Reconstitution of recycling from the phagosomal compartment in streptolysin O-permeabilized...

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Research Article

Reconstitution of recycling from the phagosomal compartmentin streptolysin O-permeabilized macrophages: Role of Rab11

Natalia Leiva, Martín Pavarotti, María I. Colombo⁎, María T. Damiani⁎

Instituto de Histología y Embriología, Facultad de Ciencias Médicas, Universidad Nacional de Cuyo-CONICET, Mendoza 5500, Argentina

A R T I C L E I N F O R M A T I O N

⁎ Corresponding authors. Fax: +1 54 261 44941E-mail addresses: [email protected]

0014-4827/$ – see front matter © 2006 Elsevidoi:10.1016/j.yexcr.2006.02.015

A B S T R A C T

Article Chronology:Received 5 September 2005Revised version received10 February 2006Accepted 15 February 2006Available online 24 March 2006

By phagocytosis, macrophages engulf large particles, microorganisms and senescent cells invesicles called phagosomes. Many internalized proteins rapidly shuttle back to the plasmamembrane following phagosome biogenesis. Here, we report a new approach to the study ofrecycling from the phagosomal compartment: streptolysin O- (SLO) permeabilizedmacrophages. In this semi-intact cell system, energy and cytosol are required toefficiently reconstitute recycling transport. Addition of GDPβS strongly inhibits thistransport step, suggesting that a GTP-binding protein modulates the dynamics of cargoexit from the phagosomal compartment. GTPases of the Rab family control vesiculartrafficking, and Rab11 is involved in transferrin receptor recycling. To unravel the role ofRab11 in the phagocytic pathway, we added recombinant proteins to SLO-permeabilizedmacrophages. Rab11:S25N, a negative mutant, strongly diminishes the release of recycledproteins from phagosomes. In contrast, wild type Rab11 and its positive mutant (Rab11:Q70L) favor this vesicular transport event. Using biochemical and morphological assays, weconfirm that overexpression of Rab11:S25N substantially decreases recycling fromphagosomes in intact cells. These findings show the requirement of a functional Rab11for the retrieval to the plasma membrane of phagosomal content. SLO-permeabilizedmacrophages likely constitute a useful tool to identify newmolecules involved in regulatingtransport along the phagocytic pathway.

© 2006 Elsevier Inc. All rights reserved.

Keywords:Recycling from phagosomesRab11Small GTPasesSLO-permeabilized macrophagesPhagocytosis

Introduction

Macrophages and neutrophils internalize large extracellularparticles by a receptor-mediated process known as phago-cytosis [1]. By fusion and fission events, phagosomesremodel themselves and acquire the ability to fuse withlysosomes [2–4]. Internalized particles are digested in theresulting phagolysosomes [5,6].

Even though large amounts of membrane are internalizedduring particle uptake, no apparent decrease in cell surfacearea is observed, suggesting effective mechanisms for the

17.du.ar (M.I. Colombo), tdam

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replenishment of plasma membrane from intracellularsources [7,8]. While transport from endosomes [9], endoplas-mic reticulum [10] and Golgi [11,12] collaborate to replacesome of the internalized plasma membrane; recycling fromthe phagosomal compartment may also be an importantsource of this membrane flux. It has been shown that multiplephagosomal proteins rapidly recycle to the plasmamembranefollowing phagosome formation [13]. Small transport vesiclescarrying phagosomal content and capable of fusing withendosomes contribute to recycling to the plasma membrane[14]. Moreover, tubular regions extending from the phago-

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http://dx.doi.org/10.1016/j.yexcr.2 006.02.015

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somes may also participate in membrane recycling [13].Proteolytically released fragments from ingested particlesmay encounter proteins (e.g. class II major histocompatibilitycomplex) responsible for antigen presentation [15,16]. Indeed,recycling from the phagosomal compartment appears to becrucial for presenting epitopes that are vulnerable to destruc-tion by proteases in the highly acidic late endocytic vesicles[17]. In addition, membrane and protein recycling as well asfission events are relevant to phagosome remodeling [18].Despite the importance of the recycling pathway in phago-some maturation and in the retrieval of immunogenicpeptides from phagosomes, the molecular mechanismsinvolved in recycling from the phagosomal compartmentremain obscure.

It is known that monomeric GTPases of the Rab familyare essential elements of the machinery controlling mem-brane traffic [19–21]. A member of this family, Rab11, isfound on a variety of subcellular membranes, including thepost-Golgi membranes [11,22], exocytic vesicles [23] and theapical tubulovesicles of parietal cells [24]. Rab11 is alsopresent in multivesicular bodies and regulates exosomerelease in an erythroleukemic cell line [25,26]. In non-polarized cells, Rab11 partially co-localized with Rab5 but itis mainly found associated with the pericentriolar recyclingcompartment [27]. Rab25 and Rab11 are also associatedwith the apical recycling system of polarized canine kidneycells [28]. Rab11 modulates transferrin receptor recyclingfrom endosomes [9,29,30] and MHC class I-related FcRnreceptor trafficking [31]. Recently, it has been reported thatRab11 regulates the recycling of CD44 from the Salmonella-containing vacuole without affecting MHC class I recyclingin HeLa cells [32]. By immunoprecipitation and proteomicassays, Rab11 has been detected on phagosomal mem-branes [3,33] and it has been shown to promote phagocy-tosis [9]. In the present study, we have focused on theparticipation of Rab11 in recycling from the phagosomalcompartment.

The lack of experimental models might explain theexistence of so few reports addressing the mechanismsunderlying recycling transport along the phagocytic pathway.We have therefore reconstituted this membrane traffickingstep in permeabilizedmacrophages. By using SLO, a toxin thatforms pores large enough to allow the passage of proteins, wehave permeabilized the plasma membrane. This semi-intactsystem enables the manipulation of the intracellular environ-ment and the incorporation of molecules that normally do notcross the plasma membrane. We have studied the require-ments for reconstituting trafficking along the phagocyticpathway in these semi-intact cells. Here, we show thatrecycling transport from phagosomes in SLO-permeabilizedmacrophages is dependent on energy and exogenouslysupplied cytosol. Addition of GTPγS, a non-hydrolyzable GTPanalogue, stimulates recycling from phagosomes, indicatingthat at least one GTPase regulates this intracellular transportstep. Moreover, our results show the requirement of the smallGTPase Rab11 for an efficient reconstitution of recyclingtransport from the phagosomal compartment in our semi-intact cell system. In this regard, our SLO-permeabilized cellresults are in agreement with our functional and morpholog-ical studies in intact cells showing that Rab11 regulates the

removal of molecules from phagosomes via recycling eventsin macrophages.

Materials and methods

Materials

All chemicals used were of reagent grade and obtained fromSigma-Aldrich (Bs. As., Argentina) unless otherwise stated.Streptolysin O was obtained from S. Bhadki (University ofMainz, Mainz, Germany). Stock solutions of SLOwere preparedat 1 mg/ml (750,000 lytic units/mg) in 0.1% BSA-10 mM HEPES(pH 7.2) and stored at −80°C. Rhodamine-actin was kindlyprovided by Dr. Gareth Griffiths (Cell Biology and BiophysicsProgram, EMBL Heidelberg, Germany). Materials for cellculture were from GIBCO (Invitrogen Argentina S.A.).

