Dissertation committee:
Γιώργος Διαλλινάς, Καθηγητής Πανεπιστημίου Αθηνών Τμήματος Βιολογίας
Σπυρίδων Ευθυμιόπουλος, Καθηγητής Πανεπιστημίου Αθηνών Τμήματος Βιολογίας
Ιωάννης Τρουγκάκος, Αναπληρωτής Καθηγητής Πανεπιστημίου Αθηνών Τμήματος Βιολογίας
Χρήστος Δελιδάκης, Καθηγητής Πανεπιστημίου Κρήτης Τμήματος Βιολογίας
Κοσμάς Χαραλαμπίδης Αναπληρωτής Καθηγητής Πανεπιστημίου Αθηνών Τμήματος Βιολογίας
Δημήτρης Στραβοπόδης, Επίκουρος Καθηγητής Πανεπιστημίου Αθηνών Τμήματος Βιολογίας
Norio Takeshita, Επίκουρος Καθηγητής University of Tsukuba, Faculty of Life and Environmental Sciences
Summary:
In eukaryotic cells, transmembrane proteins are co-translationally inserted in the lipid bilayer of the Endoplasmic Reticulum (ER). In order for these proteins to exit the ER packaged in COPII vesicles, both ER-exit motifs and interacting regulatory proteins that ensure proper cargo folding, are required. Following ER-exit,transmembrane proteins are trafficked through the Golgi network, packaged in vesicles and targeted to their final subcellular destination (plasma membrane, endosomal, mitochondrial, vacuolar etc) where they are integrated in the correct lipid bilayer. Additionally, polar cells such as neurons, muscle cells or filamentous fungi (e.g. Aspergillus) utilize cellular mechanisms that control the targeting and topology of each protein to distinct areas of the plasma membrane.
The homeostasis of cellular function depends, largely, on the continuous movement of molecules between the subcellular compartments. Several steps of this intracellular protein trafficking implicate specific coated-vesicle formation and transport on microtubules (MTs), which function as tracks in a process propelled by their associated motor proteins (kinesin, dynein). Adaptor protein complexes (APs) are important for the formation of clathrin-coated vesicles for most eukaryotes, whereas all three fungal AP complexes are universally conserved (AP-1, AP-2 and AP-3).They play key roles in the selection, traffic, endocytosis and recycling of membrane cargoes, whilst the presence of functional adaptors and clathrin is indispensable for the homeostasis and survival of eukaryotic cells.Interestingly however, in light of the discoveries of the past 10 years,the existence of specialized clathrin-independent trafficking pathwayshas also been proposed.
Transporters and apically localized cargoes, are two of the most important categories of transmembrane proteins, that participate in processes necessary for normal development. In this thesis, we study protein dimerization prior to ER-exit and investigate the role of AP complexes in transmembrane protein trafficking to the plasma membrane, but also in their rapid removal via endocytosis, as well as the role of clathrin in these processes, through the development of new genetic and biochemical tools. The filamentous fungus Aspergillus nidulans has emerged as unique, genetically tractable, system to study protein trafficking via its amenability to in vivo epifluorescence and multidimensional microscopy. The extensively studied uric acid/xanthine symporter, UapA, is used as a model cargo for studying the mechanisms of intracellular trafficking of fungal transporters, and for investigating the role of dimer formation in ER-exit. Additionally, selected apical markers are used to investigate the general trafficking pathways in which AP complexes are implicated.
More specifically,we elucidate the role of AP-1 in cargo trafficking and show that it is essential for growth due to its involvement in microtubule-based movement of vesicles towards the apex of growing hyphae. Furthermore, we identify clathrin-binding motifs in the C-terminal region of the β1 subunit, and we provide evidence that AP-1 is involved in both anterograde sorting of RabE-labeled secretory vesicles and RabA/B-dependent endosome recycling. Additionally, AP-1 is shown to be critical for microtubule and septin organization, further rationalizing its essentiality in cells that face the challenge of cytoskeleton-dependent polarized cargo traffic.
We alsoshow that AP-2 has a clathrin-independent essential role in polarity maintenance and growth of A. nidulans, which was in line with experiments showing that AP-2 does not co-localize with clathrin. We provide genetic and cellular evidence that AP-2 interacts with endocytic markers SlaB/End4 and SagA/End3 and, most importantly, the lipid flippases DnfA and DnfB, specifically in the sub-apical collar region of growing hyphae. The role of AP-2 in the maintenance of proper apical membrane lipid and cell wall composition is further supported by its functional interaction with proteins/enzymes necessary for sphingolipid biosynthesis, apical sterol-rich membrane domains formation and chitin deposition. Our findings sugggest, for the first time, that the AP-2 complex of higher fungal groups, including the most threatening fungal pathogens, has acquired, in the course of evolution, a specialized clathrin-independent function necessary for fungal polar growth.
Through this work, we gain insight on the mechanisms underlying the polarized mode of life of eukaryotic cells, with potential applications to mammalian polarized cells, such as neurons. Furthermore, given the utmost importance of membrane trafficking in cell survival, new pathways are emerging in finding targeted approaches to pathogenic fungi.
Keywords:
filamentous, fungi, traffic, exocytosis, endocytosis, AP-1, AP-2, clathrin, polar growth, oligomerization, transporters, cell biology, microtubules
