Fig. 9. Time-lapse fluorescence microscopy of cells expressing fluorescent Dsc2a chimeras depicting alterations in desmosomal cadherin distribution upon reduction of Ca⁺⁺. Cells were transferred from SCM (+ Ca⁺⁺) to LCM (-Ca⁺⁺) as indicated. (A) Confocal laser scan microscopy of MDc-2 cells using a large pinhole. The pictures are taken from Movie 9 (1 minute recording intervals) depicting the internalization of Dsc2a. YFP-containing desmosomal particles and the continued presence of diffuse non-desmosomal Dsc2a. YFP fluorescence at the cell surface. Bar, 10 µm. (B) 3D reconstruction of z-stacks each consisting of five epifluorescence micrographs showing the disintegration of large Dsc2a. GFP-labeled desmosomal structures into smaller particles in PDc-13 cells after the reduction of the Ca⁺⁺ concentration (arrows). The complete sequence is provided as Movie 10 (1 minute recording intervals; jcs.biologists.org/supplemental ). Bar, 5 µm. (C) Projection images of z-stacks, each consisting of five focal planes recording epifluorescence of Dsc2a.GFP in PDc-13 cells. Note the fusion of small fluorescent desmosomal particles after the reduction of Ca²⁺ (arrows). The entire image series is presented in Movie 11 (2 minute recording intervals). Bar, 5 µm. (D) Overlay of projected fluorescence pictures (5 focal planes) and corresponding phase contrast micrographs obtained from Movie 12 (jcs.biologists.org/supplemental ), which was recorded in PDc-13 cells. Note the overall reduction of desmosomes together with the fusion of desmosomes (black arrows) and fission of desmosomes (white arrows) after removal of calcium. Bar, 10 µm.