Zurich Open Repository and Archive University of Zurich Main Library Strickhofstrasse 39 CH-8057 Zurich www.zora.uzh.ch Year: 2016 Host–parasite coevolution drives adaptation of the giardia lamblia endocytic system Zumthor, Jon Paulin Posted at the Zurich Open Repository and Archive, University of Zurich ZORA URL: https://doi.org/10.5167/uzh-136371 Dissertation Published Version Originally published at: Zumthor, Jon Paulin. Host–parasite coevolution drives adaptation of the giardia lamblia endocytic system. 2016, University of Zurich, Vetsuisse Faculty. Endocytosis Redesigned: Host–Parasite Coevolution Drives Adaptation of the Giardia lamblia Endocytic System ___________________________________________________________________________ Dissertation zur Erlangung der naturwissenschaftlichen Doktorwürde (Dr. sc. nat.) vorgelegt der Mathematisch-naturwissenschaftlichen Fakultät der Universität Zürich von Jon Paulin Zumthor von Haldenstein GR Promotionskomitee Prof. Dr. Adrian B. Hehl (Vorsitz und Leitung der Dissertation) Prof. Dr. Urs Greber Prof. Dr. Lloyd Vaughan Zürich, 2016 Table of Contents PART I SUMMARY .................................................................................................................. 3 1. Summary ........................................................................................................................ 3 2. Zusammenfassung .......................................................................................................... 4 PART II Aim of the Thesis ..................................................................................................... 8 PART III Introduction ............................................................................................................ 9 1. Giardia lamblia ............................................................................................................... 9 1.1. Giardiasis and the life cycle of Giardia lamblia ....................................................... 9 1.2. Evolutionary background ...................................................................................... 10 1.3. The Endomembrane System in Giardia lamblia .................................................... 11 1.4. Constitutive and regulated protein secretion ....................................................... 14 2. Canonical clathrin-mediated endocytosis .................................................................... 15 2.1. Clathrin .................................................................................................................. 17 2.2. Adaptor protein compleX 2 ................................................................................... 19 2.3. Dynamin ................................................................................................................ 19 3. Endocytosis in Giardia lamblia ...................................................................................... 20 4. PX domain-containing proteins and phosphoinositides. .............................................. 24 5. Goals of the Thesis ....................................................................................................... 25 5.1. Main project: Structural and molecular investigation of the endocytic system in Giardia lamblia ................................................................................................................. 25 5.2. Subproject: Characterization of the giardial PX-domain containing protein family 26 6. Bibliography ................................................................................................................. 27 PART IV Manuscripts .......................................................................................................... 38 1. Manuscript I ................................................................................................................. 38 2. Manuscript II (Draft) ..................................................................................................... 89 3. Manuscript III ............................................................................................................. 104 PART V Discussion and future directions ......................................................................... 128 1. Discussion ................................................................................................................... 128 1.1. General ................................................................................................................ 128 1.2. Redistribution of PVs in the course of reductive evolution favors endocytic communication via the shortest and most direct route ................................................ 129 1 1.3. Cooption of giardial clathrin - from dynamic to static ........................................ 130 1.4. Does GFP alter GlCHC behavior? ......................................................................... 131 1.5. GlDRP performs a conserved role in membrane fission events .......................... 131 1.6. GlAP2 functions – some conserved, some not .................................................... 132 1.7. How is GlCHC linked to the endocytic membranes? ........................................... 133 1.8. A customized co-IP protocol involving limited crosslinking enables the detection of protein interactions in the giardial clathrin protein network .................................... 134 1.9. Conclusions ......................................................................................................... 135 2. Future directions ........................................................................................................ 137 2.1. Static GlCHC is not involved in transient membrane coat formation but its function remains elusive – suggestions to functionally analysis GlCHC ......................... 137 2.2. Testing the conserved membrane severing function of GlDRP .......................... 138 3. Bibliography ............................................................................................................... 139 Acknowledgements ........................................................................................................... 144 Curriculum Vitae ................................................................................................................ 146 2 PART I SUMMARY 1. Summary Giardia lamblia (syn. G. intestinalis and G. duodenalis) is a protozoan enteroparasite and a major cause for non-bacterial diarrhea worldwide. The disease, called Giardiasis, spreads worldwide from the Arctic [11] to the Tropics [12] and the WHO estimates that 270 million people are symptomatic, with more than 0.5 Mio new cases reported each year [10]. The simple life cycle of Giardia lamblia alternates between a flagellated trophozoite stage which proliferates in the small intestine of mammalian hosts and a cyst stage which survives in the environment and is responsible for transmission. Although giardiasis occurs most frequently in developing countries, the number of yearly endemic cases in Europe and the USA cause significant hospitalizations costs. Aside from its role as a human pathogen, Giardia has received growing attention in biology research. Phylogenetically, G. lamblia had been thought of as an ancient organism that could represent the missing evolutionary link between prokaryotes and eukaryotes. To date, the current hypothesis favors Giardia’s simplified cellular organization as the result of reductive evolution, a process that is thought to be driven by the eXtreme adaptation to a given ecological niche, as eXperienced by parasitic organisms. During host-parasite co-evolution G. lamblia has minimized cellular compleXity and adapted morphologically to the intestinal environment. In addition, Giardia is amenable to in vitro culture and has therefore become an attractive eukaryotic model for the study of complex cell biological and phylogenetic aspects in a “bare-bones” eukaryotic cellular system. One of the most striking morphological adaptations in G. lamblia to the host’s environment was the evolution of the ventral disc (VD). The VD mediates attachment of the trophozoite to the gut epithelium and prevents extrusion by peristalsis. Consequently, Giardia’s flat ventral domain is in close contact with the epithelium whilst the dome-shaped dorsal domain is eXposed to the intestinal lumen. This dorso-ventral polarization is reflected in the distribution of Giardia’s endocytic organelles, the peripheral vacuoles (PVs). PVs underly the entire dorsal plasma membrane (PM) but are excluded from the area occupied by the VD. Traces of reductive evolution are also seen in PVs, in that they present some similarities and many differences compared to canonical endocytic compartments. In line with classical endosomes PVs are the initial recipients of endocytosed material and associate with the conserved endocytic key factors clathrin (GlCHC), dynamin (GlDRP) and adaptor protein 2 (GlAP2). Although their molecular functions are not very well understood, it appears that they are not involved in the formation of highly dynamic clathrin coated vesicles (CCVs) as such PM derived transport intermediates have never been observed. In contrast to most eukaryotic endocytic systems, PVs have a fiXed position in close proximity to the PM and are of a non-fusogenic nature. Moreover they seem to undergo a regulated organelle cycle for indiscriminate 3 uptake of fluid phase material. Taken together, PV organelles maintain endocytic and lysosomal properties in parallel