Browsing by Subject "Dictyostelium discoideum"
Now showing 1 - 8 of 8
Results Per Page
Sort Options
Item A new view of culmination in dictyostelium: morphogenetic roles of cell shapes, intercellular junctions, and extracellular matrices(Texas Tech University, 2000-05) Grimson, Mark JeffreyNot availableItem Characterization of the epsin homolog EpnA in Dictyostelium discoideum(2008-05) Brady, Rebecca Jane, 1980-; O'Halloran, TheresaClathrin-coated pits on the plasma membrane invaginate into coated vesicles to internalize receptors and membrane. The clathrin adaptor epsin contains an aminoterminal ENTH domain that binds PI(4,5)P₂ and a carboxy-terminal domain that binds clathrin, and accessory proteins such as AP2. Here, we assessed how inter- and intramolecular factors affect the contribution of epsin to coated-pit function in living cells. We found Dictyostelium epsin was not required for global clathrin function, but plays an essential role in spore development. We demonstrated that clathrin, but not AP2, was critical for epsin to associate with clathrin-coated pits. We found that the carboxy-terminal region of epsin was essential, but not sufficient, for targeting epsin within clathrin-coated pits on the plasma membrane. In addition to targeting epsin to the membrane, the amino-terminal ENTH domain regulates the interaction between epsin and clathrin, an essential property that cannot be replaced by an alternate PI(4,5)P₂ binding domain. Moreover, the ENTH domain facilitates the functional interaction between clathrin and actin during late stages of endocytosis, possibly by regulating the activity of the adaptor Hip1r. Both the ability to bind PI(4,5)P₂ and another function mediated by residue T107 are critical for the activity of the ENTH domain. Our results support a model where the ENTH domain coordinates with the clathrin-binding C-terminal domain to allow a dynamic interaction of epsin with coated pits. Furthermore, we propose that the ENTH domain of epsin facilitates the membrane recruitment and phosphorylation of Hip1r, which in turn mediates the productive interaction of clathrin with the actin cytoskeleton at the plasma membrane.Item Contribution of AP2 and AP180 to clathrin function in Dictyostelium discoideum(2009-05) Wen, Yujia, 1975-; O'Halloran, TheresaAP2 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.Item Defining the function of the Chediak-Higashi syndrome related protein, LvsB, in Dictyostelium discoideum : functional interactions that antagonize vesicle fusion(2013-08) Falkenstein, Kristin Nicole; De Lozanne, ArturoLesions in the human Lyst gene are associated with the lysosomal disorder Chediak Higashi Syndrome. The absence of Lyst causes the formation of enlarged lysosome related compartments in all cells. This defect results in severe immunodeficiency, neurological dysfunction, and ultimately in death. Despite decades of research, the mechanism for how these enlarged compartments arise is not well established. Two opposing models have been proposed for Lyst function. The fission model describes Lyst as a positive regulator of fission from lysosomal compartments, while the fusion model identifies Lyst as a negative regulator of fusion between lysosomes. To date, a consensus on which model is correct has not been reached. This thesis details my investigation of Lyst function using Dictyostelium discoideum. To establish a definitive model for the function of the Dictyostelium Lyst ortholog, LvsB, we used assays that distinguish between defects in vesicle fusion versus fission. We compared the phenotype of cells defective in LvsB with that of two known fission defect mutants ([mu]3 and WASH null mutants). The temporal localization characteristics of the post-lysosomal marker vacuolin, as well as vesicular acidity and fusion dynamics of LvsB null cells are distinct from those of both fission defect mutants. These distinctions are predicted by the fusion defect model and implicate LvsB as a negative regulator of vesicle fusion. This work also presents evidence that LvsB antagonizes the function of two fusion regulatory proteins, Rab14 and dLIP5. The Dictyostelium Rab14 GTPase is known to stimulate lysosome fusion, and here we implicate dLIP5 as a promoter of Rab14 activity. Constitutive activation of Rab14 increases vesicle fusion in wild type cells but not in dLIP5 mutant cells. Thus, Rab14 activity is dependent on dLIP5. Additionally, the aberrant vesicle morphology and fusion phenotypes of LvsB mutant cells are suppressed by expression of dominant inactive Rab14 or disruption of dLIP5. This suppression suggests that LvsB antagonizes Rab14 activity to negatively regulate vesicle fusion. These studies validate the fusion model for LvsB function and provide new insights into the relationships that dictate vesicle fusion regulation. By extension, we propose that Lyst negatively regulates vesicle fusion by antagonizing the activity of a RabGTPase.Item From poles to equator: functional analysis of DdAurora during mitosis and cytokinesis in Dictyostelium discoideum(2007) Li, Hui, 1976-; De Lozanne, ArturoThe Aurora kinases are highly conserved serine/threonine kinases that play essential roles throughout mitosis. In metazoans, these functions are mediated by Aurora A and B at the spindle poles and the equatorial region respectively. I show here that Dictyostelium contains a single Aurora kinase, DdAurora that displays characteristics of both Aurora A and B. Like Aurora A, DdAurora has an extended N-terminal domain with an A-box and localizes to the spindle poles during early mitosis. Like Aurora B, DdAurora localizes to centromeres in metaphase, the central spindle during anaphase and the cleavage furrow at the end of cytokinesis. In addition to these known features of Aurora A and B, I found that DdAurora remains associated with centromeres during anaphase and telophase which has not been shown in any other organisms. INCENP