Fig. 2.

Actin preferentially concentrates in filopodia, cell-cell, and cell-substrate surfaces of ES cells. A–C Representative images of cells expressing EGFP-actin (green) and H2B-mCherry (red) exemplifying those key features of actin organization described in the text. Merge images (transmission, green and red channels) obtained at specific optical planes allow identifying the relative positions of the cells within colonies (left panels). Images obtained at specific planes of the cells as detailed in each case (central panels, in pseudocolor scale) and 3D reconstructions of the green and red channels images (right panels). White arrows point to actin-enriched structures in contact with the substrate, and the red and green arrows point to actin enrichments at cell-cell contacts and filopodia linking the cell to the substrate, respectively. The yellow arrows point to regions facing the extracellular milieu of those cells at the colony border. The white asterisks indicate actin-enriched filopodia extending from the cell showed in the dotted rectangle to a neighboring cell. D Representative examples of cells showing membrane blebs (light blue arrows) (left panels, pseudocolored images). Intensity profiles along the membrane borders (dotted, gray lines in the insets). Grey triangles show the initial and final positions of the lines along the blebs. Bleb formation may be also accompanied by changes in the nucleus shape (right panel, blue asterisk). The white asterisk indicates actin-enriched filopodia (Scale bars: 10 μm). Other examples of EGFP-actin fluorescence intensity along membrane blebs can be found in Additional file 16: Supplementary Fig. S7