Cells

J-774 E-clone and RAW 264.7, two macrophage cell lines, weregrown to confluence in D-MEM supplemented with 10% FCS.The cDNA of Rab11a and its mutants (a generous gift from Dr.David Sabatini, New York University, New York, USA) weresubcloned into the vector pEGFP as fusion proteins with greenfluorescent protein. Macrophages were transfected usingLipofectamine 2000 with pEGFP (control vector), pEGFP-Rab11:wt, pEGFP-Rab11:Q70L (a GTPase deficient mutant)and pEGFP-Rab11:S25N (a GTP-binding defective mutant).Stable transfected cells were selected with geneticin (0.5 mg/ml). The expression of the transfected proteins was visualizedby fluorescence microscopy.

Phagocytic particles

For biochemical studiesFormaldehyde-fixed Staphylococcus aureus (10% cell suspen-sion, 4 × 107 particles/ml, 2 mg of IgG/ml binding capacity)(IgG Sorb, The enzyme center, Malden, MA) were incubatedwith rabbit anti-mouse IgG polyclonal antibody (BethylLaboratories, Inc, Montgomery, Texas) for 1 h at 20°C, theparticles were washed three times with PBS-1%BSA and thenincubated with radio-iodinated monoclonal mouse anti-DNPIgG for 1 h at 20°C. In some experiments, monoclonal mouseanti-Myc antibodies replaced anti-DNP IgG. Coated S. aureuswere washed three times and suspended in PBS-1%BSA. Tolabel the antibody, the dichloramine T method was used[34].

For morphological purposesPolybead® Polystyrene Microspheres (≅3 μm diameter, 2.5%Solids-Latex, Polysciences, Inc., Warrington, PA) were coatedsequentially with bovine serum albumin and rabbit anti-albumin antibodies (Bethyl Laboratories, Inc., Montgomery,Texas) followed by Texas red mouse anti-rabbit IgG (H + L)conjugate (2 mg/ml, highly cross-adsorbed, Molecular Probes,Eugene, OR) or FITC-labeled mouse anti-rabbit IgG (BethylLaboratories, Inc, Montgomery, Texas) as indicated [35,36].Coated beads were washed three times and suspended inPBS.

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Biochemical recycling assay

To study recycling from the phagosomal compartment, apreviously described assay was used [13,37]. Briefly, 1 × 106

J-774 macrophages were suspended with ≈20 phagocyticparticles per cell (S. aureus coated with rabbit anti-mouseIgG and 125I-mouse anti-DNP IgG) and incubated for 20 minat 4°C for binding and then transferred for 15 min to 37°C toallow internalization. Afterwards, cells were washed exten-sively and trypsinized at 4°C to remove non-internalizedparticles. Cells loaded with the phagocytic marker wereincubated at 37°C for different periods of time to allow therecycling process to proceed. During phagosome matura-tion, some of the antibodies were dissociated from theinternalized particles and were transported by a vesicle-mediated process to the cell surface and finally releasedinto the efflux media. After the recycling time, cells werecentrifuged and supernatants were treated with 10% tri-chloroacetic acid (TCA). The time-dependent appearance ofacid-precipitable phagocytosed 125I-anti DNP IgG into theefflux media was indicative of recycling whereas solubleradioactivity was a consequence of the degradation pro-duced in the phagosomes. Results were expressed as thepercentage of precipitated (recycled IgG) cpm to total (effluxand cell-associated) cpm.

Morphological recycling assay

RAW 264.7 macrophages grown on 24 mm coverslips wereloaded with the beads opsonized with fluorescent antibodies(see Phagocytic Particles). After 15 min of uptake at 37°C, cellswere cooled on ice to stop phagocytosis and the externalbeads were washed away with cold PBS. Cells were thenwarmed to 37°C to allow the recycling to proceed. During thechase, the fluorescent antibodies dissociated from the beadsin a time-dependent manner and were released to themedium. Thus, by recycling, beads lose the fluorescentantibodies that decorate their surface and this event wasassessed by confocal microscopy. Images were collected on aNikon C1 laser scanning confocal unit (Nikon D-Eclipse C1,Melville, NY) attached to an upright fluorescence microscope(Nikon Eclipse E600, Melville, NY) with a 100× 1.4 planApochromat objective (Nikon, Melville, NY). Excitation onthe Nikon C1 laser confocal microscope was with an air-cooled Argon laser emitting 488 nm (manufactured bySpectra Physics, Model 161C-030) and a Helium/Neon laser543 nm output (manufactured by JDS Uniphase, model1674P). Appropriate filter sets were used to collect fluoro-phore emissions. Conditions were set to avoid photo bleach-ing. Images were acquired digitally and processed using theoperation software EZ-C1 for Nikon C1 confocal microscope(Nikon, Melville, NY).

Permeabilized cell assay

For each assay, 3 × 105 J-774 macrophages were grown in 12-well plates to approximately 70–80% confluence at one daypost-plating. The buffer used to perform the phagocytosisassay was PBS supplemented with 1% BSA, pH 7.4. The cellsinternalized S. aureus sequentially coated with rabbit anti-

mouse IgG polyclonal antibody and radio-iodinated monoclo-nal mouse anti-Myc IgG for 15 min at 37°C. Cells were thencooled at 4°C, washed extensively with PBS, trypsinized andsubsequently washed twice with cold Binding Buffer (BB)(115 mM KAc; 25 mM HEPES pH 7.2; 0.5 mM MgCl2; 0.9 mMCaCl2) and incubated with 200 μl/well 7.5 μg/ml SLO for 7 minon ice. Excess SLO was removed with BB-DTT buffer (BB with1 mM DTT) and the cells were warmed to 37°C for 2 min toallow pore formation in the presence of HomogenizationBuffer (HB) (20 mM HEPES pH 7.2; 0.25 mM sucrose). SLOinserts into membranes at 4°C but only forms pores at 37°C inthe presence of a reducing environment. The wash out ofunbound SLO toxin before incubation at 37°C ensured thatonly the plasma membrane was permeabilized [38]. Cellswere checked by light, confocal and electron microscopy formorphological characteristics of SLO permeabilization. Fol-lowing permeabilization, the cells were placed on ice andincubated in fresh HB ice-cold buffer for 10 min to removeendogenous cytoplasmic proteins. Subsequently, 200 μl of gel-filtered cytosol (2 mg/ml) in fusion buffer (FB) (115 mM KAc;25 mM HEPES pH 7.2; 2.5 mM MgCl2; 2.5 mM CaCl2; 5 mMEGTA; 1 mM DTT) and an ATP regenerating solution (1 mMATP, 8 mM creatine phosphate and 40 units/ml creatinephosphokinase) were added to the cells to reconstituterecycling from phagosomes. During the recycling incubation,the participation of GTP binding proteins was assessed by theaddition of GTPγS (50 μM) or GDPβS (50 μM) in order to lock theGTPases in a GTP or GDP state, respectively. The effect of Rabson recycling was explored by adding purified recombinantprenylated proteins to the wells at a concentration of 300 nM.Transferring permeabilized cells to a 37°C water bath restoredrecycling transport. After 15 min of incubation, the effluxmedium was collected and treated with 10% TCA to precip-itate recycled antibodies. The precipitated radioactivity (effluxIgG) and soluble radioactivity (degraded peptides) weremeasured. The cells were solubilized in 1% Triton X-100 toassess the remaining intracellular radioactivity. Recycledradioactivity (acid precipitable cpm) was compared to totalphagocytosed radioactivity (precipitable and soluble cpmreleased to the efflux media plus cpm remaining cellassociated). In some experiments, results were expressed asa percentage of recycling in control conditions (cytosol plusan ATP-regenerating system added to SLO-permeabilizedmacrophages).

