2 1

cles of constant density. These separations are performed in 27% (v/v) Percoll in isotonic sucrose buffer (0.25 M sucrose, 10 m M triethanolamine-acetic acid, pH 7.4, 1 m M EDTA, 1 mg/ml polyvinylpyrrolidone) and centrifuged at 4 ° in a 50-Ti rotor for 2 hr at 17,000 rpm. For the purposes of isolation of the banded vesicles for subsequent analysis (e.g., testing for fusion competence in the cell-free fusion assay) the inclusion of polyvinylpyrrolidone in the gradients, and in subsequent washings of vesicles with this buffer, facilitates the removal of the colloidal silica particles from the vesicles.

[4] A s s a y s f o r P h a g o s o m e - E n d o s o m e Fusion and Phagosome Protein Recycling B y ALAN PITT, LUIS S. MAYORGA, ALAN L. SCHWARTZ,

and PmLIP D. STAHL Introduction Endocytosis and phagocytosis are the two primary mechanisms employed by eukaryotic cells to internalize macromolecules from the extracellular milieu.t,~ The most comprehensively studied endocytic pathway is receptor-mediated endocytosis via clathrin-coated pits and subsequent transport to lysosomes. This constitutive process intitiates at the cell surface, where specialized receptors and their associated ligands cluster in plasma membrane domains termed clathrin-coated pits. The clathrin-coats mediate pit invagination and vesiculation into the cytoplasm. Following enzymatic removal of the clathrin coat, the intraceUular vesicle undergoes extensive fusion with other newly formed endosomes. Both the uncoated vesicles and the larger organelles formed by their multiple fusions are termed early endosomes. In the early organelles formed by their multiple fusions are termed early endosomes. In the early endosome, ligandreceptor dissociation and sorting occur. Soon afterward, receptors recycle to the plasma membrane while free ligand progresses to another population of vesicles termed late endosomes.3-5 Late endosomes are both more t A. L. Schwartz, Annu. Rev. Imrnunol. 8, 195 (1990). 2 S. C. Silverstein, S. Greenberg, F. Di Virgilio, and T. H. Steinberg, in "Fundamental Immunology" (W. E. Paul, ed.), p. 703. Raven, New York, 1989. P. D. Stahl, P. H. Schlesinger, E. Sigardson, J. S. Rodman, and Y. C. Lee, Cell 19, 207 (1980). 4 A. L. Schwartz, A. Bolognesi, and S. E. Fridovich, J. Cell Biol. 98, 732 (1984). 5 H. J. Geuze, J. W. Slot, G. J. A. M. Strous, and A. L. Schwartz, Eur. J. Cell Biol. 32, 38 (1983).


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dense and acidic and display a slight increase in hydrolytic activity compared to early endosomes. Also, late endosomes do not fuse in vitro to early endosomes.6,7 From late endosomes, endocytosed material then migrates to very dense and hydrolase-rich lysosomes where complete hydrolysis occurs. 1 Contrary to endocytosis, phagocytosis is a function of specialized cells. In vertebrates, mononuclear macrophages and polymorphonuclear granulocytes are the primary phagocytes.2 While endocytosis internalizes individual molecules or small particles (< 100 nm), phagocytosis internalizes relatively large particles (>500 nm) (e.g., bacteria). Phagocytic particle engulfment initiates on binding of phagocytic particles to Fc, complement (CR3), or mannose receptors.2 The role of clathrin in phagocytic engulfment is not fully understood; however, much evidence indicates that a combination of regional actin polymerization and receptor-ligand interactions provides the mechanical force for engulfment) Once engulfment is complete, the phagosome transforms into a degradative compartment termed a phagolysosome. In macrophages, phagolysosome formation requires extensive interactions among phagosomes and other vesicle populations. Most notably, phagosomes fuse with endosomes and then with lysosomes. 9 Thus, while endocytosis and phagocytosis employ different internalization mechanisms, the fate of internalized material is similar. The biochemical mechanisms that regulate phagolysosome biogenesis are very complex. As evidenced by a number of investigations, both membrane fusion and membrane recycling play important roles in this process) °,11 For example, many parasites that reside in phagosomes prevent their hydrolytic destruction by inhibiting hydrolase-containing compartments from fusing to phagosomes.12 Also, following degradation and processing of phagocytosed antigen, vesicle-mediated protein trafficking is required for subsequent antigen presentation on the cell surface) 3 Therefore, to better understand the mechanisms employed by phagosomes to

