ATP DEPLETION - A NOVEL METHOD TO STUDY JUNCTIONAL PROPERTIES IN EPITHELIAL TISSUES .2. INTERNALIZATION OF NA-ATPASE AND E-CADHERIN(,K+)

MDCK and JTC cells were subjected to ATP depletion by treating the cel ls with 10 mu M antimycin A and 10 mM 2-deoxyglucose. As visualized by confocal fluorescence microscopy, E-cadherin and Na+,K+-ATPase were r apidly internalized following depletion of the intracellular ATP store s, The time course of internalization was similar to the depolymerizat ion of the cortical actin network and dissolution of the actin ring (s ee companion paper, this volume, pp. 3301-3313), Cell surface biotinyl ation was used to assay the amount of surface-accessible E-cadherin an d Na+,K+-ATPase during ATP depletion, At 30 minutes of ATP depletion, 74% and 69% of E-cadherin and Na+,K+-ATPase were internalized, respect ively, in MDCK cells, By 60 minutes of ATP depletion, internalization increased to 95% and 89%, respectively, The redistribution of both pla sma membrane proteins was not microtubule dependent, Similar results w ere observed in JTC cells, Total biotinylated protein decreased by 67% and 82%, after 30 minutes and 60 minutes of ATP depletion, respective ly, The E-cadherin internalization strongly suggests that disruption o f adherens junctions occurred following ATP depletion, These results, along with the previously described loss of tight junction integrity, suggest that ATP depletion may be a useful method to study the assembl y and disassembly of junctional complexes in epithelial cells.