Universiẗat, Diss., 2003--Heidelberg.

Entamoeba histolytica is an important human intestinal parasite that has a major impact on human health and is responsible for approximately 100,000 deaths each year. Entamoeba histolytica is one of several known eukaryotes that harbour strongly reduced mitochondria, called mitosomes, which have lost the vast majority of mitochondrial pathways as well as their organellar genome. While the occurrence and function of mitosomes have been well studied, little is known about their inheritance and division. Mitochondrial division in all studied eukaryotes relies on the participation of dynamin proteins for membrane scission. The central aim of this study was to characterize the dynamin protein family in Entamoeba histolytica and to analyze if they participate in mitosomal division. In relation to this work we studied the occurrence of mitosomes in the distantly related reptilian parasite Entamoeba invadens and revisited the phylogenetic relationships among mitosomal Hsp70, a protein we used for mitosomal localization experiments. Our studies revealed that Entamoeba histolytica contains two classical and two strongly derived members of the dynamin protein family, which we called Drp1, Drp2, Drp3 and Drp4. Drp1 and Drp2 exhibit the classical dynamin protein structure with a GTPase, middle and GTPase effector domain, while Drp3 and Drp4 only appear to contain the dynamin GTPase domain. Using phylogenetic reconstructions we could not identify closely, and thus functionally related, dynamins for Drp1 and Drp2 within the eukaryotic tree of life including the mitochondria‐associated amoebozoan dynamins DymA and DymB. The structurally derived dynamins however, were closely related to amoebozoan and archaeplastidan proteins involved in cytokinesis and chloroplast division. All Entamoeba dynamins are differentially expressed in trophozoites with EhDrp2 appearing to be most abundant and Drp3 expressed the least. We conducted stage conversion experiments using E. invadens to understand the importance of dynamins during cyst formation. During encystation all dynamin expression levels increased. Interestingly, Drp3 expression is strongly upregulated in the mid cyst stages and Drp4 during the late phase of encystation. Thus, Drp3 and Drp4 appear not to be involved in cytokinesis and possibly evolved a novel function in the cyst formation process. We carried out Drp2 enzymatic characterization and localization experiments as well as complementation studies using the related amoebozoan Dictyostelium discoideum in order to understand the role and function of E. histolytica Drp2 in the cell. We found that its kinetic characteristics are comparable to other members of the eukaryotic dynamin protein family by exhibiting low substrate specificity, the ability to oligomerize to higher structures and a substrate dependent cooperative enzyme activity. Drp2 localized to abundant punctate structures in the cytosol but did not colocalize with mitosomes. In addition, Drp2 was not able to complement D. discoideum DymA. Both findings suggest that Drp2 is not directly involved in mitosomal (or mitochondrial) division. We overexpressed Drp2 in E. histolytica and D. discoideum and found a significant effect on cytoskeletal organization. Both strains showed a strong impairment in amoeboid movement, cell‐surface attachment and cell growth. Additionally, the number of nuclei was increased significantly. Our data imply that Drp2 plays an important role for cytoskeletal organization. Additionally in this study, we show that mitosomes are also abundantly present in E. invadens suggesting that mitosomes are characteristic for all Entamoeba spp. Furthermore, we demonstrate that E. invadens cysts contain mitosomes in high abundance comparable to its vegetative life stage. Our studies verify that mitosomal Hsp70 is part of the amoebozoan protein family and of mitochondrial origin as shown by in silico characterization and localization experiments using the homologous Hsp70 antibody.

Mitochondrial dynamics resulting from competing fusion and fission reactions are required for normal cellular function in eukaryotes. Mgm1, a dynamin related protein, is a key component in yeast mitochondrial fusion and is evolutionarily conserved. Previous in vivo studies suggest that the GTPase domain and oligomerization are required for Mgm1 mediated mitochondrial inner membrane fusion. This work demonstrates that purified Mgm1 forms dynamic low order oligomers, and has GTPase activity and kinetic properties consistent with a mechanoenzyme and with a role in inner membrane mitochondrial fusion. Mutations of key residues in the GTPase domain show diminished GTPase activity, while a mutation in the GTPase effector domain implicated in self-assembly results in a lower propensity to form oligomers. Together these data indicate that Mgm1 mediates fusion through oligomerization and GTP binding/hydrolysis in a manner similar to other dynamin mechanoenzymes.

