arXiv subjects

Mark C Leake

Publications and source records attributed to Mark C Leake.

15 recordsLinked to original sources

arXiv2025

The transitional kinetics between open and closed Rep structures can be tuned by salt via two intermediate states

DNA helicases undergo conformational changes; however, their structural dynamics are poorly understood. Here, we study single molecules of superfamily 1A DNA helicase Rep, which undergo conformational transitions during bacterial DNA replication, repair and recombination. We use time-correlated single-photon counting (TCSPC), fluorescence correlation spectroscopy (FCS), rapid single-molecule F\"orster resonance energy transfer (smFRET), Anti-Brownian ELectrokinetic (ABEL) trapping and molecular dynamics simulations (MDS) to provide unparalleled temporal and spatial resolution of Rep's domain movements. We detect four states revealing two hitherto hidden intermediates (S2, S3), between the open (S1) and closed (S4) structures, whose stability is salt dependent. Rep's open-to-closed switch involves multiple changes to all four subdomains 1A, 1B, 2A and 2B along the S1 to S2 to S3 to S4 transitional pathway comprising an initial truncated swing of 2B which then rolls across the 1B surface, following by combined rotations of 1B, 2A and 2B. High forward and reverse rates for S1 to S2 suggest that 1B may act to frustrate 2B movement to prevent premature Rep closure in the absence of DNA. These observations support a more general binding model for accessory DNA helicases that utilises conformational plasticity to explore a multiplicity of structures whose landscape can be tuned by salt prior to locking-in upon DNA binding.

q-bio.BM↗

arXiv2025

Single-molecule biophysics

Biological molecules, like all active matter, use free energy to generate force and motion which drive them out of thermal equilibrium, and undergo inherent dynamic interconversion between metastable free energy states separated by levels barely higher than stochastic thermal energy fluctuations. Here, we explore the founding and emerging approaches of the field of single-molecule biophysics which, unlike traditional ensemble average approaches, enable the detection and manipulation of individual molecules and facilitate exploration of biomolecular heterogeneity and its impact on transitional molecular kinetics and underpinning molecular interactions. We discuss the ground-breaking technological innovations which scratch far beyond the surface into open questions of real physiology, that correlate orthogonal data types and interplay empirical measurement with theoretical and computational insights, many of which are enabling artificial matter to be designed inspired by biological systems. And finally, we examine how these insights are helping to develop new physics framed around biology.

physics.bio-ph↗

arXiv2024

Correlating fluorescence microscopy, optical and magnetic tweezers to study single chiral biopolymers such as DNA

Biopolymer topology is critical for determining interactions inside cell environments, exemplified by DNA where its response to mechanical perturbation is as important as biochemical properties to its cellular roles. The dynamic structures of chiral biopolymers exhibit complex dependence with extension and torsion, however the physical mechanisms underpinning the emergence of structural motifs upon physiological twisting and stretching are poorly understood due to technological limitations in correlating force, torque and spatial localization information. We present COMBI-Tweez (Combined Optical and Magnetic BIomolecule TWEEZers), a transformative tool that overcomes these challenges by integrating optical trapping, time-resolved electromagnetic tweezers, and fluorescence microscopy, demonstrated on single DNA molecules, that can controllably form and visualise higher order structural motifs including plectonemes. This technology combined with cutting-edge MD simulations provides quantitative insight into complex dynamic structures relevant to DNA cellular processes and can be adapted to study a range of filamentous biopolymers.

physics.bio-ph↗

arXiv2023

The Physics of Life: No such thing as a dumb question

Physics of Life research in the UK is proving to be transformative to scientific insight and translational impact, but challenges remain. Here I discuss its disruptive potential and the barriers to interdisciplinary research seen through the lens of the activities of one of its pioneers, Tom McLeish, FRS.

cond-mat.soft↗

arXiv2022

Fluidification of entanglements by a DNA bending protein

In spite of the nanoscale and single-molecule insights into how nucleoid associated proteins (NAPs) interact with DNA, their role in modulating the mesoscale viscoelasticity of the entangled genome in vivo has been overlooked so far. By combining microrheology and molecular dynamics simulation we find that the important NAP called Integration Host Factor (IHF) lowers the viscosity of entangled $\lambda$DNA 20-fold at physiological concentrations and stoichiometries. We argue that IHF may act as a "genomic fluidiser", reducing the effective viscosity of the nucleoid $\sim$200-fold. Our results suggest a previously unappreciated key role of IHF in regulating DNA dynamics and re-organisation in vivo

cond-mat.soft↗

arXiv2022

Surviving Early Career Research and Beyond in the Physics of Life: A concise user guide

