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Characterisation and functional analysis of fission yeast tropomyosin mutants and development of quantum dot-antibody conjugates for cellular imaging

East, Daniel (2010) Characterisation and functional analysis of fission yeast tropomyosin mutants and development of quantum dot-antibody conjugates for cellular imaging. Doctor of Philosophy (PhD) thesis, University of Kent. (doi:10.22024/UniKent/01.02.94322) (KAR id:94322)


Tropomyosin (Tm) is an evolutionarily conserved dimeric a-helical coiled coil protein, which interacts end-to-end to form polymers capable of associating with and stabilising actin-filaments and thereby regulate myosin function. The fission yeast, Schizosaccharomyces pombe, possesses a single Tm, Cdc8, an essential protein which can be acetylated on its amino terminus to increase its affinity for actin and enhance its ability to regulate myosin function. During this study extensive analyses on the physical properties of acetylated and unacetylated Cdc8 protein, together with a series of novel amino terminal Cdc8 mutants were undertaken in an attempt to explore the effects of amino terminal modification on the Cdc8 protein. In addition, a series of experiments were undertaken to develop fluorescent quantum dot (QD)-antibody (IgG) conjugates for visualising Cdc8 localisation in S. pombe cells.

Modifications to the amino terminus altered the stability of the Cdc8 protein, its ability to form end-to-end interactions and its affinity for actin. Changes in actin affinity were reflected in the ability of the Cdc8 proteins to regulate myosin S1 ATPase activity. Despite changes to their biochemical properties, Cdc8 proteins expressed in a temperature sensitive S. pombe strain were capable of complementing function at the restrictive temperature. However, when expressed in a Naacetyltransferase deficient S. pombe strain, the mutant Cdc8 proteins were unable to rescue the growth defects associated with this strain. The QD-IgG conjugates produced during this study produced superior images when compared to organic fluorophores and were significantly more resistant to photobleaching.

This work has provided insights into the importance of acetylation and the structure of the amino terminus for the function of fission yeast Tm and has highlighted the importance of reaction stoichiometry, the difficulties arising from non-specific binding and quenching of fluorescence intensity when coupling QDs to IgG complexes.

Item Type: Thesis (Doctor of Philosophy (PhD))
DOI/Identification number: 10.22024/UniKent/01.02.94322
Additional information: This thesis has been digitised by EThOS, the British Library digitisation service, for purposes of preservation and dissemination. It was uploaded to KAR on 25 April 2022 in order to hold its content and record within University of Kent systems. It is available Open Access using a Creative Commons Attribution, Non-commercial, No Derivatives ( licence so that the thesis and its author, can benefit from opportunities for increased readership and citation. This was done in line with University of Kent policies ( If you feel that your rights are compromised by open access to this thesis, or if you would like more information about its availability, please contact us at and we will seriously consider your claim under the terms of our Take-Down Policy (
Subjects: Q Science > QH Natural history > QH301 Biology
Divisions: Divisions > Division of Natural Sciences > Biosciences
SWORD Depositor: SWORD Copy
Depositing User: SWORD Copy
Date Deposited: 12 Jul 2023 11:23 UTC
Last Modified: 12 Jul 2023 11:23 UTC
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