Recruitment of CD63 to Cryptococcus neoformansphagosomes requires acidificationKaterina Artavanis-Tsakonas*, J. Christopher Love*†, Hidde L. Ploegh*‡, and Jatin M. Vyas*‡§
*Whitehead Institute for Biomedical Research, Cambridge, MA 02142; †CBR Institute for Biomedical Research, Boston, MA 02115; and §Division of InfectiousDiseases, Department of Medicine, Massachusetts General Hospital, Boston, MA 02114
Communicated by Gerald R. Fink, Whitehead Institute for Biomedical Research, Cambridge, MA, August 29, 2006 (received for review May 19, 2006)
The subcellular localization of the cluster of differentiation 63 (CD63)tetraspanin and its interaction with the class II MHC antigen presen-tation pathway were examined in the context of phagocytosis by livecell imaging, by using monomeric red fluorescent protein-taggedmouse CD63 expressed in primary bone marrow-derived cell cultures.Upon phagocytosis of Cryptococcus neoformans and polystyrenebeads, CD63 was recruited selectively to C. neoformans-containingphagosomes in a MyD88-independent acidification-dependent man-ner. Bead-containing phagosomes, within a C. neoformans-contain-ing cell, acidified to a lesser extent and failed to recruit CD63 to a leveldetectable by microscopy. CD63 recruitment to yeast phagosomesoccurred independently of class II MHC and LAMP-1. These observa-tions indicate that the composition of distinct phagosomal compart-ments within the same cell is determined by phagosomal cargo andmay affect the outcome of antigen processing and presentation.
Professional antigen-presenting cells (APCs) acquire micro-bial pathogens by phagocytosis and process them for pre-
sentationbyclassIIMHCmolecules.Internalizationofparticulatematter can be mediated through a number of receptors,including Fc receptors, complement receptors, integrins, andscavenger receptors. Each mode of entry is associated withspecific morphological changes, all of which ultimately result information of the phagosome (reviewed in ref. 1). Phagosomematuration is complex and not completely understood. Onceformed, the phagosome undergoes a series of fusion and fissionevents that sequentially incorporate elements of early endo-somes, late endosomes, and lysosomes; these events result indynamic delivery and removal of material (reviewed in ref. 2).
Several pathogens successfully evade immune attack by ex-ploiting the phagosomal environment to render it favorable forreplication and survival. Examples include Leishmania donovani(3), Salmonella typhimurium (4), Mycobacterium tuberculosis (5),and Cryptococcus neoformans (6). Furthermore, internalizationof different pathogenic organisms may yield functionally distinctphagosomes. Whatever the routes involved in pathogen entryand subsequent phagosome maturation, current models wouldbenefit from a more accurate description of phagocytosis inliving cells through use of suitably tagged endosomal proteins,such as the class II MHC products that ultimately presentpathogen-derived peptides (7).
Here we investigate the cluster of differentiation 63 (CD63)tetraspanin within the endosomal pathway by tagging it withmonomeric red fluorescent protein (mRFP1) and using live cellimaging to observe its behavior in primary mouse APCs. CD63, alsoknown as LAMP-3, was characterized originally as a plateletactivation marker (8) and has been used as a marker of lateendosomes and lysosomes (9). More recently, the focus has shiftedto its interaction with class II MHC and its role in antigenpresentation. In professional APCs such as dendritic cells (DCs),subcellular fractionation and biochemical analysis show that humanCD63 segregates into class II MHC-rich domains within the endo-cytic pathway and cell surface (10–12). Furthermore, this tet-raspanin protein preferentially resides in membrane patches en-
riched for particular class II MHC–peptide complexes (13).Disruption of these microdomains in immortalized B cells signifi-cantly reduces their ability to activate T cells. The lysosomal sortingsequence in its C terminus (14) has led to the hypothesis that CD63may act as part of a tetraspanin web that chaperones class II MHCthrough the endosomal pathway and recycles between the cellsurface and late endosomes (13). How tetraspanins and class IIMHC are integrated into membrane domains conducive to antigenpresentation is not known.
