Journal: Journal of Cell Science

Article Title: Analysis of monocyte cell tractions in 2.5D reveals mesoscale mechanics of podosomes during substrate-indenting cell protrusion

doi: 10.1242/jcs.259042

Figure Lengend Snippet: Podosome-forming monocytes exert cell-wide tractions that can be one-sidedly correlated to podosomes in the presence of broad indentations. (A,B) LPS-treated ER-Hoxb8 monocytes were seeded on soft PAA gels, fixed, and then stained for (A) vinculin and (B) talin-1, in addition to labeling of F-actin using fluorescent phalloidin. Vinculin and talin-1 partially colocalize with and partially form rings around phalloidin-stained actin punctae, defining them best as podosomes. In merge images, vinculin and talin-1 are shown in magenta, and F-actin is shown in green. Images are representative of 12 and 9 cells for vinculin and talin-1, respectively. (C) A mechanical analysis of scattered actin punctae-displaying cells revealed a substantial strain energy expenditure along the z direction (E z ) compared to that in the xy direction ( Exy ). E z /E xy =0.29±0.16 (mean±s.d., n =31 cells from three independent experiments). (D) A common mechanical pattern was found across the cells. While tractions mostly aligned at the cell boundary, the indenting area spanned over the cell area. Representative images show F-actin, T xy and U z for two example cells out of 29. (E) In multiple cells, the focally resolved tractions and their dynamics could be correlated to U z and podosomes. Boxes in the example F-actin images (left) denote regions shown on the right. Cell outlines are marked in the T xy and U z maps, and arrowheads denote the location of apparent actin reorganization. Images are representative of 14 cells. (F) Quantifying the mutual k -nearest neighbor distance between a traction (red) and podosome actin peaks (blue) exposed smaller traction-to-podosome distances (T2P) than podosome-to-traction distances (P2T) over the measured time series (bar graphs show mean±s.d. of 188 frames for two representative cells out of 14). Across these cells, a one-sided colocalization for T2P (mean±s.d.: 1.8±0.4 µm) in contrast to P2T (mean±s.d.: 4.6±0.8 µm) could be shown (boxplot, n =14 cells from three independent experiments). (G) A key feature of the temporal evolution could be observed across the majority of cells: whereas actin scattered widely and apparently stochastically over the entire cell area, in-plane traction appeared and disappeared in the center while being robust at the periphery (images show the indicated time projections of F-actin and T xy for two example cells, representative of n =24 cells from three independent experiments). (H) Plotting the in-plane tractions and out-of-plane deformation as a function of distance from the cell COM revealed a circularly symmetrical centripetal traction ring at the periphery (plotted in red, middle) and a central broad indentation (plotted in blue, right). Darker colors represent later time frames; n =200 frames. Diagrams (left) illustrate circular projections by rotation around the COM for one frame each. *** P <0.001 (Mann–Whitney U-test). Boxplots in C and F show the 25th and 75th quantiles (box) in addition to the median value (line). The whiskers extend to 1.5 times the interquartile range beyond the 25th and 75th quantile, with points outside this plotted separately. Scale bars: 10 µm in A,B,D–G; 5 µm in E insets.

Article Snippet: Boxplots were generated using the MATLAB ‘boxplot’ function.

Techniques: Staining, Labeling, MANN-WHITNEY