The contractile vacuole (CV) is an osmoregulatory organelle found exclusively in algae and protists. In addition to expelling excessive water out of the cell, it also expels ions and other metabolites and thereby contributes to the cell's metabolic homeostasis. The interest in the CV reaches beyond its immediate cellular roles. The CV's function is tightly related to basic cellular processes such as membrane dynamics and vesicle budding and fusion; several physiological processes in animals, such as synaptic neurotransmission and blood filtration in the kidney, are related to the CV's function; and several pathogens, such as the causative agents of sleeping sickness, possess CVs, which may serve as pharmacological targets. The green alga Chlamydomonas reinhardtii has two CVs. They are the smallest known CVs in nature, and they remain relatively untouched in the CV-related literature. Many genes that have been shown to be related to the CV in other organisms have close homologues in C. reinhardtii. We attempted to silence some of these genes and observe the effect on the CV. One of our genes, VMP1, caused striking, severe phenotypes when silenced. Cells exhibited defective cytokinesis and aberrant morphologies. The CV, incidentally, remained unscathed. In addition, mutant cells showed some evidence of disrupted autophagy. Several important regulators of the cell cycle as well as autophagy were found to be underexpressed in the mutant. Lipidomic analysis revealed many meaningful changes between wild-type and mutant cells, reinforcing the compromised-autophagy observation. VMP1 is a singular protein, with homologues in numerous eukaryotic organisms (aside from fungi), but usually with no relatives in each particular genome. Since its first characterization in 2002 it has been associated with several cellular processes and functions, namely autophagy, programmed cell-death, secretion, cell adhesion, and organelle biogenesis. It has been implicated in several human diseases: pancreatitis, diabetes, and several types of cancer. Our results reiterate some of the observations in VMP1's six reported homologues, but, importantly, show for the first time an involvement of this protein in cell division. The mechanisms underlying this involvement in Chlamydomonas, as well as other key aspects, such as VMP1's subcellular localization and interaction partners, still await elucidation. / Die kontraktile Vakuole ist ein osmoregulatorisches Organell, das ausschließlich in Algen und Protisten vorkommt. Zusätzlich zu ihrer Rolle als Ausstoßer überflüßigen Wassers aus der Zelle heraus, stößt sie auch Ionen und andere Metaboliten aus, und trägt dabei zur metabolischen Homöostase der Zelle bei. Das Interesse an der kontraktilen Vakuole erstreckt sich über seine unmittelbare zelluläre Rolle hinaus. Die Funktion der kontraktilen Vakuole ist mit einigen grundsätzlichen zellulären Verfahren, wie Membrandynamik und Vesikelknospung und -fusion, verwandt; einige physiologische Verfahren in Tieren, zum Beispiel synaptische Neurotransmission und das Filtrieren des Blutes in den Nieren, sind mit der Funktion der Vakuole eng verwandt; und einige Pathogene—der Ursacher der Schlafkrankheit als Beispiel—besitzen kontraktile Vakuolen, die als Ziele von Medikamenten dienen könnten. Die grüne Alge Chlamydomonas reinhardtii verfügt über zwei Vakuolen. Sie sind die kleinsten bekannten in der Natur, und bleiben bisher verhältnismäßig unerforscht. Viele Gene, die in anderen Organismen als kontraktile-Vakuole-bezogen erwiesen wurden, haben Homologe in C. reinhardtii. Wir versuchten, diese Gene auszuschalten und den Einfluss auf die Vakuole zu beobachten. Die Ausschaltung eines unserer Gene, VMP1, verursachte starke, beachtliche Phänotype. Die Zellen zeigten gestörte Zytokinese und aberrante Zellformen. Die kontraktile Vakuole blieb jedoch verschont. Des Weiteren zeigten Mutantzellen einige Hinweise auf gestörte Autophagie. Einige wichtige Gene des Zellzyklus und der Autophagie waren unterexprimiert in Mutantzellen. Lipidomische Analyse zeigte mehrere bedeutsame Unterschiede zwischen Wildtyp und Mutant, die die Beobachtungen der gestörten Autophagie verstärkten. VMP1 ist ein singularisches Protein, mit Homologen in zähligen eukaryotischen Organismen (jedoch nicht in Pilzen), aber üblicherweise ohne Verwandte in den jeweiligen Genomen. Seit seiner Erstcharakterisierung 2002 wurde es mit etlichen zellulären Verfahren, wie Autophagie, programmiertem Zelltod, Sekretion, Zelladhäsion, und Biogenese der Organellen, assoziiert. Es wurde auch mit einigen menschlichen Krankheiten wie Diabetes, Pankreatitis, und einigen Arten von Krebs in Verbindung gebracht. Unsere Ergebnisse wiederholen einige Beobachtungen in anderen Organismen, zeigen dennoch zum ersten Mal eine Beteiligung von VMP1 an der Zellteilung. Die unterliegenden Mechanismen dieser Beteiligung in Chlamydomonas, sowie andere wichtige Aspekte, etwa die subzelluläre Lokalisierung von VMP1 und dessen Interaktionspartner, warten noch auf Aufklärung.

