The condensation of HP1α/Swi6 imparts nuclear stiffness.

Biomolecular condensates have emerged as major drivers of cellular organization. It remains largely unexplored, however, whether these condensates can impart mechanical function(s) to the cell. The heterochromatin protein HP1α (Swi6 in Schizosaccharomyces pombe) crosslinks histone H3K9 methylated nucleosomes and has been proposed to undergo condensation to drive the liquid-like clustering of heterochromatin domains. Here, we leverage the genetically tractable S. pombe model and a separation-of-function allele to elucidate a mechanical function imparted by Swi6 condensation. Using single-molecule imaging, force spectroscopy, and high-resolution live-cell imaging, we show that Swi6 is critical for nuclear resistance to external force. Strikingly, it is the condensed yet dynamic pool of Swi6, rather than the chromatin-bound molecules, that is essential to imparting mechanical stiffness. Our findings suggest that Swi6 condensates embedded in the chromatin meshwork establish the emergent mechanical behavior of the nucleus as a whole, revealing that biomolecular condensation can influence organelle and cell mechanics.

Effect of loops on the mean-square displacement of Rouse-model chromatin.

Chromatin polymer dynamics are commonly described using the classical Rouse model. The subsequent discovery, however, of intermediate-scale chromatin organization known as topologically associating domains (TADs) in experimental Hi-C contact maps for chromosomes across the tree of life, together with the success of loop extrusion factor (LEF) model in explaining TAD formation, motivates efforts to understand the effect of loops and loop extrusion on chromatin dynamics. This paper seeks to fulfill this need by combining LEF-model simulations with extended Rouse-model polymer simulations to investigate the dynamics of chromatin with loops and dynamic loop extrusion. We show that loops significantly suppress the averaged mean-square displacement (MSD) of a gene locus, consistent with recent experiments that track fluorescently labeled chromatin loci. We also find that loops reduce the MSD's stretching exponent from the classical Rouse-model value of 1/2 to a loop-density-dependent value in the 0.45-0.40 range. Remarkably, stretching exponent values in this range have also been observed in recent experiments [Weber et al., Phys. Rev. Lett. 104, 238102 (2010)0031-900710.1103/PhysRevLett.104.238102; Bailey et al., Mol. Biol. Cell 34, ar78 (2023)1059-152410.1091/mbc.E23-04-0119]. We also show that the dynamics of loop extrusion itself negligibly affects chromatin mobility. By studying static "rosette" loop configurations, we also demonstrate that chromatin MSDs and stretching exponents depend on the location of the locus in question relative to the position of the loops and on the local friction environment.

An ESCRT grommet cooperates with a diffusion barrier to maintain nuclear integrity.

The molecular mechanisms by which the endosomal sorting complexes required for transport (ESCRT) proteins contribute to the integrity of the nuclear envelope (NE) barrier are not fully defined. We leveraged the single NE hole generated by mitotic extrusion of the Schizosaccharomyces pombe spindle pole body to reveal two modes of ESCRT function executed by distinct complements of ESCRT-III proteins, both dependent on CHMP7/Cmp7. A grommet-like function is required to restrict the NE hole in anaphase B, whereas replacement of Cmp7 by a sealing module ultimately closes the NE in interphase. Without Cmp7, nucleocytoplasmic compartmentalization remains intact despite NE discontinuities of up to 540 nm, suggesting mechanisms to limit diffusion through these holes. We implicate spindle pole body proteins as key components of a diffusion barrier acting with Cmp7 in anaphase B. Thus, NE remodelling mechanisms cooperate with proteinaceous diffusion barriers beyond nuclear pore complexes to maintain the nuclear compartment.

Loops and the activity of loop extrusion factors constrain chromatin dynamics.

The chromosomes-DNA polymers and their binding proteins-are compacted into a spatially organized, yet dynamic, three-dimensional structure. Recent genome-wide chromatin conformation capture experiments reveal a hierarchical organization of the DNA structure that is imposed, at least in part, by looping interactions arising from the activity of loop extrusion factors. The dynamics of chromatin reflects the response of the polymer to a combination of thermal fluctuations and active processes. However, how chromosome structure and enzymes acting on chromatin together define its dynamics remains poorly understood. To gain insight into the structure-dynamics relationship of chromatin, we combine high-precision microscopy in living Schizosaccharomyces pombe cells with systematic genetic perturbations and Rouse model polymer simulations. We first investigated how the activity of two loop extrusion factors, the cohesin and condensin complexes, influences chromatin dynamics. We observed that deactivating cohesin, or to a lesser extent condensin, increased chromatin mobility, suggesting that loop extrusion constrains rather than agitates chromatin motion. Our corresponding simulations reveal that the introduction of loops is sufficient to explain the constraining activity of loop extrusion factors, highlighting that the conformation adopted by the polymer plays a key role in defining its dynamics. Moreover, we find that the number of loops or residence times of loop extrusion factors influence the dynamic behavior of the chromatin polymer. Last, we observe that the activity of the INO80 chromatin remodeler, but not the SWI/SNF or RSC complexes, is critical for ATP-dependent chromatin mobility in fission yeast. Taking the data together, we suggest that thermal and INO80-dependent activities exert forces that drive chromatin fluctuations, which are constrained by the organization of the chromosome into loops.

