Environmental chamber

RPE1 cells were plated on micro-patterned lines 16 h before imaging. In order to track the cells, nuclei were stained with NucBlue live stain (Thermo Fisher Scientific, Cat. R37605). Cells were imaged using a humidity- and temperature-controlled inverted wide-field microscope within an environmental chamber (Olympus). Cells were transferred to a live cell imaging workstation composed by an Olympus IX81 inverted microscope equipped with motorized stage and a Hamamatsu ORCA-Flash 4.0 camera. The images were collected every 20 min for a total recording time of 48–72 h using a PlanFL 4×/0.13 or a UPlanFLN ×10/0.30 phase-contrast dry objective with CellSens software (Olympus). Nuclei images were segmented and geometric centers were tracked with a global minimization algorithm. For this purpose, specific software in C++ with the OpenCV [http://opencv.willowgarage.com/wiki/] and the GSL [http://www.gnu.org/software/gsl/] libraries was developed. The migration analysis was performed by the C++ software coupled with R [www.R-project.org]¹¹ .

Time-lapse microscopy movies were recorded with a Deltavision fluorescence microscope equipped with an environmental chamber (Olympus, Applied Precision) at 10x magnification with frames every 3 minutes for 24 hours. Cells grown in 8-well chambered cover glass slides (Nunc) were shifted into phenol red-free medium (Invitrogen) supplemented with 10% fetal bovine serum and L-glutamine for imaging. For FRET signal analysis, ratio of background-subtracted CFP and YFP images were created by using ImageJ and custom plug-ins23 (link). Signals were normalized by subtracting the minimum value across all time points from each single-cell time course. IMS-RP release in cell cytoplasm was analyzed in ImageJ by visual inspection, which enabled an identification of the first frame of IMS-RFP release and subsequent time of cell death of each single-cell of the cell population analyzed.

For long-term live cell imaging, cells were grown on 1.5 Lab-Tek II 4-chambered coverslips (Thermo Fisher Scientific). Live cells maintained at 37°C in imaging media were imaged using a Zeiss Axiovert 200 inverted microscope equipped with an UltraView spinning disk confocal head (Perkin-Elmer), an Orca ER-cooled CCD camera (Hamamatsu), a 20×/0.75 N.A. Plan-Apochromat objective, and an environmental chamber (Solent Scientific). Solid-state 491 and 561 nm lasers (Spectral Applied) and ET 530/50 and ET 605/70 emission filters (Chroma) were used for excitation and emission of EGFP and RFP fluorescence, respectively. Alternatively, time-lapse images were captured using an Olympus IX71 inverted microscope equipped with an Orca ER cooled CCD camera, a 20×/0.75 N.A. UPlan SApo objective and an environmental chamber. Image acquisition was performed using Metamorph (Molecular Devices) and processing was performed using ImageJ.