FRAP and FLIP analysis

Yeast used for FRAP and FLIP were grown and collected as described above, and imaging was conducted for 1 hr after collection. Photobleaching movies were taken on an Andor spinning disk confocal microscope through a 63x oil objective (NA = 1.4). The microscope is equipped with an Andor Ultra EMCCD and Metamorph software. For FRAP measurements, a single circular ROI of 0.77 μm² that corresponds to the NVJ was selected and bleached with a 408 nm laser at 100% power and 100 ms dwell time. One image was taken before the bleach, and subsequent images were taken every 500 ms for a total movie length of 25 s. For FLIP measurements, single circular ROIs of 0.77 μm² were selected, taking care to select an area of the NE that was furthest from the NVJ. Each bleaching cycle consisted of a pre-bleach image, a single bleach with a 408 nm laser with a 100 ms dwell time, and four post-bleach images taken 500 ms apart. Each movie captured a total of 50 bleach cycles, which corresponds to 300 s movies. Fiji software was used to quantify bleaching curves and halftimes. Pre-processing of images included background subtraction as described above and 3D Gaussian smoothing (sigma = 0.5). FRAP quantification was performed using the double normalization method as previously described (Phair and Misteli, 2000). Briefly, intensity was measured for all time points in an ROI corresponding to the bleached region (I_frap) and an ROI corresponding to the whole cell (I_whole-cell), and normalized intensities were generated for each timepoint (I_normalized(t)) using Equation 1:

In the equation above, the ‘pre’ subtext indicates the timepoint preceding ROI bleaching. Intensity recovery curves were created for each movie by further normalizing values such that pre-bleach intensities were set to one and post-bleach intensity of an ROI was set to 0. Full normalization was accomplished using Equation 2 followed by subtracting the normalized I_frap-bleach value from all timepoints.

In Equation 2, I_frap-bleach indicates the intensity of the photobleached ROI at the time of photobleaching. Halftimes were calculated from individual fluorescence recovery curves using Graphpad Prism eight software and fitting the data to a one-phase exponential association. Pre-processing for FLIP images was the same as for FRAP images. FLIP movies were quantified using Fiji to monitor the intensity of NVJ-associated signal over time. Intensity measurements were normalized using Equation 3.

In Equation 3, I_FLIP(t) is the relative fluorescence at the NVJ at time t, I_NVJ(t) is the raw intensity value at the NVJ at time t, and I_{NVJ-pre-bleach} is the intensity at the NVJ before bleaching occurred. Halftimes were calculated from FLIP decay curves, which were generated in Graphpad Prism eight software by fitting the data to a one-phase exponential decay.