Dragonfly 503

For ER area quantification in fixed (F/Tx) cells, the ER was visualized by rabbit Ab to calnexin and AlexaFluor 488-conjugated anti-rabbit Ab. Samples were then incubated with AlexaFluor 555-conjugated WGA (5 μg/mL) for 30 min at room temperature to delineate cell boundary and mounted in MOWIOL 4-88 with DAPI to mark nucleus. Preparations were examined with the Andor Dragonfly 503 spinning disc confocal microscope (0.13 μm steps) equipped with HCX PL APO 63×/1.4 oil objective and Zyla 4.2 PLUS sCMOS camera. Images were deconvoluted with Huygens Professional software, and the ER area coefficient (area occupied by ER/area free of ER) was calculated from calnexin fluorescence intensity in the cytoplasm of individual cells using an in-house written macro (Text S1) for Fiji processing program. AlexaFluor 647-conjugated WGA was used when quantification was performed in phenotypic rescue experiments with C53-TagRFP.
For ER area quantification during live-cell imaging, cells were transferred to FluoroBrite™ DMEM without serum and incubated for 15 min at 37 °C with 1 µM ER-Tracker Green, AlexaFluor 555-conjugated WGA (5 μg/mL) and Hoechst 33342 (0.5 μg/mL). After washing in FluoroBrite™ DMEM, the preparations were examined with the Andor Dragonfly 503 spinning disc confocal microscope (0.5 μm steps) equipped with HC PL APO 63×/1.2 water objective, and iXon Ultra 888 EMCCD camera.

The cells were seeded on microscope cover glasses (0.15 × 10⁶ cells per 35 mm dish with the cover glass) and incubated overnight. The next day, the cells were fixed in 4% paraformaldehyde, washed with PBS, stained with membrane dye CellBrite Blue (Biotium) according to the manufacturer’s protocol, mounted in PBS, and visualised using Andor Dragonfly 503 confocal spinning disc microscope with a 63 × /1.2 NA objective.
Images were deconvolved by Huygens software and contrast was enhanced using ImageJ. The signals of AcGFP and iRFP713 were adjusted uniformly over all mutants to illustrate the intensity of expression.