P35g 0 20 c

LLC-PK1-CL4 cells were plated on No. 0 uncoated glass-bottom dishes (P35G-0-20-C; MatTek Corp., Ashland, MA) and co-transfected with equal amounts of untagged espin plasmid construct in the pcDNA3 vector (Life Technologies) and pEGFP–human beta-actin (Life Technologies). FRAP was performed 20–22 h after transfection in Leibovitz's L-15 medium (Life Technologies) containing 10% (v/v) fetal bovine serum at 37°C using a temperature-controlled stage. Initial experiments were conducted using a ×100 objective on the LSM510 META confocal microscope. Photobleaching was performed with the 488 nm laser at full power with a tube current of 6.5 A for 25–30 iterations. Post-bleach images were acquired at the designated intervals. Later experiments were carried out using a total internal reflection fluorescence ×100 NA1.49 objective (Nikon, Melville, NY) on a CSU-X1 spinning disk confocal (Andor, South Windsor, CT) equipped with FRAPPA module driven by Andor IQ2 software. The 488 nm laser was set at 100% for photobleaching at 50 µs pixel dwell and two iterations. Images were taken at 20 s intervals using an Andor iXon EMCCD camera and used to make QuickTime movies. For clarity, analysis was conducted on espin-elongated microvilli, ∼7–9 µm long, that extended out over the apical surface of a neighboring untransfected cell.

hPTCs were seeded on glass bottom dishes (P35G-0-20-C, MatTek). Following 48h of transduction with Ad-h-WIPF3, they were starved for 24h in serum-free medium. Cdc42 was subsequently activated by incubating cells with epidermal growth factor (EGF; Cytoskeleton Inc., Cat# CN02) at 0,5 units/mL for 5 min. Cells were then fixed by addition of formaldehyde (Polysciences, Cat# 18814–20) directly into cell medium to a final concentration of 3,7%. After permeabilization (0.1% Triton X-100 in PBS for 5 min) and washing, cells were incubated with PBS containing 1% BSA for 45 min, and subsequently with rabbit anti-WIPF3 (anti-WIPF3: Atlas Antibodies, Cat# HPA041145 at 1:750) for 1 hour. Secondary goat anti-rabbit Alexa Fluor 488 was applied for 30 min together with fluorescent phalloidin (Alexa Fluor 555 Phalloidin at 1:400, Cat# A34055), and nuclei were counterstained with DAPI. Mounting medium (Aqua-Poly/Mount) was applied to the glass bottom (20 mm ∅), and a 15 mm diameter coverslip (VWR, Cat# 631 1579) was placed on top. One image was taken from the center of each dish using an Olympus BX60 microscope with a 20x objective and the CellSens Dimension software. The integrated density was measured in ImageJ, and fluorescence intensity was obtained as a measure of actin filaments in the all cells of each image.

Polyacrylamide gel substrates were prepared according to Wang and Pelham [71 (link)]. Briefly, No. 0 glass-bottom culture dishes (MatTek P35G-0-20-C; Ashland, MA, USA) were treated with 0.1 M NaOH, 97% (3-aminoproyl) trimethoxysilane (Sigma-Aldrich 281778; St. Louis, MO, USA), and 0.5% glutaraldehyde (Polysciences 01909; Warrington, PA, USA). Culture dishes were stored in a desiccator for up to 2 weeks until use. A 4.6 kPa hydrogel was made as follows: 100 µL of 40% acrylamide solution (Fisher Scientific BP1402; Waltham, MA, USA), 100 µL of 2% bis-acrylamide solution (Fisher Scientific BP1404; Waltham, MA, USA), 10 µL of 1 M 4-(2-hydroxyethyl) piperazine-1-ethanesulfonic acid (HEPES, Sigma-Aldrich H6147; St. Louis, MO, USA), 790 µL of deionized water, 6 µL of 1% ammonium persulfate (Bio-Rad 161-0700), and 4 µL of 0.4% (v/v) TEMED, (Fisher Scientific BP150; Waltham, MA, USA). Next, 4 µL of polymer solution was quickly pipetted onto the activated glass culture dishes and covered with a 12 mm No. 1.5 circular coverslip (Fisher Scientific 12-545-80; Waltham, MA, USA). Then, the coverslip was removed and washed three times with 50 nM HEPES. Finally, 1 mL of 200 mg/mL of type I collagen solution was added to the gel and the hydrogel was kept at 4 °C overnight.