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Structural and Spatiotemporal Organisation of Bax and Apaf1 during Apoptosis

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cam.restrictionthesis_access_embargoed
cam.supervisorKukulski, Wanda
dc.contributor.authorBorgeaud, Alicia
dc.contributor.orcidBorgeaud, Alicia [0000-0002-7231-7424]
dc.date.accessioned2021-10-26T01:03:37Z
dc.date.available2021-10-26T01:03:37Z
dc.date.submitted2021-03-31
dc.description.abstractIn this thesis, I present insights into the structural, spatiotemporal and functional characteristics of two essential steps of apoptosis, Bax cluster formation and Apaf1 activation, using a combination of biochemical and microscopy approaches. I sought to better understand the contribution of spatial and structural protein organisation within cells towards the efficient promotion of apoptosis, an essential cellular pathway. During apoptosis, Bax oligomerises on the outer mitochondrial membrane promoting mitochondrial ruptures of several hundred nanometres wide and a complete remodelling of the mitochondrion architecture. Using cryo-electron tomography, Bax clusters were shown to form distinct cellular environments organised in a meshwork similar to a sponge-like structure that might be bound to the ruptured outer mitochondrial membrane. This suggests that Bax may sequester components of the membrane into the cytosol. In this thesis, a protein-proximity biotinylation assay in living cells (BioID/TurboID) is presented, which allowed the identification of Bax cluster interactor proteins such as the fission proteins DRP1 and MFF and the mitochondrial matrix chaperone TRAP1. This demonstrates that Bax clusters might sequester various proteins in addition to membrane pieces, possibly favouring the course of apoptosis. Furthermore, correlative light and electron microscopy of resin embedded DRP1KO cells suggest a crucial role for DRP1 in forming Bax clusters and mitochondrial ruptures during apoptosis. Bax activity leads to the release of Cytochrome c from mitochondrial cristae, which interacts with the cytoplasmic protein Apaf1, leading to the formation of a heptameric caspase activation platform called the apoptosome. Here, using fluorescence live-microscopy, it is discovered that Apaf1 forms multiple cytosolic foci during apoptosis which do not co-localise with Bax clusters. Apaf1 foci formation is triggered by the availability of Cytochrome c in the cytosol. Moreover, an Apaf1 mutant lacking the ability to bind Cytochrome c reveals that the interaction between Apaf1 and Cytochrome c is necessary for foci formation, suggesting that Apaf1 foci formation is dependent on the same process as apoptosome formation. Nonetheless, foci formation can be triggered by a constitutively active version of Apaf1 lacking the Cytochrome c binding domain. However, foci formed by this mutant are not sufficient to trigger apoptosis. Moreover, it is found that Apaf1 foci are transient when the final stages of apoptosis are inhibited, suggesting that the foci dynamics regulate cell fate. Caspase-9 is shown to be critical for Apaf1 foci formation during apoptosis. Finally, correlative light and electron microscopy techniques reveal that Apaf1 forms a high-order structure during apoptosis, rather than dispersed complexes of the structure known as the apoptosome in vitro. These results suggest that the spatial and structural organisation of the apoptotic sequence of events is essential for an efficient course towards cell death.
dc.description.sponsorshipThe MRC LMB, Cambridge Trust and Churchill Professor Sir Robert Edwards Scholarship
dc.identifier.doi10.17863/CAM.77340
dc.identifier.urihttps://www.repository.cam.ac.uk/handle/1810/329895
dc.language.isoeng
dc.publisher.collegeChurchill
dc.publisher.institutionUniversity of Cambridge
dc.rightsAll Rights Reserved
dc.rights.urihttps://www.rioxx.net/licenses/all-rights-reserved/
dc.subjectApoptosis
dc.subjectApaf1
dc.subjectBax
dc.subjectElectron microscopy
dc.subjectBioID
dc.titleStructural and Spatiotemporal Organisation of Bax and Apaf1 during Apoptosis
dc.typeThesis
dc.type.qualificationlevelDoctoral
dc.type.qualificationnameDoctor of Philosophy (PhD)
dc.type.qualificationtitlePhD in Biological life sciences
rioxxterms.licenseref.urihttps://www.rioxx.net/licenses/all-rights-reserved/
rioxxterms.typeThesis

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