Cytosol preparation

Bovine brains were washed and homogenized in a Douncehomogenizer in the presence of HB buffer containing aprotease inhibitor mixture (10 μM Leupeptin, 12 mM 1,10-o-phenanthroline, 0.5mMbenzamidine, 2 μg/ml soybean trypsininhibitor). The homogenate was centrifuged at 100,000 × g for1 h. The supernatant was assayed for protein concentrationusing the Bradford method with bovine serum albumin (BSA)as a standard (Pierce, Rockford, IL). The cytosol was frozen inliquid nitrogen and stored at −80°C until used. The averageconcentration of cytosol was approximately 4 mg/ml. Sepha-dex G-25-300 (Pharmacia Biotech Inc, Piscataway, New Jersey)columns washed sequentially with PBS, PBS-1%BSA andFusion Buffer (FB) were used for cytosol gel filtration [39].


Enzymatic activity

Lactate dehydrogenase (LDH) was quantified using pyruvateand NADH,H+ as substrates. The formation of NAD+ reflectedthe enzymatic activity and was measured spectrophotometri-

Fig. 1 – Reconstitution of recycling transport from the phagosomplasma membrane was permeabilized using increasing amountassessed by the release of the cytosolic enzyme lactate dehydroN-acetylglucosaminidase (NAG) in the efflux medium checked inpercentage of enzymatic activity found in the wash out medium cintracellular enzyme). The graph shows the curve that best fits th(B) The recycling assay was performed in J-774 macrophages asloaded with the phagocytic marker (S. aureus sequentially coatedantibodies). After 15 min of uptake, cells were exhaustively waspermeabilizedwith SLO and recycling transport was reconstituteregenerating system. Recycling was assessed by measuring theafter 15 min of incubation at 37°C. Results were expressed as a pinternalized. The graph shows the curve that best fits data fromreconstituted by addition of 2 mg/ml cytosol (Cy) and, as indicateATP-depleting mixture (DS) (see Materials and methods). Maximcytosol and ATP. Recycled IgG cpm released to the efflux mediumpercentage of total internalized cpm (values represent means ± Scomparison tests).

cally at 340 nm (Shimatzu UV160 spectrophotometer, KyotoCorporation, Japan). N-acetilglucosaminidase (NAG) was mea-sured by fluorescence spectrophotometry using methylum-belliferyl N-acetilglucosamine in citrate buffer 0.4 M pH 4.5 assubstrate (λ excitation: 360 nm and λ emission: 460 nm)

al compartment in SLO-permeabilizedmacrophages. (A) J-774s of SLO. The washout of endogenous compounds wasgenase (LDH). Measuring the intralysosomal enzymetracellular membranes integrity. Data were expressed as theompared to total enzymatic activity (released plus remainingemeans± SEM from three experiments conducted separately.described under Materials and methods. Basically, cells werewith rabbit anti-mouse and radio-iodinated mouse anti-Myc

hed to separate non-internalized particles. Next, cells wered in the presence of increasing amounts of cytosol and an ATPacid-precipitable radioactivity released into the efflux mediaercentage of the total phagocytic marker effectivelytwo independent experiments. (C) Recycling transport wasd in the figure, an ATP regenerating system (RS) or anal reconstitution of transport was achieved in the presence of

during 15 min of incubation at 37°C was expressed as theEM, n = 6, *P < 0.05, one-way ANOVA and Dunnett multiple


(Packard Fluorocount microplate reader, Hewlett-Packard).Enzymatic activity found in the extracellular media and thatremaining cell-associated constituted the total enzymaticactivity. Enzyme released into the efflux media was comparedto the total enzymatic activity andwas indicative ofmembranepermeabilization.

Purification of recombinant proteins

Rab11:wt, Rab11:Q70L and Rab11:S25N as well as Rab5:wt,Rab5:Q79L and Rab5:S34N subcloned in the pGEX plasmidvector were expressed as GST fusion proteins in BL21 E.coli (Amersham Pharmacia Biotech, Piscataway, New Jer-sey). Protein synthesis was induced by the addition of1 mM isopropyl-β-D-thiogalactopyranoside (IPTG) (Promega,Madison, WI) for 18 h at 25°C. Proteins were purifiedusing glutathione–sepharose 4B (Amersham PharmaciaBiotech, Piscataway, New Jersey) and stored at −80°Cuntil used. To be functional, these proteins had to beprenylated by incubation with 100 μM geranylgeranylpyr-ophosphate, 4 mg/ml filtered cytosol, 200 μM GDP, 1 mMDTT, 2 mM MgCl2, 1.5 mg/ml BSA and 1 mM PMSF for90 min at 37°C.

Fig. 2 – Visualization of plasma membrane permeabilization anRAWmacrophages were loadedwith 3 μmbeads coated sequentanti-rabbit antibodies. After 15min of uptake, RAW cells were permethods. Recycling transport was reconstituted in the presencetymosin-β4 (25 μM) and ATP (200 μM) for 15 min. The effectivenexogenously added actin. Fluorescent actin polymerized inside ccortex typical of macrophages (A and D). Confocal images showinternalized beads (B and E) (white arrowheads). Insets show a m

Results

Reconstitution of vesicular recycling transport fromphagosomes in SLO-permeabilized macrophages

In order to investigate the molecular machinery involved inrecycling from the phagosomal compartment, we havedeveloped an in vitro reconstitution assay using SLO-permea-bilized macrophages. For this purpose, cells were allowed tointernalize for 15 min the phagocytic probe (fixed S. aureussequentially coated with rabbit anti-mouse IgG and radio-iodinatedmouse anti-Myc antibodies) and chased for differentperiods of time. During the chase time, a fraction of 125I-mouseanti-Myc IgG detached from phagocytosed particles wasreleased from maturing phagosomes and was recovered inthe efflux media. The presence of radio-labeled antibodies inthe media was indicative of recycling from the phagosomalcompartment. Immediately after phagocytosis and before thechase (recycling period), plasma membrane pores were madeusing streptolysin O (see Materials and methods). Severalconsiderations have to be taken into account at this point:recycling occurs early during phagosome maturation and this

d transport from phagosomes in SLO-treated macrophages.ially with albumin, rabbit anti-albumin and FITC-labeled goatmeabilized using SLO toxin as described under Materials andof a buffer containing rhodamine-labeled G-actin (8.8 μM),ess of permeabilization was tested by the incorporation ofells mimicking endogenous actin, thus forming the actinvesicles carrying FITC-labeled antibodies used to decorateagnification of phagosome area. Scale bars = 3 μm.

