Image analysis and quantification.

All microscopy data were quantified using Fiji (ImageJ2). The raw image data were sum-intensity projected, and protein kinetics at the region of interest (ROI) were analyzed using the quantification workflow described previously (46). In brief, raw data were registered using the ImageJ plug-in StackReg. A polygon (ROI) was drawn to encompass the fluorescence signal at the bud neck region, and the derived intensities were corrected by background subtraction. The quantification data were extracted, normalized, and further analyzed to derive the temporal protein kinetics. The graphs representing kinetic signatures of individual proteins (Fig. 3e to ​tog)g) were plotted using Origin (2015, Sr2, 69.2.272; OriginLab, USA).

The signal-to-noise ratio (SNR) was calculated on sum-intensity-projected time-lapse data analyzed in Fiji. SNR values were obtained using the following formula (47): SNR = (maximum intensity of signal − mean intensity of background signal)/standard deviation of background signal.

A notched box plot was used to represent the SNR values (Fig. 3d [the notched box graph represents 25% and 75% intervals, the black lines represent the means, the notches represent the medians, and the whiskers denote the standard deviations]).