Colibri illumination system

MDCK cells grown on coverslips were rinsed twice in PBS and then fixed and permeabilized as previously described (Tang and Brieher, 2013 (link)). For Figs. 1 E and 5 B, images were collected with LSM700 confocal microscope (Zeiss) with a NeoFluar 20×/0.5 NA or 63×/1.4 oil differential interference contrast lenses. The Z-section step size was 0.5 µm. Cell height and spreading area were measured randomly throughout the samples while viewing only the channel showing F-actin. Cell height was measured in ImageJ Orthogonal Views xz or yz sections. For other images, fixed cell fluorescence wide-field images were collected with an Axio Imager using AxioVision Release 4.7 with the Colibri illumination system (Zeiss) using an oil 63× (NA 1.4) or 20× (NA 0.8) objective. Images were acquired using a 1,000 × 1,000 charge-coupled device camera (ORCA-ER; Hamamatsu Photonics). Cell spreading area was measured based on the apical section circumference of individual cells. Composite images were generated using ImageJ software. Images were constructed in Adobe Illustrator. Quantifications and line scans were performed with ImageJ, and OriginLab was used for statistical analyses.

Actin assembly reactions were performed in actin assembly buffer (50 mM KCl, 2 mM EGTA, 2 mM MgCl₂, and 100 mM Hepes, pH 7.8) supplemented with 2 mM buffered ATP, pH 8.0. In brief, a 20-µl reaction consisting of ∼15 µg of total proteins from the membrane fraction and 0.5 µM fluorescently labeled monomeric actin was allowed to carry out at room temperature for 30 min before incubation with primary antibodies in the presence of 0.1% TX-100. The membranes were spun through a 20% sucrose cushion and resuspended in 0.1% TX-100 in assay buffer. The membranes were incubated with secondary antibodies for 2 h, spun through a 20% sucrose cushion, and resuspended in 0.1% TX-100 in assay buffer. Stained membranes were imaged using an Axio Imager with the Colibri illumination system (both Carl Zeiss) using a 63× objective (NA 1.4) attached to a 1K × 1K ORCA-ER CCD camera (Hamamatsu Photonics).