6 j. Gruenberg and K. E. Howell, Proc. Natl. Acad. Sci. USA 84, 5758 (1987). 7 R. Diaz, L. Mayorga, and P. Stahl, J. Biol. Chem. 263, 6093 (1988). s S. Greenberg, J. E. Khoury, F. Di Virgilio, E. M. Kaplan, and S. C. Sflverstein, J. Cell Biol. 113, 757 (1991). 9 L. M. Mayorga, F. Bertini, imd P. D. Stahl, J. Biol. Chem. 266, 6511 (1991). lo W. A. Muller, R. M. Steinman, and Z. A. Cohn, J. CellBiol. 86, 304 (1980). 11 T. Lang, C. de ChasteUier, A. Ryter, and L. Thilo, Eur. J. Cell Biol. 46, 39 (1988). 12 K. A. Joiner, S. A. Fuhrman, H. M. Miettinen, I. Kasper, and I. Mellman, Science 249, 641 (1990). ,3 C. V. Harding, D. S. Collins, J. W. Slot, H. J. Geuze, and E. R. Unanue, Cell 64, 393 (1991).




process and traffic internalized material, we developed the following probes and cell-free assays. Phagocytic Probe The phagocytic particle in our assays is mouse ~25I-labeled anti-2,4dinitrophenol (DNP) IgG-rabbit anti-mouse IgG-staphylococcus aureus. 9 This probe provides many advantages to the study of phagosome processing. The exposed Fc regions of anti-DNP IgG allow for Fc receptormediated phagocytosis. Also, assaying for the radiolabel of ~2SI-labeled anti-DNP IgG throughout an experiment is both simple and sensitive. Mouse anti-DNP IgG is resistant to degradation within the hydrolasecontaining compartments and the phagocytic probe is stable to pH 4. Finally, binding of anti-DNP IgG to DNP is not significantly affected by its radioiodination, thus allowing for its subsequent use in a vesicle fusion assay as described below. Endosomes in the phagosome-endosome fusion assay are labeled with purified fl-ghicuronidase that had previously been derivatized with dinitrofluorobenzene.7 Thus, the endosomal probe is termed DNP-fl-glucuronidase. Synthesis o f l25I-labeled Anti-2, 4-Dinitrophenol IgG-Rabbit Anti-Mouse IgG-Staphylococcus aureus

1. Isolate and iodinate anti-DNP IgG as previously described ~4,15to a specific radioactivity of 2000 cpm/ng anti-DNP IgG. 2. Wash formaldehyde-fixed S. aureus (IgGsorb; The Enzyme Center, Malden, MA) with HBSA {Hanks' balanced salt solution buffered with 10 m M 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid (HEPES) and 10 m M 2-[2-hydroxy- 1,1-bis(hydroxymethyl)ethyl]aminoethanesulfonic acid (TES), pH 7.4} and supplemented with 1% (v/v) bovine serum albumin (BSA). 3. Incubate 200 al of s. aureus of a 10% (v/v) suspension (approximately 4 × 107 particles/#l, 2 mg of IgG/ml-binding capacity) with 200/lg of rabbit anti-mouse IgG polyclonal antibody (IgG fraction; Organon Teknika Corporation, West Chester, PA) for I hr at 20 °. 4. Wash the particles three times in HBSA and incubate with 25 #g of ~25I-labeled anti-DNP IgG for 1 hr at 20 ° . 5. Wash antibody-coated So aureus three times and resuspend in HBSA to its original volume. z4 R. K. Keller and O. Touster, Z Biol. Chem. 250, 4765 (1975). ~5 F. L. Otsuka, M. J. Welch, K. D. McElvany, R. A. Nicolotti, and J. B. Fleischman, J. Nucl. Med. 25, 1343 (1984).


