Csu x1

Stills of the actin and microtubule networks in epidermal pavement cells were taken by a Zeiss LSM880 microscope with a Plan-Apochromat 40 x/1.2 W objective and 488-nm argon laser for excitation, videos were taken by an inverted spinning disc confocal microscope (Yokogawa CSU-X1 on a Nikon Ti-E platform, laser box Agilent MLC400, camera Andor Ixon) with 100 x/1,45 O plan apochromatic objective, excitation laser line set at 488 nm, and image interval 1 s as described previously (Rosero et al., 2016 (link)). Cytoskeleton bundling and density were quantified as described previously (Higaki et al., 2010 (link)) with minor modifications (Rosero et al., 2013 (link); Rosero et al., 2019 (link)).
Cytoskeletal dynamics measurements were done in two biological replicates: with at least 40 cells from at least 20 videos analyzed using the QuACK method (Cvrčková and Oulehlová, 2017 (link)).
Actin networks in developing trichromes were visualized using an inverted spinning disc confocal microscope (Zeiss Axio Observer 7 microscope with a vertical stage equipped with a Yokogawa CSU-W1 spinning disk unit and Photometrics Prime 95B camera) with Zeiss Plan Apochromat 40 x/1.2 W objective and 488-nm laser excitation line.

Microscopy was performed on spinning disk confocal microscope (Yokogawa CSU-X1) set up on a Nikon Eclipse Ti inverted microscope with a 100× ApoTIRF 1.4 NA objective (Nikon) in line with 2× amplification. BODIPY 493/503 fluorophore was exited on 561 nm laser line. Fluorescence was detected by an iXon Ultra 897 EMCCD camera (Andor). Acquired images were processed using FIJI software (http://fiji.sc/Fiji).

Myosin IIA-GFP knock-in DCs migrating into 5 × 5 μm microchannels coated with 20 μg ml⁻¹ fibronectin were imaged using a spinning disk microscope consisting of Yokagawa CSU-X1 spinning head mounted on an Eclipse Ti inverted microscope (Nikon) equipped with a Coolsnap HQ2 camera (Photometrics) with a × 60 differential interference contrast 0.75 NA taking one image every 10 s. Only the cell middle plan was imaged. Macropinosome velocity was measured over 2 min post formation by detection of its initial and final positions. During the same time frame, myosin IIA enrichment was determined in a region surrounding macropinosomes. To take only into account myosin IIA enrichment above background, myosin IIA patches were considered as clusters bigger than 10 pixels above the mean GFP intensity of the cell front GFP cytoplasmic signal.