Figure 1.
The images show that Cytochalasin B affects cellular structure and morphology and disrupts the actin cytoskeleton.
Analysis of HeLa cells after 96 h of treatment. (A) Representative actin stained images of DMSO control cells (left) and 0.6 µM Cyt B-treated cells (right). B) Quantification of cellular spread area (number of cells, DMSO, n = 63; Cyt B, n = 153). (C) Quantification of cellular aspect ratio (number of cells, DMSO, n = 63; Cyt B, n = 153). (D) Cellular mean traction forces measured in 10 kPa polyacrylamide gels (number of cells, DMSO n = 79; Cyt B n = 40). Scale bar is 20 µm. ***: p<0.001.
Figure 2.
The quantification tool discerns that the effect of cytochalasin B is more pronounced on SFs than on FAs.
(A) Representative images of vinculin in DMSO control cells (top) and Cyt B-treated cells (bottom). (B) Quantification of the number of FAs per cell (a), FAs area (b), and circularity (c) (number of cells: DMSO, n = 33; Cy B, n = 29). (C) (a) The stress fibers quantification tool analyses different parabolic paths (dashed arcs) between two focal adhesions (red ellipses) and selects the parabola that fulfills the intensity requirements (green arc) (Materials and methods). (b) F-actin staining and (c) vinculin staining of a cell after filtering process. (d) Representative example of a z plane containing a stress fiber and the stress fiber in 3D detected by the tool (white line). (e) Image of the stress fibers quantified by the algorithm. (D) Quantification of the number of SFs. (E) Analysis of the number of SFs depending on the number of FAs. (F) Correlation between the number of SFs and FAs. (G) Quantification of the percentage of FAs that have at least one SF (number of cells: DMSO, n = 24; Cyt B, n = 27). Scale bars are 20 µm. ***: p<0.001.
Figure 3.
There are strong linear correlations between the number of SFs and FAs with cellular morphological features.
(A) Ratio between the number of FAs per cell divided by its spread area for both DMSO and CytB condition. DMSO control cells is normalized to one. The spread area of cells similarly defines the number of FAs in both conditions. (B) Same quantification taking into account the aspect ratio of cells. Differences between both cell types are statistically significant (*: p<0.05). (C) Corresponding correlation between the number of FAs of a cell and the spread area. (D) Likewise, same correlation analysis between FAs and the aspect ratio. (E–H) Same analysis performed with SFs instead of FAs. (E–F) Ratio between the number of SFs per cell and both cellular spread area and aspect ratio reveal that DMSO-treated cells have significantly more than CytB-treated cells (***: p<0.001) (D) (number of cells: DMSO, n = 24; Cyt B, n = 27). The trend line and the coefficient of determination can be observed for each condition.
Figure 4.
The distribution and quantification of the average intensity of critical actin SF properties reveal different protein distributions.
(A) (a) Examples of measurements of the F-actin intensity of a DMSO control cell and a cell treated with Cyt B. (b) The intensity distribution of the protein in the cell (c) is normalized to a circle taking into account the distance to the centroid and the closest edge (d) and the mean along all directions is quantified obtaining the Radial Mean Intensity (RMI). (B) Top: The Radial Mean Intensity distribution of vinculin. Bottom: The Radial Mean Intensity is divided in 5 sectors to obtain the Local Radial Mean Intensity (LRMI) of vinculin. Statistical differences between both conditions were only observed in the section closest to the edge of the cell (*: p<0.05). (C–D) Same quantification for actin and Non-Muscle Myosin II. Statistical differences are observed in both proteins between Cyt-B and DMSO-treated cells (number of cells: DMSO, n = 18; Cyt B, n = 16). (E) Quantification of cell height. (F) Quantification of the percentage of total protein along the Z-axis (number of cells: DMSO, n = 29, CytB, n = 32). Scale bars are 20 µm. (*: p<0.05; **: p<0.01; ***: p<0.001).