Mitochondrial dynamics resulting from competing fusion and fission reactions are required for normal cellular function in eukaryotes. Mgm1, a dynamin related protein, is a key component in yeast mitochondrial fusion and is evolutionarily conserved. Previous in vivo studies suggest that the GTPase domain and oligomerization are required for Mgm1 mediated mitochondrial inner membrane fusion. This work demonstrates that purified Mgm1 forms dynamic low order oligomers, and has GTPase activity and kinetic properties consistent with a mechanoenzyme and with a role in inner membrane mitochondrial fusion. Mutations of key residues in the GTPase domain show diminished GTPase activity, while a mutation in the GTPase effector domain implicated in self-assembly results in a lower propensity to form oligomers. Together these data indicate that Mgm1 mediates fusion through oligomerization and GTP binding/hydrolysis in a manner similar to other dynamin mechanoenzymes.

During early development, a series of regulated cell movements is required to set up the adult body plan of an organism. Collectively referred to as gastrulation, these coordinated cell movements organize the germ layers and establish the major body axes of the embryo. One such coordinated cell movement, epiboly, describes the thinning and spreading of a multilayered cell sheet to cover the embryo during gastrulation. The zebrafish embryo has emerged as a vital model system to study the cellular and molecular mechanisms that drive epiboly. In the zebrafish, the blastoderm undergoes epiboly to engulf the yolk cell and close the blastopore at the vegetal pole. This is achieved through the coordinated movement of the deep cells, which make up the embryo proper, and two extra-embryonic tissues, the enveloping layer and yolk syncytial layer. Epiboly is essential to the development of most organisms; however, the cellular and molecular mechanisms driving epiboly are poorly understood. Here I report the findings of two distinct projects which addressed the cellular and molecular basis for epiboly in the zebrafish. One cellular mechanism thought to be involved in driving epiboly is the removal of yolk cell membrane ahead of the advancing blastoderm margin. Using a combination of drug- and dominant-negative based approaches to inhibit Dynamin, a key component of the endocytic machinery, I demonstrated that marginal yolk cell endocytosis is dispensable for the successful completion of epiboly. Instead, I found that Dynamin primarily acts in the blastoderm where it maintains integrity of the enveloping layer (EVL) during epiboly. Dynamin maintains EVL integrity through regulation of the Ezrin/Radixin/Moesin (ERM) family of proteins and the activity of the small GTPase Rho A. With the goal of identifying genes involved in regulating epiboly, I characterized the calpain family of calcium-dependent cysteine proteases in the zebrafish and examined the developmental expression patterns of these genes. My study provided insight into the evolution of this large gene family. Furthermore, I found that most members of this family are expressed in the early embryo, suggesting that they may play a role in regulating early developmental processes such as epiboly.

During early development, a series of regulated cell movements is required to set up the adult body plan of an organism. Collectively referred to as gastrulation, these coordinated cell movements organize the germ layers and establish the major body axes of the embryo. One such coordinated cell movement, epiboly, describes the thinning and spreading of a multilayered cell sheet to cover the embryo during gastrulation. The zebrafish embryo has emerged as a vital model system to study the cellular and molecular mechanisms that drive epiboly. In the zebrafish, the blastoderm undergoes epiboly to engulf the yolk cell and close the blastopore at the vegetal pole. This is achieved through the coordinated movement of the deep cells, which make up the embryo proper, and two extra-embryonic tissues, the enveloping layer and yolk syncytial layer. Epiboly is essential to the development of most organisms; however, the cellular and molecular mechanisms driving epiboly are poorly understood. Here I report the findings of two distinct projects which addressed the cellular and molecular basis for epiboly in the zebrafish. One cellular mechanism thought to be involved in driving epiboly is the removal of yolk cell membrane ahead of the advancing blastoderm margin. Using a combination of drug- and dominant-negative based approaches to inhibit Dynamin, a key component of the endocytic machinery, I demonstrated that marginal yolk cell endocytosis is dispensable for the successful completion of epiboly. Instead, I found that Dynamin primarily acts in the blastoderm where it maintains integrity of the enveloping layer (EVL) during epiboly. Dynamin maintains EVL integrity through regulation of the Ezrin/Radixin/Moesin (ERM) family of proteins and the activity of the small GTPase Rho A. With the goal of identifying genes involved in regulating epiboly, I characterized the calpain family of calcium-dependent cysteine proteases in the zebrafish and examined the developmental expression patterns of these genes. My study provided insight into the evolution of this large gene family. Furthermore, I found that most members of this family are expressed in the early embryo, suggesting that they may play a role in regulating early developmental processes such as epiboly.

