Journal of Cell Science 113, 3531-3541 (2000) 3531 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: [email protected])

Accepted 20 July; published on WWW 13 September 2000

SUMMARY

Phagolysosome biogenesis is essential for the killing and Rab5(Q79L)-expressing macrophages, uncontrolled fusion degradation of intracellular pathogens. It involves the events occurred, leading to the appearance of giant fusion of phagosomes with various endocytic organelles, a phagosomes. These phagosomes could initiate their process known to be regulated in part by Rab proteins. We maturation and acquire LAMP1, but failed to generate generated RAW 264.7 macrophages expressing an active the microbicidal conditions needed to kill intracellular mutant of Rab5 (Rab5(Q79L)) to determine the role of parasites. These results identify Rab5 as a key molecule Rab5 in phagocytosis and phagolysosome biogenesis. Our regulating phagosome-endosome fusion and as an essential results indicate that Rab5 stimulates phagocytosis of latex component in the innate ability of macrophages to restrict beads but not Fc or C3 receptor-mediated phagocytosis. the growth of intracellular parasites. Rab5 also acts to restrict the complete fusion of phagosomes with endosomes, a phenomenon allowing exchange of solutes from the two compartments without Key words: Rab5, Phagosome, Kiss and run, Leishmania, Membrane complete intermixing of their membrane (kiss and run). In fusion

INTRODUCTION (Kornfeld and Mellman, 1989; Claus et al., 1998). Thus, the fusion of phagosomes with these organelles is fundamental for Intracellular pathogens internalized by phagocytosis are phagocytic cells like macrophages to control the spread of sequestered in compartments originating from the plasma infection. Indeed, several microorganisms have evolved membrane, the phagosomes. Newly formed phagosomes are mechanisms to inhibit phagosome-lysosome fusion and unable to kill and degrade their content and must therefore survive within their host cells (for reviews see Finlay and engage in a complex process of maturation referred to as Falkow, 1997; Sinai and Joiner, 1997; Aderem and Underhill, phagolysosome biogenesis (Berón et al., 1995; Desjardins, 1999; Méresse et al., 1999). 1995). This process involves the binding to and movement Phagolysosome biogenesis was first believed to involve the along microtubules (Blocker et al., 1997, 1998) and the complete fusion of phagosomes with lysosomes (see multiple steps required for recognition and fusion with Rabinowitz et al., 1992). However, recent studies revealed that endocytic organelles (Desjardins et al., 1997; Jahraus et al., phagosomes engage in a regulated series of fusion events, first 1998). During these interactions, phagosomes acquire several with early and late endosomes, which are necessary for the of the molecules required for their microbicidal activity. These subsequent fusion with lysosomes to proceed (Pitt et al., 1992; include proton pump ATPases involved in phagosome Desjardins et al., 1994a, 1997; Jahraus et al., 1994; Via et al., acidification (Mellman, 1992) and a variety of hydrolases 1997). Despite this dynamic fusion activity, phagosomes responsible for the digestion of phagosome content (Claus et and endosomes maintain a relatively stable size over time al., 1998). Although hydrolases are present in various (Desjardins et al., 1994a). Based on some of these populations of endocytic organelles, the cellular bulk of these observations, we proposed that phagosomes fuse with molecules are stored within late endosomes and lysosomes endocytic organelles through repeated transient fusion events, 3532 S. Duclos and others a process referred to as the ‘kiss and run’ hypothesis MATERIALS AND METHODS (Desjardins, 1995; Storrie and Desjardins, 1996). This process has several advantages. It allows organelles to exchange Cell culture and stable transfections contents without the complete mixing of their membranes, The murine macrophage cell line RAW 264.7 (American Type Culture limiting the need for large scale recycling processes. Limited Collection, Rockville, MD) was cultured in DMEM, pH 7.4, exchange of membrane molecules has the further potential to supplemented with 10% heat inactivated FBS (Life Technologies Inc., allow the gradual transformation of organelles observed along ON, Canada), 20 mM Hepes, pH 7.3-7.4, and antibiotics (100 U/ml µ the endocytic and phagocytic pathways. Evidence for ‘kiss and penicillin, 100 g/ml streptomycin), at 37°C in 5% CO2. The myc- run’ exchanges has been obtained from various biological tagged Rab5a(Q79L) cDNA was cloned in the EcoRI site of the systems including