R E C O N S T I T U T I O N IN C E L L - F R E E E X T R A C T S


As shown in Fig. l, on internalization 125I-labeled anti-DNP IgG remains associated with the S. a u r e u s for approximately 5 - l0 min. Thereafter, the majority of 125I-labeled anti-DNP IgG quickly dissociates from the particle. The low concurrent rise in trichloroacetic acid (TCA)-precipitable radioactivity indicates that the majority of the 125I-labeled anti-DNP IgG that leaves the S. a u r e u s is not degraded. Thus, nSI-labled anti-DNP IgG marks two aspects of the phagosome. At early times after internalization (up to l0 min), ~2SI-labeled anti-DNP IgG remains attached to the S. a u r e u s and labels the phagocytic particle; later, it dissociates from the S. a u r e u s and serves as a lumenal marker of the phagosome. I n V i t r o P h a g o s o m e - E n d o s o m e Fusion

The probe discussed above has been used to investigate phagosomeendosome fusion. 9 The design of the fusion assay is similar to an endosome-endosome fusion assay previously developed in our laboratory. 7 Briefly, early endosomes in one population of cells are labeled with DNP-fl-glucuronidase while the phagocytic probe described above is phagocytosed by a second population of cells. Postnuclear supernatants containing the two probes are obtained and mixed under fusogenic conditions (see below). On fusion and mixing of the vesicle contents, immune corn-



:5 ° 4 0 ft. 20



20 30 Time (rain)


FIG. 1. Characterization of the phagocyticprobe followingphagocytosis.The phagocytic probe prepared as described above was internalized for 5 rain followedby chase for up to 30 rain. 0, Cell associated;II, S. aureus associated;O, TCA soluble.




plexes can form between the DNP-fl-glucuronidase and anti-DNP IgG. Determination of the amount of fl-glucuronidase activity associated with the antibody-coated S. aureus provides a measurement of complex formation.

Phagosome Labeling 1. Preincubate 100 kd of the phagocytic probe prepared as described above with 1 X l0 s J774-E clone macrophages at 4 ° for 1 hr in a final volume of 0.5 ml. 2. Initiate phagocytosis by the addition of 2 ml of HBSA at 37 ° and incubate at 37 ° for 5 min. 3. Stop phagocytosis by the addition of 10 ml of HBSA at 4 °. With this protocol, approximately 90% of the antibody-coated S. aureus is cell associated after the 37" incubation and 70% is completely intracellular. At this early time of internalization, approximately 80% of the t25I-labeled anti-DNP IgG is attached to the S. aureus.

Endosome Labeling 1. Preincubate 1 X 108 J774-E clone macrophages with DNP-flglucuronidase (20/~g/ml) in 0.5 ml HBSA for 1 hr at 4 °. 2. Initiate endocytosis by the addition of 2 ml HBSA at 37 ° for 5 rain. 3. Stop internalization by the addition of excess HBSA at 4 °.

Preparation of Postnuclear Supernatant 1. Wash the cells (centrifuge at 200 g for 3 min at 4 °) once with HBSA, once with PBS with 5 m M EGTA, and once with homogenization buffer [ 2 5 0 m M sucrose, 0.5 m M ethylene glycol-bis (fl-aminoethylether)N,N,N',N'-tetraacetic acid (EGTA), 20 m M HEPES-KOH, pH 7.2]. Resuspend the cells in homogenization buffer to 5 X 107 cells/ml. 2. Homogenize the cells by either the syringe method as previously described 7 or by use of a ball-bearing homogenizer. Phagosomes are more fragile than endosomes and more prone to breakage during homogenization. Thus, to prevent overhomogenization, stop homogenizing when 80% of the cells are broken (15- 30 passes through either device). 3. Remove nuclei by centrifuging homogenate at 200 g for 3 min at 4 °. 4. Postnuclear supernatants (200-#1 aliquots) may be frozen in liquid nitrogen for storage.




