Figure 5: dSEPT7 reduction results in reorganization of dSTIM and dOrai in resting neurons.

(a) Representative images of immunostained endogenous dSTIM and dOrai, in an optical section of a surface view of the PM and subcellular regions in close proximity of the PM, in either resting (−TG) or store-depleted (+TG) cells of the indicated genotypes. Scale bar, 5 μM. Images shown in the top two panels were acquired using confocal microscopy and those in the bottom two panels were acquired using structured illumination microscopy (SIM). (b) A K–S plot comparing normalized dSTIM intensities near the PM in the indicated genotypes either in resting conditions or after TG treatment. The distributions for TG-treated wild-type cells is shifted significantly to the right when compared with the resting cells, indicating a greater proportion of cells with higher normalized dSTIM intensities. The distribution for resting cells with reduced dSEPT7 is shifted significantly to the right when compared with the resting wild-type cells, indicating a higher proportion of cells with higher normalized dSTIM intensities. ***P<0.0001, K–S test, WT (−TG) compared with either WT (+TG) or dSEPT7 het (−TG). Normalized dSTIM intensity near the surface=dSTIM intensity in the optical section representing the cell surface/total dSTIM intensity for the cell. Images acquired using confocal microscopy were used for the analysis in b,c. (c) Bar graph quantifying the Mander’s co-localization coefficients for the amount of STIM intensity co-localizing with Orai intensity in the indicated genotypes and treatment conditions. **P<0.001, Mann–Whitney U test with Bonferroni correction. (d–g) Representative images of dOrai protein organization on the surface of resting and TG-treated cells of the indicated genotypes. dOrai was visualized by optimal image acquisition settings with confocal microscopy. Original grey scale images were deconvoluted and pseudo-coloured for better representation of high and low dOrai intensities with warmer colours representing higher intensities. A surface plot depicting the spatial distribution of dOrai intensities accompanies each image. Calibration bar represents the grey scale intensities corresponding to each colour. Scale bar, 5 μm.