Evidence for a molecular complex consisting of Fyb/SLAP, SLP-76, Nck, VASP and WASP that links the actin cytoskeleton to Fc
receptor signalling during phagocytosis
Marc G. Coppolino1,
Matthias Krause2,*,
Petra Hagendorff2,
David A. Monner2,
William Trimble1,
Sergio Grinstein1,
Jürgen Wehland2 and
Antonio S. Sechi1,
1 Programme in Cell Biology, Research Institute, The Hospital for Sick Children, 555 University Avenue, Toronto, Ontario, M5G 1X8, Canada
2 Department of Cell Biology, Gesellschaft für Biotechnologische Forschung (GBF), Mascheroder Weg 1, D-38124 Braunschweig, Germany
* Present address: Department of Biology, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, MA 02138-4307, USA
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Fig. 1. Ena/VASP proteins and Fyb/SLAP accumulate at sites of phagocytosis in Raw264.7 macrophages. (A,C) Analysis by western blotting of protein extracts of Raw264.7 macrophages using an anti-Evl monoclonal antibody (A) and an anti-Fyb/SLAP affinity-purified polyclonal antibody (C). (B,D) Localisation of Evl and Fyb/SLAP in Raw264.7 macrophages during phagocytosis. Raw264.7 cells were incubated with IgG-opsonised sheep RBC (for Evl labelling) or IgG-coated beads (for Fyb/SLAP labelling) for approximately five minutes, fixed and stained with the above mentioned antibodies. Both Evl (B, left pa nel) and Fyb/SLAP (D, left panel) localise to phagocytic cups where they colocalise with actin (B,D; right panels). Arrows point to examples of phagocytic cups surrounding sheep RBC (B) or beads (D). Scale bar: 10 µm.
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Fig. 2. Ena/VASP proteins and Fyb/SLAP accumulate at sites of phagocytosis in primary macrophages. (A,C) Western blot analysis of protein extracts of monocyte-derived macrophages using an anti-VASP monoclonal antibody (A) and anti-Fyb/SLAP (C) affinity-purified polyclonal antibody. (B,D) Analysis of VASP and Fyb/SLAP intracellular distribution in monocyte-derived macrophages during phagocytosis. Cells undergoing phagocytosis were incubated with IgG-opsonised sheep RBC (for VASP labelling) or beads (for Fyb/SLAP labelling) for approximately five minutes, fixed and stained with the antibodies described above. In macrophages engaged in phagocytosis, both VASP (B) and Fyb/SLAP (D) localise to forming phagosomes. Again, both proteins colocalise with actin at these sites (B,D). Arrows point to examples of phagocytic cups surrounding sheep RBC (B) or beads (D). Scale bar: 10 µm.
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Fig. 3. Dynamics of GFP-VASP during early events of phagocytosis. Raw264.7 macrophages expressing GFP-VASP were incubated with IgG-opsonised sheep RBC and followed by digital video microscopy. GFP-VASP begins to accumulate at the sites of engulfment approximately one minute after SRBC binding, remains associated with phagocytic cups during until the entire internalisation process and disappears upon closure of the phagocytic cups. The positions of bound sheep RBC were observed using DIC optics (not shown). Arrowheads indicate sites of sheep RBC binding that led to complete phagosome formation. Numbers represent elapsed time (in seconds) after sheep RBC settled onto cells. Scale bar: 10 µm.
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Fig. 4. Dynamics of GFP-Fyb/SLAP during early events of phagocytosis. Raw264.7 macrophages expressing GFP-Fyb/SLAP were incubated with IgG-opsonised SRBC and followed by video microscopy. GFP-Fyb/SLAP begins to accumulate at the sites of engulfment approximately one minute after sheep RBC binding, remains associated with phagocytic cups during the entire internalisation process and disappears upon closure of the phagocytic cups. The positions of bound sheep RBC were observed using DIC optics (not shown). Arrowheads indicate sites of sheep RBC binding that led to complete phagosome formation. Numbers represent elapsed time (in seconds) after SRBC settled onto cells. Scale bar: 10 µm.
