Probing DNA interactions with proteins using a single-molecule toolbox: inside the cell, in a test tube and in a computer

Abstract

DNA-interacting proteins have roles in multiple processes, many operating as molecular machines which undergo dynamic meta-stable transitions to bring about their biological function. To fully understand this molecular heterogeneity, DNA and the proteins that bind to it must ideally be interrogated at a single molecule level in their native in vivo environments, in a time-resolved manner, fast enough to sample the molecular transitions across the free-energy landscape. Progress has been made over the past decade in utilizing cutting-edge tools of the physical sciences to address challenging biological questions concerning the function and modes of action of several different proteins which bind to DNA. These physiologically relevant assays are technically challenging but can be complemented by powerful and often more tractable in vitro experiments which confer advantages of the chemical environment with enhanced detection signal-to-noise of molecular signatures and transition events. In the present paper, we discuss a range of techniques we have developed to monitor DNA-protein interactions in vivo, in vitro and in silico. These include bespoke single-molecule fluorescence microscopy techniques to elucidate the architecture and dynamics of the bacterial replisome and the structural maintenance of bacterial chromosomes, as well as new computational tools to extract single-molecule molecular signatures from live cells to monitor stoichiometry, spatial localization and mobility in living cells. We also discuss recent developments from our laboratory made in vitro, complementing these in vivo studies, which combine optical and magnetic tweezers to manipulate and image single molecules of DNA, with and without bound protein, in a new super-resolution fluorescence microscope.

Millisecond single-molecule localization microscopy combined with convolution analysis and automated image segmentation to determine protein concentrations in complexly structured, functional cells, one cell at a time

“…3 The measured, experimental PSF is shown (upper panel) over a 2 mm range in z centred on the focal plane (z ¼ 0), alongside the analytical PSF (middle panel) and the analytical PSF convolved with localisation error and noise for qualitative comparison (lower panel). 25,26 The soware objectively identies candidate bright spots by a combination of pixel intensity thresholding and image transformation. Representative images of GFP and mCherry are shown in the le-hand panels of Fig.…”

“…The number of molecules in distinct 'spots' or 'foci' of uorescence, which have a mean effective diameter of a few hundred nm consistent with the measured point spread function (PSF) width of our microscope, was measured using our bespoke super-resolution localization microscope 25,26 which could objectively track automatically detected candidate uorescent spots over time using robust probabilistic criteria. The autouorescence background and camera noise were characterized by narroweld microscopy images of wild-type parental cells (i.e.…”

An automated image analysis framework for segmentation and division plane detection of single liveStaphylococcus aureuscells which can operate at millisecond sampling time scales using bespoke Slimfield microscopy

“…Light microscopy has developed from its inception over 300 years ago into being an invaluable biophysical tool for studying complex biological processes in living cells (25). Similarly, automated analytical and computational tools of theoretical biophysics are proving useful in the interpretation of new forms of imaging data (26,27). In particular, the use of fluorescence microscopy, and associated analyses of the resultant images for studying complex processes, has added much to our understanding of complex molecular architectures inside living cells.…”

“…Our image analysis framework uses fluorescence and brightfield image data as an input and interrogates these with automated image segmentation and watershedding algorithms to detect individual S. aureus cells, determine the location of their cell walls, and identify cell division planes in cells containing fluorescently labelled EzrA. Importantly, these techniques can be applied to imaging data from Slimfield microscopy (4,36,37,43,49) which enables tracking of single-molecule complexes over millisecond time scales which are comparable to diffusive molecular mobility inside living cells (27,56), as well as being compatible with advanced analytical methods which employ single cell copy number quantification through convolution modelling (57). S. aureus cells have EzrA-GFP located at the mid-cell position in the early stage of division, and can be visualised using millisecond Slimfield microscopy, as in this work.…”

Using Fluorescence Recovery After Photobleaching (FRAP) to Study Dynamics of the Structural Maintenance of Chromosome (SMC) Complex In Vivo

“…In general, advances in live-cell imaging in combination with the use of Green Fluorescent Protein (GFP) and its variants have facilitated the ability to study the composition and dynamics of protein complexes in a cellular context (8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19). In this chapter we describe the techniques of Fluorescence Recovery After Photobleaching (FRAP) (20) and Fluorescence Loss In Photobleaching (FLIP) to study the dynamics of YFP-tagged MukB, E or F in foci (6).…”