phagosome-endosome interaction (Wang expression vector pCIN4 (Rees et al., 1996). Stable transfections were performed by electroporation essentially as described (Stacey et al., and Goren, 1987; Desjardins et al., 1994a, 1997), endosome- 1993) except that 20 µg of linearized plasmid DNA were used for endosome interaction (Berthiaume et al., 1995), and exocytosis the transfection. Clones were selected for their ability to grow in of neurotransmitters (Alvarez de Toledo et al., 1993; Albillos 500 µg/ml G418 (Calbiochem, San Diego, CA). Clones resistant to et al., 1997). In the later case, the fusion pore formed between the antibiotic were tested for the expression of myc Rab5(Q79L) by mast cell granules and the plasma membrane could stay open RT-PCR using the following oligonucleotides primers: primer 1, for several seconds, allowing the complete transmitter release encoding the 5′ end of the c-myc epitope (5′GGAATTCG- without full fusion of the vesicle with the plasma membrane. CCATGGAACAAAAACTC-3′) and primer 2, encoding the 3′ end of Transient fusions between Golgi tubules have also been Rab5 (5′GGAATTCTTAGTTACTACAACAC-3′), as well as by proposed to occur (Weidman, 1995). The molecular immunofluorescence microscopy using the anti-myc epitope antibody mechanisms governing transient membrane interactions are 9E10 (Evan et al., 1985). poorly understood. Parasites Membrane fusion is the focus of intense investigation. Leishmania donovani promastigotes (Sudanese strain 1S) transfected Intracellular membrane trafficking requires Rab GTPases and with the luciferase expression vector pGEM72f/anealuc (St-Denis et SNARE proteins which appear to act in conjunction to allow al., 1999) were grown at 26°C in the presence of 50 µg/ml G418 in vesicle movement, docking, fusion and fission (McBride et al., RPMI 1640 with glutamine (Life Technologies Inc.) supplemented 1999). Both Rab and SNARE proteins are present on with 20% heat inactivated FBS (Hyclone, Logan, UT), 10 mM phagosomes. While the SNARE proteins syntaxins and adenine, 0.0005% hemin in 50% triethanolamine, 1 µg/ml 6-biopterin, synaptobrevins have been identified on phagosomes at all 0.0001% biotin in 95% ethanol, 20 mM MES, and antibiotics stages of their maturation (Hackham et al., 1996, 1998; (100 U/ml penicillin, 100 µg/ml streptomycin) at pH 5.5. Desjardins et al., 1997), in some studies Rab5 and Rab7 have Promastigotes were grown to stationary or late stationary phases prior been shown to associate transiently to maturing phagosomes to each experiment. (Desjardins et al., 1994a,b; Via et al., 1997; Funato et al., Antibodies and immunofluorescence microscopy 1997). Rab5 and Rab7 have first been identified on early endosomes and late endosomes, respectively (Chavrier et al., The primary antibodies used were: an affinity-purified rabbit antibody to EEA1 (Simonsen et al., 1998), a monoclonal rat anti-LAMP1 1990), and shown to be involved in endosome fusion (Gorvel (Developmental Studies Hybridoma Bank, Department of et al., 1991; Bucci et al., 1992; Feng et al., 1995; Méresse et Pharmacology and Molecular Sciences, Johns Hopkins University al., 1995; Vitelli et al., 1997). Seminal work has further allowed School of Medicine, Baltimore, MD, and the Department of us to characterize several of the Rab5 effectors, underlining the Biological Sciences, University of Iowa, Iowa City, IA, under contract complexity of membrane interaction among endovacuolar N01-HD-6-2915 from the NICHD), and a mouse monoclonal organelles (see Novick and Zerial, 1997; Gonzalez and antibody CA7AE directed against the repeating units of Scheller, 1999). Despite the increasing body of data regarding lipophosphoglycan (LPG) (Tolson et al., 1989). For all Rab5 interactions with its effectors, the role of Rab5 in the immunofluorescence experiments, cells were grown on 18 mm round interaction of phagocytic and endocytic organelles is not well coverslips. Fixation was performed either in 4% paraformaldehyde understood. Rab5 or some of its effectors have been shown followed by 0.2% Triton X-100 permeabilization, or in 80% methanol/20% acetone for 20 minutes at –20°C. After washes in cold to regulate some aspects of phagosome-early endosome PBS, cells were incubated in a blocking solution made of 2% BSA interactions (Alvarez-Dominguez et al., 1996; Jahraus et al., and 0.2% gelatin in PBS. Incubation with the primary antibodies was 1998; Alvarez-Dominguez and Stahl, 1999; Steele-Mortimer et done for 1 hour at room temperature. After washes in PBS/1% BSA, al., 1999). In the present study, we generated macrophage cell cells were incubated with the appropriate secondary antibodies (Texas lines expressing