Fig. 3 – GTPases regulate recycling from the phagosomalcompartment in a semi-intact cell system. The recyclingassay was performed as indicated in Fig. 1. Recyclingtransport was reconstituted in the presence of cytosol(2 mg/ml) and an ATP regenerating system (RS) (open bar).Addition of GDPβS (50 μM) (black bar) or GTPγS (50 μM)(diagonal hatch bar) was used to assess the participation ofGTP-binding proteins on recycling from phagosomes. Theacid-precipitable radioactivity released into the efflux mediawas indicative of recycling. Results were normalized torecycling in the presence of cytosol and an ATP-regeneratingsystem. Experiments were done in duplicate and datarepresent means ± SEM (n = 4, *P < 0.05, one-way ANOVA andDunnett multiple comparison tests).


transport event is temperature-dependent. For this reason,recycling from the phagosomal compartment was blockedduring the permeabilization step by cooling the cells at 4°Cand activating the SLO toxin for only 2 min at 37°C.

To determine the amount of SLO necessary to perforatethe plasma membrane without affecting intracellular orga-nelles, we monitored the release of a cytosolic enzyme.Estimations based in the release of lactate dehydrogenaseindicated that at least 80% of the cells were permeabilizedunder our working conditions. Measuring the leakage of thelysosomal enzyme N-acetylglucosaminidase assessed theintegrity of the intracellular membranes. At the SLO concen-tration chosen, less than a 3% of the intraorganellar enzymewas released (Fig. 1A).

The reconstitution of recycling transport in the SLO-permeabilized macrophage system was dependent on thepresence of exogenously supplied cytosol. We observed thattransport increased with protein cytosol concentration untilreaching a plateau (Fig. 1B). We also determined if thereconstitution of recycling was temperature-dependent. Forthis purpose, we measured recycling in both permeabilizedand intact cells kept at 4°C during the 15 min chase. Therelease to the efflux media of recycled peptides at 4°C inintact cells was only 9.62% of the amount recycled at 37°C,whereas in SLO-permeabilized cells there was only a slightincrease to 12.64% of the total amount recycled at 37°C inthis semi-intact system. This clearly indicates that recy-cling is hampered at 4°C in both intact and permeabilizedcells. Next, we analyzed the energy requirement of thisintracellular trafficking event. As expected, addition of anATP-regenerating system was required for efficient trans-port reconstitution. The removal of ATP using an ATP-depleting system resulted in inhibition of recycling fromphagosomes, reflecting the general requirement of ATP formembrane traffic (Fig. 1C). Addition of ATP to permeabi-lized macrophages in the absence of gel-filtered cytosolresulted in a recycling rate similar to that achieved in thepresence of buffer alone (data not shown). In summary, ourresults demonstrate that recycling from the phagosomalcompartment is temperature- and energy-dependent andreliant on exogenously supplied cytosol, in agreement withrequirements observed for in vitro reconstitution of othermembrane trafficking events.

Next, we used confocal microscopy to confirm plasmamembranepermeabilizationwithout affecting recycling trans-port fromphagosomes. Briefly,macrophageswere loadedwith3 μm beads sequentially coated with bovine serum albuminand rabbit anti-albumin antibodies followed by FITC-labeledmouse anti-rabbit IgG, and then cells were treated with SLO asdescribed in Materials and methods. After the permeabiliza-tion step, we added rhodamine-coupled G-actin (8.8 μM) to oursystem in a buffer containing tymosin-β4 (25 μM) and ATP(200 μM). The SLO treatment permeabilized the plasmamembrane enough to allow exogenous fluorescent actin tobe incorporated into the cytoskeleton in a pattern mimickingendogenous actin. Confocal images showed that the actinreached the cell interior and polymerized to form the actincortex typical of macrophages (Figs. 2A and D). These semi-intact cells retain the ability to form vesicles. Images showsmall vesicles carrying the fluorescent antibodies used to

opsonize phagocytosed particles (Figs. 2b and e, see insets).The SLO-permeabilized macrophages constitute a versatilesystem that allows intracellular environment manipulationand facilitates the study of protein or nucleotide effects onrecycling from the phagosomal compartment.

Rab11 is required for recycling from phagosomes inpermeabilized cells

Studies performed in SLO-permeabilized macrophages conferthe ability to examine the effect of molecules specifically onthe recycling transport step. In these assays, particle uptakewas carried out under equivalent conditions for the totality ofthe cells, and then the plasma membrane was perforated forthe incoming of reagents. This semi-intact system enablesdissociated analysis of the role played by a molecule onphagocytosis and on the recycling pathway from the phago-somal compartment.

To address the participation of GTP binding proteins inrecycling from phagosomes, we assayed the effect of guano-sine nucleotides in our permeabilized system. Addition ofGTPγS, a non-hydrolyzable analogue of GTP, enhanced recy-cling from the phagosomal compartment; whereas GDPβS, anucleotide that cannot bind another phosphate, significantlyreduced this vesicle transport (Fig. 3). These findings clearly


suggest that a GTP-binding protein regulates vesicle recyclingfrom the phagosomal compartment.

By proteomics and immunoprecipitation assays, the smallGTPase Rab11 has been detected on phagosomal membranes[3,33]. Moreover, Greenberg and collaborators have shown thatRab11 promotes phagocytosis by increasing membrane avail-ability via enhanced endosome recycling [9]. Therefore, wefocused on the involvement of Rab11 in membrane retrievalfrom the phagosomal compartment. To investigate the partic-ipation of Rab11 in this membrane transport step, we added toour systempurified recombinant proteins. As shown in Fig. 4A,addition of prenylatedRab11:wt or Rab11:Q70L (the active formof Rab11) increased recycling traffic from the phagosomalcompartment in SLO-permeabilizedmacrophages. In contrast,the presence of Rab11:S25N (the inactive mutant of Rab11)significantly diminished recycling. Interestingly, Rab11 pro-teins did not significantly affect the degradation pathway inmacrophages (Fig. 4B). The amount of internalized radioactiv-ity degraded in the presence of Rab11 was insignificant (lessthan 2% of total phagocytosed IgG). The effect on recycling

from the phagosomal compartment was specific to Rab11,since Rab5 did not modify this transport step. Addition ofprenylated Rab5:wt and especially its positive mutant (Rab5:Q79L) to SLO-permeabilized macrophages increased degrada-tion of the internalized particles to almost the double of thevalues observed in the presence of Rab11. On the contrary,Rab5:S34N, themutant locked in theGDP-bound state, reducedthe degradation occurring in early phagosomes (Fig. 4D). Asphagosome matures recycling process reaches a plateau,whereas degradation increases during chase time. Further-more, neither Rab5:wt nor its mutant forms significantlyaffected recycling from phagosomes (Fig. 4C). In agreementwith these results, it has recently been shown that Rab5 is notrequired for the sealing of recycling vesicles departing fromendosomes in a permeabilized cell system [40].