Endosome- Phagosome Fusion Assay A. Phagosome Preparation 1. Quick-thaw a postnuclear supernatant aliquot (200/A) containing phagocytic probe. 2. Dilute five-fold in homogenization buffer and centrifuge 1.5 min at 12,000 g at 4°. 3. Resuspend the pellet (phagosome-enriched fraction) in 100/tl homogenization buffer. 4. Add DNP-BSA (final concentration of 0.25 mg/ml) to quench mouse anti-DNP IgG present outside the vesicles. B. Endosome Preparation 1. Quick-thaw postnuclear supernatants containing labeled endosomes.

2. Dilute 10-fold in homogenization buffer and centrifuge at 35,000 g for 1 min (lysosome-enriched fraction). 3. Centrifuge the resulting supernatant at 50,000g for 5 min (endosome-enriched fraction). 4. Resuspend the endosome-enriched fraction in 100/tl homogenization buffer. C. Fusion Assay 1. Combine 4/zl each of endosome-enriched fraction and phagosomeenriched fraction. 2. Add 2/tl cytosol (10 mggml) to make the reaction 2 mg/ml cytosol. 3. Add 1/tl of a 10× solution to make the reaction sample 250 mM sucrose, 0.5 mM EGTA, 20 mM HEPES-KOH, pH 7.2, 1 m_M dithiothreitol, 1.5 mM MgC12, 100 mM KCI, 1 mM ATP, 8 mM creatinine phosphate, 31 units/ml creatinine phosphokinase, and 0.25 mg/ml DNPBSA. Fusions performed in the absence of ATP include an ATP-depleting system (5 mM 2-mannose, 25 U/ml hexokinase). 4. Incubate the fusion reaction at 37 o for 45 min. 5. Stop the reaction by returning the tube to 4 °. 6. Quantitate the amount of DNP-fl-glucuronidase associated with anti-DNP IgG-coated S. aureus by lysing samples in solubilization buffer [1% (v/v) Triton X-100, 0.2% (w/v) methylbenzethonium chloride, 1 mM ethylenediaminetetraacetic acid (EDTA), 0.1% (w/v) BSA, 0.15 M NaCI, 10 mM Tris-HC1, pH 7.4, 0.25 mg/ml DNP-BSA] followed by centrifugation at 2800 g for 5 min. Resuspend the pelleted phagocytic particles in 100~1 solubilization buffer plus t00~tl 4-methylumbelliferyl fl-D-




glucuronide (I .64 mg/ml in 0.1 M acetate buffer, pH 4.5). Incubate at 37 ° for 2 hr. 7. Add 1.0 ml glycine buffer (130 m M glycine, 60 m M NaC1, 80 mM NaCO3, pH 9.5) to stop the reaction and measure fluorescence as described. 7 Phagosome-endosome fusion is cytosol, ATP, K +, and NSF (the Nethylmaleimide-sensitive factor active in vesicular transport in the secretory pathway) dependent.7,16-~8 At low concentrations of cytosol (0.1 mg/ ml), GTPTS stimulates fusion while at high cytosol concentrations (1.5 mg/ml) GTPTS inhibits fusion markedly (Fig. 2A). Futhermore, salt concentrations also differentially affect fusion. At low salt concentrations (50 m M KC1 or NaC1), maximal fusion is observed with low cytosol and GTP),S. At high salt concentrations (125 m M KC1 or NaC1), maximal fusion is observed with high cytosol concentration without GTPyS (Fig. 2B). Electron microscopic examination of a phagosome-endosome fusion reaction reveals that multiple fusions among endosomes and phagosomes occur simultaneously (Fig. 3). Analysis of Intracellular Pathway of Phagocytosed Mouse Anti-DNP In Fig. 1, the ~2SI-labeled anti-DNP IgG that dissociates from the S. aureus is not fully accounted for in the 10% (v/v) TCA-soluble fraction of radiolabeled protein. Assaying the cell media for radioactivity during prolonged periods of phagocytic uptake reveals that some 125I-labeled antiDNP IgG recycles intact out of the cell. Thus, an intracellular pathway exists to transport phagocytosed protein from the phagosome to the cell surface. To investigate whether nonphagosomal vesicle populations contribute to the recycling pathway, we fractionated cells following phagocytic uptake of our probe. J774-E clone macrophages internalized prebound antibody-coated S. aureus for 3 min as described above followed by chase at 37 ° in HBSA for increasing times. Cells were then fractionated as described below to determine the subcellular location of ~25I-labeled antiDNP IgG. Subcellular Fractionation following Phagocytic Particle Uptake and Chase

