Browsing by Subject "Contractile vacuole"

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Contribution of AP2 and AP180 to clathrin function in Dictyostelium discoideum
(2009-05) Wen, Yujia, 1975-; O'Halloran, Theresa
AP2 complex protein is an essential clathrin adaptor protein during clathrin mediated endocytosis. However, this view has been challenged in simple organisms. To gain insight into this conflict, the role of AP2 in clathrin localization and other clathrin related processes were assessed in Dictyostelium discoideum. In Dictyostelium, deleting function AP2 caused mild phenotypes in clathrin membrane localization, cytokinesis, osmoregulation and cell development. This supported the idea that AP2 have significant roles in multicellular organisms but not in unicellular system. Clathrin mediated processes carries important function not only on the plasma membrane but also on some internal organelles. But clathrin coated vesicles on internal organelles are not as well studied as on the plasma membrane. To understand more of the clathrin coated vesicles on internal organelles, the clathrin coated vesicles on Dictyostelium discoideum contractile vacuole were studied. Contractile vacuole associated clathrin coated vesicles contained clathrin adaptor proteins AP2, AP180, and epsin but not Hip1r. The absence of AP180 or AP2 produced abnormal large vacuoles, but the absence of epsin did not cause any detectable contractile vacuole abnormality. The enlarged contractile vacuoles in AP180 minus cells were caused by excessive homotypic fusion among contractile vacuoles. Using both GST-pull down and immunostaining AP180 was identified as the possible adaptor protein for a contractile vacuole-associated SNARE protein, Vamp7B. Therefore recycling Vamp7B from contractile vacuole by AP180 through clathrin coated vesicles could be an efficient way to prevent excessive homotypic fusions among contractile vacuoles. Dictyostelium contractile vacuoles offer a valuable system to study clathrin coated vesicles on cell internal organelles.
Functional analysis of the clathrin assembly protein, AP180, in Dictyostelium discoideum
(2006) Stavrou, Irene; O'Halloran, Theresa J.
AP180, an important coat component of clathrin-coated vesicles, is known to assemble clathrin triskelia into cages of uniform size. To gain insight into the relationship between AP180 and clathrin, the gene that encodes AP180 in Dictyostelium discoideum was cloned and a mutant strain carrying a deletion in this gene was constructed using homologous recombination. Unlike clathrin mutants, AP180 null cells displayed normal pinocytosis, cytokinesis and development into fruiting bodies. However, AP180 mutant cells were osmosensitive, a phenotype also exhibited by clathrin light chain and clathrin heavy chain mutants. The contractile vacuole in AP180 mutant cells became abnormally large in a hypotonic environment and the cycle of expansion and discharge of the vacuole took twice as long compared to that of wild-type cells. Expression of GFP tagged AP180 showed that it localized to punctae at the plasma membrane, cytoplasm and perinuclear area and that it associated extensively with clathrin at these sites. AP180 also localized to the contractile vacuole and in the absence of AP180 more contractile vacuoles were labeled with clathrin. The association of AP180 with the contractile vacuole was affected in the absence of clathrin light chain and the internalization of AP180 into cytoplasmic punctae required the presence of clathrin heavy chain. This work also investigated the dynamics between clathrin, AP180 and AP-2, which is another clathrin assembly protein. A double mutant strain was constructed that had the genes for both AP180 and AP-2 deleted. Of all the clathrin mediated processes examined only osmoregulation was more severe than in the AP180 or AP-2 single mutant cells. The osmosensitivity of the double mutant was an indication that clathrin events at the plasma membrane and the contractile vacuole are linked to some extent. In the absence of both adaptor proteins, the membrane association of clathrin was decreased but not completely abolished whereas the presence of clathrin on the contractile vacuole was markedly decreased. These results present a functional relationship between clathrin, AP180 and AP-2 and suggest that endocytic events mediated by clathrin, AP180 and AP- 2 are important in the normal function of the contractile vacuole.
Localization of LvsA on the contractile vacuole in Dictyostelium discoideum
(2011-12) Cheng, Ying-Hsien; De Lozanne, Arturo; Huibregtse, Jon; Mehdy, Mona; Morgan, Jennifer; Gross, Jeff
The BEACH family proteins are conserved in all eukaryotes and are important for membrane trafficking. Defects in specific BEACH proteins have been linked to severe human disorders. For example, loss of human LYST protein causes the Chediak-Higashi Syndrome (CHS), a lethal disorder that affects lysosomal function. I postulate that different classes of BEACH proteins contribute distinct cellular functions in specific organelles. Based on this functional specificity, I hypothesize that each class of BEACH proteins must localize to their respective organelle where they are known to function. Unfortunately, the localization of most mammalian BEACH proteins is not known and no localization signal has been determined for any BEACH protein. Previous work showed that the Dictyostelium LvsA protein localizes and functions on the contractile vacuole while LvsB localizes and functions on the lysosome. Thus, Dictyostelium is a good model system to understand how BEACH proteins localize to specific organelles. Using a knock-in approach and parasexual techniques, I generated a collection of LvsA truncation mutants tagged with GFP and expressed them in different cell lines. Hence I can test the ability of each mutant protein to localize on contractile vacuoles by fluorescence microscopy. I show here that LvsA requires two regions to localize on the contractile vacuole: the N-terminal 140-457 amino acids and the BEACH. In addition, the expression of the N-terminal 651 amino acids of LvsA causes a dominant negative effect suggesting a possible functional protein-protein interaction within this region. Furthermore, sequence alignment analysis shows that this N-terminal region is only conserved within each class of BEACH family proteins. This finding supports our hypothesis and suggests that diversity within the N-terminal region may be due to the specialized targeting sequences of each class of BEACH proteins. Taken together, these results suggest that the conserved BEACH domain may serve as a general localization sequence while the N-terminal segment is responsible for targeting these proteins to their distinct organelles. This study will facilitate the identification of localization signals in other BEACH proteins which is important to dissect the molecular mechanism of their respective functions.