In summary, our results indicate that, as for othermembrane transport events, reconstitution of the recyclingpathway from phagosomes is GTP sensitive. Moreover, Rab11specifically regulates recycling transport from the phagosomalcompartment in SLO-permeabilized macrophages.

Fig. 4 – A functional Rab11 is required for recycling transportof phagocytosed proteins in permeabilized cells. Briefly, J-774macrophages (3 × 105 cells) internalized pre-boundS. aureus-125I-anti-Myc IgG for 15 min at 37°C. Cells werewashed several times with ice cold buffer and thenpermeabilized using SLO, as described in Fig. 1. After thewashout of endogenous cytosol, transport was reconstitutedat 37°C in the presence of gel-filtered donor cytosol, an ATPregenerating system and recombinant purified Rab proteins(300 nM) for 15 min. The acid-precipitable radioactivitymeasured in the efflux media indicated recycling fromphagosomes whereas the soluble radioactivity released tothemedia reflected degradation occurring in phagosomes. (Aand B) Macrophages were incubated with prenylated Rab11:wt (open bar), the GDP-bound form (Rab11:S25N) (black bar) orthe GTP-bound mutant (Rab11:Q70L) (diagonal hatch bar) for15 min at 37°C to allow phagosome maturation. Panel Ashows the recycled acid-precipitable radioactivity measuredin the efflux media expressed as a percentage of recycling inthe presence of cytosol and an ATP-regenerating system.Panel B shows the soluble radioactivity released into theextracellular media as a consequence of degradation inphagosomes normalized to degradation in the presence ofcytosol and an ATP-regenerating system. Both figures showthe means ± SEM of five independent experiments (*P < 0.05,one-way ANOVA and Dunnett multiple comparison tests). (Cand D) Permeabilized macrophages were incubated duringrecycling with purified prenylated proteins at 300 nM finalconcentration: Rab5:WT (open bar), GDP-bound Rab5 (Rab5:S34N) (black bar) and GTP-bound Rab5 (Rab5:Q79L) (diagonalhatch bar). Panel C shows acid-precipitated radioactivity(recycled IgG) expressed as a percentage of recycling in thepresence of cytosol and an ATP-regenerating system. Panel Dshows soluble radioactivity found in the efflux media(degraded peptides) normalized to degradation in thepresence of cytosol and ATP. Results are expressed asmeans ± SEM (n = 4, *P < 0.05, one-way ANOVA and Dunnettmultiple comparison tests).


Rab11 modulates recycling from the phagosomalcompartment in intact macrophages

We were next interested in determining the participation ofRab11 inmembraneandprotein retrieval fromthephagosomalcompartment in intact cells. To this end, macrophages over-expressing the wild type GTPase and its mutants were used.Basically, recycling from phagosomes was assessed by mea-suring the time-dependent appearance of phagocytosed 125I-anti-DNP IgG in the efflux media [13,37]. S. aureus, coated withrabbit anti-mouse antibody and 125I-mouse anti-DNP IgG, werephagocytosed by J-774 macrophages. As phagosomes mature,some IgG molecules dissociate from the internalized particlesand recycle to the cell surface via vesicular transport. As aconsequence, during the chase time, an increasing amount ofradioactivity was recovered in the effluxmedia.Within 15minof incubation, themajority of the intracellular radioactivity, aswell as that in the medium, was TCA precipitable, indicatingthat the anti-DNP IgGmolecules recycle almost intact (data notshown).We therefore conclude that the antibodies themselveswere resistant to degradation. We used macrophages over-expressing GFP-Rab11:wt, the negative mutant locked in theGDP-bound form (GFP-Rab11:S25N) and the active mutant inthe GTP-bound state (GFP-Rab11:Q70L). Fig. 5A shows thatoverexpression of the dominant negative mutant of Rab11significantly diminished recycling from phagosomes mea-sured as the time-dependent release of radio-iodinatedprotein. Concomitantly, in cells overexpressing the dominantnegative mutant of Rab11, radio-iodinated IgG within cellsincreased markedly (Fig. 5B). As has been shown [9], phagocy-tosis was enhanced in macrophages overexpressing Rab11:wtor its positive mutant (Rab11:Q70L) (Fig. 5C). The increase inparticle uptake was comparable to that achieved by over-expression of Rab5:wt or GTP-boundRab5 (Rab5:Q79L) (Fig. 5D).

RAW macrophages overexpressing Rab11:wt and itsdominant negative Rab11:S25N and positive Rab11:Q70Lmutants fused to the green fluorescent protein (GFP, green)

Fig. 5 – Rab11:S25N inhibits recycling from phagosomes in intacinternalized pre-bound S. aureus-125I-anti-DNP IgG for 15 min atthen incubated at 37°C for the periods of time indicated in the figperformed as described under Materials and methods. The acid-indicated recycling from the phagosomes. In panel A, the radioapercentage of the total radioactive marker internalized by the mindependent experiments. The curve slopes were calculated usianalyzed using one-way ANOVA and Newman Keuls multiple coRab11:S25N produced a significant decrease in the kinetics of recyPanel B shows the percentage of radioactivity remaining inside tfive independent experiments (*significant differences from contcomparison tests). (C) J-774 macrophages (1 × 106 cells) transfectpEGFP-11:wt (open bar), the GDP-bound mutant pEGFP-Rab11:S2(diagonal down hatch bar) internalized pre-bound S. aureus-125IAfterwards, cells were exhaustively washed several times withRadioactivity that remained cell-associated was indicative of phamarker offered to cells. The figure shows themeans ± SEMof five ecomparison tests). (D) Phagocytosis assay was performed as indtransfected with pEGFP (vector plasmid) (diagonal up hatch bar),S34N (black bar) and the GTP-bound formpEGFP-Rab5:Q79L (diagoand normalized to total radioactive phagocytic particles offered texperiments (*P < 0.05, one-way ANOVA and Dunnett multiple c

were analyzed by confocal microscopy. These cells wereloaded with beads sequentially coated with bovine serumalbumin, rabbit anti-albumin IgG and Texas red labeledmouse anti-rabbit antibodies (red) as described in Materialsand methods [35,36]. Images were obtained after 15 minchase. Rab11:wt (Fig. 6a) displays a vesicular punctuatepattern throughout the cytoplasm. Some Rab11-positivediscrete patches labeling the phagosomal membranes wereobserved (see arrow). Small vesicles containing red fluores-cent antibodies detached from the phagocytosed beads werealso depicted (panel b). The GDP-bound mutant Rab11:S25N(panel d) was both cytosolic and membrane-associated. Thismutant seems to be concentrated at the perinuclear region.Neither association to the phagosomal compartment nor tovesicular structures was observed. In contrast, the GTP-bound form Rab11:Q70L (panel g) was mainly associated tomembranes. Some enlarged vesicles, probably formed byfusion between vesicles departed from the phagosomalcompartment (carrying red fluorescent antibodies used tocoat phagocytosed beads) and early endosomes were ob-served (see panel h and arrowheads). It is likely that thesevesicles cannot fuse with the plasma membrane to releasetheir content to the extracellular media. It has been shownthat GTP hydrolysis is required for the exit of Rab11- andtransferrin receptor-positive vesicles from the pericentriolarrecycling compartment but not from sorting endosomes inTRVb cells [30]. Furthermore, we have demonstrated thatoverexpression of the GTP-bound mutant of Rab11 in anerythroleukemic cell line generates large multivesicularbodies. However, the release of the internal vesicles to theextracellular media was inhibited indicating that fusion ofthe multivesicular bodies with the plasma membrane washampered [25]. These observations could explain the pres-ence of the large vesicles containing antibodies detachedfrom internalized beads in macrophages overexpressingthe GTP-bound form of Rab11 (Figs. 6G–I) without acorresponding increase in the biochemical recycling data