1. Following the procedure outlined above, allow J774-E clone macrophages to phagocytose antibody-coated S. aureus for 3 min at 37 °. 16 R. Diaz, L. S. Mayorga, P. J. Weidman, J. E. Rothman, and P. D. Stahl, Nature (London) 339, 398 0989). l~ L. Mayorga, R. Diaz, and P. Stahl, Science244, 1475 (1989). is L. S. Mayorga, R. Diaz, M. I. Columbo, and P. D. Stahl, CellRegul. 1, l l 3 (1989).





A 15

lO 8



~'1o C

0 -~ LL


1.1_ 2 i

' 0'2

' 04

' 0'6

' 0'8

Cytosolic Proteins (mg/ml)





[KCI] (raM)

FIG. 2. In vitro fusion of phagosomeswith early endosomes.(A) Effectof GTPyS at different cytosol concentrations:e, GTPyS; i GTP; O, control. (B) Effect of KCI: A, concentratedcytosol;0, dilutecytosol+ GTPyS;O, no cytosol.

2. Wash the cells two times in HBSA and incubate again at 37 ° in HBSA for increasing lengths of time (0-30 min). 3. Obtain postnuclear supernatants as described above from cells representing each of the chase times. 4. Dilute the postnuclear supernatant five-fold in homogenization buffer and centrifuge 1.5 min at 12,000 g at 4 °. 5. Save the pellet (Phagosome-enriched fraction) and centrifuge the supernatant at 35,000 g for 1 min at 4 °. 6. Save the pellet (lysosome-enriched fraction) and centrifuge supernatant at 50,000 g for 5 min (endosome-enriched fraction). As shown in Fig. 4A, 125I-labeled anti-DNP IgG in the phagosomeenriched fraction decreases with chase times of up to 30 min. Assaying the remaining vesicles in the cell for ~25I-labeledanti-DNP IgG reveals that the endosome-enriched fraction displays a time-dependent increase in radioactivity (Fig. 4B). The majority of radioactivity in this fraction is detergent soluble, trypsin insensitive, and precipitable by 10% (v/v) TCA. The amount of nSI-labeled anti-DNP IgG in the lysosome-enriched membrane pellet does not change with increasing chase times. Characterization of the nSI-labeled anti-DNP IgG-containing vesicles (transport vesicles) in the endosome-enriched membrane pellet reveals that they share features characteristic of early endosomes. As determined by Percoll density gradient centrifugation, the transport vesicles display a buoyant density distribution




FIG. 3. Electron micrograph demonstrating multiple fusions among endosomes and phagosomes. Early endosomes (5 min at 37 °) from three separate populations of J774-E clone macrophages were loaded with 5-, 10-, and 20-nm gold particles coated with mannose-BSA. Endosome fractions of postnuclear supernatants from these cells were incubated under fusogenic conditions (0.1 mg/ml cytosol, 20 p.M GTPyS) with the phagosome fraction isolated as described in text. Bar: 0.2/~m.

similar to that observed for material endocytosed in the fluid phase similar to those of early endosomes (data not shown). Employing the DNPbinding property of t25I-labeled anti-DNP IgG, we determined that the fusogenicity of transport vesicles is similar to that described previously for early endosome fusion. Early endosomes labeled with DNP-flglucuronidase as described above were mixed under fusogenic conditions (i.e., ATP, cytosol, and KCI) with transport vesicles. Following incubation at 37 ° for 1 hr, fusion samples were lysed in the presence of the scavenger protein, DNP-BSA. Immune complexes were immunoprecipitated with immobilized rabbit anti-mouse IgG and immunoprecipitated 13-





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Assays for phagosome-endosome fusion and phagosome protein recycling.

[4] ASSAYS FOR P H A G O S O M E - E N D O S O M E F U S I O N 2 1 cles of constant density. These separations are performed in 27% (v/v) Percoll i...
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