Extrusion of chromatin loops by a composite loop extrusion factor.

Chromatin loop extrusion by structural maintenance of chromosome (SMC) complexes is thought to underlie intermediate-scale chromatin organization inside cells. Motivated by a number of experiments suggesting that nucleosomes may block loop extrusion by SMCs, such as cohesin and condensin complexes, we introduce and characterize theoretically a composite loop extrusion factor (composite LEF) model. In addition to an SMC complex that creates a chromatin loop by encircling two threads of DNA, this model includes a remodeling complex that relocates or removes nucleosomes as it progresses along the chromatin, and nucleosomes that block SMC translocation along the DNA. Loop extrusion is enabled by SMC motion along nucleosome-free DNA, created in the wake of the remodeling complex, while nucleosome rebinding behind the SMC acts as a ratchet, holding the SMC close to the remodeling complex. We show that, for a wide range of parameter values, this collection of factors constitutes a composite LEF that extrudes loops with a velocity, comparable to the velocity of remodeling complex translocation on chromatin in the absence of SMC, and much faster than loop extrusion by an isolated SMC that is blocked by nucleosomes.

Interconnecting solvent quality, transcription, and chromosome folding in Escherichia coli.

All cells fold their genomes, including bacterial cells, where the chromosome is compacted into a domain-organized meshwork called the nucleoid. How compaction and domain organization arise is not fully understood. Here, we describe a method to estimate the average mesh size of the nucleoid in Escherichia coli. Using nucleoid mesh size and DNA concentration estimates, we find that the cytoplasm behaves as a poor solvent for the chromosome when the cell is considered as a simple semidilute polymer solution. Monte Carlo simulations suggest that a poor solvent leads to chromosome compaction and DNA density heterogeneity (i.e., domain formation) at physiological DNA concentration. Fluorescence microscopy reveals that the heterogeneous DNA density negatively correlates with ribosome density within the nucleoid, consistent with cryoelectron tomography data. Drug experiments, together with past observations, suggest the hypothesis that RNAs contribute to the poor solvent effects, connecting chromosome compaction and domain formation to transcription and intracellular organization.

Covariance distributions in single particle tracking.

Several recent experiments, including our own experiments in the fission yeast, Schizosaccharomyces pombe, have characterized the motions of gene loci within living nuclei by measuring the locus position over time, then proceeding to obtain the statistical properties of this motion. To address the question of whether a population of such single-particle tracks, obtained from many different cells, corresponds to a single mode of diffusion, we derive theoretical equations describing the probability distribution of the displacement covariance, assuming the displacement itself is a zero-mean multivariate Gaussian random variable. We also determine the corresponding theoretical means, variances, and third central moments. Bolstering the theory is good agreement between its predictions and the results obtained for various simulated and measured data sets, including simulated particle trajectories undergoing simple and anomalous diffusion, and the measured trajectories of an optically trapped bead in water, and in a viscoelastic polymer solution. We also show that, for sufficiently long tracks, each covariance distribution in all of these examples is well-described by a skew-normal distribution with mean, variance, and skewness given by the theory. However, for the experimentally measured motion of a gene locus in S. pombe, we find that the first two covariance distributions are wider than predicted, although the third and subsequent covariance distributions are well-described by theory. This observation suggests that the origin of the theory-experiment discrepancy in this case is associated with localization noise, which influences only the first two covariances. Thus, we hypothesized that the discrepancy is caused by locus-to-locus heterogeneity in the localization noise, of independent measurements of the same tagged site. Indeed, simulations implementing heterogeneous localization noise revealed that the excess covariance widths can be largely recreated on the basis of heterogeneous noise. Thus, we conclude that the motion of gene loci in fission yeast is consistent with a single mode of diffusion.

Subcellular Organization: A Critical Feature of Bacterial Cell Replication.

Spatial organization is a hallmark of all living systems. Even bacteria, the smallest forms of cellular life, display defined shapes and complex internal organization, showcasing a highly structured genome, cytoskeletal filaments, localized scaffolding structures, dynamic spatial patterns, active transport, and occasionally, intracellular organelles. Spatial order is required for faithful and efficient cellular replication and offers a powerful means for the development of unique biological properties. Here, we discuss organizational features of bacterial cells and highlight how bacteria have evolved diverse spatial mechanisms to overcome challenges cells face as self-replicating entities.

Replication fork passage drives asymmetric dynamics of a critical nucleoid-associated protein in Caulobacter.

In bacteria, chromosome dynamics and gene expression are modulated by nucleoid-associated proteins (NAPs), but little is known about how NAP activity is coupled to cell cycle progression. Using genomic techniques, quantitative cell imaging, and mathematical modeling, our study in Caulobacter crescentus identifies a novel NAP (GapR) whose activity over the cell cycle is shaped by DNA replication. GapR activity is critical for cellular function, as loss of GapR causes severe, pleiotropic defects in growth, cell division, DNA replication, and chromosome segregation. GapR also affects global gene expression with a chromosomal bias from origin to terminus, which is associated with a similar general bias in GapR binding activity along the chromosome. Strikingly, this asymmetric localization cannot be explained by the distribution of GapR binding sites on the chromosome. Instead, we present a mechanistic model in which the spatiotemporal dynamics of GapR are primarily driven by the progression of the replication forks. This model represents a simple mechanism of cell cycle regulation, in which DNA-binding activity is intimately linked to the action of DNA replication.