t cells. J-774 transfected-macrophages (1 × 106 cells)37°C. Cells were washed several times with cold buffer andure. Recycling from the phagosomal compartment wasprecipitable radioactivity measured in the efflux mediactivity recycled into the efflux media was expressed as aacrophages. The figure shows the means ± SEM of fiveng linear regression analysis. Comparison of the slopes,mparison tests, indicated that overexpression of the mutantcling transport from the phagosomal compartment (*P < 0.05).he cells after recycling. The figure shows the means ± SEM ofrol at P < 0.05, one-way ANOVA and Dunnett multipleed with pEGFP (vector plasmid) (diagonal up hatch bar),5N (black bar) and the GTP-bound form pEGFP-Rab11:Q70L-anti-Myc IgG (≈20 particles per cell) for 15 min at 37°C.ice cold buffer to remove non-internalized particles.gocytosis and expressed as a percentage of total phagocyticxperiments (*P < 0.05, one-wayANOVAandDunnettmultiple

icated for panel C using J-774 macrophages (1 × 106 cells)pEGFP-5:wt (open bar), the GDP-bound mutant pEGFP-Rab5:nal downhatch bar). Intracellular radioactivitywasmeasuredo cells. Results represent means ± SEM of five independentomparison tests).


(Fig. 5A). Taken together, these results show that Rab11 actsalong the phagocytic pathway by regulating the efflux ofmembrane and proteins from the phagosomal compartmentto the cell surface.

Discussion

Phagocytosis and phagosome biogenesis involve a series ofmembrane fusion and budding events [2,4]. During the processof phagosome maturation, new proteins are added to phago-somes, either by recruitment from the cytosol or from fusionwith organelles of the endocytic pathway, and other mole-cules are removed from phagosomes via recycling events.

Protein and membrane recycling from the phagosomalcompartment are also important for antigen presentation.

We have previously explored some of the molecularcomponents involved in regulating recycling transport fromphagosomes in intact cells. We have shown that hetero-trimeric G proteins regulate the recycling pathway from thephagosomal compartment [37]. We have also reported thattransport of recycling vesicles requires an adequate balancebetween both microfilament- and microtubule-dependentcytoskeleton [35]. Moreover, we have recently shown that arab coupling protein (RCP) is required for efficient proteinand membrane recycling from the phagosomal compart-ment in macrophages [36]. However, further reports de-scribing the molecular machinery implicated in recycling

Fig. 6 – GTP-bound Rab11 is associated as discrete patches to early phagosomes. RAW 264.7 macrophages overexpressingRab11 and its mutant proteins fused to the green fluorescent protein (GFP) were analyzed by confocal microscopy (leftpanels). Rab11:wt (A) displays a punctuate vesicular pattern throughout the cell. Some restrained patches of Rab11 arefound associated to early phagosomes (see arrow). In contrast, Rab11:S25N (the negative mutant) is mainly cytosolic andconcentrates at the perinuclear region (D). The GTP-bound form of Rab11 (Rab11:Q70L) (G) is associated to membranes andlarge vesicles (see arrowheads). Middle panels show internalized beads decorated with albumin, rabbit anti-albumin IgGand Texas red-labeled mouse anti-rabbit antibodies (as described in Materials and methods) and vesicles containing redfluorescent antibodies detached from phagocytosed beads. Merged images are shown at the right panels. Images weretaken after 15 min of phagocytosis and 15 min of chase at 37°C. Images are representative of at least six independentexperiments. Scale bars = 3 μm.


from phagosomes have been hampered by the lack oftechniques that specifically study this transport step. Here,we introduce an approach to the reconstitution of recyclingtransport from the phagosomal compartment in SLO-per-meabilized macrophages. This method enables addressingquestions regarding this membrane transport step by allow-ing control of the intracellular environment. Many vesicle-mediated membrane-trafficking steps have been reconsti-tuted in cell-free and semi-intact cell systems. However,

recycling from the phagosomal compartment presents addi-tional technical difficulties. One of the major drawbacks withreconstituting recycling from phagosomes in permeabilizedcells is that this transport step occurs soon after phagocyto-sis. Reported standard protocols have to be adjusted to avoidrecycling during the permeabilization step. In our experi-ments, we optimized the amount of SLO required to formpores that allow the entry of large molecules with a veryshort enzyme warming activation time. Under our working


conditions, SLO-induced permeabilization and cytosol deple-tion occur fast. The use of lower SLO concentrations requiredlonger enzyme activation at 37°C and significantly reducedthe efficiency in recycling transport reconstitution. Weconfirmed effective plasma membrane permeabilization byshowing rapid entry into the cells of fluorescent-labeled G-actin visualized by confocal microscopy. Using our experi-mental protocols, we also observed the presence of vesiclescontaining the fluorescent antibodies used to opsonize theinternalized beads. These vesicles are likely formed by fusionbetween vesicles departed from the phagosomal compart-ment carrying the phagocytosed fluorescent antibodies andcompartments of the endocytic pathway. Thus, SLO-treatedmacrophages retained the ability to recycle molecules fromphagosomes by vesicle-mediated transport. Therefore, per-meabilized cell systems are a valuable experimental linkbetween in vitro cell free studies and in vivo assays on intactcells. The development of this new versatile system to studymembrane trafficking will likely facilitate the description ofnew molecules important for regulation of recycling alongthe phagocytic pathway.

Next, we investigated the requirements to reconstituterecycling transport from the phagosomal compartment. Therecycling rate was dependent on the concentration ofexogenously supplied cytosol until reaching a plateau. Thecytosol provides soluble factors required for faithfully retrievalof recycled IgG from phagosomes to the plasma membrane.Our results showed that recycling transport from the phago-somal compartment was an ATP-dependent process. As hasbeen described for other membrane transport steps recon-stituted in cell free and semi-intact cell systems, energy is ageneral requirement for intracellular trafficking [41].

To address the effect of guanosine nucleotides on thisvesicle transport, we incubated permeabilized macrophageswith thenon-hydrolyzable analogueofGTPorGDPβS.Additionof GDPβS substantially inhibited recycling from the phagoso-mal compartment, whereas GTPγS stimulated this transportstep. Previous reports have shown that multiple steps duringvesicle trafficking are GTP-dependent [42]. In neuroendocrineand mast cells, GTPγS stimulates exocytosis, although hydro-lysis of GTP is necessary for adequate membrane transport[41,43]. Reconstituted transferrin recycling from endosomes ina permeabilized cell system is also enhanced by GTPγS,indicating that GTP hydrolysis is not strictly required [44].Moreover, GTPγS stimulates in vitro endosome–endosomefusion only in the presence of suboptimal amounts of addeddonor cytosol [34,45,46]. In contrast, addition of GTPγS reducesendosome to TGN retrograde transport [47] and transport fromthe TGN to the cell surface[48]. Our findings assign a role ofGTP-binding proteins in controlling the shuttle of moleculesfrom phagosomes back to the plasma membrane.