Because APCs largely acquire source materials for antigenpresentation by phagocytosis, we investigated the behavior of CD63in relation to class II MHC in primary cells that actively phagocy-tose particulate matter. APCs derived from the bone marrow ofeither C57BL�6 (B6) or class II MHC-eGFP (enhanced GFP)knockin mice were transduced with a lentivirus encoding mRFP1-tagged CD63. We used Cryptococcus neoformans (CN) to inducephagosome formation. These phagosomes recruited CD63 onlywhen the phagosomes successfully acidified, presumably by fusionwith vesicles containing vacuolar ATPase. Phagocytosis of polysty-rene beads by the same cells that acquired CN failed to show CD63localization on the phagosomal membrane and acidified to a lesserextent. Within one and the same cell, phagosomal content deter-mines phagosomal membrane composition and may ultimatelyinfluence antigen processing and presentation.
ResultsCD63-mRFP1 and HA-CD63 Fusion Proteins Mature and Show an Intra-cellular Distribution Comparable to Their Untagged Counterparts. Al-though antibodies exist for many human tetraspanins, anti-mouseCD63 antibodies that effectively recognize native fully glycosylatedCD63 are not available. To characterize the behavior of CD63 andvalidate the use of tagged CD63 derivatives for live cell imaging, weused two epitope-tagged fusion constructs. For biochemical anal-ysis, including the identification of associated proteins, we used anN-terminal affinity tag comprising a HA epitope. For visualizationof the subcellular localization and trafficking patterns of CD63 byoptical microscopy, we tagged mouse CD63 at its C terminus withmRFP1, a fluorophore derived from dsRED but engineered toprevent the pronounced tetramerization of its precursor (15).
CD63 contains three predicted N-linked glycosylation siteswithin its large extracellular loop. Maturation of CD63 in humanDCs involves the acquisition of complex-type N-linked glycans;this modification manifests by a gradual transition over a 1-hperiod from a discrete 37-kDa band to a heterodisperse smearcharacteristic of terminal glycosylation (16). Given the lack of
Author contributions: K.A.-T., J.C.L., H.L.P., and J.M.V. designed research; K.A.-T., J.C.L., andJ.M.V. performed research; K.A.-T., J.C.L., H.L.P., and J.M.V. analyzed data; and K.A.-T.,J.C.L., H.L.P., and J.M.V. wrote the paper.
proper antibodies, we were unable to test the efficacy of ourfusion proteins by comparing them to endogenous CD63. Bymaking use of epitope tags grafted onto CD63, however, we wereable to compare the characteristics of the fusion proteins to thoseof the closely related human CD63. Using retroviral vectors, wetransduced each CD63-encoding construct into the mouse mac-rophage-like cell line RAW 264.7. Pulse–chase analysis andimmunoprecipitation with either anti-HA or -mRFP1 antibody(Fig. 1A) showed that after 90 min of chase, both fusion proteinsacquired endoglycosidase H (EndoH) resistance, which is indic-ative of proper folding and transport from the endoplasmicreticulum to the Golgi. Treatment of immunoprecipitates con-taining CD63-mRFP1 (obtained from cells labeled for 3 h) withPNGase F caused collapse to a single band identical in size tothat of the EndoH digestion products (Fig. 1B). To determinethe extent of trafficking beyond the Golgi and subcellularlocalization at steady state, we reasoned that the highly homol-ogous mouse and human forms of CD63 should behave similarly.We introduced the mouse CD63-mRFP1 construct into U373human astrocytoma cells through lentiviral infection. Using ananti-human CD63 monoclonal antibody, we observed nearlyperfect colocalization with the tagged mouse CD63 (Fig. 1C);small amounts of mouse CD63-mRFP1 were detectable at thecell surface as well. Addition of the mRFP1 tag thus preservesthe subcellular localization of CD63. Taken together, these datajustify the use of both fusion proteins to derive meaningful dataon the role of mouse CD63 within the endocytic pathway by livecell imaging.