Autophagy is an evolutionarily conserved process across eukaryotes that is responsible for degradation of cargo such as aggregate-prone proteins, pathogens, damaged organelles, macromolecules etc. via its delivery to lysosomes. The process is known to involve the formation of a double-membraned structure, called autophagosome, that engulfs the cargo destined for degradation and delivers its contents by fusing with lysosomes. This process involves several proteins at its core which include two transmembrane proteins, ATG9 and VMP1. While ATG9 and VMP1 has been discovered for about a decade and half, the trafficking and function of these proteins remain relatively unclear. My work in this thesis identifies and characterises a novel trafficking route for ATG9 and VMP1 and shows that both these proteins traffic via the dynamin-independent ARF6-associated pathway. Moreover, I also show that these proteins physically interact with each other. In addition, the tools developed during these studies helped me identify a new role for the most common autophagy receptor protein, p62. I show that p62 can specifically associate with and sequester LC3-I in autophagy-impaired cells (ATG9 and ATG16 null cells) leading to formation of LC3-positive structures that can be misinterpreted as mature autophagosomes. Perturbations in the levels of p62 were seen to affect the formation of these LC3-positive structures in cells. This observation, therefore, questions the reliability of LC3-immunofluorescence assays in autophagy-impaired cells as method of assessing autophagy and points towards the homeostatic function played by p62 in autophagy-impaired cells.

Autophagy is the delivery of cytoplasmic cargo to lysosomes for degradation. Defects in autophagy are responsible for various diseases, including neurodegenerative diseases and cancer. While studies in yeast have largely characterized autophagy in response to nutrient starvation, these elegant studies do not account for autophagy in other contexts, including selective autophagy of organelles. A previous screen identified Vps13D as a gene required for the autophagic removal of mitochondria, mitophagy. Vps13D is highly conserved and essential in animals, and Vps13d loss-of-function mutants have enlarged mitochondria and mitophagy deficiencies in both cell and animal models. However, the mechanism by which Vps13D regulates these processes has not been defined. Here, I use mitochondrial clearance in the developing Drosophila intestine and fibroblasts from VPS13D mutant patients as experimental models to investigate the function of Vps13D. I discover that Vps13D is a regulator of ubiquitin and Atg8a/LC3/GABARAP localization around mitochondria. These functions are dependent on Pink1, a ubiquitin kinase, and the core autophagy machinery, respectively. Furthermore, Vps13D regulates mitochondria and endoplasmic reticulum (ER) contact sites downstream of Vmp1, a repressor of mitochondria and ER contact sites. I find that Marf, a mitochondria and ER tether and regulator of mitochondrial fusion, acts downstream of both Vmp1 and Vps13D. These findings explain the phenotypes in Vps13d mutants, as dysregulation of ubiquitin, Atg8a, and mitochondria and ER membrane contact sites impair regulation of both autophagy and mitochondria morphology.