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Using Affimers to dissect a multiprotein complex in living cells
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Shelford, James (2021) Using Affimers to dissect a multiprotein complex in living cells. PhD thesis, University of Warwick.
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Official URL: http://webcat.warwick.ac.uk/record=b3817715
Abstract
Accurate cell division depends on the precise segregation of chromosomes to generate two genetically identical daughter cells. High fidelity in this process is achieved by the formation of a bipolar mitotic spindle, an array of microtubules that are organised and regulated by the concerted action of motor and non-motor proteins. One known non-motor protein complex composed of TACC3, ch-TOG, clathrin and GTSE1, has been implicated in mitotic spindle stability. This complex is important for the organisation of microtubules that form the kinetochore fibres, which facilitate the movement of chromosomes during mitosis. Owing to the importance of this complex in ensuring accurate mitosis, it is an attractive target to disrupt in a cancer context.
Affimers are novel synthetic binding proteins based on the consensus sequence of a phytocystatin cysteine protease inhibitor found in plants. Here, we used Affimers as a research tool to dissect the TACC3{ch-TOG{clathrin{GTSE1 complex in human cells, with a goal of developing specific inhibitors to disrupt cell division in an anti-cancer context. Expression of Affimers in HeLa cells as mCherry-fusion proteins led to specific disruption of the TACC3{ch-TOG interaction at the mitotic spindle, displacing ch-TOG while leaving the localisation of the other complex components intact. Inhibition of TACC3{ch-TOG had no effect on a number of functions previously ascribed to these proteins, such as MT growth and plus-end tracking. Inhibition of this interaction led to unexpected fragmentation of the pericentriolar material (PCM) in metaphase cells following the formation of a bipolar spindle, coupled with a delayed transition to anaphase. This effect was not observed with a control Affimer. Thus, this work has uncovered a novel role of TACC3{ch-TOG in maintaining PCM integrity during mitosis to ensure timely cell division. More broadly, this thesis demonstrates that Affimers are useful tools for dissecting the functional properties of multiprotein complexes in living cells.
Item Type: | Thesis (PhD) | ||||
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Subjects: | Q Science > QH Natural history Q Science > QH Natural history > QH426 Genetics Q Science > QP Physiology |
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Library of Congress Subject Headings (LCSH): | Protein binding, Microtubules, Cell division, Spindle (Cell division), Mitosis | ||||
Official Date: | December 2021 | ||||
Dates: |
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Institution: | University of Warwick | ||||
Theses Department: | School of Life Sciences | ||||
Thesis Type: | PhD | ||||
Publication Status: | Unpublished | ||||
Supervisor(s)/Advisor: | Royle, Stephen J. ; Bayliss, Richard | ||||
Sponsors: | Midlands Integrative Biosciences Training Partnership ; Biotechnology and Biological Sciences Research Council (Great Britain) | ||||
Format of File: | |||||
Extent: | xi, 148, 10, leaves : illustrations | ||||
Language: | eng |
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