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Development of a new genetically-encoded tag for correlative light electron microscopy
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Clarke, Nicholas I. (2018) Development of a new genetically-encoded tag for correlative light electron microscopy. PhD thesis, University of Warwick.
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Official URL: http://webcat.warwick.ac.uk/record=b3169095~S15
Abstract
To understand cell biology in detail, we must explore subcellular organization in 3D and locate proteins at high resolution. To achieve this, the most popular approach is to use two complimentary imaging techniques; light and electron microscopy. Combining these techniques by correlative or correlated light and electron microscopy (CLEM) allows cellular events to be observed first by fluorescence microscopy and then the same event can be tracked and visualised at high-resolution using electron microscopy. However, a current challenge is to develop probes to precisely visualize proteins in cells using this method.
This thesis introduces FerriTag, a new genetically-encoded chemically-inducible tag for correlative light-electron microscopy. FerriTag is a fluorescent recombinant electron- dense ferritin particle that can specifically label target proteins rapidly and efficiently using rapamycin-induced heterodimerization. The processing protocol described for CLEM is simple yet robust and can potentially be used for tagging any protein-of- interest. FerriTag is easily distinguished from background in electron micrographs due to its high signal to noise ratio and also provides a labelling resolution of 10 ± 5 nm. These qualities make FerriTag an ideal probe for CLEM.
FerriTag can be used to perform contextual nanoscale mapping of protein location relative to a subcellular structure. This was utilised to study the distribution and conformation of huntingtin-interacting protein 1 related (HIP1R) in and around clathrin-coated pits, providing insight to the roles played by HIP1R, clathrin and actin during clathrin-mediated endocytosis. FerriTag offers great potential for future CLEM applications and will be a useful discovery tool for cell biology.
| Item Type: | Thesis or Dissertation (PhD) | ||||
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| Subjects: | Q Science > QH Natural history > QH301 Biology | ||||
| Library of Congress Subject Headings (LCSH): | Cytology, Proteins, Electron microscopy, Microscopy | ||||
| Official Date: | March 2018 | ||||
| Dates: |
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| Institution: | University of Warwick | ||||
| Theses Department: | Centre for Mechanochemical Cell Biology | ||||
| Thesis Type: | PhD | ||||
| Publication Status: | Unpublished | ||||
| Supervisor(s)/Advisor: | Royle, Stephen J. ; Cross, Robert, A. | ||||
| Sponsors: | Cancer Research UK | ||||
| Extent: | 139 leaves : illustrations, charts. | ||||
| Language: | eng |
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