Computational protocol: Arrestins regulate cell spreading and motility via focal adhesion dynamics

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Protocol publication

[…] Serum-starved cells were plated on eight-well slides coated with 1.25 μg/ml FN or 0.1 mg/ml PDL. Cells were fixed with 4% paraformaldehyde, permeabilized with 0.4% Triton X-100, and blocked with 2% BSA in phosphate-buffered saline (PBS). Most cells were stained with rhodamine–phalloidin. Rescue experiments in which DKO cells were infected to express HA-tagged arrestins were also stained with anti-HA antibody to detect expression. Images were taken on a Nikon TE2000-E automated inverted microscope (Nikon, Melville, NY) with 40× oil objective with additional 1.5 optical magnification. Focal adhesion numbers and size were quantified from confocal images taken on LSM 510 Meta Confocal (Zeiss, Jena, Germany) with 40× oil objective and analyzed with ImageJ (National Institutes of Health, Bethesda, MD). [...] To examine clathrin dynamics, TIRFM live-cell videos were acquired on a Nikon TE2000E microscope with Nikon TIRF2 system using TIRFM 100×/1.49 numerical aperture oil lens, Cascade 512B camera (Photometrics, Tucson, AZ), and IPLab software (Scanalytics, Fairfax, VA). To analyze the velocity of clathrin-coated vesicles at focal adhesions in WT and DKO MEFs coexpressing GFP-paxillin and mCherry-clathrin, 2-min double-channel TIRFM sequences (3 s/frame) were used. Analysis was limited to a region of interest (ROI) at focal adhesions defined by paxillin localization to examine clathrin velocity parameters specifically within this area. Individual clathrin-coated vesicles were automatically tracked within the ROI for the duration of the time sequence using Imaris software (Bitplane, South Windsor, CT). The average velocity of 72 clathrin tracks/condition (WT or DKO) and 1519 instantaneous velocities/condition were analyzed. These data were used to calculate average velocity and average instantaneous velocity of clathrin vesicles within the ROI, respectively. [...] Cell size analysis was from 10–15 randomly selected fields/experiment and measured for area using ImageJ or Nikon NIS software. Cells with moderate expression of either HA-arrestin were selected for cell size and focal adhesion number rescue measurements. To measure arrestin colocalization with paxillin, cells were transfected with HA-tagged arrestins and stained for HA and endogenous paxillin. Cells were imaged on an LSM 510 Meta Confocal with 40× oil objective. The Pearson coefficient was determined using Coloc2 function in ImageJ after background was corrected to eliminate nonspecificity. Focal adhesion size and number were measured from confocal images using ImageJ with qualifications for focal adhesion area: 0.5–100 μm2. Focal adhesion lifetimes were calculated using focal adhesions containing GFP-paxillin that assembled and disassembled from the leading edge of the cell. Because most data sets significantly deviated from normal distribution and thus violated assumption for parametric statistical tests, they were analyzed by nonparametric Kruskal–Wallis analysis of variance, followed by posthoc pairwise comparisons between groups of interest. To minimize the probability of type I error, Bonferroni correction for multiple comparison was applied. Focal adhesion size and lifetime distributions were compared using nonparametric Kolmogorov–Smirnov test. Velocity and instantaneous velocity of clathrin particles were compared using an unpaired Student's t test. In all experiments, p < 0.05 was considered significant. […]

Pipeline specifications

Software tools ImageJ, Imaris, Coloc
Applications SPIM, Microscopic phenotype analysis
Chemicals Nocodazole