Biology subjects

Murray, S. M.

Publications and source records attributed to Murray, S. M..

12 recordsLinked to original sources

bioRxiv2023

Putting the Par back into ParABS: Plasmid Partitioning Driven by ParA Oscillations

The ParABS system is crucial for the faithful segregation and inheritance of many bacterial chromosomes and low-copy number plasmids. However, despite extensive research, the spatio-temporal dynamics of the ATPase ParA and its connection to the dynamics and positioning of the ParB-coated cargo has remained unclear. In this study, we utilise high-throughput imaging, quantitative data analysis, and computational modelling to explore the in vivo dynamics of ParA and its interaction with ParB-coated plasmids and the nucleoid. As previously observed, we find that F-plasmid ParA undergoes collective migrations ( flips) between cell halves multiple times per cell cycle. We reveal that a constricting nucleoid is required for these migrations and that they are triggered by a plasmid crossing into the cell half with greater ParA. Using simulations, we show that these dynamics can be explained by the combination of nucleoid constriction and cooperative ParA binding to the DNA, in line with the behaviour of other ParA proteins. We further show that these ParA flips act to equally partition plasmids between the two lobes of the constricted nucleoid and are therefore important for plasmid stability, especially in fast growth conditions for which the nucleoid constricts early in the cell cycle. Overall our work identifies a second mode of action of the ParABS system and deepens our understanding of how this important segregation system functions.

microbiology↗

bioRxiv2023

Mid-cell migration of the chromosomal terminus is coupled to origin segregation in Escherichia coli

Bacterial chromosomes are dynamically and spatially organised within cells. In slow-growing Escherichia coli, the chromosomal terminus is initially located at the new pole and must therefore migrate to midcell during replication to reproduce the same pattern in the daughter cells. Here, we use high-throughput time-lapse microscopy to quantify this transition, its timing and its relationship to chromosome segregation. We find that terminus centralisation is a rapid discrete event that occurs ~25 min after initial separation of duplicated origins and ~50 min before the onset of bulk nucleoid segregation but with substantial variation between cells. Despite this variation, its movement is tightly coincident with the completion of origin segregation, even in the absence of its linkage to the divisome, suggesting a coupling between these two events. Indeed, we find that terminus centralisation does not occur if origin segregation away from mid-cell is disrupted, which results in daughter cells having an inverted chromosome organisation. Overall, our study quantifies the choreography of origin-terminus positioning and identifies an unexplored connection between these loci, furthering our understanding of chromosome segregation in this bacterium.

microbiology↗

bioRxiv2022

Star Track: Inferred counting and tracking of replicating DNA loci

Fluorescent microscopy is the primary method to study DNA organization within cells. However the variability and low signal-to-noise commonly associated with live-cell time lapse imaging challenges quantitative measurements. In particular, obtaining quantitative or mechanistic insight often depends on the accurate tracking of fluorescent particles. Here, we present [*]Track, an inference method that determines the most likely temporal tracking of replicating intracellular particles such DNA loci while accounting for missing, merged and spurious detections. It allows the accurate prediction of particle copy numbers as well as the timing of replication events. We demonstrate [*]Tracks abilities and gain new insight into plasmid copy number control and the volume dependence of bacterial chromosome replication initiation. By enabling the accurate tracking of DNA loci, [*]Track can help to uncover the mechanistic principles of chromosome organisation and dynamics across a range of systems. SignificanceMicroscopy is one of the main tools for studying the intracellular organisation of cells. In particular, fluorescent proteins allow us to study the dynamics of many cellular processes. However, this requires the accurate tracking of fluorescent foci. Here, we present [*]Track a tool tailored to the tracking of replicating persistent subcellular particles such as DNA loci. [*]Track provides accurate predictions of particle copy number and replication timing even in the presence of substantial noise. The knowledge of these quantities are critical for uncovering the mechanisms behind many cell-cycle dependent processes, such the control of chromosome and plasmid replication initiation.

