Supplemental Material

This article contains the following supporting material:

• Movie01 - Wild type cells expressing GFP-tagged clathrin light chain were transferred to water and imaged using fluorescence microscopy. Dynamic clathrin punctae are seen moving in the cytoplasm and also assembling on the contractile vacuole bladder. The top right cell (t=36 seconds) and the bottom left cell (t=48-57 seconds) show the most obvious labeling. Dynamic clathrin punctae persist in the vicinity of the contractile vacuole as it discharges. Images were taken every 3 seconds and the movie is played at 6 frames per second.
• Movie02 - AP180 null cells expressing GFP-tagged clathrin light chain were transferred to water and imaged using fluorescence microscopy. Clathrin punctae appear prominentently on the membrane of an enlarged contractile vacuole bladder (bottom cell) and remain in the vicinity of the bladder as it discharges. The top cell also shows a contractile vacuole labeled with GFP-clathrin light chain but this bladder does not discharge completely in the duration of this movie. Images were taken every 3 seconds and the movie is played at 6 frames per second.
• Movie03 - Wild type cells were transferred from media into water and imaged with DIC microscopy. The cell to the left shows an active contractile vacuole discharging at the beginning of this movie. Soon after, the cell to the right shows two contractile vacuole expanding and discharging. Images were taken every 3 seconds and the movie is played at 6 frames per second.
• Movie04 - AP180 null cells were transferred from media into water and imaged with DIC microscopy. The three cells in this movie show expanded contractile vacuole bladders pressed against the plasma membrane. These contractile vacuoles are enlarged but eventually discharge. Additional enlarged contractile vacuoles appear. Images were taken every 3 seconds and the movie is played at 6 frames per second.
• Figure S1 - Wildtype (Ax2) and AP180 null cells were fixed, stained with Texas Red phalloidin, and imaged with Differential Interference Contrast (DIC) and fluorescence (TxRD-Phalloidin) microscopy. The pattern and organization of the actin cytoskeleton in AP180 null cells was similar to wildtype cells. Scale bar, 10 microns.