Figure 9.

Digital video microscopy of a mechanically induced intercellular Ca²⁺ wave in HeLa cells expressing Cx43–GFP in the presence of 40 IU/ml apyrase. (A) A phase-contrast image of Cx43–GFP HeLa cells superimposed with the corresponding fluorescent image of Cx43–GFP showing plaques of GFP-labeled gap junctions (arrows, white patches) that formed between individual cells. To discard irrelevant background information and before superimposition, the fluorescent GFP image was processed to set the pixel values to black if less than a selected threshold value. (B) An outline of the cell boundaries (gray lines) as well as the positions of the Cx43–GFP plaques (red dots, arrows) were mapped from the phase-contrast and fluorescence images. (C and D) Digital video microscopy of the sequential changes in [Ca²⁺]_i induced by mechanically stimulating a single Cx43–GFP HeLa cell (white arrow). The time each image was recorded is indicated at the bottom right. The changes in [Ca²⁺]_i are mapped onto the cell outlines (the expanding gray shaded zone) to correlate the propagation route of the intercellular Ca²⁺ wave with the location of the Cx43–GFP. Close inspection reveals that a Ca²⁺ wave only propagates at sites where two cells are coupled by a gap junction (red dots). Pseudocolor bar represents changes in [Ca²⁺]_i, as measured from the changes in fura-2 fluorescence, in arbitrary values.