Quantification of phagosomal recruitment and comparison of SnxA and 2xPX. (A) Shows automated methodology to quantify fluorescent reporter recruitment to a phagocytic event. Channel containing yeast (red) is thresholded and particles larger than 1.5 μm2 are segmented (white circles). (B) This area is used to monitor fluorescence change of pHrodo-labeled-yeast, a proxy for phagosomal pH. (C) The perimeter is enlarged by 0.2 μm to generate a ring of 0.3 μm thickness over the phagosomal membrane (inner ring). As a control, an additional ring further away from the yeast (outer ring, expanded by 0.85 μm from the yeast) was also generated to give a measure of background cytosol fluorescence that should remain constant. (D) Timelapse of 2xPX-GFP recruitment to a pHrodo-labeled yeast containing phagosome in WT cells. (E) An identical experiment in ΔPIKfyve mutants. (F) Quantification of phagosomal enrichment in both cell lines. N indicates the total number of phagosomes measured, across three independent experiments. *P < 0.05, ***P < 0.005. Graph shows the mean ± SEM across independent experiments; thin lines show each of the individual repeats. (G) Timelapse of phagocytosis in WT cells coexpressing SnxA-GFP and 2xPX-RFP (see Video 3 for full timeseries). (H) Quantification of probe intensities over time in this video.
