Development of a method for monitoring the clathrin-dependent protein trafficking in the parasite Plasmodium bergheiGluhić, Zala (Avtor) Marc, Janja (Mentor) Waters, Andrew P. (Komentor) Plasmodium bergheiknocksidewaysclathrinfluorescent taggingMalaria is caused by the Apicomplexan parasite Plasmodium, which has a very complex life cycle involving various changes and modifications to the parasite itself as well as to the cells it resides in. To better understand how parasite invades red blood cells, obtains nutrients and remains hidden from its host, it is important to deepen our knowledge in parasite cell biology. Changes in the host cell are mediated by a wide array of exported proteins, but the specific role and transportation pathways of a large proportion is not yet well understood. Clathrin is a vesicular coating protein expected to act in Plasmodium intracellular trafficking but knowledge about proteins that are being transported in clathrin-coated vesicles is very limited. In this work we have addressed nine Plasmodium berghei proteins which are exported or localised in various cell locations and might be transported in a clathrin-dependent manner. To obtain more information how these proteins are trafficked, we prepared plasmids for C-terminal tagging of endogenous Plasmodium proteins with fluorescent protein TagBFP and HA epitope tag. The plasmids have been transfected in a genetically modified parasite line which enables the use of the molecular technique knocksideways. Upon addition of rapamycin, the clathrin in knocksideways parasites is relocalised to the parasite membrane, thus inactivating clathrin-dependent trafficking pathways. This allows investigation of the effect of inactivating clathrin-mediated protein trafficking on the protein of interest. We have endogenously tagged nine proteins in the rodent malaria parasite P. berghei and inspected how successful the process of integration and protein expression has been. Integration screening was followed by analyses with flow cytometry and imaging flow cytometry to visualise protein expression and localisation. One of the nine studied is Zn finger protein of an unknown function (PBANKA_141590), which has been examined further in our work. We determined how the expression of the protein changes during intra-erythrocytic development and obtained information about protein localisation. Live imagestream and microscopy studies indicate that it is a nuclear protein. Knocksideways experiments indicate the protein does not depend on clathrin for its nuclear localisation. The studies on this protein illustrate the range of information that can be obtained about a previously unstudied protein using these methods. We have generated plasmids and parasite lines that provide new tools to study and contribute towards understanding of protein functions and Plasmodium protein transportation pathways.20192019-09-25 11:55:12Magistrsko delo/naloga111169sl