an active form of Rab5 (Rab5(Q79L)) to study Red-conjugated anti-mouse IgM (BIO/CAN Scientific, Mississauga, the role of this molecule in phagocytosis, phagosome- ON, Canada), ALEXA-conjugated anti-rabbit IgG or ALEXA- endosome interaction, and phagolysosome biogenesis. Our conjugated anti-rat IgG (Molecular Probes, Eugene, OR)) for 30 results indicate that Rab5 activity controls the occurrence of minutes in the dark, at room temperature. Cells were mounted on transient fusion events between phagosomes and endosomes. Gelvatol (Air Products & Chemicals, Allentown, PA) and observed at The deregulation of Rab5 activity induces the formation of the Zeiss inverted epifluorescence microscope. latex beads- or Leishmania donovani-containing giant Opsonization of latex beads and measurement of phagosomes that are able to mature by acquiring hydrolases phagocytic rates and late endosome/lysosome membrane markers, but unable to The 0.8 µm latex beads (Sigma, St Louis, MO) were briefly sonicated efficiently kill the intracellular parasite L. donovani. The to disrupt aggregates, then diluted 1/10 in bidistilled water (ddH2O) involvement of Rab5 in the acquisition of phagosomal and washed by centrifugation 3 times 5 minutes at 12,000 g. For IgG leishmanicidal properties identifies it as a key component in opsonization, beads were incubated for 2 hours at 37°C with 5 mg/ml the ability of macrophages to restrain the spread of infection. 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, 1997). Proteins were separated according to their isoelectric point and pH 7.4), and once in ddH2O. The beads were further incubated with molecular mass using 18 cm immobilized pH gradient (IPG) strips, a mouse IgG antibody against BSA (Sigma) for 1 hour at 37°C, pH 3-10 (Amersham), for the first dimension following the followed by an overnight incubation at 4°C, with constant agitation. manufacturer procedures. The second dimension was performed on Beads were then washed 3 times and resuspended in BSS. 12% SDS acrylamide gels with 10% sucrose allowing separation of Opsonization was controlled by incubation of the beads with an anti- proteins between 130 kDa and 15 kDa approximately. At the end of mouse antibody coupled to the fluorochrome ALEXA (Molecular the migration, gels were fixed and silver stained following standard Probes) for 5 minutes at room temperature and observation at the procedures. The gel polypeptide patterns were then analyzed using the Zeiss epifluorescence microscope. For complement coating, beads package software MELANIE II (Bio-Rad, Glattbrugg, Switzerland; were first washed in ddH2O. Mouse serum (Sigma) was centrifuged Appel et al., 1997). For mass spectrometry analysis, gels were stained in order to pellet contaminating cells. The serum was then diluted 1/5 with zinc acetate without fixation. The protein spots were excised with in ddH2O and incubated 1:1 with the beads for 2 hours at 37°C, the acrylamide and analyzed by MALDI-TOF-MS (matrix-assisted followed by an overnight incubation at 4°C. Afterwards, beads were laser-desorption ionization time-of-flight mass spectrometry) as washed several times and resuspended in BSS. Opsonization was described previously (Rabilloud et al., 1998). controlled with a mouse anti-C3 antibody coupled to the FITC fluorochrome (ICN Biomedicals Inc., Aurora, OH). Characterization of endosomal markers on To evaluate the level of phagocytosis of control and mutant cells, Leishmania containing-phagosomes µ 0.8 m naked, IgG-opsonized, or C3-opsonized latex beads diluted Cells were infected with L. donovani parasites (3×107/ml in DMEM) 1/100 in DMEM were internalized for 1 hour at 37°C, followed by a for 30 minutes at 37°C. Cells were then washed and further incubated µ chase of 1 hour. Naked latex beads of 3 m were also internalized. or not for 120 minutes to form early phagosomes (30′/0) or late Cells were then processed for electron microscopy. The number of phagosomes (30′/120′). The presence of the early (EEA1) and late beads per cell profile was then counted on thin section at the electron (LAMP1) endosomal markers was assessed in different experiments microscope. A minimum of 50 cells per sample was evaluated. Each using the corresponding antibodies, while parasites were labeled with experiment was repeated at least 3 times. To allow comparison the anti-LPG monoclonal antibody CA7AE. Quantification of the between each experiment, the values obtained for control cells were number of Leishmania-containing phagosomes positive for the arbitrarily set at 1. The index for the mutant cells was subsequently different markers was done as follow. Cells were first examined in