Department of Molecular and Cell Biology, University of Connecticut, Storrs, CT 06269, USA1
Author for correspondence: David Knecht. Tel: +1 860 486 2200. Fax: +1 860 486 4331. e-mail: knecht{at}uconn.edu
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ABSTRACT |
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Keywords: phagocytosis, myosin II
Abbreviations: DIC, differential interference contrast; DsRed-Ec, DsRed Escherichia coli; FIU, fluorescence intensity unit(s)
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INTRODUCTION |
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Phagocytic ability is not limited to multicellular eukaryotes. Simpler organisms, like the social amoeba Dictyostelium, are very agile phagocytes. The process of particle uptake in Dictyostelium looks remarkably similar to macrophage phagocytosis (Aizawa et al., 1997 ; Rupper & Cardelli, 2001
). The evidence suggests that phagocytosis is driven by a receptor-activated signal transduction cascade leading to the actin cytoskeleton, similar to the system observed in mammalian cells. However, in Dictyostelium it is possible to take a genetic approach to understanding the process of phagocytosis (Cornillion et al., 2000
; Janssen & Schleicher, 2001
; Robinson & Spudich, 2000
). To initiate a genetic analysis of phagocytosis, it was important to have a robust and versatile method of characterizing the process. In this paper we report the use of Escherichia coli expressing the 28 kDa fluorescent protein DsRed (Baird et al., 2000
) for a Dictyostelium phagocytosis assay. This phagocytosis substrate provides a bright signal that is digestible by cells, resistant to photobleaching and provides a fluorescence emission that is stable at physiological pH (Aubry et al., 1993
; Baird et al., 2000
). To separate the process of binding from internalization, we have added a washing step that removes bound bacteria from the surface of the cells. This optimized assay allows examination of rates of surface binding, internalization and degradation of prey particles
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METHODS |
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DsRed-expressing E. coli.
Fluorescent bacteria for phagocytosis assays were prepared by introducing a DsRed expression vector into E. coli cells. The E. coli line XL-1 Blue MFR' (Stratagene) was transformed with the vector pDG75 (kindly provided by Dr Daniel Gage, University of Connecticut, Storrs, CT, USA). The vector was created by amplifying the DsRed gene from pDsRed (Clontech) using a primer containing a ribosome-binding site (as described in Bringhurst et al., 2001 ) and the fragment was cloned into pGemTeasy (Promega). E. coli cells containing the vector were grown in LB medium with 1·0 mM IPTG and 75 mg ampicillin ml-1 at 37 °C with shaking. After the cells reached stationary phase the cultures were shaken for an additional 24 h during which time the DsRed became maximally fluorescent. Cultures were concentrated by centrifugation and resuspended at an OD600 of 8·0 in 20 mM sodium phosphate buffer (pH 6·3) and stored at 4 °C for no more than 3 weeks (1:200 dilution in above buffer measured in a BioRad SmartSpec 3000).
Phagocytosis assay.
Dictyostelium cells were harvested by centrifugation in the cold for 5 min at 200 g, washed once in 20 mM sodium phosphate buffer, pH 6·3, and resuspended at a concentration of 4x106 cells ml-1. Cells were then incubated in 20 mM sodium phosphate buffer, pH 6·3, at 22 °C and shaken at 130 r.p.m. in a 50 ml flask for 15 min. After this initial incubation, 2x1010 DsRed E. coli (DsRed-Ec) cells were added for every 4x106 Dictyostelium cells. Alternatively, 2x1010 YG fluorescent plain latex beads (Polysciences) were used. DsRed-Ec or latex beads were briefly sonicated and vortexed vigorously to break-up any aggregates before addition to the Dictyostelium suspension. This gave a final ratio of 5000 prey particles to each Dictyostelium cell. At each time point in the assay, 0·5 ml cells were transferred from the flask to 5 ml ice cold 20 mM sodium phosphate buffer in a 15 ml conical tube. Cells were washed free of unbound bacteria by centrifugation at 200 g for 5 min at 4 °C in a swinging bucket rotor (Beckman JH6). The pellet was then resuspended in 5 ml fresh buffer by vortexing for 5 s at maximum speed. The pellets were washed two additional times and after the third wash the pellet was resuspended in 200 µl buffer and transferred to a 96-well microtitre plate (Falcon). Fluorescence was assessed in a plate fluorimeter (CytoFluor Series 400; PerSeptive Biosystems) using 530 nm excitation and 580 emission for the DsRed signal or 485 nm excitation, 508 nm emission for YG beads. Each time point was done in triplicate from the same flask unless otherwise indicated. Background signal was established by adding 2x1010 DsRed-expressing bacteria or 2x1010 YG beads per millilitre of 20 mM sodium phosphate buffer to flasks without cells and prepared as above.