Early Career Researcher (ECR) development is a dynamic challenge that tensions the urge to perform ground-breaking research against an ultimate practical aspiration of establishing an acceptable level of job security. There is no typical career path for an ECR, least of all in the area of the Physics of Life or Biophysics/Biological Physics. Being explicitly interdisciplinary across the physical-life sciences interface presents more opportunities for a multiplicity of career trajectories through different home academic institutions and departments, as well as offering a broader range of alternative future career trajectories in non-academic sectors. That said, there are key common features, such as the transient nature of fixed-term postdoc contracts, the substantial research and domestic challenges that these present, and the often overwhelming pressures of the realities of competition in the job market. In this short article I outline the key challenges to ECRs in the Physics of Life and discuss simple strategies to manage and potentially overcome them.

physics.ed-ph↗

arXiv2021

Investigating molecular crowding during cell division in budding yeast with FRET

Cell division, aging, and stress recovery triggers spatial reorganization of cellular components in the cytoplasm, including membrane bound organelles, with molecular changes in their compositions and structures. However, it is not clear how these events are coordinated and how they integrate with regulation of molecular crowding. We use the budding yeast Saccharomyces cerevisiae as a model system to study these questions using recent progress in optical fluorescence microscopy and crowding sensing probe technology. We used a Förster Resonance Energy Transfer (FRET) based sensor, illuminated by confocal microscopy for high throughput analyses and Slimfield microscopy for single-molecule resolution, to quantify molecular crowding. We determine crowding in response to cellular growth of both mother and daughter cells, in addition to osmotic stress, and reveal hot spots of crowding across the bud neck in the burgeoning daughter cell. This crowding might be rationalized by the packing of inherited material, like the vacuole, from mother cells. We discuss recent advances in understanding the role of crowding in cellular regulation and key current challenges and conclude by presenting our recent advances in optimizing FRET-based measurements of crowding whilst simultaneously imaging a third color, which can be used as a marker that labels organelle membranes. Our approaches can be combined with synchronised cell populations to increase experimental throughput and correlate molecular crowding information with different stages in the cell cycle.

q-bio.CB↗

arXiv2021

The End Restraint Method for Mechanically Perturbing Nucleic Acids in silico

Far from being a passive information store, the genome is a mechanically dynamic and diverse system in which torsion and tension fluctuate and combine to determine structure and help regulate gene expression. Much of this mechanical perturbation is due to molecular machines such as topoisomerases which must stretch and twist DNA as part of various functions including DNA repair and replication. While the broad-scale mechanical response of nucleic acids to tension and torsion is well characterized, detail at the single base pair level is beyond the limits of even super-resolution imaging. Here, we present a straightforward, flexible, and extensible umbrella-sampling protocol to twist and stretch nucleic acids in silico using the popular biomolecular simulation package Amber -- though the principles we describe are applicable also to other packages such as GROMACS. We discuss how to set up the simulation system, decide forcefields and solvation models, and equilibrate. We then introduce the torsionally-constrained stretching protocol, and finally we present some analysis techniques we have used to characterize structural motif formation. Rather than define forces or fictional pseudoatoms, we instead define a fixed translation of specified atoms between each umbrella sampling step, which allows comparison with experiment without needing to estimate applied forces by simply using the fractional end-to-end displacement as a comparison metric. We hope that this easy to implement solution will be valuable for interrogating optical and magnetic tweezers data on nucleic acids at base pair resolution.

q-bio.BM↗

arXiv2021

PySTACHIO: Python Single-molecule TrAcking stoiCHiometry Intensity and simulatiOn, a flexible, extensible, beginner-friendly and optimized program for analysis of single-molecule microscopy

