Plan apo 60 1.40 na

Confocal images were obtained using a laser scanning confocal fluorescent microscope (A1R, Nikon) equipped with a fluorescent unit (ECLIPSE Ti), objective lens (Plan Apo ×60 1.40 NA, oil immersion), and NIS Elements software (version 3.10; Nikon). In BiFC assay (Fig. 2D), images were acquired using ×20 objective lens (2048 × 2048 pixels, 0.31 μm/pixel). The averaged fluorescence intensity of complimented Venus in the images was divided by that of the Hoechst signals. The Venus/Hoechst ratio of the mutants ([4-ii], [4-iii], and [5]) was normalized to that of [4-i].

Vegetative DicMaLionR cells were seeded on a 35‐mm glass‐bottom dish (P35G‐1.5‐14‐C, MatTek, Ashland, MA, USA) and washed twice with KK2 buffer after attachment, then filled with KK2 buffer for observation. Five‐minutes later from the start of observation, oligomycin (mixture of A, B, and C isomers; O‐500, Alomone Labs, Jerusalem, Israel), an inhibitor of ATP production, was added at the final concentration of 2.5 μM. Time‐lapse images were acquired every 10 s using 60× oil immersion (PlanApo 60×/1.40 NA, Nikon, Tokyo, Japan) objective lens and Dragonfly200 (Andor, Tokyo, Japan) equipped with EMCCD (iXonUltra 888, Andor, Tokyo, Japan) camera. A solid‐state laser (ILE‐400, Andor, Tokyo, Japan) was used as the source for providing 561‐nm (RFP excitation) wavelength lights.

Cells were fixed in 4 % paraformaldehyde/PBS for 15 min at room temperature, followed by three washes in PBS. Permeabilization was achieved by incubation in 0.2 % Triton X-100/PBS for 15 min on ice, followed by three PBS washes. Cells were incubated in blocking solution (3% Bovine Serum Albumin/PBS) overnight, followed by staining with anti-HA antibody conjugated to AlexaFluor647 (BioLegend, #682404, 1:1000) and Hoechst (1:2000) in blocking solution overnight. Finally, cells were washed three times in PBS and imaged using confocal microscopy. Images were collected on a Yokogawa spinning disc confocal on a Nikon Eclipse-Ti inverted microscope (Nikon) equipped with a PLAN APO 60×1.40 N.A. oil immersion objective. Images were acquired with a Photometrics Prime 95B sCMOS camera controlled with NiS-Elements software, using 1 × 1 binning, in single z-plane. Images were exported and fluorescence in the EGFP (490 nm) and Cy5 (645 nm) channels was quantified manually using ImageJ 1.53c software.