In vivo high content analyses of protein-protein interactions at budding yeast kinetochores using a wide-field time-gated FLIM-based platform openFLIM-HCA
File(s)
Author(s)
Guo, Wenjun
Type
Thesis
Abstract
This thesis describes my work to develop and apply an automated fluorescence lifetime imaging (FLIM) microscopy-based method for high-content analysis (HCA) of protein- protein interactions (PPIs) at the budding yeast kinetochore in live cells via Förster resonance energy transfer (FRET). FLIM provides a robust readout of FRET, since the fluorescence lifetime is independent of the fluorophore concentration, excitation power and the spectral properties of the sample and instrument, but it requires relatively high signal to noise. Here I demonstrated, for the first time, that FLIM HCA could be applied in live cells to study endogenous proteins labelled with fluorescent proteins and developed new protocols and data acquisition and analysis tools to address the challenges associated with the low copy number of kinetochore proteins (KPs) and high (autofluorescence) background.
The openFLIM-HCA platform developed at Imperial College London implements automated multiwell plate FLIM using wide-field, optically-sectioned, time-gated imaging with open source software to analyse FLIM data. I adapted the instrumentation and software tools to map (sub-10 nm) co-localisation of KPs using FRET in live yeast cells and validated this methodology by showing that it can detect known KP interactions. I then explored the potential of this FLIM HCA methodology to query the kinetochore localisation of Rdh54p as a means to investigate how this DNA repair protein promotes the adaptation to the DNA damage-induced cell cycle arrest. I also forced the associations of Rdh54p (and mutants) with most of the proteins in the proteome using the high- throughput synthetic physical interaction (SPI) method to identify associations that affect cell growth. Both the FLIM and SPI data converged on the CTF19/COMA complex at the kinetochore, suggesting that Rdh54p constitutively localises adjacent to this complex, which may have functional significance. The SPI screens also identified hits related to the DNA damage checkpoint that are interesting for future investigations.
The openFLIM-HCA platform developed at Imperial College London implements automated multiwell plate FLIM using wide-field, optically-sectioned, time-gated imaging with open source software to analyse FLIM data. I adapted the instrumentation and software tools to map (sub-10 nm) co-localisation of KPs using FRET in live yeast cells and validated this methodology by showing that it can detect known KP interactions. I then explored the potential of this FLIM HCA methodology to query the kinetochore localisation of Rdh54p as a means to investigate how this DNA repair protein promotes the adaptation to the DNA damage-induced cell cycle arrest. I also forced the associations of Rdh54p (and mutants) with most of the proteins in the proteome using the high- throughput synthetic physical interaction (SPI) method to identify associations that affect cell growth. Both the FLIM and SPI data converged on the CTF19/COMA complex at the kinetochore, suggesting that Rdh54p constitutively localises adjacent to this complex, which may have functional significance. The SPI screens also identified hits related to the DNA damage checkpoint that are interesting for future investigations.
Version
Open Access
Date Issued
2019-06
Date Awarded
2019-09
Copyright Statement
Creative Commons Attribution NonCommercial Licence
License URL
Advisor
Thorpe, Peter
French, Paul
Dunsby, Christopher
Sponsor
Imperial College London
Publisher Department
Francis Crick Institute
Publisher Institution
Imperial College London
Qualification Level
Doctoral
Qualification Name
Doctor of Philosophy (PhD)