Determination of the expression level of mEos3.2-paxillin in the clonal T24 cells stably expressing mEos3.2-paxillin used in this study (0.9× the endogenous paxillin; i.e., total paxillin ≈1.9× of the endogenous paxillin), determination of the thresholding density factor for the Voronoï segmentation analysis, and the correction method in the estimate of the FA-protein island diameters for limited single-molecule localization precisions of PALM and dSTORM images (using Voronoï segmentation analysis). (A) The mEos3.2-paxillin–expressing T24 cell clone used in this work expresses mEos3.2-paxillin at ≈0.9× of the endogenous paxillin in non-transfected cells, and thus the total paxillin amount in these cells will be ≈1.9× the endogenous paxillin without transfection, assuming that the endogenous paxillin expression level is unchanged after the mEos3.2-paxillin expression. The figure shows that the number of mEos3.2 on-events (the number of fluorescent spots in the raw PALM image = the number of localizations) detected in the entire view-field was 330,000 ± 38,000 (n = 9 cells). Based on this value, the amount of expressed mEos3.2-paxillin was roughly estimated in the following way. Assuming that the view-fields employed here represent about two-thirds of the entire basal PM (including the FAs) and that 70∼90% of expressed mEos3.2-paxillin molecules are recruited to the basal PM, and since the mean number of on-events per each mEos3.2 molecule = 1.4 (Fig. 2 A-e) and only ≈60% of mEos3.2 is fluorescent (Baldering et al., 2019), the number of expressed mEos3.2-paxillin molecules is estimated to be 655,000–842,000 copies/cell (3,300,00×[3/2]/[0.7–0.9]/1.4/0.60). The copy number of endogenous paxillin expressed in a T24 cell is unknown, but since 920,000 copies of zyxin and 810,000 copies of VASP, which are both essential components of FAs, are expressed in a T24 cell (Tsunoyama et al., 2021Preprint), it would not be unreasonable to assume that an approximately similar number of endogenous paxillin copies exists in a T24 cell (say, ≈850,000 copies). Therefore, after the expression of mEos3.2-paxillin, the total paxillin might be over-expressed by factors of 1.8–2.0 ([655 k + 850 k]/850 k∼[842 k + 850 k]/850 k; assuming that endogenous paxillin expression was not decreased due to the overexpression of mEos3.2). Therefore, we will employ the copy number at which the expressed mEos3.2-paxillin is 0.9× the level of endogenous paxillin and the total number of paxillin molecules is 1.9× the level of endogenous paxillin in non-transfected T24 cells. (B and C) Determinations of the proper Voronoï polygon thresholding density factor (B) and the effects of single-molecule localization precisions (19 and 29 nm) on the island diameters evaluated by the Voronoï tessellation analysis (Levet et al., 2015) for the PALM (29-nm precision) and dSTORM (19-nm precision) images (the relationship of the evaluated diameters with true diameters; C), using Monte Carlo simulations of the PALM and dSTORM images of the paxillin islands. Details of simulation and analysis: A circular paxillin island with a given diameter between 20 and 120 nm was placed at the center of a square with a side length 10 times the circle diameter (except for the 20-nm diameter circle where a side length of 20 times the diameter was employed). Single molecule localization errors of 0 nm (B) and 19 and 29 nm (C; for dSTORM of HMSiR and PALM of mEos3.2) were employed. The positions of the fluorescent spots in the PALM/STORM raw images outside the circle were randomly placed at a number density of 0.002/nm² (a typical number of on-events [fluorescent spots in the PALM/STORM raw images] inside the FA in the experimental images), with added Gaussian noise to account for the localization errors (0, 19, and 29 nm). The locations of the fluorescent spots in the PALM/STORM raw images inside the circle were generated in the same way, with the exception that the number density was increased to 0.02/nm², 10× greater than that of the outside density, which is typical of the number density found in experimentally observed islands (0.019/nm² for the identified islands with diameters in the range of 30∼100 nm). The average number densities of fluorescent molecules will be [1/1.4]x and [1/2.7]x of these on-event densities (the densities of the fluorescent spots) for mEos3.2 and HMSiR, respectively (Fig. 2 A-e and Fig. S3 A-d), but since the actual blinking number for each molecule will follow the probability distribution described by the geometric function (Hummer et al., 2016), we included this effect in the simulation. 30 images were generated for each condition, and the islands in each image were detected by the Voronoï tessellation analysis with a minimum diameter of 13 nm, and the diameters of the detected circles were determined (see the main text). The diameters evaluated in this manner (mean ± SEM) are plotted against the true diameters used in the simulation. (B) Determination of the optimal Voronoï polygon thresholding density factor based on the best estimate of island diameters, showing that a thresholding density factor of 1.45 provides the most accurate estimation of the island diameters. The figure shows the evaluated diameters plotted against the true diameters (diameters used for simulation) for various Voronoï polygon thresholding density factors. The single-molecule localization error was set at 0 nm for this determination. From these plots, the closeness of the estimated diameter (d_Estimated) to the true diameter (d_True; dashed line represents d_Estimated = d_True) was estimated as the sum of $([dEstimated−dTrue]2/dTrue2)$ determined every 10 nm in the range of 30–100 nm (orange text). See the table on the right. (C) The relationship of the evaluated island diameters with the true diameters (open circles). The curves represent the best-fit quadratic functions for the plots of open circles in the range of d_True ≥20 nm, which were used to estimate the true mean diameters of the FA-protein islands in Fig. 4 G, Fig. 5 D, and Fig. S4 A and B. The dashed linear lines indicate the ideal case of d_Estimated = d_True. The filled circles show d_Estimated−d_True (mean ± SEM; for both open and filled circles). The differences increase with an increase of the true diameter, but appear to level off from around d_True ≈100 nm.