Figure 5.

SWAP70 Promotes Phagosomal RAC1 Activity

(A) Confocal micrographs and quantification of RAC1 (green in merge) and SWAP70 (magenta) positive phagosomes for seven donors (∼100 cells/donor). Aver., average ± SEM.

(B) Venn diagram showing phagosomal distribution of RAC1, F-actin, and SWAP70.

(C) Live cell imaging of dendritic cells expressing SWAP70-GFP (green in merge) and RAC1-RFP (magenta). The inset shows a time series during zymosan uptake. BF, bright field. See also Movie S14.

(D) Quantification from (C). The histogram shows the time difference of peak recruitment of SWAP70-GFP and RAC1-RFP based on fluorescence intensities (88 phagosomes). Negative values indicate that RAC1 was recruited prior to SWAP70 and positive values later than SWAP70.

(E and F) Multicolor 3D-STED microscopy of zymosan-pulsed dendritic cells immunostained for RAC1 (green) and SWAP70 (magenta; E) or F-actin (F). Left: cross-section and orthogonal sections (indicated by dashed yellow lines). Middle: maximum intensity height maps. Right: maximum intensity surface projection. Yellow arrowheads, overlap of RAC1 with SWAP70 (E) or F-actin (F) on the surface of the phagosomes. See also Movies S15 and S16.

(G) RAC1 activation upon control (siControl) and SWAP70 siRNA (siSWAP70) after zymosan addition by G-LISA assay (Abs, absorbance units at 490 nm; mean ± SEM). The background (t = 0) is from cells without zymosan.

(H) RAC1-positive phagosomes upon siControl and siSWAP70 counted by microscopy (>20 cells/donor/condition analyzed; individual donors shown).

(I) Phagosomes positive for F-actin and/or RAC1 with siControl or siSWAP70.

(J) Model of F-actin cage formation by SWAP70. SWAP70 is recruited to phagosomes by coincidence detection. Scale bars: 10 μm (A and C), 2 μm (E and F).