CD63 Recruitment to the Phagosome Is Rapid and Depends on Con-tent. DC maturation can be determined by the level of class II MHCon the cell surface. Upon LPS stimulation, these cells rearrangetheir endosomal compartments and form tubular structures thatdeliver class II MHC to the cell surface (17–19). In class IIMHC-eGFP bone marrow-derived DCs lentivirally transduced to
express CD63-mRFP1, we observed partial overlap between CD63and class II MHC in immature DCs, whereas in fully mature cells,the majority of CD63 was present in intracellular compartments,clearly segregated from surface class II MHC (Fig. 6, which ispublished as supporting information on the PNAS web site). Thesedata suggest that if CD63 reaches the cell surface, then it mustseparate from the associated class II MHC and recycle back to anintracellular location. Because we demonstrate by immunoprecipi-tation–Western blot that these molecules do interact in mouse cells(Fig. 7, which is published as supporting information on the PNASweb site), these data collectively raise the question of where thesemolecules first interact.
Phagocytosis represents an important means by which mostmicrobial pathogens gain intracellular access and enter the antigen-processing pathway. To study CD63 and class II MHC interactionswithin the context of this pathway, we chose the phagosome as alikely environment where these proteins might initially interact. Theprocess of phagocytosis has been studied by using inert polystyrenebeads, and we therefore sought to study the behavior of thesemolecules within the context of this widely used model. In CD63-mRFP1-expressing bone marrow-derived cells, time-lapse experi-ments examining both early (within minutes of ingestion) and late(24 h after ingestion) time points failed to reveal detectable changesin CD63 distribution on bead phagosomes (Fig. 2A). Class II MHCalso seemed unresponsive to the presence of the bead, with onlytrace amounts being recruited to the phagosomal membrane after24-h incubation. Opsonizing beads or coating them with ovalbuminalso failed to produce any detectable change in the phagosomes(data not shown). Complete ingestion of beads was confirmed byphase-contrast microscopy as well as z-stack images of cells. In allcases, only cells with clear internalization of beads were used foranalysis.
Given the ability of certain intracellular pathogens to modify thearchitecture of the phagosome (3–5), we hypothesized that, byexposing the bone marrow DCs to a pathogenic organism, we might
Fig. 1. CD63 fusion proteins mature normally. (A andB) RAW 264.7 cells were transduced to stably expressmouse CD63-mRFP1 or HA-CD63. Cells were pulse-labeled for 15 min with [35S]cysteine and methionineand chased for 0, 90, or 180 min (A) or labeled for 3 h(B). Cells were lysed in 1% Brij58 and proteins immu-noprecipitated with anti-mRFP1 or anti-HA antibody.At each time point, half of the sample was treated withendoglycosidase H (A) or F (B) for 1 h at 37°C. Sampleswere run on a 12% SDS-polyacrylamide gel andpolypeptides visualized by autoradiography. The‘‘CHO’’ labels indicate the number of N-linked glycansand complex-type sugars (C.T.) present. (C) Humanastrocytoma U373 cells expressing mouse CD63-mRFP1were fixed, permeabilized, and stained with anti-human CD63 antibody. Localization of mouse CD63-mRFP1 (Left) and human CD63 (Center) and themerged image (Right).
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observe differences in CD63 and class II MHC distribution. Weselected CN, a pathogenic encapsulated yeast (20), as our modelorganism (H99, Serotype A), for its clinical importance and ease ofdetection. Upon phagocytosis of CN, a bright fluorescent ringappeared on the phagosome, indicating robust recruitment ofCD63-mRFP1 or class II MHC-eGFP (Fig. 2B). This observationwas not limited only to CN but was also seen when cells phagocy-tosed Saccharomyces cerevisiae (Fig. 8, which is published as sup-porting information on the PNAS web site; ref. 21).
