ATP DEPLETION - A NOVEL METHOD TO STUDY JUNCTIONAL PROPERTIES IN EPITHELIAL TISSUES .1. REARRANGEMENT OF THE ACTIN CYTOSKELETON

The effect of cellular injury caused by depletion of intracellular ATP stores was studied in the Madin-Darby canine kidney (MDCK) and JTC ce ll lines. In prior studies, it was shown that ATP depletion uncouples the gate and fence functions of the tight junction. This paper extends these observations by studying the changes in the actin cytoskeleton and tight junction using electron microscopy and confocal fluorescence microscopy in combination with computer-aided three-dimensional recon struction, Marked regional differences in the sensitivity to the effec ts of ATP depletion were observed in the actin cytoskeleton. Actin dep olymerization appears to first affect the cortical actin network runni ng along the apical basal axis of the cell. The next actin network tha t is disrupted is the stress fibers found at the basal surface of the cell. Finally, the actin ring at the level of the zonulae occludens an d adherens is compromised. The breakup of the actin ring correlates wi th ultrastructural changes in tight junction strands and the loss of t he tight junction's role as a molecular fence. During the process of a ctin network dissolution, polymerized actin aggregates form in the cyt oplasm, The changes in the junctional complexes and the potential to r everse the ATP depletion suggest that this may be a useful method to s tudy junctional complex formation and its relationship to the actin cy toskeletal network.