1 Department of Biomedicine - Physiology and Biophysics, Department of Biomedicine, Health, Aarhus University2 Aarhus BSS Administrative Centre - Aarhus BSS HR and PhD Administration, Aarhus BSS Administrative Centre, Aarhus BSS, Aarhus University3 Department of Clinical Medicine - Molekylær Medicinsk afdeling (MOMA), Department of Clinical Medicine, Health, Aarhus University4 Department of Biomedicine - Forskning og uddannelse, Vest, Department of Biomedicine, Health, Aarhus University5 Aarhus BSS Administrative Centre - Aarhus BSS HR and PhD Administration, Aarhus BSS Administrative Centre, Aarhus BSS, Aarhus University6 Department of Clinical Medicine - Molekylær Medicinsk afdeling (MOMA), Department of Clinical Medicine, Health, Aarhus University7 Department of Biomedicine - Forskning og uddannelse, Vest, Department of Biomedicine, Health, Aarhus University
Translocation of vesicles within the cytoplasm is essential to normal cell function. The vesicles are typically transported along the microtubules to their destination. The aim of this study was to characterize the vesicular movement in resting and stimulated renal epithelial cells. MDCK cells loaded with either quinacrine or acridine orange, dyes taken up by acidic vesicles, were observed at 37°C in semiopen perfusion chambers. Time-lapse series were analyzed by Imaris software. Our data revealed vigorous movement of stained vesicles in resting MDCK cells. These movements seem to require intact microtubules because nocodazole leads to a considerable reduction of the vesicular movements. Interestingly, we found that extracellular ATP caused the vesicular movement to cease. This observation was obvious in time lapse. Similarly, other stimuli known to increase the intracellular Ca²⁺ concentration ([Ca²⁺]i) in MDCK cells (increment in the fluid flow rate or arginine vasopressin) also reduced the vesicular movement. These findings were quantified by analysis of single vesicular movement patterns. In this way, ATP was found to reduce the lateral displacement of the total population of vesicles by 40%. Because all these perturbations increase [Ca²⁺]i, we speculated that this increase in [Ca²⁺]i was responsible for the vesicle arrest. Therefore, we tested the effect of the Ca²⁺ ionophore, ionomycin (1 μM), which in the presence of extracellular Ca²⁺ resulted in a considerable and sustained reduction of vesicular movement amounting to a 58% decrease in average lateral vesicular displacement. Our data suggest that vesicles transported on microtubules are paused when subjected to high intracellular Ca²⁺ concentrations. This may provide an additional explanation for the cytotoxic effect of high [Ca²⁺]i.
Journal of Membrane Biology, 2011, Vol 244, p. 43-53