the adjusted by using the same factor of conversion. red channel to localize parasites. The selected field was then observed in the green channel to determine if the parasites were present or not Morphology of phagosomes by electron microscopy in EEA1 or LAMP1 positive phagosomes. Quantification was To determine the effect of Rab5(Q79L) on phagosome morphology, performed on 100 phagosomes for each time point and each marker cells were fed with latex beads or infected with Leishmania parasites in 3 different sets of experiments. (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 Survival of L. donovani in RAW 264.7 macrophages observe the formation of early phagosomes, Leishmania parasites To determine the survival rate of L. donovani in macrophages, were internalized for 20 minutes without a chase. In some cases, adherent cells were infected with luciferase-expressing parasites in 16 nm gold particles coated with BSA were internalized using DMEM for one hour at 37°C, at a ratio of ten parasites per standard procedures (Rabinowitz et al., 1992) for 30 minutes in order macrophage. Uningested Leishmania were removed by three washes to load endosomes, or internalized prior to infection for 30 minutes with cold PBS. Macrophages were then incubated for one hour in followed by an overnight chase to load lysosomes. Cells were fixed complete medium containing either 500 nM bafilomycin A1 (Kamiya in 2% glutaraldehyde, post-fixed in 1% OsO4, dehydrated in alcohol, Biomedical Compary) in DMSO or only DMSO. After few washes in processed for flat embedding in Epon 812 and observed at the Zeiss cold PBS, cells were further incubated in complete medium and CEM 902 electron microscope as described previously (Desjardins et survival rates were determined after 1, 6, 24, 48 and 72 hours post- al., 1994a). infection by measuring luciferase activity in cell extracts. Luciferase activity was measured in Leishmania-infected cells Size selective transfer experiment (‘kiss and run’) extracts using the Promega Luciferase Assay system as recommended To determine whether phagosomes and endosomes engage in by the manufacturer (Promega Corp, Madison, WI). Briefly, host cells complete or transient fusion events, and if Rab5 is involved in this were lysed in 100 µl of 1× Cell Culture Lysis Reagent. Then, 20 µl process, we adopted the following strategy. We used endocytic tracers of cells extracts were mixed with 100 µl Luciferase Assay Reagent at of three different sizes and evaluated if they were all transferred to room temperature, and light emission was quantified in a luminometer phagosomes at once (complete fusion) or if there was a size-selective (Berthold, Nashua, NH). Since only living parasites express the transfer indicative of narrow transient openings between the luciferase gene, the light emission produced by the reaction of phagosome and the endosome membrane. Cells were infected with L. luciferase with its substrate is proportional to the number of living × 7 donovani (3 10 /ml in DMEM) for 1 hour at 37°C, washed in cold Leishmania. 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 RESULTS Epon 812 and observed at the electron microscope. Quantitation was performed on 50 phagosomes in 3 independent experiments. Rab5 is a small GTPase that plays important roles in membrane 2-D gel electrophoresis and protein identification interactions and fusion (see Mohrmann and van der Sluijs, 1999; Mills et al., 1999). This protein, first localized to early Phagosomes were formed by the internalization of 0.8 µm latex beads for 60 minutes in culture medium. Cells were then incubated for 60 endocytic organelles (Chavrier et al., 1990), is also present on minutes in culture medium with or without 500 nM bafilomycin A1 phagosomes (Desjardins et al., 1994a; Via et al., 1997). Its (Kamiya Biomedical Company, Tukwila, WA). Latex bead-containing function during phagocytosis, in phagosome properties and in phagosomes were isolated from control, bafilomycin-treated cells or phagolysosome biogenesis is poorly understood. In the present Rab5(Q79L) cells as described previously (Desjardins et al., 1994a, study, we transfected the mouse macrophage cell line RAW 3534 S. Duclos and others

5 internalizing both 0.8 µm and 3 µm naked beads. In contrast, no Control significant difference was observed in the internalization of Rab5(Q79L) 0.8 µm latex beads opsonized with IgG or complement. These 4 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 3 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 LB3 PB1 2 endocytosis in other cell types (Stenmark et al., 1994). Interestingly, overexpression of Rab5 in HeLa cells has also been