It is well known that members of the Rab family, smallGTPases that control membrane traffic, are important indelivering vesicles to the appropriate intracellular targets andin the regulation of this process [19–21]. Increasing evidencesuggests that at least some Rabs may have a role in vesicleformation, as well as in docking and fusion [19,49]. Theinvolvement of common components in vesicle formationand docking/fusion events may provide a means to ensure aclose coupling between these processes. This phenomenon

would be particularly important in the phagocytic pathway,where rapid plasma membrane internalization in the form ofphagosomes must be followed by a compensatory retrieval ofmembrane in order to prevent the reductionof cell surface areaand the cessation of uptake of the phagocytic particles. Themaintenance of membrane organization is strikingly impor-tant during high levels of membrane flux. Rab proteins,recruiting different effectors molecules, may act as thefunctional link between coupled processes. To assess theeffect of Rab proteins on recycling transport separately fromthe internalization process, we use the SLO-permeabilizedmacrophage system. Phagocytosis is performed under identi-cal conditions for all cells prior to the perforation of the plasmamembrane to allow the passage of proteins. By this means,Rabs can be added to these semi-intact cells during recycling.Our experiments with SLO-permeabilized macrophages dem-onstrated the requirement for functional Rab11 for recyclingalong the phagocytic pathway, given that addition of purifiedRab11:S25N significantly reduced this membrane traffickingevent. In agreement with these data, our results in intact cellsshowed that overexpression of Rab11:S25N, a dominantnegative mutant preferentially locked in the GDP-boundstate, also significantly decreased recycling from the phago-somal compartment. These studies point to a function for thesmall GTPase Rab11 in regulating vesicular recycling transportfrom the phagosomal compartment. It has been shown thatRab11 promotes phagocytosis by enhancing endosomal mem-brane recycling [9]. Our results suggest that Rab11 additionallycontrols phagocytosis by regulating membrane recycling fromthe phagosomal compartment. Furthermore, we present thefirst evidence of a Rab protein regulating recycling from thephagosomal compartment in professional phagocytes. Futurestudies using the reconstituted recycling assay in permeabi-lized and in intact macrophages will provide further insightinto themolecular mechanisms regulating recycling transportfrom the phagosomal compartment.

Acknowledgments

We thank Dr. Mark Kuehnel and Dr. Gareth Griffiths forsuggestions and helping with the Rhodamine-actin experi-ments. We are grateful to Dr. Walter Berón for helpful adviceand to Dr. Luis Mayorga for critically reading the manuscript.This work was partially supported by Sepcyt, UJAM, PIP andPICTs 2002 # 1-11004 grants to M.I.C. and to M.T.D.

R E F E R E N C E S

[1] A. Aderem, D.M. Underhill, Mechanisms of phagocytosis inmacrophages, Annu. Rev. Immunol. 17 (1999) 593–623.

[2] M. Desjardins, L.A. Huber, R.G. Parton, G. Griffiths, Biogenesisof phagolysosomes proceeds through a sequential series ofinteractions with the endocytic apparatus, J. Cell Biol. 124(1994) 677–688.

[3] M. Desjardins, J.E. Celis, G. vanMeer, H. Dieplinger, A. Jahraus,G. Griffiths, L.A. Huber, Molecular characterization ofphagosomes, J. Biol. Chem. 269 (1994) 32194–32200.

[4] W. Beron, C. Alvarez-Dominguez, L. Mayorga, P.D. Stahl,


Membrane trafficking along the phagocytic pathway, TrendsCell Biol. 5 (1995) 100–104.

[5] T.E. Tjelle, T. Lovdal, T. Berg, Phagosome dynamics andfunction, Bioessays 22 (2000) 255–263.

[6] O.V. Vieira, R.J. Botelho, S. Grinstein, Phagosome maturation:aging gracefully, Biochem. J. 366 (2002) 689–704.

[7] I. Mellman, Quo vadis: polarized membrane recycling inmotility and phagocytosis, J. Cell Biol. 149 (2000) 529–530.

[8] A. Aderem, How to eat something bigger than your head, Cell110 (2002) 5–8.

[9] D. Cox, D.J. Lee, B.M. Dale, J. Calafat, S. Greenberg, ARab11-containing rapidly recycling compartment inmacrophages that promotes phagocytosis, Proc. Natl. Acad.Sci. U. S. A. 97 (2000) 680–685.

[10] E. Gagnon, S. Duclos, C. Rondeau, E. Chevet, P.H. Cameron, O.Steele-Mortimer, J. Paiement, J.J. Bergeron, M. Desjardins,Endoplasmic reticulum-mediated phagocytosis is amechanism of entry into macrophages, Cell 110 (2002)119–131.

[11] S. Urbe, L.A. Huber, M. Zerial, S.A. Tooze, R.G. Parton, Rab11, asmall GTPase associated with both constitutive and regulatedsecretory pathways in PC12 cells, FEBS Lett. 334 (1993)175–182.

[12] W. Chen, Y. Feng, D. Chen, A. Wandinger-Ness, Rab11 isrequired for trans-golgi network-to-plasma membranetransport and a preferential target for GDP dissociationinhibitor, Mol. Biol. Cell 9 (1998) 3241–3257.

[13] A. Pitt, L.S. Mayorga, A.L. Schwartz, P.D. Stahl, Assays forphagosome–endosome fusion and phagosome proteinrecycling, Methods Enzymol. 219 (1992) 21–31.

[14] A. Pitt, L.S. Mayorga, A.L. Schwartz, P.D. Stahl, Transportof phagosomal components to an endosomalcompartment, J. Biol. Chem. 267 (1992) 126–132.

[15] C.V. Harding, Phagocytic processing of antigens forpresentation by MHC molecules, Trends Cell Biol. 5 (1995)105–109.

[16] J.M. Escola, F. Deleuil, E. Stang, J. Boretto, P. Chavrier, J.P.Gorvel, Characterization of a lysozyme-majorhistocompatibility complex class II molecule-loadingcompartment as a specialized recycling endosome in murineB lymphocytes, J. Biol. Chem. 271 (1996) 27360–27365.

[17] G. Sinnathamby, L.C. Eisenlohr, Presentation by recyclingMHC class II molecules of an influenzahemagglutinin-derived epitope that is revealed in the earlyendosome by acidification, J. Immunol. 170 (2003)3504–3513.

[18] W.L. Lee, M.K. Kim, A.D. Schreiber, S. Grinstein, Role ofubiquitin and proteasomes in phagosome maturation, Mol.Biol. Cell 16 (2005) 2077–2090.

[19] V.M. Olkkonen, H. Stenmark, Role of Rab GTPases inmembrane traffic, Int. Rev. Cytol. 176 (1997) 1–85.

[20] M. Zerial, H. McBride, Rab proteins as membrane organizers,Nat. Rev., Mol. Cell Biol. 2 (2001) 107–117.

