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First published online 12 September 2006
doi: 10.1242/jcs.03190

Journal of Cell Science 119, 4079-4087 (2006)
Published by The Company of Biologists 2006
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Selective membrane exclusion in phagocytic and macropinocytic cups

Valentina Mercanti1,*, Steve J. Charette1,*, Nelly Bennett2, Jean-Jeacques Ryckewaert3, François Letourneur4 and Pierre Cosson1,{ddagger}

1 Université de Genève, Centre Médical Universitaire, Département de Physiologie Cellulaire et Métabolisme, 1 rue Michel Servet, CH-1211 Genève 4, Switzerland
2 Laboratoire de Biochimie et Biophysique des Systèmes Intégrés, Département de Réponse et Dynamique Cellulaires, CEA-Grenoble, 17 rue des Martyrs, 38054 Grenoble Cedex 9, France
3 Laboratoire de Chimie des Protéines, ERM 201 INSERM/CEA/UJF, 17 rue des Martyrs, 38054 Grenoble Cedex 9, France
4 Institut de Biologie et Chimie des Protéines (IBCP UMR 5086), CNRS Université de Lyon, IFR128 BioSciences Lyon-Gerland, 7 passage du Vercors, 69367 Lyon Cedex 07, France


Figure 1
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  		Fig. 1. Phagosome formation in cells expressing the CRAC-GFP protein. Cells expressing CRAC-GFP (green) were allowed to engulf labeled yeast (red) for 0, 1, 2, 3, 5 or 10 minutes. The cells were then fixed and processed for immunofluorescence analysis. (A) Representative examples of a phagocytic cup, of a newly formed phagosome and of a maturing early phagosome. A white line indicates the delimitation of each cell. Bar, 4 µm. (B) The graph indicates the percentage of each structure compared with the total number of CRAC-GFP positive cells. At each time point, at least 60 cells engaged in phagocytosis and expressing CRAC-GFP were considered. The experiment was repeated two times with identical results. After 1 minute, phagocytic cups (triangle) were visible. After 2 minutes, CRAC-GFP-positive newly formed phagosomes (square) were detectable while maturing early phagosomes without CRAC-GFP staining (circle) were observed after 3 minutes.

 

Figure 2
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  		Fig. 2. H+-ATPase and protein disulfide isomerase are not delivered to phagocytic cups. Cells expressing CRAC-GFP (green) were allowed to engulf yeast particles (red) for 10 minutes. The cells where then fixed and processed for total immunofluorescence to detect H+-ATPase (A) or protein disulfide isomerase (PDI) (B) H+-ATPase (white) was detected in maturing early phagosomes but not in phagocytic cups (data not shown) or in newly formed phagosomes. Protein Disulfide Isomerase (white) was not detected in newly formed or maturing early phagosomes. Bars, 4 µm.

 

Figure 3
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  		Fig. 3. Surface H72 and H161 proteins are captured in newly formed phagosomes. (A-C) Cells expressing CRAC-GFP (green) were allowed to engulf yeast particles (red) for 10 minutes. They were then fixed and processed for immunofluorescence using H72 or H161 antibodies. (A) Representative pictures of H72 labeling (white) are shown for shallow (1) and deep (2) phagocytic cups as well as newly formed (3) and maturing early (4) phagosomes. (B,C) Relative intensity of the H72 (B) or H161 (C) staining in each type of phagocytic structure (1, 2, 3 and 4). Each dot indicates the H72 (B) or H161 (C) fluorescence intensity in the membrane of one phagosome relative to the intensity at the cell surface in the same cell. A line indicates the average. (D,E) Cells were incubated with H72 or H161 antibodies for 5 minutes at 4°C, washed and allowed to engulf yeast particles (red) for 3 minutes. Cells were then fixed, permeabilized and incubated with a fluorescent secondary antibody (white). (D) Schematic representation of the experiment. (E) Surface H72 and H161 (white) are incorporated in newly formed phagosomes. Bars, 4 µm.

 

Figure 4
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  		Fig. 4. Membrane proteins H36 and PM4C4 are excluded from phagocytic cups. (A) Immunofluorescence analysis of fixed cells with H36 and PM4C4 antibodies revealed a mostly surface localization of the corresponding proteins. (B) Cellular proteins were separated on acrylamide gel, transferred to a nitrocellulose membrane and detected with H36 and PM4C4 antibodies. (C-E) Cells expressing CRAC-GFP were allowed to engulf yeast particles for 10 minutes before fixation and then processed for total immunofluorescence using H36 or PM4C4 antibodies. (C) Representative pictures are shown for shallow (1) and deep (2) phagocytic cups and for newly formed (3) or early (4) phagosomes. CRAC-GFP is shown in green, the yeast particle in red and H36 staining in white. H36 staining was strongly reduced in the phagocytic cups (1,2), and in the newly formed (3) and maturing early (4) phagosomes. (D,E) Relative intensity of the H36 (D) or PM4C4 (E) staining in phagocytic cups or in phagosomes (1-4). The relative intensity of H36 and PM4C4 was determined and plotted as described in the legend to Fig. 3. Bars, 4 µm.