Within an individual cell that had ingested both beads and CN,the acquisition of CD63 (Fig. 2C) and of class II MHC (data notshown) by the phagosomes remained dependent on their contents.Phagosomes containing CN developed a bright fluorescent ring ofCD63-mRFP1, whereas those containing beads remained dark.Acquisition of CD63, therefore, does not seem to be readilytriggered by the act of phagocytosis alone but requires additionalstimuli absent from inert beads but present on yeast. Furthermore,this observation demonstrates the ability of different phagosomalcontents to elicit differential recruitment of CD63, class II MHC,and almost certainly other molecules to the phagosome. Entry ofbeads and CN into membrane-delimited compartments was veri-fied by observation of actin flashing in cells expressing actin-GFP(22) and by electron microscopy (Fig. 9, which is published assupporting information on the PNAS web site).
Class II MHC Is Recruited to the Phagosome Before CD63. By trans-ducing bone marrow-derived cells from a class II MHC-eGFPmouse with CD63-mRFP1 lentivirus, we confirmed that bothmolecules are recruited to the same CN phagosome (Fig. 3A). Inaddition, through time-lapse microscopy, we were able to examinewhether these routes of vesicular transport emanate from sourcesof different membrane composition or from a single homogeneoussource. Time-lapse analysis revealed that class II MHC arrives at thephagosomal membrane separately and slightly earlier than CD63(Fig. 3B). We examined the kinetics of recruitment more closely bydoing a quantitative analysis of increasing fluorescence intensity ofmultiple independent phagosomal events. Analysis was done in
single-labeled cells to avoid bleedthrough of red fluorescence intothe green channel. We also used Cap59��� CN, an acapsularmutant (23) showing enhanced uptake by phagocytes, to facilitatecollection of a large number of phagocytic events. Cap59��� yeastinduced the same endosomal rearrangements as wild-type CN, theonly difference being that terminal levels of CD63 and class II MHCwere slightly lower (Fig. 10, which is published as supportinginformation on the PNAS web site), a point that did not interferewith resolution of recruitment kinetics. By identifying nascentphagosomes in cells expressing either class II MHC-eGFP orCD63-mRFP1, we could monitor the recruitment of each molecule
BCD63 Beads Merge
Phase
Class II MHCCD63
CD63 Beads Merge
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Fig. 2. CD63 and class II MHC are recruited selectively to yeast phagosomes.(A) B6-derived bone marrow cultures expressing CD63-mRFP1 were exposedto ‘‘dragon green’’ polystyrene beads. Still images show CD63-mRFP1 distri-bution in the cell. (B) Bone marrow cells from B6 mice expressing CD63-mRFP1(Left) or from class II MHC-eGFP-expressing mice (Right) were incubated withCN. The images display the phagosomal distribution of CD63-mRFP1 and classII MHC-eGFP. (C) B6-derived bone marrow cultures were transduced withCD63-mRFP1 and incubated with both CN and dragon green polystyrenebeads. An example of CD63-mRFP1 distribution in a cell having taken up twoyeast, and one bead is shown. The far-right bright-field image demonstratesthe presence of CN within the cell. (Scale bars, 10 �m.)
Fig. 3. Class II MHC recruitment to the phagosome precedes that of CD63. (A)Within 10 min after addition of CN to bone marrow cultures derived from classII MHC-eGFP mice and transduced to express CD63-mRFP1, phagosomes ac-quire both CD63-mRFP1 and class II MHC (Left, CD63-mRFP1; Center, class IIMHC; and Right, merge). (Scale bar, 10 �m.) (B) Time-lapse images in both redand green channels show that class II MHC-eGFP appears around the phago-somes (indicated by the arrows) slightly before CD63-mRFP1. (Scale bar, 5 �m.)(C) Kinetic analysis of fluorescence acquisition around phagosomes. Each datapoint represents the average intensity measured at each time point for at leastnine independent Cap59��� CN phagocytic events for CD63-mRFP1 (■ ) andclass II MHC-eGFP (E) recruitment. The linear fits indicate the initial rates ofrecruitment for each protein to the phagosome.
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over time (Movies 1 and 2, which are published as supportinginformation on the PNAS web site).