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Fig. 5. Clostridium difficile toxin B inhibits the recruitment of VASP. Raw264.7 macrophages expressing GFP-VASP were incubated with 10 ng/ml C. difficile toxin B for two hours prior to use in phagocytosis assays with IgG-opsonised sheep RBC. Toxin B-treated cells bind sheep RBCs but do not internalise them (arrowheads in A). Note that there was no accumulation of GFP-VASP on the membrane abutting the bound particles (arrowheads in B). Scale bar: 10µm.
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Fig. 6. Inhibition of actin cup formation by ActA repeats. Raw264.7 cells transiently transfected with GFP-tagged ActA repeats (A-aA'') or with the mutated form of the repeats (B-B'') were incubated with antibody-coated sheep RBC, fixed and stained with Texas Red-conjugated phalloidin. Cells expressing the active form of the ActA repeats were able to bind sheep RBC but did not form actin cups (arrows in A, A''). In contrast, the formation of actin cups was not affected in control (untransfected) cells and in cells that expressed the mutated form of the ActA repeats (arrows in B, B''). A, B: phalloidin lebelling; A', B': GFP signal; A'', B'': phase contrast. Scale bar: 10 µm.
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Fig. 7. Influence of ActA repeats on the rate of phagocytosis. Raw264.7 cells were transiently transfected with wild-type or mutated GFP-tagged ActA repeats and then incubated with antibody-coated sheep RBC. After incubation at 37°C for 15 minutes, non-internalised sheep RBC were lysed with distilled water and the cells fixed. The phagocytic efficiency is expressed as the number of internalised sheep RBC per macrophage. Error bars indicate one standard deviation from the mean.
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Fig. 8. Scanning electron microscopy analysis of the interaction between Raw264.7 cells and sheep erythrocytes. Untransfected Raw cells (A), cells expressing GFP-tagged ActA repeats (B), or the mutated GFP-tagged ActA repeats (C) were incubated with opsonised sheep RBC, fixed and processed for scanning electron microscopy. Raw cells expressing GFP-tagged ActA repeats were still able to bind to SRBC but did not form lamellipodia-like extentions, which, in contrast, are formed both by control cells and cells expressing the inactive form of the ActA repeats. Scale bar: 2 µm.
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Fig. 9. Localisation of signalling proteins to phagocytic cups. Raw264.7 cells were incubated with antibody-coated sheep RBC, fixed and co-stained with Texas Red-conjugated phalloidin (A, C) and with antibodies against Nck (A'), Vav (B') or SLP-76 (C'). The three signalling proteins all localise to the phagocytic cups (arrowheads in A’, C’) where they colocalise with actin (arrowheads in A, C). Scale bar: 5 µm.
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Fig. 10. (A) Analysis by western blotting of protein extracts of monocyte-derived macrophages and Jurkat T-cells with an anti-WASP monoclonal antibody. (B) Localisation of WASP in Raw264.7 cells undergoing phagocytosis. In macrophages engulfing IgG-opsonised beads, WASP colocalises with actin at phagocytic cups. Arrowheads point to sites where opsonised beads induced the formation of phagocytic cups. Stars indicate opsonised beads that have bound to a cell but have not induced the accumulation of actin or WASP. Scale bar: 10 µm. (C) Fyb/SLAP, SLP-76, Nck, Ena/VASP proteins and WASP are located in a multi-protein complex in stimulated Raw264.7 cells. Cell lysates from control and stimulated Raw264.7 cells were incubated with the immobilised WASP monoclonal antibody 67B4. Immunoprecipitates were resolved by SDS-PAGE, blotted and probed with antibodies to Fyb/SLAP, SLP-76, Nck and Evl. The stimulation of Raw264.7 cells results in the association of Fyb/SLAP, SLP-76, Nck and Evl with WASP. E, full Raw264.7 cell lysate; WASP IP, WASP immunoprecipitate.
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© The Company of Biologists Ltd 2001