Phagocytic index shown to stimulate receptor-mediated adenovirus endocytosis, 1 by as yet unknown mechanisms (Rauma et al., 1999). Rab5(Q79L) induces the formation of giant phagosomes 0 3 µm 0.8 µm 0.8 µm 0.8 µm Next, we determined whether the Rab5(Q79L) mutation had "naked" IgG C3 any effect on the morphology of phagosomes containing either Fig. 1. Rab5(Q79L) increases phagocytosis of serum proteins- latex beads or the intracellular protozoan parasite Leishmania opsonized latex beads but not C3- or IgG-opsonized beads. donovani. Cells were fed latex beads or infected with L. Macrophages expressing Rab5(Q79L) or expressing the vector alone donovani promastigotes for 60 minutes followed by a chase were allowed to internalize 3 µm or 0.8 µm ‘naked’ latex beads period of 60 minutes, and prepared for electron microscopy. (opsonized by serum proteins), 0.8 µm IgG- or complement- In normal cells, latex beads or Leishmania were observed opsonized (C3) latex beads for 1 hour followed by a 1 hour-chase. individually in phagosomes with the membrane tightly Cells were then prepared for electron microscopy and the number of surrounding the particle (not shown) or the parasite (Fig. 2A). beads per cell profiles was counted at the electron microscope. To The presence of two parasites in the same phagosome was allow comparison between each experiment, the values obtained for never observed. In Rab5(Q79L)-expressing cells, latex beads control cells were arbitrarily set at 1 and the index for Rab5(Q79L)- expressing cells adjusted using an appropriate conversion factor. or Leishmania were observed in very large lucent vacuoles Rab5(Q79L)-expressing cells internalized 3 to 4 times more naked with a loose membrane. In many cases (over 25% of the latex beads than control cells. However, the internalization of beads phagosomes observed), these vacuoles contained two or more by either the FcR (IgG-coated beads) or the CR (C3-coated beads) parasites (Fig. 2B). These results indicate that Rab5 can also was similar in both cell types. These values represent the mean of the modulate the fusion properties of phagosomes, including analysis of 100 cell profiles in at least 3 independent experiments. phagosome-phagosome fusion, the only way by which two L. Error bars indicate the standard deviation. 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 264.7 with an active form of Rab5 (Q79L, with reduced these large vacuoles were formed at the entry step, or were the GTPase activity) to study the role of Rab5 in these processes. result of subsequent interactions with intracellular organelles. For this experiment, we internalized Leishmania donovani Rab5(Q79L) stimulates non-specific phagocytosis parasites for 20 minutes without a chase in order to form very but not Fc or C3 receptor-mediated phagocytosis early phagosomes. At the electron microscope, the parasites The role of Rab5 during phagocytosis and on the subsequent were observed in phagosomes of normal size (not shown), properties of phagosomes is poorly understood. To determine indicating that the giant structures were not formed during whether Rab5 is involved in phagocytosis per se, we measured the internalization step, but were rather generated from the rate of internalization of latex beads in normal and in phagosome interaction with an internal pool of membranes, Rab5(Q79L)-expressing cells. Three types of internalization after their formation. were analyzed: (a) non-specific internalization of ‘naked’ latex Electron microscopy clearly shows that large endosomes beads through undefined receptors; (b) internalization of latex fuse with phagosomes. However, the possibility that beads opsonized with IgG through the Fc receptor (FcR); and lysosomes, which are able to fuse with phagosomes in certain internalization of complement-opsonized latex beads through conditions, also constitute a source of membrane deserves the complement receptor (CR). In the case of so-called ‘naked’ some consideration. To determine which compartments beads, biochemical analysis using 2-D gel electrophoresis contribute membranes to generate the large phagosomes, we has shown that these beads are in fact rapidly opsonized by first infected cells with Leishmania for 60 minutes followed by serum proteins present in the culture medium prior to their a 60 minutes-incubation in culture medium to get rid of the internalization by macrophages, the main one being bovine non-internalized parasites. BSA-gold particles were then serum albumin (unpublished observation). Quantification at the internalized for 30 minutes, which is sufficient to fill early and electron microscope of the number of beads present in