Browsing by Subject "Dictyostelium"
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Item APβ1/2 and Hip1r : insights into early and late stage clathrin adaptors in Dictyostelium discoideum(2012-05) Sosa, Ramiro Thomas; O'Halloran, Theresa; Gross, Jeffrey M.; Johnson, Arlen W.; Mehdy, Mona; Morgan, Jennifer R.Clathrin-mediated endocytosis is the process whereby specific cargoes are internalized into coated vesicles from the plasma membrane. Numerous clathrin adaptors facilitate this process by linking the coat protein clathrin to the plasma membrane by associating with PI(4,5)P2 and binding to membrane-bound cargo. Here, I investigated the role of two clathrin adaptors, APβ1/2 and Hip1r, in clathrin-mediated endocytosis. I found that Dictyostelium APβ1/2 functions in both the AP1 and AP2 complexes, unlike vertebrates, which have distinct β subunits for each AP complex. I found that APβ1/2 function is required for several clathrin-dependent processes, including cytokinesis, development and osmoregulation. I also uncovered a role for APβ1/2 in the stability other subunits of the AP1 and AP2 complexes. Finally, phenotypic comparisons of APβ1/2 mutant cells with cells missing subunits that are specific to the AP1 or AP2 complex allowed me to distinguish between endocytic defects and endosomal trafficking defects in clathrin mutants. My investigation of Hip1r centered on the known requirement for Hip1r in actin dynamics during endocytosis and a possible role for Hip1r phosphorylation in regulating actin. To determine how phosphorylation contributes to Hip1r function, I identified a specific serine residue that serves as a Hip1r phosphorylation site. I also identified a novel role for the kinase PKB in Hip1r phosphorylation. I determined that phosphorylation is not required for Hip1r localization to the plasma membrane. Similar to Hip1r, PKB is required for proper actin dynamics during endocytosis. My results support a model in which epsin recruits Hip1r to the plasma membrane during formation of clathrin-coated vesicles. Here, Hip1r functions as both a clathrin adaptor and a negative regulator of actin polymerization. I propose that phosphorylation of Hip1r by PKB triggers a reduction in the affinity of Hip1r for clathrin, which may stimulate actin polymerization and tethering of clathrin-coated vesicles with the actin cytoskeleton.Item Functional analysis of Abp1 in Dictyostelium(2006-08) Wang, Yanqin, 1974-; O'Halloran, TheresaThis work identified an ortholog of Abp1 (actin binding protein 1) in Dictyostelium (Dabp1). In order to analyze the functions of Dabp1 in Dictyostelium, loss-of–function studies and gain-of-function studies were performed by generating cells that either deleted the Dabp1 gene from the genome or overexpressed the Dabp1 protein. In these mutants, most actin-based processes were intact. However, cell motility was altered during early development. During chemotactic streaming, more than 90% of wild type cells had a single leading pseudopodium and a single uropod, whereas more than 27% of Dabp1 null cells projected multiple pseudopodia. Similarly, ~ 90% of cells that overexpressed Dabp1 projected multiple pseudopodia during chemotactic streaming, and displayed reduced rates of cell movement. Expression of the SH3 domain of Dabp1 showed this domain to be an important determinant in regulating pseudopodium number. These results suggest that Abp1 controls pseudopodium number and motility in early stages of chemotactic aggregation in Dictyostelium. This work also revealed an interplay between Dabp1 and MyoB, one of the Myosin I proteins, in controlling pseudopodia formation in Dictyostelium. These two proteins colocalize partially at the cortex in growing cells. The peripheral localization of MyoB was dependent on Dabp1. Depletion of both Dabp1 and MyoB caused defects in organization of the actin cytoskeleton and actin related activities such as formation of small F-actin filled spikes on the cell cortex of growing cells, a higher percentage of multinucleated cells, and an increased number of pseudopodia branching extensively. When MyoB was overexpressed in Dabp1 null mutants, cells had similar phenotypes as Dabp1/MyoB double null mutants, and displayed an increased number of pseudopodia with many branches. Overexpression of Dabp1 in MyoB null mutants rescued the defects in pseudopodia formation. The SH3 of Dabp1 was shown to be important for the rescue of defects caused by depletion of MyoB. Collectively, these data suggest that MyoB and Dabp1 work cooperatively to regulate the uniformity and integrity of the actin extensions during chemotaxis. MyoB requires Dabp1 to function in this process. Dabp1 may function as a scaffold to recruit MyoB to the proper localization. These studies of Dabp1 in Dictyostelium raise broad question about functions of actinassociated proteins in pseudopodia formation and the importance of uniformity and integrity for actin structures in chemotaxis.Item Linking Two Seemingly Unrelated Diseases, Cancer and Acute Respiratory Distress Syndrome, Through a Dictyostelium Secreted Protein(2014-06-05) Herlihy, Sarah EThe work in this dissertation links two diseases through a protein secreted by Dictyostelium discoideum cells. The protein, AprA, inhibits cell proliferation and induces chemorepulsion (movement away) of Dictyostelium cells. This has implications in both cancer research and the study of Acute Respiratory Distress Syndrome. Cancer is a misregulation of cellular proliferation. Often the removal of a primary tumor results in rapid metastatic cell proliferation. The rapid proliferation of metastatic cells indicates the presence of a factor, called a chalone, secreted by the primary tumor cells, that inhibits metastatic cell proliferation. The ability of AprA to inhibit proliferation of the cells that secretes it classifies it as a chalone. Using the model organism Dictyostelium and the protein AprA allows us to study chalone signaling mechanisms. Acute Respiratory Distress Syndrome (ARDS) is characterized by an excess influx of neutrophils into the lungs. Neutrophils damage the lung tissue and ultimately recruit more neutrophils that repeat the process. A need exists to remove these cells and allow resolution to occur. One way to accomplish this is through chemorepulsion, the directional movement of cells away from an external cue. We can use AprA to study the mechanisms of chemorepulsion. In this dissertation, I have found that the PTEN-like protein CnrN, which is an inhibitor of proliferation and chemotaxis, is involved in both AprA proliferation inhibition and chemorepulsion of Dictyostelium cells. I have shown that the human protein DPPIV, which is structurally similar to AprA, causes chemorepulsion of human neutrophils. Additionally, aspirated DPPIV reduces the accumulation of neutrophils in the lungs of a mouse model of ARDS. Work shown in the appendices suggests that AprA signals through specific G protein-coupled receptors. The work in this dissertation studies the role of chalones and chemorepellents. It allows the unique opportunity to study chemorepulsion in both Dictyostelium and human cells. The hope and goal is that the work in this dissertation could lead to novel therapies for diseases such as cancer and ARDS.