Necroptosis is a pathway of inflammatory cell death that is associated with several pathologies and is induced by ligation of surface TLR or cytokine receptors in macrophages. Many signaling pathways depend on endocytosis, a process mediated by GTPases such as dynamin. We evaluated the role of dynamin-dependent endocytosis in the necroptosis of macrophages using various dynamin inhibitors. Using flow cytometry, we confirmed that during necrosome signaling, various dynamin inhibitors (e.g. Dyngo 4a and Dynasore) blocked the internalization of TLR4, which also resulted in the inhibition of cytokine production. Despite the similar impact of Dynasore and Dyngo 4a on TLR4 endocytosis and cytokine production, only Dyngo 4a prevented TLR4-induced necroptosis of macrophages. Further studies indicated that Dyngo 4a was a potent stimulator of the p38 MAPK pathway, and activation of this pathway by Dyngo 4a was responsible for the inhibition of necroptosis of macrophages following TLR4 signaling. Thus, these studies reveal the previously unknown role of the p38 MAPK pathway in regulating the activation of necrosome signaling.

No description available.

Mitochondria are dynamic organelles that constantly undergo opposing fission and fusion events which impact many aspects of mitochondrial and cellular homeostasis including bioenergetic activity, calcium buffering and organelle transport. The large GTPase dynamin-related protein 1 (Drp1) acts as a mechanoenzyme to catalyze fission of mitochondria. Drp1 activity is regulated through a series of reversible posttranslational modifications. Phosphorylation of the conserved serine residue, S656, by cAMP dependent protein kinase A (PKA) acts as a master regulator of Drp1 activity. Two phosphatases oppose PKA by dephosporylating Drp1 S656, a mitochondrial isoform of protein phosphatase 2A and the calcium-calmodulin dependent phosphatase calcineurin (CaN). Here I report the characterization of a conserved CaN docking site on Drp1, an LxVP motif, just upstream of the Drp1 S656 site. Mutational modification of the Drp1 LxVP motif resulted in selective bidirectional modulation of formation of the CaN:Drp1 complex. Stability of the CaN:Drp1 LxVP motif mutant complexes was qualitatively described by affinity purification and quantitatively described by isothermal titration calorimetry. Stability of the CaN:Drp1 complex was found to directly correlate with Drp1 S656 dephosphorylation kinetics as demonstrated by studies conducted in vitro and in intact cells. Further, the CaN:Drp1 signaling axis was shown to shape basal mitochondrial morphology in a heterologous cell line system and in primary hippocampal neurons. Finally, disruption of the CaN:Drp1 signaling axis was found to protect neurons from oxygen-glucose deprivation, an in vitro model of ischemic injury. While these results suggest that the CaN:Drp1 signaling axis may be a potential target for neuroprotective therapeutic exploitation, the mechanism by which disruption of the CaN:Drp1 signaling axis specifically and mitochondrial elongation generally results in resistance to ischemic injury remains unknown. Additional studies reported here demonstrate that mitochondrial fragmentation remains a prominent feature of injured neurons regardless of the fidelity of the CaN:Drp1 signaling axis. Mitochondrial fragmentation at the time of injury was found to occur in a Drp1-independent manner. Chronic mitochondrial elongation was also found to leave unaltered the ability of neurons to detoxify reactive oxygen species, buffer intracellular calcium and supply ATP for homeostatic function.

Given the debilitating effect that mitochondrial dysfunction has on human health, it is important to understand mitochondrial dynamics that are vital for the maintenance of mitochondrial function, genome, morphology, and quality control. Mitochondrial dynamics result from a balance in mitochondrial fusion and fission. Although the mechanism and regulation of mitochondrial fission are largely elucidated, less is known about mitochondrial fusion. Mgm1 is a protein that mediates mitochondrial fusion in yeast. However, the molecular mechanism of Mgm1 function in mediating mitochondrial fusion is unclear. In this thesis, first, I show that Mgm1 contains a lipid-binding domain by demonstrating that purified Mgm1 has lipid-binding activity and by identifying mutations in conserved residues that abrogate these interactions. Second, I show that Mgm1 assembles into hexameric rings and undergoes nucleotide-dependent structural transitions that, I believe, initiate membrane fusion. Lastly, I demonstrate that Mgm1 exhibits membrane-remodeling activities that are crucial for the tethering and lipid-mixing steps in the membrane fusion event. Together, I propose a mechanistic model of Mgm1 function in mediating mitochondrial fusion that advances the fields of mitochondrial biology, cellular protein-membrane dynamics, and the etiology of neurodegenerative diseases.