipophosphoglycan. J. Exp. Med. 185, 20612068.
Desjardins, M., Nzala, N. N., Corsini, R. and Rondeau, C. (1997).
Maturation of phagosomes is accompanied by changes in their fusion
properties and size-selective acquisition of solute materials from
endosomes. J. Cell Sci. 110, 2303-2314.
Evan, G. I., Lewis, G. K., Ramsay, G. and Bishop, J. M. (1985). Isolation
of monoclonal antibodies specific for human c-myc proto-oncogene product.
Mol. Cell. Biol. 5, 3610-3616.
Feng, Y., Press, B. and Wandinger-Ness, A. (1995). Rab 7: an important
regulator of late endocytic membrane traffic. J. Cell Biol. 131, 1435-1452.
Finlay, B. B. and Falkow, S. (1997). Common themes in microbial
pathogenicity revisited. Microbiol. Mol. Biol. Rev. 61, 136-169.
Funato, K., Beron, W., Yang, C. Z., Mukhopadhyay, A. and Stahl, P. D.
(1997). Reconstitution of phagosome-lysosome fusion in streptolysin Opermeabilized cells. J. Biol. Chem. 272, 16147-16151.
Geppert, M. and Sudhof, T. C. (1998). RAB3 and synaptotagmin: the yin
and yang of synaptic membrane fusion. Annu. Rev. Neurosci. 21, 75-95.
Gonzalez, L. Jr and Scheller, R. H. (1999). Regulation of membrane
trafficking: structural insights from a Rab/effector complex. Cell 96, 755-758.
Regulation of phagosome-endosome fusion by Rab5 3541
Gorvel, J. P., Chavrier, P., Zerial, M. and Gruenberg, J. (1991). rab5
controls early endosome fusion in vitro. Cell 64, 915-925.
Hackam, D. J., Rotstein, O. D., Bennett, M. K., Klip, A., Grinstein, S. and
Manolson, M. F. (1996). Characterization and subcellular localization of
target membrane soluble NSF attachment protein receptors (t-SNAREs) in
macrophages. Syntaxins 2, 3, and 4 are present on phagosomal membranes.
J. Immunol. 156, 4377-4383.
Hackam, D. J., Rotstein, O. D., Sjolin, C., Schreiber, A. D., Trimble, W. S.
and Grinstein, S. (1998). v-SNARE-dependent secretion is required for
phagocytosis. Proc. Nat. Acad. Sci. USA 95, 11691-11696.
Jahraus, A., Storrie, B., Griffiths, G. and Desjardins, M. (1994). Evidence
for retrograde traffic between terminal lysosomes and the prelysosomal/late
endosome compartment. J. Cell Sci. 107, 145-157.
Jahraus, A., Tjelle, T. E., Berg, T., Habermann, A., Storrie, B., Ullrich, O.
and Griffiths, G. (1998). In vitro fusion of phagosomes with different
endocytic organelles from J774 macrophages. J. Biol. Chem. 273, 3037930390.
Kornfeld, S. and Mellman, I. (1989). The biogenesis of lysosomes. Annu.
Rev. Cell Biol. 5, 483-525.
McBride, H. M., Rybin, V., Murphy, C., Giner, A., Teasdale, R. and Zerial,
M. (1999). Oligomeric complexes link Rab5 effectors with NSF and drive
membrane fusion via interactions between EEA1 and syntaxin 13. Cell 98,
377-386.
Mellman, I. (1992). The importance of being acid: the role of acidification in
intracellular membrane traffic. J. Exp. Biol. 172, 39-45.
Méresse, S., Gorvel, J. P. and Chavrier, P. (1995). The rab7 GTPase resides
on a vesicular compartment connected to lysosomes. J. Cell Sci. 108, 33493358.
Méresse, S., Steele-Mortimer, O., Moreno, E., Desjardins, M., Finlay, B.
B. and Gorvel, J. P. (1999). Controlling the maturation of pathogencontaining vacuoles: A matter of life or death. Nature Cell Biol. 1, E183E188.
Mills, I. G., Jones, A. T. and Clague, M. J. (1999). Regulation of endosome
fusion. Mol. Membr. Biol. 16, 73-79.
