Spinning disk

Still images of the intestinal lumen were taken with an Olympus IX81 microscope, csu-xi Yokogawa spinning disk, and Andor iXon EM CCD camera. They were analyzed using Metamorph software (version 7.8.0.0, Molecular Devices, Sunnyvale, CA). Images of a micrometer taken at the same magnification were used to calibrate the measuring tool. This tool was then used to determine the width of the intestinal lumen.

Images were taken with either an Olympus BX51 microscope and Hamamatsu ImagEM Electron multiplier (EM) CCD camera or with a Olympus IX81 microscope, csu-xi Yokogawa spinning disk, and Andor iXon EM CCD camera.

L4 stage nematodes were paralyzed with 25 mM Levamisole (AppliChem) in M9 buffer, mounted on 2% agarose pads on glass slides and closed with coverslips. The FRAP assay was performed on an inverted fully motorized Nikon microscope, in association with a Yokogawa Spinning Disk connected to a back‐illuminated EMCCD camera (Andor iXON DU‐897, 512 × 512 pixels, 16 bit, 35 frames/s). The setup was equipped with a 100× oil immersion lens and with 488‐nm emission lasers. Mitochondrial continuity was determined in body wall muscle of transgenic nematodes overexpressing myo‐3::GFP(mit). Bleaching was performed with a 488‐nm laser in regions of interest (ROIs) of 4 × 13.33 μm in size. Within the bleached area, mitochondria were selected and the mean fluorescence was quantified over time, while the mean cytoplasmic fluorescence was used for the background subtraction. Images were obtained every 100 ms for a total of 20 s and analyzed by using Fiji imaging software (Open Source). All values were normalized to the pre‐bleached intensity. The mobile fraction M(f) (or percentage of recovery) was calculated from the normalized recovery curves in function of the difference between the photobleached point and the maximum recovery state.