We treated the data using a first-order kinetics model anddetermined initial rates of recruitment using linear regression. Theratio of the recruitment rates of class II MHC-eGFP to CD63-mRFP1 was 1.8 (P � 2 � 10�12), demonstrating, in a statisticallysignificant manner, that recruitment into phagosomes of class IIMHC proceeds at a consistently different and more rapid pace thanthat of CD63-mRFP1 (Fig. 3C). The increase in fluorescenceobserved for CD63-mRFP1 and class II MHC-eGFP is gradual, notsaltatory, and thus is consistent with the involvement of transportvesicles far smaller than the target organelles. The near-constantsize of the phagosome in the course of maturation further impliesa balance of fusion and fission events. These observations suggestthat these reporters must have their origin in membrane vesicles ordistinct membrane microdomains, and that multiple discrete inputpathways exist for gradual modification of the composition of thephagosomal membrane.
Phagosome Acidification Coincides with CD63 Recruitment. We at-tempted to change the CD63 recruitment phenotype of either beadsor CN by manipulation of cellular surface molecules known to beinvolved in yeast phagocytosis. Adsorption of mannosylated BSA orIgG onto beads to force engagement of the mannose and Fc�IIreceptors did not result in CD63 recruitment. Furthermore, uptakeof CN by bone marrow DCs derived from a MyD88��� mousefailed to abrogate recruitment of this tetraspanin, excluding in-volvement of MyD88-dependent Toll-like receptors (TLR; see Fig.11, which is published as supporting information on the PNAS website). Conversely, polystyrene beads with adsorbed LPS failed torecruit CD63 (data not shown), suggesting that if TLR4 is involvedin this process, its ligation alone is insufficient to induce theendosomal rearrangement observed with yeast. We next focused onidentifying physical changes to the phagosome itself in an effort tounderstand the mechanism whereby this tetraspanin traffics. Be-cause CD63 is widely accepted as a lysosomal marker, it seemedlogical that it may be delivered to the phagosome through fusionwith lysosomal compartments, thereby leading to a change in pH.By incubating CD63-mRFP1-transduced cells with Lysosensor, apH indicator that accumulates in acidic vesicles and fluoresces whenprotonated, in conjunction with CN and�or beads, we assessed thekinetics of phagosome acidification as it relates to CD63 recruit-ment. Consistently, the appearance of CD63 in the phagosomecoincided with that of Lysosensor, which reached maximal fluo-rescence by 10 min after internalization (Fig. 4A). Beads generateda significantly lower level of acidification (Fig. 4B), suggesting thatperhaps trace amounts of CD63 are recruited to these phagosomesat levels not detectable by live cell imaging.
Acidification of the Phagosome Is Required for CD63 Recruitment butNot for Other Lysosomal Markers. Because phagosome acidificationand CD63 recruitment largely coincided, we asked whether theseevents were interdependent. Bafilomycin is a selective and potentinhibitor of vacuolar ATPase (24), the enzyme complex responsiblefor pumping protons across the membrane, and hence vacuolaracidification. We added bafilomycin to cell cultures (1 �M) andeffectively blocked vacuolar acidification. In each experiment, weverified lack of acidification by absence of Lysosensor dye fromphagosomes (data not shown). In cells transduced to expressCD63-mRFP1, bafilomycin successfully blocked CD63 recruitmentto CN-containing phagosomes (Fig. 5A). We repeated this exper-iment using cells derived from a class II MHC-eGFP knockinmouse as well as with B6 bone marrow-derived cells transduced toexpress LAMP-1-GFP. We observed unabated recruitment of bothclass II MHC and LAMP-1 to the yeast phagosomes despite thepresence of bafilomycin. These results further support our findingthat CD63 and class II MHC traffic to the phagosome indepen-dently as a result of different subcellular signals. Furthermore, that
acidification is required for the recruitment of CD63, but not forLAMP-1, distinguishes these molecules as residents of differentendosomal subcompartments, an argument bolstered by their non-identical subcellular distribution when probed by immunofluores-cence (Fig. 5B). To date, CD63 and LAMP-1 have been largely usedinterchangeably as lysosomes markers.