To remove surface-bound bacteria or beads, the first two washes were performed in 20 mM sodium phosphate buffer containing 5 mM sodium azide, a modification of the method of Glynn (1981) . This azide concentration causes release of surface-bound bacteria and beads, but does not significantly reduce cell viability if it is subsequently washed out (data not shown). The final wash did not contain azide and the cells were resuspended in buffer without sodium azide before they were analysed in the fluorescent plate reader.
Binding without uptake.
To measure binding in the absence of uptake, flasks containing Dictyostelium were incubated in an ice bucket of packed shaved ice on a rotary shaker (100 r.p.m.) for 15 min before ice-cold bacteria were added. The mixture was then incubated on ice while shaking for 10 min and then washed as described above. All steps were performed at as close to 0 °C as possible.
DsRed breakdown.
To measure breakdown of fluorescent bacteria, cells were allowed to take up bacteria for 30 min as described above. The cells were then washed twice in ice-cold sodium phosphate buffer, pH 6·3, with 5 mM sodium azide and once in buffer without sodium azide. Washed cells were resuspended in HL-5 at 4x106 cells ml-1. To one flask, unlabelled Klebsiella aerogenes was added to a final concentration of 1x1010 ml-1 and an equal volume of buffer was added to the other. The cells were shaken at 130 r.p.m. and aliquots were removed at specified time points and centrifuged at 2400 g for 5 min at 4 °C to separate cells from any secreted DsRed-labelled material. Three hundred microlitres of supernatant was removed and added to a 96-well microtitre plate. The rest of the supernatant was discarded and then the cells were resuspended and added to another well. The fluorescence of the cells and supernatant was measured on a fluorescence plate reader as described above.
Confocal microscopy.
Images of cells and bound bacteria were acquired using a Bio-Rad MRC600 laser scanning confocal microscope. To minimize internalization of bound particles during imaging, the cells were placed in 20 mM sodium phosphate buffer in a Rose chamber cooled by a 20/20 Technologies stage controller set at 0 °C on the stage of a Zeiss Axiovert Microscope. Dual channel imaging was used with a rhodamine filter to visualize the DsRed bacteria and differential interference contrast (DIC) to image the whole cells and bound particles. Optical sections of the rhodamine channel were made using a x63 1·25 NA oil immersion objective. Z-Series images were collected at 0·5 µm intervals. Single slices from the Z-stacks are presented with the two channels overlaid. The DsRed bacteria are pseudocoloured red in the merged image. Images were processed with NIH image (developed at the US National Institutes of Health and available at http://rsb.info.nih.gov/nih-image/) and Photoshop (Adobe).
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RESULTS |
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Several experiments were performed to confirm that azide was removing surface-bound bacteria. To visualize the effects of azide treatment, cells were incubated for various times with DsRed-Ec and then washed with or without azide. The cells were then imaged with a confocal microscope. This allowed us to use optical sectioning to determine if bacteria were near the surface of the cell or inside. Cells incubated for 5 min or more had many bacteria associated with the surface when washed with buffer alone and some bacteria appeared to have been internalized (Fig. 3). When washed with azide, all the DsRed-Ec cells were found inside the cell. When cells that had taken up bacteria for 40 min were examined, there was a significant increase in the amount of bacteria associated with the surface as well as inside the cell. All of the surface-bound bacteria appeared to have been removed by the azide wash.
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The rounding and detachment of cells from surfaces in the presence of azide are mediated by myosin II, since cells lacking myosin II contractile activity remain attached to the substrate when exposed to azide (Pasternak et al., 1989 ). To assess if the release of bound bacteria from the surface of the cell was mediated by a generalized cell shape change, we analysed the effect of azide on phagocytosis by mutant cells lacking myosin II heavy chain (mhcA-). mhcA- cells were able to phagocytose bacteria nearly as well as wild-type cells (Fig. 5
). Surprisingly, the release of bacteria from mhcA- cells was equivalent to that of wild-type cells (Fig. 5
). This result indicates that myosin II is not required for phagocytosis of bacteria and that the azide-induced bacterial release is not functioning through a generalized cell shape change or other myosin II-dependent mechanism.