As camera pixel arrays have grown larger and faster, and optical microscopy techniques ever more refined, there has been an explosion in the quantity of data acquired during routine light microcopy. At the single-molecule level, analysis involves multiple steps and can rapidly become computationally expensive, in some cases intractable on office workstations. Complex bespoke software can present high activation barriers to entry for new users. Here, we redevelop our quantitative single-molecule analysis routines into an optimized and extensible Python program, with GUI and command-line implementations to facilitate use on local machines and remote clusters, by beginners and advanced users alike. We demonstrate that its performance is on par with previous MATLAB implementations but runs an order of magnitude faster. We tested it against challenge data and demonstrate its performance is comparable to state-of-the-art analysis platforms. We show the code can extract fluorescence intensity values for single reporter dye molecules and, using these, estimate molecular stoichiometries and cellular copy numbers of fluorescently-labeled biomolecules. It can evaluate 2D diffusion coefficients for the characteristically short single-particle tracking data. To facilitate benchmarking we include data simulation routines to compare different analysis programs. Finally, we show that it works with 2-color data and enables colocalization analysis based on overlap integration, to infer interactions between differently labelled biomolecules. By making this freely available we aim to make complex light microscopy single-molecule analysis more democratized.

q-bio.BM↗

arXiv2021

Correlative approaches in single-molecule biophysics: a review of the progress in methods and applications

Here, we discuss a collection of cutting-edge techniques and applications in use today by some of the leading experts in the field of correlative approaches in single-molecule biophysics. A key difference in emphasis, compared with traditional single-molecule biophysics approaches detailed previously, is on the emphasis of the development and use of complex methods which explicitly combine multiple approaches to increase biological insights at the single-molecule level. These so-called correlative single-molecule biophysics methods rely on multiple, orthogonal tools and analysis, as opposed to any one single driving technique. Importantly, they span both in vivo and in vitro biological systems as well as the interfaces between theory and experiment in often highly integrated ways, very different to earlier traditional non-integrative approaches. The first applications of correlative single-molecule methods involved adaption of a range of different experimental technologies to the same biological sample whose measurements were synchronised. However, now we find a greater flora of integrated methods emerging that include approaches applied to different samples at different times and yet still permit useful molecular-scale correlations to be performed. The resultant findings often enable far greater precision of length and time scales of measurements, and a more understanding of the interplay between different processes in the same cell. Many new correlative single-molecule biophysics techniques also include more complex, physiologically relevant approaches as well as increasing number that combine advanced computational methods and mathematical analysis with experimental tools. Here we review the motivation behind the development of correlative single-molecule microscopy methods, its history and recent progress in the field.

q-bio.QM↗

arXiv2021

Combining single-molecule super-resolved localization microscopy with fluorescence polarization imaging to study cellular processes

Super-resolution microscopy has catalyzed valuable insights into the sub-cellular, mechanistic details of many different biological processes across a wide range of cell types. Fluorescence polarization spectroscopy tools have also enabled important insights into cellular processes through identifying orientational changes of biological molecules typically at an ensemble level. Here, we combine these two biophysical methodologies in a single home-made instrument to enable the simultaneous detection of orthogonal fluorescence polarization signals from single fluorescent protein molecules used as common reporters on the localization of proteins in cellular processes. These enable measurement of spatial location to a super-resolved precision better than the diffraction-limited optical resolution, as well as estimation of molecular stoichiometry based on the brightness of individual fluorophores. In this innovation we have adapted a millisecond timescale microscope used for single-molecule detection to enable splitting of fluorescence polarization emissions into two separate imaging channels for s- and p- polarization signals, which are imaged onto separate halves of the same high sensitivity back-illuminated CMOS camera detector. We applied this fluorescence polarization super-resolved imaging modality to a range of test fluorescent samples relevant to the study of biological processes, including purified monomeric green fluorescent protein, single combed DNA molecules, and protein assemblies and complexes from live Escherichia coli and Saccharomyces cerevisiae cells. Our findings are qualitative but demonstrate promise in showing how fluorescence polarization and super-resolved localization microscopy can be combined on the same sample to enable simultaneous measurements of polarization and stoichiometry of tracked molecular complexes, as well as the translational diffusion coefficient.