DiscussionSpecialized membrane microdomains stabilized by proteins of thetetraspanin family are functionally important, but their origin andorganization in living cells have not been well studied. The impor-tance of ‘‘tetraspanin webs’’ in a wide array of cellular processes isbecoming increasingly clear (reviewed in ref. 25), including theirrole in antigen presentation (26). Here we have addressed thebehavior of CD63 in the maturation of phagosomes using live cellimaging. To date, characterization of CD63 has been accomplishedin human cell lines, largely through biochemical analysis (12, 16, 27).The data presented here focus on the behavior of CD63 and classII MHC in live primary cultures of mouse APCs in the context ofantigen capture by phagocytosis.
Our observation that phagosomal composition depends on theidentity of the ingested particle, even when two different particlesare within a single cell, may have important implications for antigenprocessing and presentation. This specificity may be due, in part, tothe initial interaction of a phagocyte with a target pathogen. Thecombination of surface molecules involved depends on the patho-gen itself. For example, Leishmania relies on complement andscavenger receptors to gain entry (28), whereas yeast engage themannose receptor to trigger cellular uptake (29). Because phago-somes are, at least in part, plasma membrane-derived (30), theinitial composition of the phagosomal environment must differdepending on the selective engagement of surface molecules. Thesedifferences become more pronounced as phagosomes mature and
lysosensor CD63 merge
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Fig. 4. CD63 recruitment and phagosome acidification coincide. Bone mar-row cultures derived from B6 mice and transduced to express CD63-mRFP1were incubated with CN and the pH indicator, Lysosensor. (A) Time-lapseimaging of phagosome acidification as determined by Lysosensor recruitment(Upper) and of CD63-mRFP1 recruitment (Lower) are shown. Representativeimages have been selected at 0, 200, and 500 sec. (Scale bar, 5 �m.) (B)Colocalization of Lysosensor (Left) and of CD63-mRFP1 (Center) in the CNphagosome after at least 10-min incubation is apparent in the merged image(Right). (Scale bar, 10 �m.)
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pathogens manipulate their immediate surroundings to their ad-vantage (28, 31, 32).
Pathogens that are able to interfere specifically with the vacuolarATPase proton pump may be capable of manipulating antigenpresentation through a direct effect not only on endosomal pH andthus lysosomal protease activity but also on phagosomal compart-ment composition. CN can thrive in an acidic milieu and showsreduced viability in the absence of phagosome acidification (33).Histoplasma capsulatum, another fungal pathogen, actively recruitsvacuolar ATPase and thereby controls the pH of its immediateenvironment (34). Likewise, Helicobacter pylori blocks proper acid-ification by inhibiting fusion of its compartment with late lysosomalcompartments. This obstruction interferes with antigen presenta-tion, presumably because efficient antigen processing cannot takeplace in the absence of functional proteases (35). We propose thateach pathogen, by virtue of its unique surface architecture, may bedelivered to a cellular compartment with distinct processing pro-teases and with different routes of access to the class II MHCmolecules themselves.
Blocking acidification inhibited recruitment of CD63 to theCN-containing phagosome but did not affect that of class II MHCor LAMP-1. This result suggests that the acquisition of lysosomalproteins by the phagosome is a multistep process consisting first ofclass II MHC and LAMP-1 recruitment and acidification and thenCD63 recruitment. This tetraspanin must be directly responsive tosignals triggered upon acidification, serving either as part of adelivery complex or as a structural purpose once within the confinesof the phagosome. Signaling by the vacuolar ATPase (vATPase)itself has been shown to influence the recruitment of AOP ribo-sylation factor 6 (Arf6) and ADP ribosylation factor nucleotide-binding site opener (ARNO) (36).
The observed robust recruitment of CD63 may indicate optimalaccess to antigen-loading machinery and subsequently efficientprocessing and presentation. Immunoprecipitations with theCDw78 antibody from cells lysed in weak detergents identified acomplex of associated proteins, including CD63, CD82, HLA-DR,and HLA-DM, which catalyzes exchange of class II-associatedinvariant chain peptide (CLIP) peptide with antigenic peptide (13).Selective recruitment of CD63 to CN-containing phagosomes mayrepresent a physiological phenomenon whereby antigens destinedfor processing and presentation gain more efficient access tocomponents of the antigen-presentation pathway. CD63, as part ofa family of molecules that characteristically play a structural rolewithin the cell, could be recruited to phagosomes to tether theantigen-loading machinery together. The notion that phagosomalcontents influence their processing and subsequent presentationhas been proposed in other systems as well (37, 38).