 

Figure 5
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  		Fig. 5. Membrane sorting in macropinosomes. (A,B,D) Cells expressing CRAC-GFP were fixed and processed for total immunofluorescence using different antibodies. (A) Representative pictures of newly formed macropinosomes (green) stained with H36, PM4C4, H72 or H161 antibodies (white). (B) Relative intensity of H36, PM4C4, H72 and H161 staining in newly formed macropinosomes was determined and plotted as described in the legend to Fig. 3. H36 and PM4C4 staining was strongly reduced in newly formed macropinosomes. H72 and H161 staining was not. (C) H72 and H161 proteins found in the macropinosomes originate from the plasma membrane. Cells were incubated with H72 or H161 antibodies for 5 minutes at 4°C, washed and allowed to perform macropinocytosis for 3 minutes. Cells were then fixed, permeabilized and processed for immunofluorescence. (D) H+-ATPase (white) was not detected in newly formed macropinosomes (green). (E) The cell surface was biotinylated and cells incubated for 0 or 1 hour. Cells were then lysed, the H36 protein was immunoprecipitated, migrated on a polyacrylamide gel, transferred to nitrocellulose and revealed with the H36 antibody (total H36), or with avidin (biot. H36). A control where no antibody was used for the immunoprecipitation (w/o Ab) confirmed the specificity of the immunoprecipitation. Biotinylated surface H36 was not degraded during macropinocytosis. Bars, 4 µm.

 

Figure 6
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  		Fig. 6. H36 and PM4C4 proteins are excluded from macropinocytic cups. (A) Macropinocytosis in living cells expressing CRAC-GFP. Pictures of closed macropinosomes as well as pictures of macropinocytic cups at 6 and 12 seconds before closure are shown. Schematic representations of the criteria used to identify macropinocytic cups are shown on the right of each corresponding series of pictures. w: width, h: height. U-shaped CRAC-GFP structures designate structures where either the width is smaller than the height (top drawing) or where the width at the aperture is smaller than the width at the bottom of the cup (bottom drawing). 82% of U-shaped CRAC-GFP structures observed by live microscopy closed within 20 seconds and thus represented bona fide macropinocytic cups. (B,C) Cells expressing CRAC-GFP were fixed and processed for total immunofluorescence using different antibodies. (B) Relative intensity for H36, PM4C4, H72 and H161 staining in macropinocytic cups. H36 and PM4C4 were depleted in macropinocytic cups, H72 and H161 were not. (C) Macropinocytic cups in cells stained with H36 or H72 antibodies (white), and a pseudopod stained with H36. H36 was not depleted in the pseudopod. Graphs on the right represent the relative intensity of staining in cups or pseudopods compared with plasma membrane staining. Each graph is the average of a line scanning (8 µm long) done on the membrane of four cups or four pseudopods. The region scanned is delimited by the two red squares while the red arrows indicate the middle of the cups and of the pseudopods. Bars, 4 µm.

 

Figure 7
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  		Fig. 7. Composition of newly formed macropinosomes in mutant cells. Mutant cells (µ1, µ2, µ3, myoVII, myoK and myoB) expressing CRAC-GFP were fixed and processed for immunofluorescence using H36 (A) or PM4C4 (B) antibodies. Graphs present the relative intensity of H36 and PM4C4 in newly formed macropinosomes, determined and plotted as described in legend to Fig. 3.

 

Figure 8
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  		Fig. 8. Absence of massive vesicular transport around phagocytic cups. Cells were allowed to engulf yeast particles for ten minutes, then fixed and processed for electron microscopy. The left panel is a representative image of a cell (C) with a phagocytic cup containing a yeast particle (Y). The right panel is a magnification of the box in the left panel. The phagocytic cup is surrounded by a continuous layer of F-actin (arrows) and presents no evidence for fusion or fission of vesicles. An endoplasmic reticulum cistern (arrowheads) is visible in the vicinity of the phagocytic cup but no direct contact was observed. Bar, 0.5 µm.

 

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© The Company of Biologists Ltd 2006