biophysics↗

bioRxiv2022

Partition complex structure can arise from sliding and bridging of ParB dimers

Chromosome segregation is vital for cell replication and in many bacteria is controlled by the ParABS system. A key part of this machinery is the association of ParB proteins to the parS-containing centromeric region to form the partition complex. Despite much work, the formation and structure of this nucleoprotein complex has remained unclear. However, it was recently discovered that CTP binding allows ParB dimers to entrap and slide along the DNA, as well as leading to more efficient condensation through ParB-mediated DNA bridging. Here, we use semi-flexible polymer simulations to show how these properties of sliding and bridging can explain partition complex formation. We find that transient ParB bridges can organise the DNA into either a globular state or into hairpins and helical structures, depending on the bridge lifetime. In separate stochastic simulations, we show that ParB sliding reproduces the experimentally measured multi-peaked binding profile of Caulobacter crescentus, indicating that bridging and other potential roadblocks are sufficiently short-lived that they do not hinder ParB spreading. Indeed, upon coupling the two simulation frameworks into a unified sliding and bridging model, we find that short-lived ParB bridges do not hinder ParB sliding and the model can reproduce both the ParB binding profile as well as the condensation of the nucleoprotein complex. Overall, our model clarifies the mechanism of partition complex formation and predicts its fine structure. We speculate that the DNA hairpins produced by ParB bridging underlie the secondary function of ParB to load the Structural Maintenance of Chromosome (SMC) complex onto the DNA.

biophysics↗

bioRxiv2022

DNA bridging explains sub-diffusive movement of chromosomal loci in bacteria

Chromosomal loci in bacterial cells show a robust sub-diffusive scaling of the mean square displacement, MSD({tau}) [~]{tau} , with < 0.5. On the other hand, recent experiments have also shown that DNA-bridging Nucleoid Associated Proteins (NAPs) play an important role in chromosome organisation and compaction. Here, using polymer simulations we investigate the role of DNA bridging in determining the dynamics of chromosomal loci. We find that bridging compacts the polymer and reproduces the sub-diffusive elastic dynamics of monomers at timescales shorter than the bridge lifetime. Consistent with this prediction, we measure a higher exponent in a NAP mutant ({Delta}H-NS) compared to wild-type E. coli. Furthermore, bridging can reproduce the rare but ubiquitous rapid movements of chromosomal loci that have been observed in experiments. In our model the scaling exponent defines a relationship between the abundance of bridges and their lifetime. Using this and the observed mobility of chromosomal loci, we predict a lower bound on the average bridge lifetime of around 5 seconds. Significance StatementThe bacterial chromosome exhibits dynamics that cannot be explained by simple polymer models. In particular, the mean square displacement of individual chromosomal loci exhibits a power law scaling with an exponent less than that predicted by polymer theory. Here, we use polymer simulations and experiments to show that DNA bridging by Nucleoid Associated Proteins can explain these anomalous dynamics. Consistent with this, we show that in the absence of the bridging protein H-NS, the scaling exponent increases. Chromosomal loci also display rare rapid movements not explainable by polymer theory, even accounting for the viscoelasticity of the cytoplasm. Our simulations show that bridging can additionally explain this behaviour. Finally, we predict a lower bound on the average bridge lifetime within cells.

biophysics↗

bioRxiv2022

CCR4-NOT subunit CCF-1/CNOT7 interacts with the PAL-1/CDX-1 transcription factor to regulate multiple stress responses in Caenorhabditis elegans