Mohrmann, K. and van der Sluijs, P. (1999). Regulation of membrane
transport through the endocytic pathway by rabGTPases. Mol. Membr. Biol.
16, 81-87.
Novick, P. and Zerial, M. (1997). The diversity of Rab proteins in vesicle
transport. Curr. Opin. Cell Biol. 9, 496-504.
Pitt, A., Mayorga, L. S., Stahl, P. D. and Schwartz, A. L. (1992). Alterations
in the protein composition of maturing phagosomes. J. Clin. Invest. 90,
1978-1983.
Pizarro-Cerdà, J., Moreno, E., Desjardins, M. and Gorvel, J. P. (1997).
When intracellular pathogens invade the frontiers of cell biology and
immunology. Histol. Histopathol. 12, 1027-1038.
Rabilloud, T., Kieffer, S., Procaccio, V., Louwagie, M., Courchesne, P. L.,
Patterson, S. D., Martinez, P., Garin, J. and Lunardi, J. (1998). Twodimensional electrophoresis of human placental mitochondria and protein
identification by mass spectrometry: toward a human mitochondrial
proteome. Electrophoresis 19, 1006-1014.
Rabinowitz, S., Horstmann, H., Gordon, S. and Griffiths, G. (1992).
Immunocytochemical characterization of the endocytic and phagolysosomal
compartments in peritoneal macrophages. J. Cell Biol. 116, 95-112.
Rauma, T., Tuukkanen, J., Bergelson, J. M., Denning, G. and Hautala, T.
(1999). Rab5 GTPase regulates adenovirus endocytosis. J. Virol. 73, 96649668.
Rees, S., Coote, J., Stables, J., Goodson, S., Harris, S. and Lee, M. G.
(1996). Bicistronic vector for the creation of stable mammalian cell lines
that predisposes all antibiotics-resistant cells to express recombinant protein.
BioTechniques. 20, 102-110.
Roberts, R. L., Barbieri, M. A., Pryse, K. M., Chua, M., Morisaki, J. H.
and Stahl, P. D. (1999). Endosome fusion in living cells overexpressing
GFP-rab5. J. Cell Sci. 112, 3667-3675.
Rybin, V., Ullrich, O., Rubino, M., Alexandrov, K., Simon, I., Seabra, C.,
Goody, R. and Zerial, M. (1996). GTPase activity of Rab5 acts as a timer
for endocytic membrane fusion. Nature 383, 266-269.
St-Denis, A., Caouras, V., Gervais, F. and Descoteaux, A. (1999). Role of
protein kinase C-α in the control of infection by intracellular pathogens in
macrophages. J. Immunol. 163, 5505-5511.
Scianimanico, S., Desrosiers, M., Dermine, J. F., Méresse, S., Descoteaux,
A. and Desjardins, M. (1999). Impaired recruitment of the small GTPase
rab7 correlates with the inhibition of phagosome maturation by Leishmania
donovani promastigotes. Cell. Microbiol. 1, 19-32.
Simonsen, A., Lippe, R., Christoforidis, S., Gaullier, J. M., Brech, A.,
Callaghan, J., Toh, B. H., Murphy, C., Zerial, M. and Stenmark, H.
(1998). EEA1 links PI(3)K function to Rab5 regulation of endosome fusion.
Nature 394, 494-498.
Sinai, A. P. and Joiner, K. A. (1997). Safe haven: the cell biology of
nonfusogenic pathogen vacuoles. Annu. Rev. Microbiol. 51, 415-462.
Sonnichsen, B., De Renzis, S., Nielsen, E., Rietdorf, J. and Zerial, M.
(2000). Distinct membrane domains on endosomes in the recycling pathway
visualized by multicolor imaging of Rab4, Rab5, and Rab11. J. Cell Biol.
149, 901-914.
Stacey, K. J., Ross, I. L. and Hume, D. A. (1993). Electroporation and DNAdependent cell death in murine macrophages. Immunol. Cell Biol. 71, 7585.
Steele-Mortimer, O., Méresse, S., Gorvel, J. P., Toh, B. H. and Finlay, B.
