Regulation of the contractile vacuole in Dictyostelium amoebae

The contractile vacuole (CV) is a protozoan organelle network that is responsible for osmoregulation by actively expelling water from the cell in hypotonic environments. However the mechanism by which the CV interacts with the plasma membrane to discharge its contents is poorly understood. While little is known about the biogenesis of this organelle, knockout studies in Dictyostelium discoideum have demonstrated the requirement of several proteins such as clathrin for normal CV formation. However, clathrin-mediated endocytosis may not be the directly responsible for the organization and recycling of CV components. With fluorescence microscopy and fluorescently-labeled proteins, 1-butanol, a reversible inhibitor of clathrin function, was applied to observe the direct role of clathrin in CV function. The involvement of actin was also examined by blocking polymerization with latrunculin. While actin inhibition does not immediately perturb CV function, 1-butanol treatment results in a rapid widespread disruption of the CV network. Because 1-butanol is a known PLD inhibitor, phosphatidic acid, the immediate product of PLD, was applied in the presence of 1-butanol. Phosphatidic acid resulted in only modest recovery, suggesting the fine balance in signaling required for appropriate CV function. These results implicate the role of PLD activity in CV assembly and maintenance. Understanding the pathway by which CV function is regulated will yield insights into the function of proteins in the endocytic pathway as well as provide a framework for similar studies in human macrophages.