Figure 1. Identification of SnxA as a... | Rockefeller University Press

Figure 1.

Identification of SnxA as a PI(3,5)P2reporter. (A and B) Expression of GFP-fusions of PX-domain-containing proteins in WT and ΔPIKfyve Ax3 Dictyostelium. DDB_G0285711 (A) localized to large vesicles in both cells, whereas DDB_G0289833 (B) was completely cytosolic in ΔPIKfyve. (C) Timelapse of DDB_G0289833 (SnxA)-GFP localization in Ax2 cells after addition of the PIKfyve inhibitor apilimod (3 μM). (D) Quantification of the number of SnxA-GFP vesicles per cell. >90 cells measured per time-point; data show mean ± SEM of three independent experiments. (E) Representative PIP array using lysates from WT cells expressing SnxA-GFP and probed with anti-GFP antibody. (F) Schematics of SnxA-GFP, 1xPX-GFP, and 2xPX-GFP. (G) Representative images of their localization in the cells indicated. 1xPX-GFP remained predominately cytosolic in WT cells, whereas 2xPX-GFP localized strongly to large vesicles, which was lost in ΔPIKfyve and apilimod treated cells. All scale bars = 2 μm. Source data are available for this figure: SourceData F1.

Identification of SnxA as a PI(3,5)P ₂ reporter. (A and B) Expression of GFP-fusions of PX-domain-containing proteins in WT and ΔPIKfyve Ax3 Dictyostelium. DDB_G0285711 (A) localized to large vesicles in both cells, whereas DDB_G0289833 (B) was completely cytosolic in ΔPIKfyve. (C) Timelapse of DDB_G0289833 (SnxA)-GFP localization in Ax2 cells after addition of the PIKfyve inhibitor apilimod (3 μM). (D) Quantification of the number of SnxA-GFP vesicles per cell. >90 cells measured per time-point; data show mean ± SEM of three independent experiments. (E) Representative PIP array using lysates from WT cells expressing SnxA-GFP and probed with anti-GFP antibody. (F) Schematics of SnxA-GFP, 1xPX-GFP, and 2xPX-GFP. (G) Representative images of their localization in the cells indicated. 1xPX-GFP remained predominately cytosolic in WT cells, whereas 2xPX-GFP localized strongly to large vesicles, which was lost in ΔPIKfyve and apilimod treated cells. All scale bars = 2 μm. Source data are available for this figure: SourceData F1.