Date of Award

6-10-2015

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Applied Science

First Advisor

John Bush

Abstract

Dictyostelium discoideum is a primitive eukaryote that has recognized useful in studying various aspects of lysosomal enzymes as well as development since it can differentiate from unicellular amoebae into a multicellular.In this study, we investigated the localization, ecological and molecular function of two lysosomal enzymes, hexosaminidase (NagA) and,,-glucosidase (GluA) proteins, by overexpressing the genes encoding them in D. discoideum. Separate cell-lines overexpressing a GFP-tagged versions of the full length DdNagA gene (GFP-NagA), mutant version of the NagA gene (GFP-NagA (N214S)), a GFP-tagged version of the full length DdGluA gene (GFP-GluA) and a mutant version of first 83 amino acids DdGluA fused with the GFP gene. Analysis of the experimental results from microscopic localization studies in the cells overexpressing DdNagA and DdGluA proteins have revealed that both of the GFP-tagged NagA, GluA and NagA (N214S) localize and function within the Dictyostelium lysosomes and endosomes. GFP-GluA∆,84-821 showed partial localization to both lysosome and endosome as well. Functional analysis of DdNagA overexpression, revealed a role in vesicular transport, specifically in phagocytosis, and pinocytosis. Furthermore, cells overexpressing the DdNagA gene displayed an increased rate of multicellular development and cell-cell cohesion as well as a reduced rate of programmed cell death. Cells over-expression NagA (N214S) displayed rates of phagocytosis and pinocytosis similar to the control cells, but interestingly had increased levels of programmed cell death. Furthermore, it was detected that cells overexpressing GFP-GluA∆,84-821 could not complete the development cycle, and are defective in both aggregation from both reduced cell-cell cohesion, and decreased sensitivity to cAMP. These mutant cells also proved to have an increase in induced cell death of nearly 37.02%. Both DdNagA and DdGluA overexpressing cell-lines over-secreted lysosomal glycosidase enzymes. The ecological study of the GFP-NagA, GFP-NagA (N214S), GFP-GluA and GFP-GluA∆,84-821cell-lines showed that there is decreasing of growth rates in low temperature, pH and sugar percentage conditions. GFP-NagA and GFP-GluA cell-lines showed increases in growth rate more than other cell-lines at all studied temperature conditions. Furthermore, the aggregation rates were increased under low sugar percentage and high temperatures. GFP-GluA∆,84-821 displayed low growth rates and failed to aggregation in all conditions studied. The data suggest that GFP-NagA and GFP-GluA cell-lines arwe more resistant to the environmental conditions while GFP-GluA∆,84-821 are less resistant to the environmental conditions. These data support a role for DdNagA and DdGluA in trafficking along the endocytic, phagocytes, and particular the biosynthetic pathways. In summary, cells overexpressing the two lysosomal enzymes had significant alterations in several cellular processes including vesicular trafficking, cell signaling, cell-cell contact, and differentiation. Furthermore, the first 83 amino acid in DdGluA contained the lysosome-targeting signal for this enzyme in Dictyostelium We also suggest that both the overexpression of DdNagA and DdGluA would give cells and ecological advantage. However, the overexpression of the first 83 aa of DdGluA did in fact cause a negative ecological selection most likely due to multiple effects, including alterations in lysosomal enzyme secretions and blocked developmental aggregation under both starvation and other ecological stress conditions.

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