Development of a single-cell fluorescence-based sensor to investigate Myc protein stability
type: CreativeWork
description: The transcription factor Myc is expressed in cells in response to pro-growth signals and drives expression of a plethora of genes that promote proliferation. This pleiotropic activity renders loss of Myc regulation highly beneficial to tumour cells and, in fact, Myc is deregulated in most human cancers. While its contributions to cancer have made Myc a thoroughly studied protein and an extremely desirable drug target, over fifty years of research have failed to produce an inhibitor that has successfully reached the clinic. Drug design against Myc has been limited by its lack of a defined structure with exploitable druggable sites. Recent strategies have been aimed at re-establishing post-translational regulation of Myc protein levels through inhibition of interacting partners that promote its accumulation in the cell. This exciting new therapeutic avenue is hindered by limited availability of high-throughput in vitro assays to measure changes in Myc stability in response to drug treatment. While many studies have been conducted to investigate regulation of Myc protein stability at the population level (for example, by immunoblotting for Myc), none have employed live imaging assays that are able to report on protein stability at the single cell level. The work presented in this study has aimed to fill this gap in the molecular toolbox with the use of a tandem Fluorescent Protein Timer (tFT). tFTs are novel microscopy-based tools that act as sensors of protein age, and therefore stability, at a single-celllevel. They make use of differing maturation kinetics of fluorophores tagged in tandem to the protein of interest, where the ratio of the fluorescence signals of the fluorophores functions as a read-out for protein stability. This offers a high through-put assay employable in drug screening for compounds that affect Myc stability, but also allows for the investigation of single-cell regulation of Myc. In this thesis, the generation and validation of Myc-tFT is descri…
abstract: The transcription factor Myc is expressed in cells in response to pro-growth signals and drives expression of a plethora of genes that promote proliferation. This pleiotropic activity renders loss of Myc regulation highly beneficial to tumour cells and, in fact, Myc is deregulated in most human cancers. While its contributions to cancer have made Myc a thoroughly studied protein and an extremely desirable drug target, over fifty years of research have failed to produce an inhibitor that has successfully reached the clinic. Drug design against Myc has been limited by its lack of a defined structure with exploitable druggable sites. Recent strategies have been aimed at re-establishing post-translational regulation of Myc protein levels through inhibition of interacting partners that promote its accumulation in the cell. This exciting new therapeutic avenue is hindered by limited availability of high-throughput in vitro assays to measure changes in Myc stability in response to drug treatment. While many studies have been conducted to investigate regulation of Myc protein stability at the population level (for example, by immunoblotting for Myc), none have employed live imaging assays that are able to report on protein stability at the single cell level. The work presented in this study has aimed to fill this gap in the molecular toolbox with the use of a tandem Fluorescent Protein Timer (tFT). tFTs are novel microscopy-based tools that act as sensors of protein age, and therefore stability, at a single-celllevel. They make use of differing maturation kinetics of fluorophores tagged in tandem to the protein of interest, where the ratio of the fluorescence signals of the fluorophores functions as a read-out for protein stability. This offers a high through-put assay employable in drug screening for compounds that affect Myc stability, but also allows for the investigation of single-cell regulation of Myc. In this thesis, the generation and validation of Myc-tFT is descri…
author: Ascanelli, Camilla
language: eng
keywords: Myc, Cancer, Tandem Fluorescent Timers, Live cell imaging
license: https://creativecommons.org/licenses/by/4.0/
date published: 2022-03-28
date modified: 2026-07-22T22:24:03Z
provider: Cambridge University
publisher: Networked Digital Library of Theses and Dissertations
type: WebPage
source organization: University of Cambridge

PropertyValue
property ID: DOI
value: 10.17863/CAM.87163