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Title: Reorganization of mitochondrial architecture during apoptosis and mitophagy as investigated by correlative microscopy
Author: Ader, Nicholas
ISNI:       0000 0004 8497 5090
Awarding Body: University of Cambridge
Current Institution: University of Cambridge
Date of Award: 2020
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In this thesis, I detail the use of various forms of correlative microscopy to investigate reorganization of mitochondrial architecture during two processes, apoptosis and mitophagy, in an effort to better understand the contribution of membrane remodeling to these biological processes. During apoptosis, Bax/Bak oligomerize and accumulate in and adjacent to the outer mitochondrial membrane, resulting in outer membrane ruptures several hundred nanometers wide. Using correlative microscopy, it was found that Bax clusters accumulated next to outer membrane ruptures and appeared to be amorphous. Electron cryo-tomography of vitreous cells was used to discern a higher-order organization to cluster components. In these near- native conditions, Bax clusters appeared to contain a sponge-like meshwork, suggesting that components of the outer membrane may be bound by Bax and then sequestered in the cytosol. Mitochondria with outer membrane ruptures presented a diverse array of inner membrane morphologies, such as dilution of the matrix and cristae unfolding. This unfolding was found to be linked to a loss of typical ATP synthase dimer organization. A new, comprehensive model of Bax-mediated apoptosis is presented, linking molecular reorganizations of the inner membrane with sequestration of outer membrane components into Bax clusters in an interplay to drive rupture formation. In mitophagy, the PINK1 kinase is stabilized on the outer mitochondrial membrane, where it recruits Parkin. Parkin then ubiquitinates a host of outer membrane proteins, targeting a mitochondrion for autophagy. While most literature details a wholesale removal of all mitochondria from a cell, recent work describes a localized form of mitophagy that removes selective parts of the network. Mitochondria targeted by Parkin during localized mitophagy were observed, and this process was compared between cells intact for Drp1, the mitochondrial fission protein, and those with Drp1 knocked out. Fluorescently-tagged Parkin was found on mitochondrial fragments, mitochondria-containing autophagosomes, and mitochondria-containing lysosomes. In both lines, the fragmentation that led to isolation of a mitochondrion targeted for degradation occurred prior to the recruitment of autophagic machinery. In some cases, mitochondria surrounded by multiple autophagosomes were observed. These results reveal new details on the tight link between mitochondrial membrane dynamics and the functional state of the mitochondrion. Overall, I present a thesis that describes the insights into mitochondrial biology gained from various correlative microscopy approaches.
Supervisor: Kukulski, Wanda Sponsor: Marshall Aid Commemoration Commission ; International Biomedical Research Alliance ; NIH OxCam Program ; MRC LMB ; Hughes Hall
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
Keywords: cell biology ; structural biology ; apoptosis ; electron cryo-tomography ; CLEM ; Bax ; cryo focused ion beam ; mitochondria ; parkin ; mitophagy ; autophagy