Ratiometric Microscopy of Arf6-YFP, ARF1-YFP, and YFP-NGAT at the Phagosome

Ratiometric fluorescence microscopy of macrophages expressing ARF1-YFP, ARF6-YFP, or YFP-NGAT chimeras with soluble CFP during phagocytosis.

(A, C, and E) Phase contrast (PC) and ratiometric images of macrophages during the phagocytosis of IgG-opsonized erythrocytes. Scale bars are 3 μm. Color bars indicate the molar ratio of YFP-chimera/CFP in the adjacent RM images.

(B, D, and F) Plots of the average recruitment index (Ri) for temporally aligned phagocytic events. Error bars correspond to ± SEM.

(A) ARF6-YFP rapidly accumulated at sites of phagocytosis; the region of high ARF6-YFP concentration advanced over the particle during pseudopod extension.

(B) Analysis of multiple phagosomes indicated that the level of ARF6-YFP on the phagosome peaked during the first 2.5 min of engulfment. Following this initial rise, localization to the phagosome gradually decreased but ARF6-YFP remained associated with the phagosome well after closure at 7–8 min; n = 10 phagosomes.

(C) ARF1-YFP localization to the phagosome was less pronounced than ARF6-YFP. ARF1-YFP generally accumulated over the entire phagosome and was also slightly enriched in membrane ruffles formed following closure.

(D) Averaged particle-tracking results indicated ARF1-YFP association increased transiently during the first 2 min of phagosome formation, then increased further after 5 min. ARF1-YFP levels at the phagosome plateaued shortly after closure and remained elevated over the course of observation; n = 11 phagosomes.

(E) The concentration of YFP-NGAT did not increase at sites of phagocytosis relative to the cytosol, indicating the level of activated ARF GTPases at the phagosome was too low to detect using YFP-NGAT ratiometric imaging.

(F) Aggregate particle-tracking data never indicated recruitment of YFP-NGAT to phagosomes in the presence or absence of the ARF GEF inhibitor BFA. The time course of phagosomes formed without BFA pretreatment is the average of seven phagosomes. The plot of phagosomes formed in the presence of BFA was generated from ten phagocytic events.



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