PSD-95 is an abundant scaffolding protein found in the postsynaptic densities (PSDs) of excitatory synapses throughout the mammalian brain, and plays a critical role in innate and learned behaviours. PSD-95 assembles with numerous other proteins, including glutamate receptors, adhesion molecules and signalling proteins, into postsynaptic supercomplexes that are then organised into nanoclusters that comprise the postsynaptic density of excitatory synapses. While the subcellular localisation of PSD-95 has been widely studied, much less is known about its turnover. In this thesis, I present novel insights into PSD-95 synthesis and degradation at synapses of cultured primary neurons gained using the HaloTag technology. The HaloTag consists of an engineered bacterial protein domain that covalently binds synthetic ligands labelled with fluorescent and affinity moieties. Hence, cells expressing proteins fused to the HaloTag can be used to study protein levels, complexes and turnover using these different ligands. This project was based upon a knock-in mouse line expressing the HaloTag fused to endogenous PSD-95 using gene targeting. After demonstrating that these mice were phenotypically normal and that PSD95-HaloTag fusion proteins normally assembled into supercomplexes in the PSD, hippocampal primary cultures were grown from this mouse line. Fluorescent HaloTag ligands were then used to label live neurons, allowing for the visualisation of PSD-95 at synapses by confocal microscopy. Next, I established a pulse-chase labelling method, where one ligand is used to label all existing PSD-95 first, before a second ligand can then be used to label any newly synthesised PSD-95. This allows for the identification and characterisation of subpopulations of PSD-95, which can be separately analysed. I find that PSD-95 has a half-life of 36 hours at synapses, consistent with previous literature. I was also able to observe synaptic heterogeneity in PSD-95 turnover, and classify synapses into types according to their PSD-95 expression profile. Finally, a range of chemical compounds known to modulate protein turnover and neuronal activity was applied over a 24-hour period, and their effects on PSD-95 turnover analysed. It was found that inhibiting either the proteasome or protein synthesis led to significant reductions in PSD-95 degradation as well as inhibiting PSD-95 synthesis. Thus, this project established a method offering a unique way of investigating the turnover of a specific, tagged protein, as well as gaining novel insights into the turnover of PSD-95 at individual synapses.

Stochastic fluctuations in mRNA and protein copy number between cells are inevitable during the process gene expression, even when cells carry identical chromosomes. Such fluctuations are able to impact the phenotypic fate of the cell, and are known to have greater impact when the copy number of the molecule involved is low. Additionally, up to 50% of proteins in Escherichia coli are present in the cell at a level of 10 molecules per cell or fewer (Taniguchi et al. 2010). As such, quantification of low copy number gene expression products and their distribution in cellular populations is key in understanding the process of gene expression. Currently, there are few techniques that allow investigation with the single cell and single molecule resolution required to study low copy number gene expression products. This work presents a novel method for protein quantification at the single molecule level, Quantitative HaloTag-TMR labelling, and uses the technique to quantify the absolute numbers of the low copy number RecB, RecC and RecD subunits of the bacterial DNA repair enzyme RecBCD, finding each subunit is present at between two and eight molecules per cell with mean numbers per cell of 4.9, 4.7 and 4.5 respectively. Additionally single molecule mRNA FISH was used to quantify the mRNA levels of recB and recD within cells, with means of 0.21 and 0.31 mRNA per cell being observed respectively. Finally this work presents a new method for use detecting both mRNA and protein simultaneously in individual cells by combining the HaloTag and FISH protocols to give HaloFISH. This work introduces two novel techniques that allow for single cell examination of gene expression, and investigates RecBCD expression at the single molecule level.

In recent years, semiconductor quantum dots (QDs) have arisen as a new class of fluorescent probes that possess unique optical and electronic properties well-suited for single-molecule imaging of dynamic live cell processes. Nonetheless, the large size of conventional QD-ligand constructs has precluded their widespread use in single-molecule studies, especially on cell interiors. A typical QD-ligand construct can range upwards of 35 nm in diameter, well exceeding the size threshold for cytosolic diffusion and posing steric hindrance to binding cell receptors. The objective of this research is to develop tagging strategies that allow QD-ligand conjugates to specifically bind their target proteins while maintaining a small overall construct size. To achieve this objective, we utilize the HaloTag protein (HTP) available from Promega Corporation, which reacts readily with a HaloTag ligand (HTL) to form a covalent bond. When HaloTag ligands are conjugated to size-minimized multidentate polymer coated QDs, compact QD-ligand constructs less than 15 nm in diameter can be produced. These quantum dot-HaloTag ligand (QD-HTL) conjugates can then be used to covalently bind and track cellular receptors genetically fused to the HaloTag protein. In this study, size-minimized quantum dot-HaloTag ligand conjugates are synthesized and evaluated for their ability to bind specifically to purified and cellular HTP. The effect of QD-HTL surface modifications on different types of specific and nonspecific cellular binding are systematically investigated. Finally, these QD-HTL conjugates are utilized for single-molecule imaging of dynamic live cell processes. Our results show that size-minimized QD-HTLs exhibit great promise as novel imaging probes for live cell imaging, allowing researchers to visualize cellular protein dynamics in remarkable detail.

The cilium/flagellum is a complex organelle protruding from the cell body and functioning in motility, sensing, and signalling. It is composed of hundreds of protein constituents, the majority of which comprise the flagellar cytoskeleton - the microtubule-based axoneme. Because the flagellum lacks ribosomes, its protein constituents have to be imported from the cell body and delivered to proper locations. Moreover, these proteins have to retain their function over a considerable length of time, despite the mechanical stress caused by flagellar beating and due to environmental exposure. This raises the question whether and where protein turnover occurs. Previously, it was established that Chlamydomonas reinhardtii flagella are dynamic structures (Marshall & Rosenbaum, 2001). In contrast, in the Trypanosoma brucei flagellum axonemal proteins are remarkably stable (Vincensini et al., 2018). However, the questions of axonemal assembly and stability were so far investigated only for a small number of proteins and during relatively short periods. Moreover, in these experiments expression of studied proteins was controlled by non-native regulatory elements. To elucidate the site of incorporation of proteins from all major axonemal complexes and to find out if and where the protein turnover occurs, T....