CCR4-NOT is a versatile eukaryotic protein complex that controls multiple steps in gene expression regulation from synthesis to decay. In yeast, CCR4-NOT has been implicated in stress response regulation, though this function in other organisms remains unclear. In a genome-wide RNAi screen, we identified a subunit of the CCR4-NOT complex, ccf-1, as a requirement for the C. elegans transcriptional response to cadmium and acrylamide stress. Using whole-transcriptome RNA sequencing, we show that knockdown of ccf-1 attenuates the activation of a broad range of stress protective genes in response to cadmium and acrylamide, including those encoding heat shock proteins and glutathione s-transferases. Consistently, survival assays show that knockdown of ccf-1 decreases C. elegans stress resistance. A yeast-2-hybrid screen using a CCF-1 bait identified the homeobox transcription factor PAL-1 as a physical interactor. Knockdown of pal-1 inhibits the activation of ccf-1 dependent stress genes and reduces C. elegans stress resistance. Gene expression analysis reveals that knockdown of pal-1 down-regulates the mRNA levels of elt-2 and elt-3, which serves as the master transcriptional co-regulators of stress response in the C. elegans intestinal and epidermal tissues respectively. These results reveal a new role for CCR4-NOT in stress response regulation with PAL-1 through the transcriptional control of elt-2 and elt-3 in C. elegans.

genetics↗

bioRxiv2022

High-throughput imaging and quantitative analysis uncovers the nature of plasmid positioning by ParABS

The faithful segregation and inheritance of bacterial chromosomes and low-copy number plasmids requires dedicated partitioning systems. The most common of these, ParABS, consists of ParA, a DNA-binding ATPase and ParB a protein that binds to centromeric-like parS sequences on the DNA cargo. The resulting nucleoprotein complexes are believed to move up a self-generated gradient of nucleoid-associated ParA. However, it remains unclear how this leads to the observed cargo positioning and dynamics. In particular, the evaluation of models of plasmid positioning has been hindered by the lack of quantitative measurements. Here, we use high-throughput imaging, analysis and modelling to determine the dynamical nature of these systems. We find that F plasmid is actively brought to specific subcellular home positions within the cell with dynamics akin to an over-damped spring. We identify ParA hopping on the nucleoid as the primary determinant of this geometry-sensing and develop a unified computational model that quantitatively explains the observed behaviour. Furthermore, we confirm our model prediction that cells with the lowest plasmid concentration transition to oscillatory dynamics and find similar behaviour in a distantly-related ParABS system. Our results indicate that ParABS regularly positions plasmids across the nucleoid but operates just below the threshold of an oscillatory instability, minimising ATP consumption. Overall, this work uncovers the dynamical nature of plasmid positioning by ParABS and provides insights relevant for chromosome-based systems.

biophysics↗

bioRxiv2019

Pal stabilises the bacterial outer membrane during constriction by a mobilisation-and-capture mechanism

Coordination of outer membrane constriction with septation is critical to faithful division in Gram-negative bacteria and vital to the barrier function of the membrane. Recent studies suggest this coordination is through the active accumulation of the peptidoglycan-binding outer membrane lipoprotein Pal at division sites by the Tol system, but the mechanism is unknown. Here, we show that Pal accumulation at Escherichia coli division sites is a consequence of three key functions of the Tol system. First, Tol mobilises Pal molecules in dividing cells, which otherwise diffuse very slowly due to their binding of the cell wall. Second, Tol actively captures mobilised Pal molecules and deposits them at the division septum. Third, the active capture mechanism is analogous to that used by the inner membrane protein TonB to dislodge the plug domains of outer membrane TonB-dependent nutrient transporters. We conclude that outer membrane constriction is coordinated with cell division by active mobilisation-and-capture of Pal at division septa by the Tol system.

microbiology↗

bioRxiv2018

Reduced gray matter volume in the orbitofrontal cortex is associated with greater body mass index: a coordinate-based meta-analysis.

