Fluorescent recovery after photobleaching experiments

For analysis of protein turnover, cells were prepared as described in Sample Preparation and Live Cell Imaging. Cells were imaged using a spinning-disk confocal microscope (Andor TuCam imaging system, equipped with a 100× oil immersion 1.45NA Nikon Plan Apo lambda, confocal unit Yokogawa CSU-X1, Andor iXon Ultra EMCCD, FRAPPA Andor bleaching unit, and Andor iQ software). Images were acquired at a resolution of 69 nm/pixel. The fluorophores were photobleached using a 455-nm laser line. For each cell line, the minimum intensity and duration of the bleaching pulse were determined in order to minimize photobleaching of the cytosolic pool of target protein. The bleaching region of interest (ROI) was constructed as a 1 µm–wide line crossing one side of the AMR parallel to the longitudinal axis of the cell, generating an approximate 1 × 0.5–µm bleaching ROI that bleaches 30–50% of the AMR, depending on the AMR diameter. We chose a line ROI for bleaching primarily because of the speed of bleaching. Two laser lines of wavelength 488 and 561 nm were used for excitation. For measurement of the mobile fraction, all images were recorded as the 1-Z plane in the middle of the cell. For analysis of protein movement along the AMR, a Z-stack with intraslice spacing 0.3 µm was used (18 slices in total covering a Z range of 5.4 µm). First, the baseline of the protein signal in the AMR was recorded by three consecutive images taken as fast as possible (0.3 s on average for mobility measurement and 2–5 s for FRAP measurements of Z-stacks). Next, part of the AMR was bleached over 50–200 ms depending on the number of bleaching ROIs per field of view (five on the average). Finally, the FRAP signal was recorded as a sequence of 150 images taken at intervals of 0.3–1 s for mobility measurement and 2–5 s for FRAP measurements of Z-stacks (depending on fluorophore intensity and recovery half-life) for a total duration of 2–3 min. The time window of 2–3 min was chosen to maximize recovery of the proteins after bleaching (recovery half-life is on the order of seconds) and to minimize the impact of AMR contraction. To measure the impact of imaging-induced photobleaching, first some of the fields-of-view were selected to serve as control. We next conducted a FRAP experiment with one change: three to five FRAP ROIs were drawn at least 2 µm away from any cell containing AMR. FRAP ROIs were introduced to measure effect of FRAP-­induced bleaching on neighburing cells in addition to imaging-­induced photobleaching. This procedure was repeated until 20–30 cells with AMR were recorded. Finally, all images were saved as 16-bit .tiff raw data files.

The recorded data were then quantified using custom-written scripts in Fiji and Matlab. First, camera noise in the raw images was suppressed by applying a 2 × 2–pixel average filter (Fiji → Process → Filters → Mean). The mean intensity of the AMR was calculated in each image using a circular ROI 1 µm in diameter centered on either a bleached or unbleached cross-section of the AMR. In imaging-­induced bleaching control recordings, both sides of the AMR were measured. A circular ROI was chosen to ensure measurement of intensity even when AMR had shifted in the image during acquisition (we observed minor submicrometer changes in the position of AMR, most probably due to AMR contraction). For each image, the mean intensity of the signal in the selected ROIs was then recorded and saved as an annotated CSV file. Finally, using a custom-written Matlab script and measurements from FRAP experiments, model parameters (such as mobile fraction) were extracted for each recording.

Spindle length information was collected for each cell in the FRAP analysis as 13 z slices of thickness 0.5 µm taken for the mCherry channel immediately before each FRAP sequence. The length of the spindle and the AMR diameter were measured in Fiji. Both the spindle length and the diameter of the AMRs were measured as the lengths of straight lines connecting ends of spindles or opposite sides of the AMR in the image. The measured data in CSV format were loaded into custom-written Matlab scripts.