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.