Dictyostelium discoideum is a soil dwelling amoeba which has been widely used as a model organism to study cellular processes such as signal transduction, chemotaxis, endocytosis and exocytosis. The process of phagocytosis in Dicytostelium is largely comparable to that of neutrophils and macrophages in the mammalian system. Neutrophils and macrophages are cells of the innate immune system and they engulf infectious bacteria through phagocytosis. Dictyostelium cells uptake yeast and bacteria for their nutrition through phagocytosis, which is an actin dependent mechanism and is a target of multiple signaling inputs. Recent studies have uncovered different proteins involved in the signaling of particle and further studies are required to decipher the intricate mechanism leading to the F-actin rearrangement. Two of the proteins have previously known to be involved in the pathways regulating the F- actin rearrangement name PP2A phosphatase and LKB1 kinase
The main objective of this project was to determine how these proteins are affecting the two actin driven particle uptake processes, phagocytosis and fluid uptake. We showed that ablation of PsrA gene which codes the regulatory subunit of PP2A resulted in a defective phagocytosis, whereas the fluid uptake was normal. We also showed for the first time that there was an increase in the phosphorylation of some of the PKB substrate proteins in wild type cell. Cells lacking PsrA gene displayed an aberrant phosphorylation of PKB substrate protein when compared to the wild type cells further confirming the involvement of PKB substrate in phagocytosis.
Further, we looked at the effects of LKB1 kinase on phagocytosis by using a LKB1 knockdown construct introduced into wild type cells. The knock down of LKB1 resulted in a higher rate of phagocytosis while introduction of a LKB1 over expressing construct severally decreased the rate of phagocytosis indicating an inhibitory effect of LKB1. Furthermore there was an increase in the PKB substrate protein but a different pattern compared to the psrA- cells. We also carried out adhesion assays on LKB1 knockdown cells and the results showed a higher substrate adhesion as compared to the wild type cells, while psrA- cells had no adhesion defect.
| Identifer | oai:union.ndltd.org:fiu.edu/oai:digitalcommons.fiu.edu:etd-3815 |
| Date | 01 July 2016 |
| Creators | Sharief, Mujataba Rahiman |
| Publisher | FIU Digital Commons |
| Source Sets | Florida International University |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | FIU Electronic Theses and Dissertations |
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