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Volume 5 issue 5 May 2004
Structure-Function Analysis on the Beach Protein LvsA
Wei-I Wu, Juhi Yajnik, Michael Siano, and Arturo De Lozanne@
Section of Molecular Cell & Development Biology and Institute for Cellular and Molecular Biology, University of Texas at Austin, Austin, TX 78712.
Running Title: Structure-Function Analysis of a Beach Protein
Keywords: beige, Lyst, CHS, cytokinesis, Beach domain
@ Address all correspondence to:
Arturo De Lozanne,
Dept. of Molecular Cell & Development Biology,
University of Texas at Austin,
1 University Station C-0930,
Austin, TX 78712-0253.
Tel (512) 232-6100. Fax (512) 471-3878
Email a.delozanne@mail.utexas.edu
Movie 1
GFP-LvsA is localized on the contractile vacuole during the expulsion phase.
The coding region of GFP was inserted by recombination into the single-copy LvsA gene in the Dictyostelium genome. Cells were incubated in dH20 and epifluorescence images (100 msec exposure) were taken every 5 sec. In this condition the contractile vacuole is very active swelling with water and expelling the water out of the cell. Notice that GFP-LvsA does not label a contractile vacuole that is expanding. GFP-LvsA becomes associated with the contractile vacuole as soon as the vacuole reaches its maximum diameter. The vacuole remains brightly labeled by GFP-LvsA throughout its expulsion phase and then becomes unlabeled to start a new cycle. Numbers indicate time elapsed in minutes and seconds. Movie is compressed to 50X real time.
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Movie2
GFP-Dajumin is a marker of the contractile vacuole network.
GFP-Dajumin was introduced into these wild-type cells in a high-copy number episomal plasmid that yields abundant expression of this protein. Notice the difference in background in this movie compared to Movie 1. GFP-Dajumin labels the network of tubules and sacs that constitute the contractile vacuole. Cells were incubated in dH20 and epifluorescence images (100 msec exposure) were taken every 4 sec. Numbers indicate time elapsed in minutes and seconds. Movie is compressed to 20X real time.
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Movie3
GFP- N689-LvsA cells contain small functional contractile vacuoles.
Dictyostelium mutant cells containing the N689-truncated form of LvsA were transformed with the GFP-dajumin vector used in Movie 2. GFP-dajumin distribution shows that these mutant cells are able to form contractile vacuoles that can expel water. However, their vacuoles are much smaller than those found in wild-type cells. These vacuoles are usually found at the periphery of the cells. Cells were incubated in dH20 and epifluorescence images (100 msec exposure) were taken every 5 sec. Movie is compressed to 50X real time.
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Movie 4
The dynamic distribution of GFP-LvsA is disrupted by the loss of the protein drainin.
Drainin is a peripheral membrane protein associated with the membranes of the contractile vacuole and is required for the expulsion phase of the vacuole. We disrupted the gene coding for drainin in our GFP-LvsA expressing cells and monitored the distribution of GFP-LvsA in hypotonic conditions as in Movie 1. These drainin null cells display giant contractile vacuoles that fail to expel. They also have smaller contractile vacuoles that behave normally. Notice that GFP-LvsA is permanently bound to the large paralyzed vacuoles while it binds transiently to the normal vacuoles. Thus, contraction of the vacuole is necessary to disengage LvsA from the vacuole membrane. Numbers indicate time elapsed in minutes and seconds. Movie is compressed to 50X real time.
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