Volume 3 Issue 1 January 2002
The Dictyostelium LvsA Protein is Localized on the Contractile Vacuole and is Required for Osmoregulation.
Noel J. Gerald*, Michael Siano and Arturo De Lozanne

Movie 2. GFP-LvsA is localized on the contractile vacuole.
Time lapse imaging of cells expressing GFP-LvsA. Cells were incubated in dH20 and epifluorescence images (100 msec exposure) were taken every 5 sec. GFP-LvsA was clearly visible outlining the contractile vacuoles that lacked internal fluorescence. These compartments gradually expanded and contracted to a small spot that persisted briefly after contraction. GFP-LvsA is also found in the cytoplasm. Numbers indicate time elapsed in minutes and seconds. Movie is compressed to 50X real time.

Fig2.mov

Movie 7A. The contractile vacuole activity of wild-type cells. Cells were flattened with agar overlay under dH2O, and observed by low-light microscopy. Images were taken every 1 second. These wild-type cells show rapid filling and discharging of contractile vacuoles. Numbers indicate time elapsed in minutes and seconds. The movie is shown at 30X real time.

Fig7a.mov

Movie 7B. Most LvsA mutant cells contain phase lucent vacuoles that do not discharge.
Mutant lvsA- cells under hypoosmotic stress contained several swollen phase lucent vacuoles. However, most of these vacuoles showed no size changes or evidence of discharge for periods of observation up to 30 minutes.
Cells were flattened with agar overlay under dH2O, and observed by low-light microscopy. Images were taken every 1 second. Numbers indicate time elapsed in minutes and seconds. The movie is shown at 30X real time.

Fig7b.mov

Movie 7C. A few LvsA mutant cells display vacuoles that discharge but not normally.
Occasionally, two types of intermittent vacuole discharge events were observed in LvsA mutant cells. One type of mutant vacuole discharge resembled wild-type discharge as the phase lucent vacuoles contracted within 3 to 4 seconds (see vacuole that disappears at time frames 1:05-1:13). However, most other discharges appeared to be caused the sudden fusion of the vacuole with the plasma membrane. This is suggested by the formation of a bleb at the site of vacuole discharge (see vacuole that disappears at time frames 5:15-5:20).
Cells were flattened with agar overlay under dH2O, and observed by low-light microscopy. Images were taken every 1 second. Numbers indicate time elapsed in minutes and seconds. The movie is shown at 30X real time.

Fig7c.mov

Movie 7D. Contractile vacuoles of LvsA mutant cells appear to fuse with the plasma membrane.
Two more examples of vacuoles that appear to discharge by fusing with the plasma membrane. Notice that a bleb is formed at the site of discharge suggesting the rapid addition of new membrane.
Cells were flattened with agar overlay under dH2O, and observed by low-light microscopy. Images were taken every 1 second. Numbers indicate time elapsed in minutes and seconds. The movie is shown at 30X real time.

Fig7d.mov

Movie 8. GFP-LvsA associates with the membrane of the contractile vacuole only during the discharge phase.
One of the cells in Figure 2 is magnified here. Notice how the vacuole on the left side of the cell is not labeled by GFP-LvsA during the first minute. Beginning at timeframe 1:20 GFP-LvsA begins to clearly label the contractile vacuole which then starts to contract at timeframe 1:50. Contraction is complete by timeframe 2:15 when the contractile vacuole is visible as a bright spot of GFP-LvsA fluorescence. Subsequently, the fluorescence rapidly diminishes from the contracted site. Beginning timeframe 3:25 the contractile vacuole begins to expand again and continues to expand until timeframe 5:40. Throughout this time the vacuole is not labeled by GFP-LvsA. At timeframe 5:45 the vacuole becomes marked by GFP-LvsA for a new cycle of contraction.
Time lapse imaging of cells expressing GFP-LvsA. Cells were incubated in dH20 and epifluorescence images (100 msec exposure) were taken every 5 sec. Numbers indicate time elapsed in minutes and seconds. Movie is compressed to 50X real time.

Fig8.mov