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.

7 ABSTRACT Mitochondria of opisthokonts undergo permanent fusion and fission throughout the cell cycle. Keeping these two processes in balance is vital for various aspects of mitochondrial and cellular homeostasis. Both mitochondrial fusion and division mechanisms are controlled by highly conserved dynamin-related GTPases that are present in all kingdoms of life. The aspects of mitochondrial dynamics outside the opisthokonts is, however, almost completely unexplored phenomenon. In our work, we introduced a tool for live imaging of the reduced forms of mitochondria into model organisms Giardia intestinalis and Trichomonas vaginalis, anaerobic protist parasites from the Excavata supergroup of Eukaryotes. Using this technique, we investigated the dynamics of the mitosomes, the simplest forms of mitochondria, of G. intestinalis. The division of mitosomes is restricted to Giardia mitosis and is absolutely synchronized with the process. The synchrony of the nuclear and the mitosomal division persists also during the encystation of the parasite. Surprisingly, the sole dynamin-related protein of the parasite seems not to be involved in mitosomal division. However, throughout the cell cycle mitosomes associate with the...

SNARE proteins play essential role in most membrane fusions taking place in eukaryotic cell. They are responsible for all fusions that occur across endocytic and secretory pathways. Apart from these processes stand mitochondria and plastids. Fusion of these organelles is directed by specific protein machineries. In this work we review up-to-date information on SNARE mediated membrane fusion and fusion of outer and inner mitochondrial membranes with an emphasis on situation in flagellated protozoan parasite Giradia intestinalis. It was suggested that one of typical SNARE protein in Giardia (GiSec20) is localised to its highly reduced mitochondria called mitosomes. This protein is also essential for surviving of Giardia trophozoites. In this work we show that mitosomal localization of Gisec20 is caused by episomal expression however the protein is localised to endoplasmic reticulum under physiological conditions. Using GFP tag we were able to characterize its targeting signal which showed to be localised in transmembrane domain of GiSec20. This signal targets the protein to mitosomes of G. intestinalis and S. cerevisiae, respectively. Mitosomal localization was prevented by adding 3'UTR to gene sequence and its episomal expression. This suggests existence of targeting mechanism based on information...