Image analysis
The image processing package FIJI was used for image processing and analysis (Schindelin et al., 2012). Processing of raw images included: image cropping and rotation; bleach correction; background subtraction; adjustment of brightness/contrast; color switching; selection of images from z-stacks; intensity threshold adjustments; measurements of parameters including areas, mean gray values, and integrated densities.
To quantify Dop1p-mNeon fluorescence intensity in the CV bladder vs whole cell, all images were taken using the same image capturing settings. The raw images were used to select the ROIs of the area of Dop1p-labeled bladder and whole cell. The mean fluorescence intensities of these ROIs were then measured. To quantify Vma4p-mNeon localized spongiome distribution, the raw images were used to measure the area of Vma4p-labeled spongiome and whole cell. For FRAP analysis, the time-lapse images were separately adjusted by bleach correction for the pre-photobleaching series and after-photobleaching series. The intensity thresholds were adjusted for background subtraction, and intensity changes over time were measured in ROIs. Intensities were plotted using GraphPad Software Prism.