Visualization of Actin Polymerization in Invasive Structures of Macrophages and Carcinoma Cells Using Photoconvertible β-Actin – Dendra2 Fusion Proteins (original) (raw)
Figure 2
Inhibition of incorporation of photoconverted D2BA into macrophage podosomes by cytochalasin D.
(A–F) Cells were treated with DMSO (A–C) or 2µM cytochalasin D (D–F) and D2BA was photoconverted in the indicated areas (circled). Kymographs of pseudocolor pixel intensity indicate that there is no incorporation of photoconverted D2BA in the podosomes of cytochalasin D compared to DMSO-treated cells. Scale bar, 10 µm. Pseudocolor scale - blue indicates low and red high intensity. (G) Graph indicating the kinetics of D2BA incorporation into podosomes of DMSO or cytochalasin D-treated cells. n = 80 podosomes from 8 cells per condition analysed in two independent experiments. Cytochalasin D treatment effectively blocks incorporation of D2BA into podosomes, indicating that polymerization occurs at barbed ends in podosomes. Statistical analysis was performed at the 30 sec time point comparing intensities in podosomes between cells that received DMSO and those that received cytochalasin D; n.s., not significant. (H) The amount of the total photoconverted species (561 nm) is not altered between DMSO or cytochalasin D treated cells. Pixel intensity at 561 nm was analyzed in widefield in order to capture the photoconverted species of the entire cell; n = 7 cells from a representative experiment.