cell late endosomes but too short to significantly fill lysosomes. In sections of the control and mutant cells indicated that three to these conditions, gold particles were observed in large four times as many naked latex beads were internalized in endosomes and in 98% of the phagosomes indicating that Rab5(Q79L)-expressing cells than in untransfected cells (Fig. 1). fusion between these two organelles occurred. In a second set Rab5(Q79L)-expressing cells were more efficient at 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). Giant phagosomes engage in a maturation process Fig. 2. Rab5(Q79L) induces the formation of giant phagosomes. In control RAW 264.7 cells (A), after 60 minutes of internalization Next, we asked whether the biogenesis of phagolysosomes was followed by a 60 minutes-chase, Leishmania promastigotes (L) are altered in the transfected cells. Based on several studies, it is now found individually in phagosomes (P) with the membrane tightly well established that phagosome maturation is accompanied by surrounding the parasite. In Rab5(Q79L)-expressing RAW 264.7 the loss of early endocytic markers and the acquisition of cells (B), parasites are observed in large vacuoles with loose molecules of late endocytic nature (Pitt et al., 1992; Desjardins membrane, that can contain several parasites. Bars, 1 µm. 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 30 minutes followed by an overnight chase to load lysosomes. the nature of their soluble proteins, mostly made of hydrolases. After this procedure, gold particles were observed in small This was done by isolating non opsonized latex bead-containing dense vesicles while the large endosomes were mostly empty phagosomes and comparing their protein content by 2-D gel (not shown). When cells were then infected with Leishmania electrophoresis. Phagosomes from control and Rab5(Q79L)- parasites and incubated long enough to allow extensive transfected cells displayed protein patterns of overall similar interactions between phagosomes and endocytic organelles, complexity (Fig. 5A). In contrast, phagosomes isolated from gold particles were observed in less than 28% of the large bafilomycin-treated cells, a drug that inhibits endosome 3536 S. Duclos and others maturation (or endosomal transport; Clague et al., 1994; van generate conditions inside phagosomes that efficiently killed a Weert et al., 1995; van Deurs et al., 1996) and phagosome great proportion of the Leishmania parasites within 72 hours maturation (C. Rondeau and M. Desjardins, unpublished (Fig. 6). In contrast, Rab5(Q79L)-expressing macrophages were observations), displayed simplified 2-D patterns where several 5 to 10 times less efficient at killing Leishmania in 4 distinct major protein spots were present in much lower quantity (Fig. experiments. By comparison, the maturation-deficient 5A). Some of these spots excised from the 2-D gels were bafilomycin A1-treated macrophages were unable to kill 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 Leishmania- containing 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 Fig. 3. Rab5(Q79L) stimulates phagosome-phagosome fusion. (A) Multiple fusion events donovani promastigotes expressing between endosomes (E) loaded with 16 nm gold particles (small black dots) and Leishmania- luciferase in control and Rab5(Q79L)- containing phagosomes (P). Arrowheads indicate all the fusion necks formed between the expressing cells. The results obtained different compartments. (B) An example of fusion between two Leishmania-containing indicated that normal cells were able to phagosomes. Bars, 1 µm. Regulation of phagosome-endosome fusion by Rab5 3537 ABC

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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 the sequential interaction of newly formed phagosomes with biogenesis results in the inability to generate microbicidal early endosomes, late endosomes and lysosomes (Pitt et al., conditions within phagosomes. These results indicate that 1992; Desjardins et al., 1994a, 1997; Jarhaus et al., 1998). In despite the apparent maturation of phagosomes in Rab5(Q79L)- the present study, we provide evidence that Rab5 regulates the transfected cells, the proper function of Rab5 is required for the kiss and run fusion occurring between phagosomes and acquisition of leishmanicidal properties by macrophages. 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, DISCUSSION 1997). Although Rab5 was also shown to be involved in phagosome-endosome fusion in vitro (Alvarez-Dominguez et Phagolysosome biogenesis is a regulated process that involves 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.