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DISCUSSION |
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The mechanism of sodium-azide-mediated release of surface-bound particles remains unclear. It may act through depletion of ATP from the cells; however, the fact that particles are released even when the treatment is done in the cold argues that the phenomenon may be more complex. In addition, the release of surface-bound particles requires some mechanical agitation. Since both beads and bacteria are released by azide and the uptake of these particles is mediated by different receptors, it seems unlikely that the azide release is due to a direct effect on receptor affinity. A more likely explanation is that azide has an effect on cytoskeletal protein(s) that modulate receptor function. It is clear that the release of particles from the surface of the cells is not mediated by myosin II, although myosin II has been found in the phagocytic cup of lung macrophages (Stendahl et al., 1980 ). Cells lacking myosin II function show the same azide-induced release of bacteria as wild-type cells. Myosin VII is present at the early stages of phagocytic cup formation and is another possible candidate for the molecule affected by sodium azide (Tuxworth et al., 2001
). Mutants that lack myosin VII are defective in uptake of DsRed-Ec, but the small amount of surface binding that does occur is partially azide-sensitive, suggesting that myosin VII is not essential for azide release. When cells are treated with 2,4-dinitrophenol, an uncoupler of oxidative phosphorylation, the cells round up and the cortical actin filament system is disrupted (Jungbluth et al., 1994
). The rearrangement of the actin cytoskeleton induced by starving cytoskeletal components for ATP combined with the shear forces generated by vortexing the cells may cause the release of incompletely internalized bacteria.
There are several advantages to using E. coli expressing DsRed as a phagocytosis uptake marker. A major consideration is alteration of the surface chemistry of the bacteria. The surface chemistry of the prey particle is important to how Dictyostelium interacts with the particle (Cohen et al., 1994 ; Cornillion et al., 2000
; Vogel et al., 1980
). Since the DsRed bacteria are collected from growth medium and used for the assay, changes in surface chemistry caused by handling are negligible. The use of live E. coli also prevents loss of any attractant factors or signals that are secreted by living bacteria and any modification to the surface of the cell wall that is the result of preparation and labelling of bacteria using a heat killing step. A second advantage is that the fluorescence emission from DsRed protein is very pH-stable (Baird et al., 2000
). The stability of DsRed works in tandem with live E. coli which have an intact membrane, so the DsRed is protected from the environment of the phagolysosome until the bacterial membrane is disrupted. This situation differs from artificially labelled bacteria, where the fluorescence marker is accessible to the acidic environment of the phagosome immediately after internalization. Loss of the E. coli-derived DsRed fluorescence signal is likely to be the direct result of the degradation of the DsRed protein and not a pH quenching effect. A final advantage is that unlike chemical labelling procedures, it is very simple and inexpensive to prepare large amounts of bacteria for phagocytosis assays. Thus E. coil expressing DsRed has great utility as a target particle for phagocytosis assays. Expression of the DsRed protein in other bacteria with known surface characteristics will give us the opportunity to examine the role of surface chemistry on phagocytosis.
Using DsRed-Ec, we observed a half-life of the fluorescence signal of 45 min. This suggests that after 30 min incubation, when the fluorescence signal begins to plateau, we are observing the equilibrium between uptake and degradation of the DsRed signal. In this plateau period, the difference between the sodium-azide-washed samples and untreated samples indicates the binding capacity of the surface of the cell for bacteria. This binding capacity represents approximately 50% of the total signal at equilibrium. This result suggests that binding of bacteria is not the rate-limiting step in phagocytosis, since abundant surface-bound bacteria are available for internalization. The ability to remove bacteria from the surface with azide is critical to the efforts we have initiated to genetically screen for phagocytosis mutants. Mutants that bind, but do not internalize bacteria would be difficult to identify if we were not able to remove the surface-bound bacteria.
The combination of DsRed with a mild sodium azide wash to release surface-bound bacteria produces a versatile phagocytosis assay. This assay is well suited for examination of the kinetics of particle binding and uptake, and for screening for mutants defective in both the binding and subsequent stages of phagocytosis. Understanding the biochemical mechanisms of phagocytosis will be aided by our ability to separate the process into discrete steps and to identify the molecular components involved.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Aizawa, H., Fukui, Y. & Yahara, I. (1997). Live dynamics of Dictyostelium cofilin suggests a role in remodeling actin latticework into bundles. J Cell Sci 110, 2333-2344.
Aubry, L., Klein, G., Martiel, J. L. & Satre, M. (1993). Kinetics of endosomal pH evolution in Dictyostelium discoideum amoebae. J Cell Sci 105, 861-866.
Baird, G. S., Zacharias, D. A. & Tsien, R. Y. (2000). Biochemistry, mutagenesis, and oligomerization of DsRed, a red fluorescent protein from coral. Proc Natl Acad Sci USA 97, 11984-11989.
Bringhurst, R. M., Cardon, Z. G. & Gage, D. J. (2001). Galactosides in the rhizosphere: Utilization by Sinorhizobium meliloti and development of a biosensor. Proc Natl Acad Sci USA 98, 4540-4545.
Cohen, J. C., Bacon, R., Clarke, M., Joiner, K. & Mellman, I. (1994). Dictyostelium discoideum mutants with conditional defects in phagocytosis. J Cell Biol 126, 955-966.[Abstract]
Cornillion, S., Pech, E., Benghezal, M., Ravanel, K., Gaynor, E., Letourneur, F., Brucker, F. & Cosson, P. (2000). Phg1p is a nine-transmembrane protein superfamily member involved in Dictyostelium adhesion and phagocytosis. J Biol Chem 275, 34287-34292.
