MEES Theses and Dissertations
Permanent URI for this collectionhttp://hdl.handle.net/1903/19655
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Item New Insights into the Role of F-actin in Regulation of Mitochondrial Fission(2015) Li, Sunan; Karbowski, Mariusz; Marine-Estuarine-Environmental Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Mitochondrial dynamics, including fusion and fission, are vital for supplying cellular energy as well as controlling other tasks including apoptosis, aging and cellular differentiation. Defects of mitochondrial fission pathway have been implicated in a wide spectrum of human diseases such as Parkinson’s disease and Alzheimer’s disease. Although recent findings point to a role of the actin cytoskeleton in regulating mitochondrial division, little is known about the mechanism. Here, I report that transient de novo polymerization of F-actin on the outer mitochondrial membrane contributes to Drp1-dependent mitochondrial division in mammalian cells. Transient de novo F-actin assembly on the mitochondria occurs upon induction of mitochondrial fission and F-actin accumulates on the mitochondria without forming detectable submitochondrial foci. Impairing mitochondrial division through Drp1 knockout or inhibition prolonged the time of mitochondrial accumulation of F-actin and also led to abnormal mitochondrial accumulation of the actin regulatory factors cortactin, cofilin, and Arp2/3 complex, suggesting that disassembly of mitochondrial F-actin depends on Drp1 activity. Furthermore, downregulation of actin regulatory proteins Arp2/3 complex, cortactin and cofilin led to abnormal elongation of mitochondria, associated with mitochondrial accumulation of Drp1. In addition, depletion of cortactin inhibited Mfn2 downregulation- or FCCP- induced mitochondrial fragmentation. These data indicate that the dynamic assembly and disassembly of F-actin on the mitochondria participates in Drp1-mediated mitochondrial fission. Moreover, I also discovered a novel F-actin involved mechanism of mitochondrial fission regulated by deubiquitinase Usp30. Overexpression of Usp30CS predicted to lack deubiquitinase activity induced abnormal elongation and thinning of mitochondrial tubules. Furthermore, expression of Usp30CS preferably binds to Drp1, inducing a dramatic redistribution of Drp1 from the cytosol to the mitochondria, and accumulation of high molecular weight Drp1 species. Importantly, FCCP induced a gradual tubulation of Drp1-containing structures, accompanied with mitochondrial associated F-actin in a similar timeframe in Usp30CS-expressing cells, suggesting that inhibition of Usp30 deubiquitnase activity stalls progression of Drp1-dependent mitochondrial division. In sum, here I report that mitochondrial F-actin polymerization is a required step of mitochondrial fission, regulated by actin-modifying proteins and deubiquitinase Usp30, providing in-depth vision and a novel mechanism of actin cytoskeleton participated mitochondrial fission.Item CHANGES IN eIF2α PHOSPHORYLATION IN RESPONSE TO NUTRIENT DEFICIENCY AND OTHER STRESSORS IN FISH(2015) Liu, Chieh-lun; Jagus, Rosemary; Place, Allen R; Marine-Estuarine-Environmental Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The present study investigates the response of two teleost species to stressors as measured by phosphorylation of the α-subunit of the translational initiation factor, eIF2. The phosphorylation of the translational initiation factor, eIF2, on its α-subunit is an adaptive response to a variety of stressors in eukaryotes from protists to vertebrates. There are four eIF2α-specific kinases in most vertebrates, GCN2, PERK, PKR and HRI, each of which can be activated by different stressors. Two of these eIF2α-kinases, GCN2 and PERK, respond to changes in nutritional status. It was of particular interest to determine whether eIF2α phosphorylation can be used as an early marker to evaluate diets in fish. However, eIF2α phosphorylation could also be of use in monitoring other stressors of fish in aquaculture. The increase in global aquaculture production of fish has increased interest in the optimization of fish diets and health to increase production and sustainability. Studies were initiated in a zebrafish Danio rerio cell line, ZFL cells, to lay the groundwork for looking at eIF2α phosphorylation in fish and in species of more interest to aquaculture. All the eIF2α-kinases are present in the zebrafish genome and are expressed in ZFL cells. Two forms of eIF2α are expressed in ZFL cells, eIF2α-a and eIF2α-b, with eIF2α-b transcripts ~5-fold higher than those of eIF2α-a. The two gene products are 96 % identical at the amino acid level and are identical at the phosphorylation and kinase docking sites. Phosphorylation of eIF2α in ZFL cells is increased by a variety of agents/conditions; starvation, leucinol, endoplasmic reticulum stress, poly(I)poly(C) and N-methylprotoporphyrin, consistent with activation of the eIF2α kinases GCN2, PERK, PKR and HRI, respectively. Application of the same analyses to a new cell line from a marine fish, cobia Rachycentron canadum, shows that these cells are also responsive to activators of GCN2 and PERK. Cloning of eIF2α cDNA from cobia has shown close identity to the zebrafish eIF2αs. Although cobia has two eIF2α transcripts, the coding sequence of each is identical. Preliminary studies have shown that in cobia juveniles, diet, probiotics and water temperature all affect the phosphorylation state of eIF2α.