Meta-analyses of neuroimaging studies have not found a clear relationship between the orbitofrontal cortex and obesity, despite animal and human studies suggesting the contrary. Our primary meta-analysis examined what regions are associated with reduced gray matter volume, given increased body mass index. We identified 23 voxel-based morphometry studies examining the association between gray matter volume and body mass index. In a sample of 6,788 participants, we found that greater body mass index is associated with decreased gray matter volume in the right Brodmanns area 10 and 11, forming part of the right orbitofrontal cortex (FWE, p=0.05). Use of Brodmanns areas 10 and 11 as seeds in a Neurosynth network coactivation and text decoding analysis revealed that these regions are associated with studies of emotional regulation and processing, clinical symptoms and disorder, mentalizing and social cognition, and the Default mode network. Our finding uniquely contributes to the literature in showing a relationship between the orbitofrontal cortex and obesity and showing the wide-ranging impact these differences may have on social, mental, and emotional functioning as well as on the Default mode network. Exploratory analyses suggest the need for studies examining the effect of age on these findings.

neuroscience↗

bioRxiv2018

Self-organised segregation of bacterial chromosomal origins

In spite of much effort, many aspects of chromosome organisation and segregation in bacteria remain unclear. Even for Escherichia coli, the most widely studied bacterial model organism, we still do not know the underlying mechanisms. Like many other bacteria, the chromosomal origin of replication in E. coli is dynamically positioned throughout the cell cycle. Initially maintained at mid-cell, where replication occurs, origins are subsequently partitioned to opposite quarter positions. The Structural Maintenance of Chromosomes (SMC) complex, MukBEF, which is required for correct chromosome compaction and organisation, has been implicated in this behaviour but the mode of action is unknown. Here, we build on a recent self-organising model for the positioning of E. coli MukBEF, to propose an explanation for the positioning and partitioning of origins. We propose that a specific association of MukBEF with the origin region, results in a non-trivial feedback between the self-organising MukBEF gradient and the origins, leading to accurate positioning and partitioning as an emergent property. We compare the model to quantitative experimental data of origin dynamics and their colocalisation with MukBEF clusters and find excellent agreement. Overall, the model suggests that MukBEF and origins act together as a self-organising system for chromosome segregation and introduces protein self-organisation as an important consideration for future studies of chromosome dynamics.

systems biology↗

bioRxiv2017

A Tunable Protein Oscillator Controls Directional Movements In Myxococcus xanthus

Dynamic control of cell polarity is of critical importance for many aspects of cellular development and motility. In Myxococcus xanthus, a G-protein and its cognate GTPase-activating protein establish a polarity axis that defines the direction of movement of the cell and which can be rapidly inverted by the Frz chemosensory system. Although vital for collective cell behaviours, how Frz triggers this switch has remained unknown. Here, we use genetics, imaging and mathematical modelling to show that Frz controls polarity reversals via a gated relaxation oscillator. FrzX, which we newly identify as the primary Frz output, provides the gating and thus acts as the trigger for reversals. Slow relocalisation of the polarity protein RomR then creates a refractory period during which another switch cannot be triggered. A secondary Frz output, FrzZ, decreases this delay allowing rapid reversals when required. This architecture thus results in a highly tunable switch that allows a wide range of motility responses.

microbiology↗

bioRxiv2017

Self-Organisation And Positioning Of Bacterial Protein Clusters

Many cellular processes require sub-cellular positioning of proteins. This can be due to passive mechanisms such as recruitment by existing landmarks or curvature sensing. However, in bacteria active self-positioning is likely to play a role in multiple processes, including the positioning of the future division site and cytoplasmic protein clusters. How can such dynamic clusters be formed and positioned? Here, we present a model for the self-organization and positioning of dynamic protein clusters into regularly repeating patterns based on a phase-locked Turing pattern. A single peak in the concentration is always positioned at mid-domain (mid-cell) while two peaks are positioned one at each quarter-position etc. Furthermore, domain growth results in peak-splitting and pattern doubling. We argue that the model may explain the regular positioning of the highly conserved Structural Maintenance of Chromosomes (SMC) complexes on the bacterial nucleoid and provides an attractive mechanism for the self-positioning of dynamic protein clusters in other systems.

biophysics↗