Exploring Single-Cell Phenotyping and In-Cell EPR to Investigate Stress Physiology in Saccharomyces cerevisiae

Abstract

This thesis investigates the role of the vacuolar mechanosensitive Ca²⁺ channel TRPY1(YVC1) in Saccharomyces cerevisiae on cellular survival and morphological adaptation under hyperosmotic and oxidative stress conditions. Flow cytometry was optimised to resolve stress phenotypes at a single-cell level in yeast to quantify heterogeneity in cell viability (via Propidium Iodide and Fluorescein Diacetate dual-staining), growth kinetics, and morphological size shifts (using forward / side scatter parameters).

Functional complementation of Wild-type (WT), TRPY1 knock-out (KO), and plasmid complemented strains suggested that absence of TRPY1 confers acute survival advantage under moderate hyperosmotic shock and oxidative stress conditions, likely via reduced Ca2+overload; WT showed increased long-term-survival advantage by comparison. A stress dependant bimodal distribution of cell size was observed, highlighting that acute osmoregulation (< 30 s) is dominated by mechanical cell wall changes and delayed structural collapse, rather than uniform population shrinkage; the contribution of TRPY1 to the phenotypic differences observed between WT and TRPY1 KO were evaluated.

To bridge the cellular phenotypes with molecular structural dynamics, the feasibility of utilising in-cell paramagnetic resonance (EPR) was explored, by utilising genetic code expansion and orthogonal translation systems (OTS) from methanogenic archaea (Methanomethylophilus alvus and Methanosarcina mazei) to incorporate biorthogonal non-canonical amino acids(ncAA) into a yeast surface display reporter. Efficiency and fidelity of ncAA incorporation(Boc-l-lysine) was quantified using Relative Read through Efficiency (RRE) and Maximum Misincorporation Frequency (MMF) metrics. RRE was shown to be ~ 10 % in both OTS, suggesting small levels of ncAA incorporation, but high corresponding levels of MMF indicate heterogeneous canonical and non-canonical amino acid incorporation. Evaluation determined the system is insufficient to achieve the spin concentrations typically required for in-cell EPR. Ultimately, this thesis establishes a single-cell framework to resolve yeast stress phenotypes and defines the thresholds and limitations to overcome to investigate mechanosensitive channel dynamics in vivo.

Date of Award	Sept 2025
Original language	English
Awarding Institution	Aston University
Supervisor	Alan Goddard (Supervisor) & Roslyn Bill (Supervisor)