[21] F. Schimmoller, I. Simon, S.R. Pfeffer, Rab GTPases, directorsof vesicle docking, J. Biol. Chem. 273 (1998) 22161–22164.

[22] M. Wilcke, L. Johannes, T. Galli, V. Mayau, B. Goud, J.Salamero, Rab11 regulates the compartmentalization of earlyendosomes required for efficient transport from earlyendosomes to the trans-golgi network, J. Cell Biol. 151 (2000)1207–1220.

[23] M.V. Khvotchev, M. Ren, S. Takamori, R. Jahn, T.C. Sudhof,Divergent functions of neuronal Rab11b in Ca2+-regulatedversus constitutive exocytosis, J. Neurosci. 23 (2003)10531–10539.

[24] J.R. Goldenring, J. Smith, H.D. Vaughan, P. Cameron, W.Hawkins, J. Navarre, Rab11 is an apically located smallGTP-binding protein in epithelial tissues, Am. J. Physiol. 270(1996) G515–G525.

[25] A. Savina, M. Vidal, M.I. Colombo, The exosome pathway in

K562 cells is regulated by Rab11, J. Cell Sci. 115 (2002)2505–2515.

[26] A. Savina, C.M. Fader, M.T. Damiani, M.I. Colombo, Rab11promotes docking and fusion of multivesicular bodies in acalcium-dependent manner, Traffic 6 (2005) 131–143.

[27] M. Trischler, W. Stoorvogel, O. Ullrich, Biochemicalanalysis of distinct Rab5- and Rab11-positive endosomesalong the transferrin pathway, J. Cell Sci. 112 (Pt. 24) (1999)4773–4783.

[28] J.E. Casanova, X. Wang, R. Kumar, S.G. Bhartur, J. Navarre, J.E.Woodrum, Y. Altschuler, G.S. Ray, J.R. Goldenring, Associationof Rab25 and Rab11a with the apical recycling system ofpolarized Madin–Darby canine kidney cells, Mol. Biol. Cell 10(1999) 47–61.

[29] O. Ullrich, S. Reinsch, S. Urbe, M. Zerial, R.G. Parton, Rab11regulates recycling through the pericentriolar recyclingendosome, J. Cell Biol. 135 (1996) 913–924.

[30] M. Ren, G. Xu, J. Zeng, C. Lemos-Chiarandini, M. Adesnik,D.D. Sabatini, Hydrolysis of GTP on rab11 is required forthe direct delivery of transferrin from the pericentriolarrecycling compartment to the cell surface but not fromsorting endosomes, Proc. Natl. Acad. Sci. U. S. A. 95 (1998)6187–6192.

[31] E.S. Ward, C. Martinez, C. Vaccaro, J. Zhou, Q. Tang, R.J. Ober,From sorting endosomes to exocytosis: association of Rab4and Rab11 GTPases with the Fc receptor, FcRn, duringrecycling, Mol. Biol. Cell 16 (2005) 2028–2038.

[32] A.C. Smith, J.T. Cirulis, J.E. Casanova, M.A. Scidmore, J.H.Brumell, Interaction of the Salmonella-containing vacuolewith the endocytic recycling system, J. Biol. Chem. 280 (2005)24634–24641.

[33] S. Brunet, P. Thibault, E. Gagnon, P. Kearney, J.J. Bergeron, M.Desjardins, Organelle proteomics: looking at less to see more,Trends Cell Biol. 13 (2003) 629–638.

[34] L.S. Mayorga, F. Bertini, P.D. Stahl, Fusion of newly formedphagosomes with endosomes in intact cells and in a cell-freesystem, J. Biol. Chem. 266 (1991) 6511–6517.

[35] M.T. Damiani, M.I. Colombo, Microfilaments andmicrotubules regulate recycling from phagosomes, Exp. CellRes. 289 (2003) 152–161.

[36] M.T. Damiani, M. Pavarotti, N. Leiva, A.J. Lindsay,M.W. McCaffrey, M.I. Colombo, Rab coupling proteinassociates with phagosomes and regulates recyclingfrom the phagosomal compartment, Traffic 5 (2004)785–797.

[37] M.T. Damiani, M.I. Colombo, Involvement of heterotrimeric Gproteins in phagocytosis and recycling from the phagosomalcompartment, Exp. Cell Res. 271 (2001) 189–199.

[38] G. Ahnert-Hilger, W. Mach, K.J. Fohr, M. Gratzl, Porationby alpha-toxin and streptolysin O: an approach toanalyze intracellular processes, Methods Cell Biol. 31(1989) 63–90.

[39] K. Funato, W. Beron, C.Z. Yang, A. Mukhopadhyay, P.D.Stahl, Reconstitution of phagosome-lysosome fusion instreptolysin O-permeabilized cells, J. Biol. Chem. 272 (1997)16147–16151.

[40] A. Pagano, P. Crottet, C. Prescianotto-Baschong, M. Spiess, Invitro formation of recycling vesicles from endosomesrequires adaptor protein-1/clathrin and is regulated by rab4and the connector rabaptin-5, Mol. Biol. Cell 15 (2004)4990–5000.

[41] M.G. Mozhayeva, M.F. Matos, X. Liu, E.T. Kavalali, Minimumessential factors required for vesicle mobilization athippocampal synapses, J. Neurosci. 24 (2004) 1680–1688.

[42] J. Krijnse-Locker, M. Ericsson, P.J. Rottier, G. Griffiths,Characterization of the budding compartment of mousehepatitis virus: evidence that transport from the RER to theGolgi complex requires only one vesicular transport step,J. Cell Biol. 124 (1994) 55–70.


[43] P. De Camilli, R. Mirsky, Neuronal and glial cell biology, Curr.Opin. Neurobiol. 7 (1997) 595–597.

[44] J.L. Martys, T. Shevell, T.E. McGraw, Studies of transferrinrecycling reconstituted in streptolysin O permeabilizedChinese hamster ovary cells, J. Biol. Chem. 270 (1995)25976–25984.

[45] L.S. Mayorga, R. Diaz, P.D. Stahl, Plasma membrane-derivedvesicles containing receptor–ligand complexes are fusogenicwith early endosomes in a cell-free system, J. Biol. Chem. 263(1988) 17213–17216.

[46] M.I. Colombo, L.S. Mayorga, P.J. Casey, P.D. Stahl, Evidence of a

role for heterotrimeric GTP-binding proteins in endosomefusion, Science 255 (1992) 1695–1697.

[47] F. Mallard, B.L. Tang, T. Galli, D. Tenza, A. Saint-Pol, X. Yue, C.Antony, W. Hong, B. Goud, L. Johannes, Early/recyclingendosomes-to-TGN transport involves two SNARE complexesand a Rab6 isoform, J. Cell Biol. 156 (2002) 653–664.

[48] S.G. Miller, H.P. Moore, Reconstitution of constitutivesecretion using semi-intact cells: regulation by GTP but notcalcium, J. Cell Biol. 112 (1991) 39–54.

[49] P. Woodman, Vesicle transport: more work for the Rabs? Curr.Biol. 8 (1998) R199–R201.

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