Figure 7.
Figure 7. iEAAR assembled after mCherry-MRCKα 1–478 transduction is a dynamic structure determining centripetal forces in multiple actin bundles. (A) 3D reconstruction of F-actin cytoskeleton detected by phalloidin-488 staining in a MCF10A cell transfected with mCherry-MRCKα 1–478. The 3D reconstruction was performed starting from a multistack 3D STED acquisition. Surface rendering was obtained on the base of F-actin fluorescence intensity with Imaris software using the following parameters: smooth = true, surface grain size = 0.104666, diameter of largest sphere = 0.392498, manual threshold, and number of voxels above 10,000. In the representation we used the following color code: red, iEAAR; blue, upward bundles; green, downward bundles; gray, basal stress fibers. (B) Artistic 3D representation of an iEAAR (red), upward bundles (blue), and downward bundles (green). (C) STED nanoscopy image of an iEAAR assembled in a cell expressing mCherry-MRCKα 1–478. (D) F-Actin density was calculated as the mean fluorescence intensity as a function of the distance from iEAAR center. The radial profile was obtained by averaging intensity radial profile of several iEAARs (n = 51). Dark dotted line represents the mean values; light shades represent the area included between +SD and −SD. Insets: Distributions of distances from iEAAR center of maximum fluorescence intensity and half decay length, respectively. Blue dots, samples; black dot, mean. (E) Subconfluent MCF10A cells were cotransfected with GFP-actin and mCherry-MRCKα 1–478. Actin bundles were bleached in different regions, and movements of the bleached regions were monitored by time-lapse confocal microscopy. (F) Kymograph representation of the flow of bleached regions in the actin bundle shown in E. (G) Measurements of the length of the bleached regions as a function of time at different distances from iEAAR center (different colors) of the experiment in E. The segment represented by red line is shortening, because it is the closest one to the iEAAR and is absorbed into the iEAAR itself. (H) F-actin flow velocity in the dashed bundle of the experiment in E. (I) Comparison of distributions of actin flow velocities in iEAAR-associated bundles as obtained from bleaching experiments (Bundles) and velocity of F-actin patches coalescing during the assembly of aEAAR occurring in apoptotic epithelial extrusion (Patches). Box plots depict the median values (central line), 75th and 25th percentiles (upper and lower hinges), and maximum and minimum values (upper and lower whiskers). (J) Bleaching experiment of an iEAAR in a MCF10A cell cotransfected with GFP-actin and mCherry-MRCKα 1–478 showing recovery of fluorescence (quantification in Fig. 8 J).

iEAAR assembled after mCherry-MRCKα 1–478 transduction is a dynamic structure determining centripetal forces in multiple actin bundles. (A) 3D reconstruction of F-actin cytoskeleton detected by phalloidin-488 staining in a MCF10A cell transfected with mCherry-MRCKα 1–478. The 3D reconstruction was performed starting from a multistack 3D STED acquisition. Surface rendering was obtained on the base of F-actin fluorescence intensity with Imaris software using the following parameters: smooth = true, surface grain size = 0.104666, diameter of largest sphere = 0.392498, manual threshold, and number of voxels above 10,000. In the representation we used the following color code: red, iEAAR; blue, upward bundles; green, downward bundles; gray, basal stress fibers. (B) Artistic 3D representation of an iEAAR (red), upward bundles (blue), and downward bundles (green). (C) STED nanoscopy image of an iEAAR assembled in a cell expressing mCherry-MRCKα 1–478. (D) F-Actin density was calculated as the mean fluorescence intensity as a function of the distance from iEAAR center. The radial profile was obtained by averaging intensity radial profile of several iEAARs (n = 51). Dark dotted line represents the mean values; light shades represent the area included between +SD and −SD. Insets: Distributions of distances from iEAAR center of maximum fluorescence intensity and half decay length, respectively. Blue dots, samples; black dot, mean. (E) Subconfluent MCF10A cells were cotransfected with GFP-actin and mCherry-MRCKα 1–478. Actin bundles were bleached in different regions, and movements of the bleached regions were monitored by time-lapse confocal microscopy. (F) Kymograph representation of the flow of bleached regions in the actin bundle shown in E. (G) Measurements of the length of the bleached regions as a function of time at different distances from iEAAR center (different colors) of the experiment in E. The segment represented by red line is shortening, because it is the closest one to the iEAAR and is absorbed into the iEAAR itself. (H) F-actin flow velocity in the dashed bundle of the experiment in E. (I) Comparison of distributions of actin flow velocities in iEAAR-associated bundles as obtained from bleaching experiments (Bundles) and velocity of F-actin patches coalescing during the assembly of aEAAR occurring in apoptotic epithelial extrusion (Patches). Box plots depict the median values (central line), 75th and 25th percentiles (upper and lower hinges), and maximum and minimum values (upper and lower whiskers). (J) Bleaching experiment of an iEAAR in a MCF10A cell cotransfected with GFP-actin and mCherry-MRCKα 1–478 showing recovery of fluorescence (quantification in Fig. 8 J).

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