physics.bio-ph↗

arXiv2021

Localization microscopy: a review of the progress in methods and applications

Here, we report analysis and summary of research in the field of localization microscopy for optical imaging. We introduce the basic elements of super-resolved localization microscopy methods for PALM and STORM, commonly used both in vivo and in vitro, discussing the core essentials of background theory, instrumentation and computational algorithms. We discuss the resolution limit of light microscopy and the mathematical framework for localizing fluorescent dyes in space beyond this limit, including the precision obtainable as a function of the amount of light emitted from a dye, and how it leads to a fundamental compromise between spatial and temporal precision. The properties of a "good dye" are outlined, as are the features of PALM and STORM super-resolution microscopy and adaptations that may need to be made to experimental protocols to perform localization determination. We analyse briefly some of the methods of modern super-resolved optical imaging that work through reshaping point spread functions and how they utilize aspects of localization microscopy, such as stimulated depletion (STED) methods and MINFLUX, and summarize modern methods that push localization into 3D using non-Gaussian point spread functions. We report on current methods for analyzing localization data including determination of 2D and 3D diffusion constants, molecular stoichiometries, and performing cluster analysis with cutting-edge techniques, and finally discuss how these techniques may be used to enable important insight into a range of biological processes.

physics.optics↗

arXiv2020

Molecular crowding in single eukaryotic cells: using cell environment biosensing and single-molecule optical microscopy to probe dependence on extracellular ionic strength, local glucose conditions, and sensor copy number

The physical and chemical environment inside cells is of fundamental importance to all life but has traditionally been difficult to determine on a subcellular basis. Here we combine cutting-edge genomically integrated FRET biosensing to readout localized molecular crowding in single live yeast cells. Confocal microscopy allows us to build subcellular crowding heatmaps using ratiometric FRET, while whole-cell analysis demonstrates crowding is reduced when yeast is grown in elevated glucose concentrations. Simulations indicate that the cell membrane is largely inaccessible to these sensors and that cytosolic crowding is broadly uniform across each cell over a timescale of seconds. Millisecond single-molecule optical microscopy was used to track molecules and obtain brightness estimates that enabled calculation of crowding sensor copy numbers. The quantification of diffusing molecule trajectories paves the way for correlating subcellular processes and the physicochemical environment of cells under stress.

q-bio.CB↗

arXiv2019

The emergence of sequence-dependent structural motifs in stretched, torsionally constrained DNA

The double-helical structure of DNA results from canonical base pairing and stacking interactions. However, variations from steady-state conformations result from mechanical perturbations in cells. These different topologies have physiological relevance but their dependence on sequence remains unclear. Here, we use molecular dynamics simulations to show that sequence differences result in markedly different structural motifs upon physiological twisting and stretching. We simulated overextension on four different sequences of DNA ((AA)12, (AT)12, (GG)12 and (GC)12) with supercoiling densities within the physiological range. We found that DNA denatures in the majority of stretching simulations, surprisingly including those with overtwisted DNA. GC-rich sequences were observed to be more stable than AT-rich, with the specific response dependent on base pair ordering. Furthermore, we found that (AT)12 forms stable periodic structures with non-canonical hydrogen bonds in some regions and non-canonical stacking in others, whereas (GC)12 forms a stacking motif of four base pairs independent of supercoiling density. Our results demonstrate that 20-30% DNA extension is sufficient for breaking B-DNA around and significantly above cellular supercoiling, and that the DNA sequence is crucial for understanding structural changes under mechanical stress. Our findings have important implications for the activities of protein machinery interacting with DNA in all cells.

q-bio.BM↗

arXiv2018

Tricks of the light: the remarkable power of laser tweezers to dissect complex biological questions

On 2 October 2018 Goran Hansson, Secretary General of the Royal Swedish Academy of Sciences, announced that the Nobel Prize in Physics would be jointly awarded to Arthur Ashkin, Gerard Mourou and Donna Strickland, for their groundbreaking inventions in the field of laser physics. Strickland and Mourou shared one half of the prize for their pioneering work in generating high-intensity, ultra-short optical pulses. The recipient of the other half was Arthur Ashkin for his seminal work leading to the development of optical tweezers, also referred to as optical traps or laser tweezers, and their applications to an enormous range of biological systems. As discussed below, laser tweezers are a remarkable class of optical force transduction tools which have had a profound effect in enabling several complex biological questions to be addressed impenetrable using other existing technologies.

physics.bio-ph↗