CD63 and other tetraspanins can be coimmunoprecipitated withtype II phosophoinositide 4-kinase (39, 40) and �-glutamyltranspeptidase (41). The presence of these enzymes suggests thattheir recruitment to specific tetraspanin-rich areas of membranesmay mediate regulated signaling events. In the context of thephagosome, recruitment of CD63 may, in turn, attract signalingmolecules responsible for regulating downstream antigen-processing events. Signal initiation could result in up-regulatedtranscription as well as recruitment of preexisting molecules to thephagosome.
That polystyrene beads ultimately do recruit modest amounts ofclass II MHC molecules, albeit with considerable delay (�24 h), andpossibly of CD63 below the level of detection, suggests that bothbead and CN may gain access to the class II MHC compartment.In support of this argument, ratiometric imaging showed markeddifferences in the nature of macrophage phagosomes containingeither opsonized erythrocytes or beads (42). Specifically, the en-doplasmic reticulum (ER) membrane was not detected aroundphagocytosed erythrocytes, whereas it was abundant around beads.Although we did not probe for phagosomal ER markers in thisstudy, the discrepancy observed in CD63 and class II MHCrecruitment between beads and CN underscores the obvious dif-ference between synthetic beads and real microorganisms. Theidentity of markers to assess maturation of phagosomes to phagoly-sosomes is probably best determined using microorganisms. Al-though electron microscopy and immunofluorescence demonstratethe presence of molecules expected to participate in antigen pro-cessing and presentation within bead phagosomes, the modestquantities of CD63 and class II MHC recruited to these phago-somes make polystyrene beads a less effective tool to study phago-some maturation events in living cells.
Functional antigen-presentation assays using tetraspanin knock-out models are needed to determine the relative importance oftetraspanins to the class II MHC pathway. How the phagosomalenvironment is defined, either by surface interactions that imme-diately direct entry into a defined pathway or by the nature of thephagocytosed particle inducing selective recruitment of molecules,remains to be determined.
Materials and MethodsPlasmids and Constructs. Mouse CD63 and mRFP1 cDNA werekind gifts from H. Hotta (Department of Microbiology, KobeUniversity School of Medicine, Hyogo, Japan) and R. Tsien (De-partment of Pharmacology, University of California at San Diego,La Jolla, CA), respectively. The HA-TEV-SBP affinity purificationtag has been described (43). HATEVSBP-CD63 and CD63-mRFP1 were cloned in the pGEM-T vector (Promega, Madison,WI) for sequence verification, and HATEVSBP-CD63 was trans-ferred into pMIG-W, a mouse stem-cell retroviral vector with adownstream IRES-GFP sequence (44). CD63-mRFP1 and actin-GFP were subcloned into the pHAGE vector, a third-generationlentiviral self-inactivating nonreplicative vector used for transduc-
Fig. 5. Phagosomal recruitment of CD63, but not class II MHC or LAMP-1, isblocked by inhibition of vacuolar ATPase. Bone marrow-derived cell culturesfrom B6 mice were lentivirally transduced with CD63-mRFP1 or LAMP1-GFP.Alternatively, bone marrow cultures were derived from class II MHC-eGFPmice. (A) Cells were incubated with CN in the absence or presence of 1 �Mbafilomycin and followed for CD63-mRFP1 (Left), class II MHC-eGFP (Center),or LAMP1-GFP (Right) recruitment. Images shown were taken after 30-minincubation. (Scale bars, 5 �m.) (B) B6 bone marrow cells were transduced withCD63-mRFP1 and paraformaldehyde-fixed. Analysis by immunofluorescencewith anti-mRFP1 (Left) and LAMP-1 (Center) antibodies shows colocalizationof these two molecules (Right). (Scale bars, 10 �m.)