is known to be an important binding partner of Aurora B. In Dictyostelium the conserved C-terminal IN-box domain of DdINCENP is essential for its interaction with DdAurora and for the localization of DdAurora to the central spindle. In contrast, the centromeric and spindle pole localization of DdAurora does not require an interaction with DdINCENP. Surprisingly, a truncated DdINCENP protein lacking the IN-box domain can still localize on centromeres and the central spindle even though it does not bind to DdAurora. I also found that the localization of DdAurora to the central spindle requires Kif12, a protein similar to mitotic kinesin like protein 2 (MKLP2). However, this requirement is suppressed by the overexpression of GFP-DdINCENP. GFP-DdINCENP can localize to the central spindle in the absence of Kif12 and it probably recruits DdAurora to the same location through their strong interaction. Finally, I demonstrated that Myosin II heavy chain is important for the proper localization of the DdAurora/DdINCENP complex to the cleavage furrow during late cytokinesis. With the exception of DdINCENP, no other binding partner or substrate of DdAurora has been identified in Dictyostelium. By performing large-scale immunoprecipitation in wild-type cells, I identified several potential binding partners/substrates of DdAurora, including topoisomerase B and HspA. Future esearch on these proteins may help to elucidate DdAurora function in different stages of M phase.Item 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.Item Study of Hip1r: insights from a Dictyostelium discoideum clathrin adaptor(2007) Repass, Shannon Lea, 1970-; O'Halloran, TheresaThe transport of material across the plasma membrane and between subcellular compartments is a major trafficking event by which cells communicate, regulate developmental processes and maintain homeostasis. Clathrin-coated vesicles select and traffic proteins to specific compartments in eukaryotic cells. Recently a large number of proteins have been identified that serve as clathrin adaptors and accessory proteins. Information regarding the interaction between individual clathrin adaptors and accessory proteins during coated pit formation is currently lacking. Here we investigated the intracellular role of one clathrin adaptor, Dictyostelium discoideum Hip1r, and identified a functional relationship between Hip1r and a second clathrin adaptor, epsin. Hip1r is phosphorylated and localizes to punctae on the plasma membrane that also contain epsin. Moreover, expression of the NH₂-terminal ENTH domain of epsin alone was sufficient to restore both the phosphorylation and the restricted localization of Hip1r to the plasma membrane. Analysis of the individual Hip1r domains demonstrated the phosphorylation event is also dependent upon the expression of the central coiled-coil region of the Hip1r. During development, Hip1r null cells form mature fruiting bodies that yield defective spores. While the mutant spores contain both cellulose and at least one protein secreted from prespore vesicles, spore coats lack the organized fibrils typical of wild type spores. Moreover, Hip1r spores are round, rather than ovoid, and exhibit decreased viability. Domain analysis of Hip1r in conjunction with investigation of phenotypes associated with a Hip1r/epsin double mutant reveal a requirement for full length Hip1r in the production of robust spores. Results from this study suggest that the Hip1r protein functions with epsin during cellular events in both growing and developing Dictyostelium cells and reveals a previously unidentified interaction between two clathrin adaptors.Item Study of LvsB in Dictyostelium discoideum provides insights into the Chediak-Higashi syndrome(2007-12) Kypri, Elena, 1980-; De Lozanne, ArturoThe Chediak-Higashi Syndrome is a disorder affecting lysosome biogenesis. At the cellular level, the Chediak-Higashi syndrome is characterized by the presence of grossly enlarged lysosomes in every tissue. Impaired lysosomal function in CHS patients results in many physiological problems, including immunodeficiency, albinism and neurological problems. The Chediak-Higashi syndrome is caused by the loss of a BEACH protein of unknown function named Lyst. In this work, I have studied the function of the Dictyostelium LvsB protein, the ortholog of mammalian Lyst and a protein that is also important for lysosomal function. Using a knock-in approach we tagged LvsB with GFP and expressed it from its single chromosomal locus. GFP-LvsB was observed on endocytic and phagocytic compartments. Specific analysis of the endocytic compartments labeled by LvsB showed that they represented late lysosomes and postlysosomes. The analysis of LvsB-null cells revealed that loss of LvsB resulted in enlarged postlysosomes, in the abnormal localization of proton pumps on postlysosomes and their abnormal acidification. This work demonstrated that the abnormal postlysosomes in LvsB-null cells were produced by the inappropriate fusion of lysosomes with postlysosomal compartments. Furthermore, this work provided the first evidence that LvsB is a functional antagonist of the GTPase Rab14 in vesicle fusion events. In particular, we demonstrated that reduction of Rab14 activity suppressed the LvsB-null phenotype by reducing the enlarged post-lysosomes and the enhanced rate of heterotypic fusion. In contrast, expression of an active form of Rab14 enhanced the LvsB-null phenotype by causing an even more severe enlargement of endosome size. The results provided by this work support the model that LvsB and Lyst proteins act as negative regulators of fusion by limiting the heterotypic fusion of early with late compartments and antagonize Rab GTPases in membrane fusion. The LvsB localization studies and the functional assessment of the LvsB-null phenotype helped make unique contributions to the understanding of the molecular function of Lyst proteins.