B. (1999). Biogenesis of Salmonella typhimurium-containing vacuoles in
epithelial cells involves interactions with the early endocytic pathway. Cell.
Microbiol. 1, 33-49.
Stenmark, H., Parton, R. G., Steele-Mortimer, O., Lütcke, A., Gruenberg,
J. and Zerial, M. (1994). Inhibition of rab5 GTPase activity stimulates
membrane fusion in endocytosis. EMBO J. 13, 1287-1296.
Stenmark, H., Vitale, G., Ullrich, O. and Zerial, M. (1995). Rabaptin-5 is
a direct effector of the small GTPase Rab5 in endocytic membrane fusion.
Cell 83, 423-432.
Storrie, B. and Desjardins, M. (1996). The biogenesis of lysosomes: Is it a
kiss and run, continuous fusion and fission process? BioEssays 18, 895-903.
Sturgill-Koszycki, S., Schlesinger, P. H., Chakraborty, P., Haddix, P. L.,
Collins, H. L., Fok, A. K., Allen, R. D., Gluck, S. L., Heuser, J. and
Russell, D. G. (1994). Lack of acidification in Mycobacterium phagosomes
produced by exclusion of the vesicular proton-ATPase. Science 263, 678-681.
Tolson, D. L., Turco, S. J. and Pearson, T. W. (1990). Expression of a
repeating phosphorylated disaccharide lipophosphoglycan epitope on the
surface of macrophages infected with Leishmania donovani. Infect. Immun.
58, 3500-3507.
Ullrich, H. J., Beatty, W. L. and Russell, D. G. (1999). Direct delivery of
procathepsin D to phagosomes: implications for phagosome biogenesis and
parasitism by Mycobacterium. Eur. J. Cell Biol. 78, 739-748.
van Deurs, B., Holm, P. K. and Sandvig, K. (1996). Inhibition of the vacuolar
H(+)-ATPase with bafilomycin reduces delivery of internalized molecules
from mature multivesicular endosomes to lysosomes in HEp-2 cells. Eur. J.
Cell Biol. 69, 343-350.
van Weert, A. W., Dunn, K. W., Gueze, H. J., Maxfield, F. R. and
Stoorvogel, W. (1995). Transport from late endosomes to lysosomes, but
not sorting of integral membrane proteins in endosomes, depends on the
vacuolar proton pump. J. Cell Biol. 130, 821-834.
Veras, P. S., de Chastellier, C. and Rabinovitch, M. (1992). Transfer of
zymosan (yeast cell walls) to the parasitophorous vacuoles of macrophages
infected with Leishmania amazonensis. J. Exp. Med. 176, 639-646.
Veras, P. S., de Chastellier, C., Moreau, M. F., Villiers, V., Thibon, M.,
Mattei, D. and Rabinovitch, M. (1994). Fusion between large phagocytic
vesicles: targeting of yeast and other particulates to phagolysosomes that
shelter the bacterium Coxiella burnetii or the protozoan Leishmania
amazonensis in Chinese hamster ovary cells. J. Cell Sci. 107, 3065-3076.
Via, L. E., Deretic, D., Ulmer, R. J., Hibler, N. S., Huber, L. A. and Deretic,
V. (1997). Arrest of mycobacterial phagosome maturation is caused by a
block in vesicle fusion between stages controlled by rab5 and rab7. J. Biol.
Chem. 272, 13326-13331.
Vitelli, R., Santillo, M., Lattero, D., Chiariello, M., Bifulco, M., Bruni, C.
B. and Bucci, C. (1997). Role of the small GTPase Rab7 in the late
endocytic pathway. J. Biol. Chem. 272, 4391-4397.
Wang, Y. L. and Goren, M. B. (1987). Differential and sequential delivery
of fluorescent lysosomal probes into phagosomes in mouse peritoneal
macrophages. J. Cell Biol. 104, 1749-1754.
Warren, G. and Mellman, I. (1999). Bulk flow redux? Cell 98, 125-127.
Weidman, P. J. (1995). Anterograde transport through the Golgi complex: do
Golgi tubules hold the key? Trends Cell Biol. 5, 302-305.