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tion of bone marrow stem cells (45). LAMP1-GFP (fusion con-structs were kind gifts from Ira Mellman (Department of CellBiology, Yale University, New Haven, CT) and Maggie So (De-partment of Molecular Microbiology and Immunology, OregonHealth and Science University, Portland, OR) was also subclonedinto pHAGE.
Cell Lines and Tissue Culture. The mouse macrophage cell line RAW264.7, human U373 astrocytoma cells, and viral packaging cell lineHEK293T cells were all grown in DMEM (Gibco, Grand Island,NY) supplemented with 10% FCS (HyClone, Logan, UT) andpenicillin�streptomycin (Gibco). A20 mouse B cells were grown inRPMI medium 1640 (Gibco) supplemented with 10% FCS�50 �M2-mercaptoethanol�penicillin�streptomycin. RAW 264.7 and A20cells stably expressing HATEVSBP-CD63 or CD63-mRFP1 weregenerated through retroviral and lentiviral infection, respectively(see below).
Mouse Strains and Bone Marrow Cultures. Class II MHC-eGFPknockin mice and wild-type mice on a mixed 129SVJ-B6 back-ground were killed according to approved protocols, and bonemarrow cells cultures were prepared as described (7) and seeded inequal aliquots of 6 � 105 cells�200 �l on eight-well Labtek IIchambered coverglass wells (Nalge Nunc, Naperville, IL). Mediawere replaced every 48 h.
Retroviral and Lentiviral Transduction of Cell Lines and Bone MarrowCultures. Lentivirus were made as described (45). Cellular trans-duction was done in 96-well U-bottom (A20 cells) or 96-wellflat-bottom (RAW 264.7 cells) plates whereby 100 �l of viralsupernatant supplemented with 8 �g�ml Polybrene (Sigma, St.Louis, MO) was placed onto plated cells, spun at 500 � g for 2 hat room temperature, and placed at 37°C overnight. Supernatantswere replaced with normal media the next day, and cells were
cultured as usual. Successful infection was determined by exami-nation under an epifluorescence microscope for the presence ofgreen or red fluorescence.
Antibodies and Reagents. For immunoprecipitation, anti-HA mousemonoclonal 3F10 antibody was used (Roche, Basel, Switzerland).We raised polyclonal antisera against mRFP1 by bacterial expres-sion of mRFP1 and subsequent immunization of rabbits.
Immunofluorescence was done with monoclonal rat-anti-mouseLAMP-1 (PharMingen, San Diego, CA). Secondary antibodiesused were anti-rat or -rabbit Alexafluor 647 (Molecular Probes,Eugene, OR). Additional details regarding immunofluoresence,pulse–chase, and immunoprecipations are found in SupportingText, which is published as supporting information on the PNASweb site.
Image Acquisition and Analysis. Images were acquired by using aspinning disk confocal microscope (Perkin–Elmer Ultraview RS;Perkin–Elmer, Boston, MA), and either the Ultraview ERS systemor a Prairie 3-W laser with Acousto-optical tunabe filter (PrairieTechnologies, Middleton, WI) was used. Details are found inSupporting Text.
We thank Gustavo Mostoslavsky (Department of Genetics, HarvardMedical School, Boston, MA) for the pHAGE lentiviral vector, Elefthe-rios Mylonakis (Division of Infectious Diseases, Massachusetts GeneralHospital) for the H99 CN isolate and the Cap59��� CN mutant, andGeorge Bell for helpful discussions. K.A.-T. is a National Institutes ofHealth National Research Service Award fellow (1F32CA105862-01).J.C.L. is a Gilead Fellow of the Life Sciences Research Foundation(Baltimore, MD). J.M.V. is supported by the Ellison Medical Foundationand the National Institutes of Health (5K08AI57999), and a portion ofthis research was conducted while J.M.V. was a Pfizer postdoctoral fellowin Infectious Diseases. H.L.P. is supported by the National Institutes ofHealth (5R01AI034893-13).
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