Dokument: Cryogenic electron tomography studies on cellular autophagy membranes
| Titel: | Cryogenic electron tomography studies on cellular autophagy membranes | |||||||
| URL für Lesezeichen: | https://docserv.uni-duesseldorf.de/servlets/DocumentServlet?id=66396 | |||||||
| URN (NBN): | urn:nbn:de:hbz:061-20250804-084525-9 | |||||||
| Kollektion: | Dissertationen | |||||||
| Sprache: | Englisch | |||||||
| Dokumententyp: | Wissenschaftliche Abschlussarbeiten » Dissertation | |||||||
| Medientyp: | Text | |||||||
| Autor: | Ortmann de Percin Northumberland, Claire [Autor] | |||||||
| Dateien: |
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| Beitragende: | Prof. Dr. Sachse, Carsten [Gutachter] Prof. Dr. Schröder, Gunnar [Gutachter] | |||||||
| Dewey Dezimal-Klassifikation: | 500 Naturwissenschaften und Mathematik » 570 Biowissenschaften; Biologie | |||||||
| Beschreibung: | During autophagy, a double membrane enwraps cellular cargo to deliver it to the lysosome. The aim of this PhD work was to characterise autophagic membranes, also called phagophores, during their elongation and to investigate how two autophagy proteins, the scramblase Atg9 and the lipid transported Atg2, contribute to phagophore biogenesis. To address this research question, I used cryogenic correlative light and electron microscopy (cryoCLEM). In this cryoCLEM workflow, I vitrified Saccharomyces cerevisiae cells and generated ultra-thin lamellae by FIB-milling. Regions of interest on these cellular lamellae were identified using cryogenic fluorescence microscopy prior to acquiring tomographic data on these sites of interest. For this approach, I identified a suitable fluorescent label for Atg9 and chose a deletion mutation, Δsnf7, to stall the progression of phagophores to fully formed autophagosomes in order to accumulate the structures of interest. In order to understand how the function of Atg9 and the interaction of Atg9 and Atg2 affect phagophore morphology, two known mutants, Atg9-basic and Atg2-PM4 were chosen to be studied with cryoCLEM. Atg9-basic is a triple mutant with reduced phosphatidylinositol-3-phosphate translocation activity while Atg2-PM4 is a variant with a mutation disrupting the interaction between Atg2 and Atg9. I segmented the phagophoric membranes to quantify morphological differences across the different cell types. Tomographic data from the Atg9-basic mutant show no detectable differences in phagophore morphology compared to wild-type phagophores. The tomographic data from the Atg2-PM4 mutant show that more than half of the phagophores imaged, had significantly enlarged rims. The in situ tomograms revealed potential modes of phagophore membrane extensions such as vesicle fusion and lipid transfer. The insights gained in this thesis work, contextualise previously reported interaction studies of Atg9 with Atg2 and contribute to our overall understanding of autophagosome biogenesis in Saccharomyces cerevisiae. | |||||||
| Lizenz: |  Dieses Werk ist lizenziert unter einer Creative Commons Namensnennung 4.0 International Lizenz | |||||||
| Fachbereich / Einrichtung: | Mathematisch- Naturwissenschaftliche Fakultät | |||||||
| Dokument erstellt am: | 04.08.2025 | |||||||
| Dateien geändert am: | 04.08.2025 | |||||||
| Promotionsantrag am: | 22.05.2024 | |||||||
| Datum der Promotion: | 02.07.2024 |