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 poorly understood. The finding that alteration of Rab5 GTPase could perform their functions in focal area of the phagosome activity leads to the formation of giant phagosomes indicates membrane where fusion pore formation or bridges between that Rab5 regulates the nature of the fusion events occurring endocytic organelles occur. between phagosomes and endocytic organelles. In normal cells, In the present study, we provide further evidence that Rab5 endovacuolar organelles are dynamic structures exchanging is indeed involved in restricting the complete fusion of molecules and displaying intense fusion activities. Despite phagosomes and endosomes. In normal cells, around half of these interactions, endosomes maintain their integrity and do the Leishmania-containing phagosomes (cell profiles at the Regulation of phagosome-endosome fusion by Rab5 3539

Control neurotransmitter release (Geppert and Sudhof, 1998). Interestingly, ‘kiss and run’ type of interactions between Rab5(Q79L) neurotransmitter-containing vesicles and the plasma membrane Control + Baf have been shown to occur in chromaffin cells (Alés et al., 1999). Indeed, patch amperometry experiments have shown Rab5(Q79L) + Baf that high concentrations of calcium induce the release of 107 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. 106 Rab5(Q79L) also induced the formation of giant phagosomes containing several parasites, a phenomenon not normally observed in Leishmania donovani-infected cells. This

5 further indicates that inhibition of Rab5 GTPase activity also 10 influences phagosome-phagosome fusion, as suggested by

Luciferase (RLU/s) images of fusion intermediates seen at the electron microscope (see Fig. 3). Normally, the division of L. donovani in infected 104 cell is accompanied by the fission of the phagosome resulting in the separation of the two daughter parasites in distinct tight 0244872 membrane organelles. Interestingly, some species of Time post-infection (h) Leishmania such as L. amazonensis, which are internalized in tight phagosomes are eventually observed in giant Fig. 6. Survival rate of Leishmania donovani parasites in control and phagosomes, referred to as parasitophorous vacuoles, few Rab5(Q79L)-expressing macrophages. Control (squares) and hours after infection (Veras et al., 1992, 1994). The molecular Rab5(Q79L)-expressing (circles) cells were infected for 60 minutes mechanisms involved at the membrane level to allow the with Luciferase-expressing L. donovani. Cells were then incubated sudden formation of these giant structures is not understood. for 1 hour with 500 nM bafilomycin A1 in DMSO (filled symbols) or DMSO only (open symbols), and chased in DMEM for the indicated Inhibition of the Rab5 GTPase activity has important time points. After cell lysis, the luciferase activity (corresponding to consequences in the ability of phagosomes to kill intracellular living parasites) was quantitated in a luminometer. Phagosomes of pathogens. Despite our observations that phagosomes from control cells (open squares) gradually kill their content, as seen by both control and Rab5(Q79L)-expressing cells engage in a the constant decrease of luciferase activity over time. In contrast, similar maturation process, intracellular survival of phagosomes from Rab5(Q79L)-expressing cells (open circles) can be Leishmania donovani promastigotes was increased by 5- to 10- as much as ten times less efficient at killing parasites over a 72-hour fold in the latter. The reduced leishmanicidal activity of period. As a control, phagosomes from bafilomycin A1-treated cells Rab5(Q79L)-expressing macrophages may be related to the (which do not mature) do not acquire the microbicidal properties increased phagosome size, which might dilute microbicidal needed to kill parasites in control (filled squares) and Rab5(Q79L)- molecules, such as hydrolases, to concentrations below their transfected (filled circles) macrophages. These results are representative of three independent experiments made in triplicate. effective level. Enlargement of endosomes in Rab5(Q79L)- Error bars represent the standard deviation. 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 electron microscope) interacted and fused with early endocytic induce budding after a ‘complete’ fusion would have direct organelles, as shown previously (Desjardins and Descoteaux, consequence on the capacity of endocytic organelles to 1997; Scianimanico et al., 1999). These interactions are, concentrate their lumenal molecules. A very similar hypothesis however, regulated in such a way that only partial fusions has been put forward to explain the persistence of Salmonella occur. This is obvious from the observation that tracers of three typhimurium within spacious phagosomes after infection of different sizes (5, 35 and 100 nm) present in the same macrophages (Alpuche-Aranda et al., 1994). At that time, it endosomes are not transferred to phagosomes in bulk, as was speculated that the macrophage microbicidal activity expected from complete fusion. Instead, a preferential transfer requires a close-fitting phagosome in order to efficiently of the 5 nm and the 35 nm particles occurs, but not