Franc, N. C., White, K. & Ezekowitz, R. A. (1999). Phagocytosis and development: back to the future. Curr Opin Immunol 11, 47-52.[Medline]
Glynn, P. J. (1981). A quantitative study of the phagocytosis of Escherichia coli by myxamoebae of the slime mould Dictyostelium discoideum. Cytobios 30, 153-166.[Medline]
Greenberg, S. (1995). Signal transduction of phagocytosis. Trends Cell Biol 5, 93-99.
Greenberg, S. (2001). Diversity in phagocytic signalling. J Cell Sci 114, 1039-1040.
Janssen, K. P. & Schleicher, M. (2001). Dictyostelium discoideum: A genetic model system for the study of professional phagocytes Profilin, phosphoinositides and the 1mp gene family in Dictyostelium. Biochim Biophys Acta 1525, 228-233.[Medline]
Jungbluth, A., von Arnim, V., Biegelmenn, E., Humbel, B., Schweiger, A. & Gerisch, G. (1994). Strong increase in the tyrosine phosphorylation of actin upon inhibition of oxidative phosphorylation: correlation with reversible rearrangements in the actin skeleton of Dictyostelium cells. J Cell Sci 107, 117-125.
Knecht, D. A., Cohen, S. M., Loomis, W. F. & Lodish, H. F. (1986). Developmental regulation of Dictyostelium discoideum actin gene fusions carried on low-copy and high-copy transformation vectors. Mol Cell Biol 6, 3973-3983.[Medline]
Kwiatkowska, K. & Sobota, A. (1999). Signaling pathways in phagocytosis. Bio Essays 21, 422-431.[Medline]
Loomis, W. F. (1971). Sensitivity of Dictyostelium discoideum to nucleic acid analogues. Exp Cell Res 64, 484-486.[Medline]
Maniak, M., Rauchenberger, R., Albrecht, R., Murphy, J. & Gerisch, G. (1995). Coronin involved in phagocytosis: Dynamics of particle-induced relocalization visualized by a green fluorescent protein Tag. Cell 83, 915-924.[Medline]
Pasternak, C., Spudich, J. A. & Elson, E. L. (1989). Capping of surface receptors and concomitant cortical tension generated by conventional myosin. Nature 341, 549-551.[Medline]
Rabinovitch, M. (1967). The dissociation of the attachment and ingestion phases of phagocytosis by macrophages. Exp Cell Res 46, 19-28.[Medline]
Robinson, D. N. & Spudich, J. A. (2000). Dynacortin, a genetic link between equatorial contractility and global shape control discovered by library complementation of a Dictyostelium discoideum cytokinesis mutant. J Cell Biol 150, 823-838.
Ruppel, K. M., Uyeda, T. Q. & Spudich, J. A. (1994). Role of highly conserved lysine 130 of myosin motor domain. In vivo and in vitro characterization of site specifically mutated myosin. J Biol Chem 269, 18773-18780.
Rupper, A. & Cardelli, J. (2001). Regulation of phagocytosis and endo-phagosomal trafficking pathways in Dictyostelium discoideum. Biochim Biophys Acta 1525, 205-216.[Medline]
Shelden, E. & Knecht, D. A. (1995). Mutants lacking myosin II cannot resist forces generated during multicellular morphogenesis. J Cell Sci 108, 1105-1115.
Stendahl, O. I., Hartwig, J. H., Brotschi, E. A. & Stossel, T. P. (1980). Distribution of actin-binding protein and myosin in macrophages during spreading and phagocytosis. J Cell Biol 84, 215-224.[Abstract]
Titus, M. A. (1999). A class VII unconventional myosin is required for phagocytosis. Curr Biol 9, 1297-1303.[Medline]
Tuxworth, R. I., Weber, X., Wessels, D. A., Addicks, G. C., Soll, D. R., Gerisch, G. & Titus, M. A. (2001). A role for myosin VII in dynamic cell adhesion. Curr Biol 11, 318-329.[Medline]
Vogel, G., Thilo, L., Schwarz, H. & Steinhart, R. (1980). Mechanism of phagocytosis in Dictyostelium discoideum: Phagocytosis is mediated by different recognition sites as disclosed by mutants with altered phagocytic properties. J Cell Biol 86, 456-465.
Watts, D. J. & Ashworth, J. M. (1979). Growth of myxamoebae of the cellular slime mould Dictyostelium discoideum in axenic culture. Biochem J 119, 171-174.
Received 10 July 2001;
revised 8 October 2001;
accepted 19 October 2001.