Fig. 2.
Fig. 2. Defective formation of phagocytic cups in WAS macrophages and recruitment of WASp during IgG-mediated phagocytosis. (A-C and D-F) Two examples of phagocytic cup formation in normal macrophages incubated with IgG-coated latex beads (arrowheads). Cells were stained with rhodamine-phalloidin to show the distribution of F-actin (A and D, red) and FITC-PY20 to show tyrosine phosphorylation (B and E, green). Merged images (C and F) show co-localization (yellow) of the 2 signals. (G-I and J-L) Two examples of phagocytic cup formation (arrowheads) in WAS macrophages showing F-actin. (G and J, rhodamine phalloidin) and tyrosine phosphorylation (H and K, FITC-PY20). Merged images are also shown (I and L). In contrast to normal phagocytic cups, those of WAS macrophages are deficient in F-actin, giving the appearance of black disks surrounded by a less well-formed rim. Furthermore, there is no apparent accumulation of tyrosine phosphorylated proteins in the region of the phagocytic cup. (M and N) Expression of EGFP (M) and EGFP-WASp fusion protein (N) in Cos7 cells transiently transfected with plasmid vectors pEGFP-C2 and pEGFP-WASp, respectively. (O-U) Distribution of EGFP-WASp and actin in transfected normal human macrophages. Primary human macrophages transfected with plasmid vector pEGFP-WASp show accentuated cortical staining and co-localization of EGFP-WASp with F-actin (EGFP-rhodamine phalloidin merged image (O). (P-R and S-U) Two examples of transfected macrophages incubated with IgG-coated latex beads are shown. (P and S) EGFP-WASp fusion protein, visualized using a FITC filter. (Q and T) Rhodamine-phalloidin staining of F-actin. (R and U) The merged images and sites of co-localization of EGFP-WASp fusion protein and F-actin (yellow). An untransfected cell is also shown alongside a cell expressing EGFP-WASp (S-U). In cells containing phagocytosed latex beads, EGFP-WASp and F-actin both accumulate around the particles, although some remained co-localized with F actin-rich cortical areas in these cells. The scale bar represent 5μm.

Defective formation of phagocytic cups in WAS macrophages and recruitment of WASp during IgG-mediated phagocytosis. (A-C and D-F) Two examples of phagocytic cup formation in normal macrophages incubated with IgG-coated latex beads (arrowheads). Cells were stained with rhodamine-phalloidin to show the distribution of F-actin (A and D, red) and FITC-PY20 to show tyrosine phosphorylation (B and E, green). Merged images (C and F) show co-localization (yellow) of the 2 signals. (G-I and J-L) Two examples of phagocytic cup formation (arrowheads) in WAS macrophages showing F-actin. (G and J, rhodamine phalloidin) and tyrosine phosphorylation (H and K, FITC-PY20). Merged images are also shown (I and L). In contrast to normal phagocytic cups, those of WAS macrophages are deficient in F-actin, giving the appearance of black disks surrounded by a less well-formed rim. Furthermore, there is no apparent accumulation of tyrosine phosphorylated proteins in the region of the phagocytic cup. (M and N) Expression of EGFP (M) and EGFP-WASp fusion protein (N) in Cos7 cells transiently transfected with plasmid vectors pEGFP-C2 and pEGFP-WASp, respectively. (O-U) Distribution of EGFP-WASp and actin in transfected normal human macrophages. Primary human macrophages transfected with plasmid vector pEGFP-WASp show accentuated cortical staining and co-localization of EGFP-WASp with F-actin (EGFP-rhodamine phalloidin merged image (O). (P-R and S-U) Two examples of transfected macrophages incubated with IgG-coated latex beads are shown. (P and S) EGFP-WASp fusion protein, visualized using a FITC filter. (Q and T) Rhodamine-phalloidin staining of F-actin. (R and U) The merged images and sites of co-localization of EGFP-WASp fusion protein and F-actin (yellow). An untransfected cell is also shown alongside a cell expressing EGFP-WASp (S-U). In cells containing phagocytosed latex beads, EGFP-WASp and F-actin both accumulate around the particles, although some remained co-localized with F actin-rich cortical areas in these cells. The scale bar represent 5μm.

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