the 100 nm, concentrate toxic compounds. This idea is further supported by as expected from transient fusion of the two compartments. the recent finding that the increased ability of macrophages to Narrow membrane bridges connecting endosomes and kill Listeria monocytogenes following Rab5 overexpression is phagosomes containing either latex beads, Leishmania linked to a decrease of the size of phagosomes (Alvarez- donovani or the intracellular pathogen Brucella abortus have Dominguez and Stahl, 1999). In this case, the overexpressed been documented (Desjardins et al., 1997; Desjardins and native Rab5 protein still displays its GTPase activity and its Descoteaux, 1997; Pizarro-Cerdà et al., 1997). Complex ability to stimulate both fusion and fission. Interestingly, a selective fusion of phagosomes with endosomes containing process of cargo protein concentration by membrane retrieval only small gold particles was ruled out since most of the is also proposed in the biosynthetic pathway (Warren and endosomes contained more than one tracer (see Fig. 4 and Mellman, 1999). Desjardins et al., 1997). A role in the limitation of membrane Other conditions that could reduce the overall microbicidal fusion has been suggested for other Rab proteins. In neurons, activity of vacuoles include alteration of the acidification Rab3 might be involved in modulating the levels of process. It was shown for example that phagosomes containing 3540 S. Duclos and others

Mycobacteria were unable to acidify properly, a condition Alvarez-Dominguez, C., Barbieri, A. M., Berón, W., Wandinger-Ness, A. linked to the absence of the proton pump ATPases responsible and Stahl, P. D. (1996). Phagocytosed live Listeria monocytogenes for endovacuolar organelle acidification (Sturgill-Koszycki et influences rab5-regulated in vitro phagosome-endosome fusion. J. Biol. Chem. 271, 13834-13843. al., 1994). In the present study, we were able to show that Alvarez-Dominguez, C. and Stahl, P. D. (1999). Increased expression of phagosomes from Rab5(Q79L)-expressing cells acquire the A Rab5a correlates directly with accelerated maturation of Listeria and B subunits of the V-ATPase to levels similar to those of monocytogenes phagosomes. J. Biol. Chem. 274, 11459-11462. control cells, indicating that acidification is likely to occur 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 third- normally in both cell types. This goes along previous generation software package for analysis of two-dimensional observations showing that acidification in Rab5 GTP- electrophoresis images: I. Features and user interface. Electrophoresis 18, expressing cells occurs normally (D’Arrigo et al., 1997). 2724-2734. Furthermore, the presence of cleaved forms of cathepsin D, as Araki, N., Johnson, M. T. and Swanson, J. A. (1996). A role for well as an accumulation of LAMP molecules, in phagosomes phosphoinositide 3-kinase in the completion of macropinocytosis and phagocytosis by macrophages. J. Cell Biol. 135, 1249-1260. from control and Rab5(Q79L)-expressing cells indicates that Berón, W., Alvarez-Dominguez, C., Mayorga, L. and Stahl, P. D. (1995). maturation of these organelles is not altered. In contrast, the Membrane trafficking along the phagocytic pathway. Trends Cell Biol. 5, group of Russell (Ullrich et al., 1999) has shown recently that 100-104. Mycobacteria-containing phagosomes accumulate the Berthiaume, E. P., Medina, C. and Swanson, J. A. (1995). Molecular size- fractionation during endocytosis in macrophages. J. Cell Biol. 129, 989-998. unprocessed 51 kDa proform of cathepsin D. Blocker, A., Severin, F. F., Burkhardt, J. K., Bingham, J. B., Yu, H., Olivo, Finally, another interesting finding of our study is that Rab5 J. C., Schroer, T. A., Hyman, A. A. and Griffiths, G. (1997). Molecular was shown to stimulate phagocytosis of serum-opsonized latex requirements for bi-directional movement of phagosomes along beads, but not phagocytosis mediated through the FcR or CR. microtubules. J. Cell Biol. 137, 113-129. Phagocytic uptake by these receptors has been shown to be 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 regulated in part by small GTPases of the Rho family. Indeed, Sci. 111, 303-312. Caron and Hall (1998) have demonstrated recently that Fcγ- Bucci, C., Parton, R. G., Mather, I. H., Stunnenberg, H., Simons, K., induced phagocytosis is mediated by Cdc42 and Rac, while Hofflack, B. and Zerial, M. (1992). The small GTPase rab 5 functions as complement-induced phagocytosis depends on the activity of a regulatory factor in the early endocytic pathway. Cell 70, 715-728. Caron, E. and Hall, A. (1998). Identification of two distinct mechanisms of Rho. Accordingly, our results suggest that Rab5 might act in a phagocytosis controlled by different Rho GTPases. Science 282, 1717-1721. similar way, albeit with receptors others than the FcR or CR, Chavrier, P., Parton, R. G., Hauri, H. P., Simons, K. and Zerial, M. (1990). that have not been identified at this point. Further studies will Localization of low molecular weight GTP binding proteins to exocytic and be required to firmly establish the role of Rab5 in this process. endocytic compartments. Cell 62, 317-329. 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