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Reconstitution of intracellular clathrin-coated vesicle formation
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Kuey, Cansu (2021) Reconstitution of intracellular clathrin-coated vesicle formation. PhD thesis, University of Warwick.
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Official URL: http://webcat.warwick.ac.uk/record=b3759487
Abstract
Clathrin-coated vesicle (CCV) formation is a major membrane remodelling process important for membrane traffic in eukaryotic cells. CCVs are formed at the plasma membrane during endocytosis, and at early/recycling endosomes and the trans-Golgi network during intracellular traffic. The plasma membrane is easier to access experi- mentally in vivo from the cell exterior, while intracellular compartments lack this ad- vantage. To overcome this barrier and to study intracellular CCV dynamics and func- tional requirements, we have reconstituted intracellular CCV formation on-demand, using minimal machinery. The clathrin-binding region of the b2 subunit of AP-2 was used as a `hook' which can be attached inducibly to an `anchor' protein on a membrane surface. Rerouting the hook to an anchor by chemical dimerisation was sufficient to form CCVs at mitochondria, ER, Golgi and lysosomes. As mitochondria are not part of canonical membrane trafficking, I investigated synthetic clathrin-coated pit forma- tion on mitochondria in detail. CCPs on the mitochondria (termed mitoPits), form within minutes after induction. Electron microscopy and live cell imaging revealed that initiation, maturation and scission steps of CCV formation were faithfully recon- stituted. MitoPits are double membraned invaginations that tend to form on surfaces with higher curvature. These observations suggests that enough force is generated by our synthetic system to deform both the inner and outer mitochondrial membranes and for budding of the mitoPits. Vesicle budding was shown not to depend on any scission molecule tested (dynamin, Drp1, Vps4a, actin), suggesting that intracellular CCVs do not need a scission factor. To conclude, unlike endocytosis, clathrin-coating may be sufficient for intracellular CCV budding. Given the differences in phospholipid profiles of mitochondrial membranes and plasma membrane, the phospholipid composition of the membrane may have a negligible role in CCV formation.
| Item Type: | Thesis (PhD) | ||||
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| Subjects: | Q Science > QH Natural history Q Science > QH Natural history > QH301 Biology Q Science > QP Physiology |
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| Library of Congress Subject Headings (LCSH): | Coated vesicles -- Research, Endocytosis -- Research, Biological transport, Eukaryotic cells, Cell membranes, Membrane proteins, Mitochondrial membranes | ||||
| Official Date: | September 2021 | ||||
| Dates: |
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| Institution: | University of Warwick | ||||
| Theses Department: | Warwick Medical School | ||||
| Thesis Type: | PhD | ||||
| Publication Status: | Unpublished | ||||
| Supervisor(s)/Advisor: | Royle, Stephen J. ; Smith, Corinne J. | ||||
| Sponsors: | Engineering and Physical Sciences Research Council ; Biotechnology and Biological Sciences Research Council (Great Britain) | ||||
| Format of File: | |||||
| Extent: | multiple pagings : illustrations | ||||
| Language: | eng |
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