As previously discussed, the single-molecule localisation microscopy has so far shown great promise in the study of DNA replication and repair processes. However, the majority of studies have been limited to prokaryotic model organisms. Extending these methodologies so they can be used in eukaryotic systems has the potential to greatly benefit the field. Application of such technologies will rely on demonstration of their capabilities and development of a robust methodological toolkit that warrants investment in either collaborations or the technology itself.
In this chapter, data demonstrating a methodological adaptation to PALM imaging which enables visualisation and quantification of chromatin-associated proteins in unfixed fission yeast have been presented. The development of this technique stemmed from limitations associated with chemical fixation and detergent extraction approaches, which made it difficult to identify single molecule localisations that arose from chromatin-associated molecules. This methodology utilises long camera exposure times and the subsequent effect of motion blurring to filter out fluorescent localisations from diffusing molecules while retaining those of static chromatin-associated proteins. The exact camera exposure time was optimised via computer simulations and the resulting procedure used to detect changes in DNA binding of replication proteins in distinct cell cycle stages and genetic backgrounds.
This is not the first time that long exposure times have been used to monitor DNA-association of proteins. Previous studies have used a similar approach to monitor transcription factor dynamics and the detection of DNA bound repair proteins in bacteria (Elf 2007, Uphoff 2013). Long exposure times have also been coupled with time-lapse imaging to track single molecules moving extremely slowly along actin filaments (Kim et al 2006). At the time of this study, a group used long exposure times to measure the DNA dissociation kinetics of transcription factors inside live human cells (Chen et al 2014). The data presented here demonstrate that this well documented phenomenon can be used slightly differently to study changes in the global association of proteins to the chromatin inside a eukaryotic model organism.
This development is of importance for various reasons. Firstly, this study demonstrates that SMLM approaches can be used to great effect in organisms that present significant challenges for such microscopy approaches. For example, bacterial models are favourable for studies like these due to their small size meaning that majority of the cell, and therefore the fluorophores, are in focus. In the yeast system, this is not the case as the larger cell size means a large proportion of the cell is out of focus. In SMLM techniques, widefield illumination and the stochastic nature of fluorophore activation prevents the user from collecting light from several Z-planes. However, as demonstrated here, although only a section of the cell can be visualised, this is still enough to collect meaningful data. The larger size of yeast cells also has potential effects on sptPALM-based experiments and this will be discussed in the next chapter.
A second benefit of this study is it provides a sensitive assay for measuring changes in chromatin association without disruption to the cell. Current biochemical techniques involve chemical crosslinking and breaking open cells to study chromatin association.
Although such techniques are informative, the methodology presented here can be seen as a complementary approach that is much less perturbative. Its ability to quantitatively measure global changes in chromatin association could be potentially crucial in revealing previously unknown protein behaviour.
As with all methodologies, there are limitations and this approach is no exception. The main drawback of this methodology is that it does not provide accurate spatial information on protein localisation. One advantage of SMLM techniques is the ability to create super-resolution images and is one reason that this project was initiated. However, because the cells are not chemically fixed during the procedure it allows for the diffusion of chromatin and potential rotation of the nucleus. Thus, coupled with the fact that the activation of single fluorophores is stochastic, any grouping/clustering of localisations may be an artefact of chromatin diffusion. This therefore limits the output of the approach to the relative measurement of DNA association. Efforts are still ongoing to develop an S.
pombe extraction technique that is compatible with PALM.
Despite the mobility of the chromatin during imaging, this does not have a significant effect on fitting the PSFs of chomatin-associated molecules. Diffusion of any one chromosomal locus has been shown to lie between 10-4 and 10-3 m2/s (Dion and Gasser
2013). This movement would equate to a 45nm displacement during a 350ms exposure which is well within the size equating to a single pixel (usually 100-110nm). Although in this study S. pombe was the model system chosen to establish this method, there is no reason why this cannot be extended to other eukaryotic systems. In situations where the chromatin is more mobile (such as DNA damage or certain cell types) or the molecule size is greater than 100 kDa, it is recommended that users explore altering exposure times or a localisation procedure that enables fitting slightly enlarged PSFs. In addition, there is no reason why other photoconvertible/photoactivatable fluorescent proteins that are compatible with PALM could be used.
An observation made during this imaging is the presence of a low number of localisations in situations where it would be predicted that there are no chromatin-bound molecules, e.g. Mcm4-mEos3.1 localisations in G2 cells. These localisations could be due to a multitude of reasons. Primarily, random collision with nuclear matrix proteins or indeed the chromatin itself inside the crowded nucleus could render molecules static for a short period of time. Alternatively, molecules that diffuse vertically within the focal plane within the 350ms exposure time could be detected. In the case of Mcm4, the possibility exists that random association with an ORC complex and origins of replication could result in transient association with the DNA. Recent in vitro data have shown that association of only one MCM hexamer with an origin can lead to ATP-dependant release of the helicase (Coster 2014). During sample preparation for the experiments in this chapter, sodium azide is added to prevent ATP-dependent DNA loading/unloading of molecules during imaging. Thus, the observed localisations in G2 cells could be MCMs that have transiently associated with ORC complexes and are unable to be released due to the lack of ATP. In the case of PCNA, the G2 localisations detected could well be due to post replicative repair which is known to require PCNA (Daigaku et al 2013)
In conclusion, the simple approach presented in this chapter provides a means to complement biochemical studies of protein chromatin association using PALM based localisation microscopy. In the next chapter, I will describe how this methodology has been used to complement such investigations further demonstrating the potential for its use in future discoveries.
Chapter 5
Investigating the role of ubiquitylation of the replication sliding clamp during DNA synthesis using photoactivated localisation microscopy
5.
5.1. Introduction
The replication sliding clamp, PCNA, is an essential scaffold protein that is required for many DNA metabolism processes. Most notable, is its role as a processivity factor and docking platform for the replicative polymerases during DNA synthesis. PCNA has also been well studied for its role in DNA damage bypass mechanisms that involve translesion synthesis and template switching. Central to its role in DNA damage tolerance is the post-translational modification of PCNA by addition of one or multiple ubiquitin moieties to lysine 164. This modification of PCNA and its subsequent roles in repair have been studied extensively, and its presence was considered to be solely as a result of the formation of DNA damage (discussed in Introduction).
This chapter focuses on work that was performed as part of a collaboration with Dr Yasukazu Daigaku in the Carr lab. A large amount of biochemical and genetic data had been collected by Dr Daigaku prior to the experiments described in this chapter, which allude to a damage independent role for PCNA ubiquitylation during unperturbed S-phase. These data provide evidence to suggest that ubiquitylation of PCNA promotes a stable association with the DNA. Preventing ubiquitylation by removing the E3 ubiquitin ligase, RAD18 (S. pombe Rhp18), had consequences for the progression of replication, predicted to be due to lagging strand synthesis defects. In order to support these observations PALM based imaging was utilised to investigate the role of PCNA ubiquitylation in the chromatin association of PCNA and the replicative polymerases in vivo.