DNA-relay mechanism is sufficient to explain ParA-dependent intracellular transport and patterning of single and multiple cargos.

Spatial ordering of macromolecular components inside cells is important for cellular physiology and replication. In bacteria, ParA/B systems are known to generate various intracellular patterns that underlie the transport and partitioning of low-copy-number cargos such as plasmids. ParA/B systems consist of ParA, an ATPase that dimerizes and binds DNA upon ATP binding, and ParB, a protein that binds the cargo and stimulates ParA ATPase activity. Inside cells, ParA is asymmetrically distributed, forming a propagating wave that is followed by the ParB-rich cargo. These correlated dynamics lead to cargo oscillation or equidistant spacing over the nucleoid depending on whether the cargo is in single or multiple copies. Currently, there is no model that explains how these different spatial patterns arise and relate to each other. Here, we test a simple DNA-relay model that has no imposed asymmetry and that only considers the ParA/ParB biochemistry and the known fluctuating and elastic dynamics of chromosomal loci. Stochastic simulations with experimentally derived parameters demonstrate that this model is sufficient to reproduce the signature patterns of ParA/B systems: the propagating ParA gradient correlated with the cargo dynamics, the single-cargo oscillatory motion, and the multicargo equidistant patterning. Stochasticity of ATP hydrolysis breaks the initial symmetry in ParA distribution, resulting in imbalance of elastic force acting on the cargo. Our results may apply beyond ParA/B systems as they reveal how a minimal system of two players, one binding to DNA and the other modulating this binding, can transform directionally random DNA fluctuations into directed motion and intracellular patterning.

The Slow Mobility of the ParA Partitioning Protein Underlies Its Steady-State Patterning in Caulobacter.

In bacteria, ParABS systems mediate intracellular transport of various cargos, including chromosomal regions in Caulobacter crescentus. Transport of the ParB/parS partition complex requires the DNA-binding activity of ParA, which transiently tethers the partition complex during translocation. In C. crescentus, the directionality of the transport is set up by a gradient of ParA whose concentration gradually increases from one end of the cell (old pole) to the other (new pole). Importantly, this ParA gradient is already observed before DNA replication and segregation are initiated when the partition complex is anchored at the old pole. How such micron-scale ParA pattern is established and maintained before the initiation of chromosome segregation has not been experimentally established. Although the stimulation of ParA ATPase activity by the localized ParB/parS partition complex is thought to be involved, this activity alone cannot quantitatively describe the ParA pattern observed inside cells. Instead, our experimental and theoretical study shows that the missing key component for achieving the experimentally observed steady-state ParA patterning is the slow mobility of ParA dimers (D ∼10(-3)µm(2)/s) due to intermittent DNA binding. Our model recapitulates the entire steady-state ParA distribution observed experimentally, including the shape of the gradient as well as ParA accumulation at the location of the partition complex. Stochastic simulations suggest that cell-to-cell variability in ParA pattern is due to the low ParA copy number in C. crescentus cells. The model also accounts for an apparent exclusion of ParA from regions with small spacing between partition complexes observed in filamentous cells. Collectively, our work demonstrates that in addition to its function in mediating transport, the conserved DNA-binding property of ParA has a critical function before DNA segregation by setting up a ParA pattern required for transport directionality.

Oufti: an integrated software package for high-accuracy, high-throughput quantitative microscopy analysis.

With the realization that bacteria display phenotypic variability among cells and exhibit complex subcellular organization critical for cellular function and behavior, microscopy has re-emerged as a primary tool in bacterial research during the last decade. However, the bottleneck in today's single-cell studies is quantitative image analysis of cells and fluorescent signals. Here, we address current limitations through the development of Oufti, a stand-alone, open-source software package for automated measurements of microbial cells and fluorescence signals from microscopy images. Oufti provides computational solutions for tracking touching cells in confluent samples, handles various cell morphologies, offers algorithms for quantitative analysis of both diffraction and non-diffraction-limited fluorescence signals and is scalable for high-throughput analysis of massive datasets, all with subpixel precision. All functionalities are integrated in a single package. The graphical user interface, which includes interactive modules for segmentation, image analysis and post-processing analysis, makes the software broadly accessible to users irrespective of their computational skills.

A constant size extension drives bacterial cell size homeostasis.

Cell size control is an intrinsic feature of the cell cycle. In bacteria, cell growth and division are thought to be coupled through a cell size threshold. Here, we provide direct experimental evidence disproving the critical size paradigm. Instead, we show through single-cell microscopy and modeling that the evolutionarily distant bacteria Escherichia coli and Caulobacter crescentus achieve cell size homeostasis by growing, on average, the same amount between divisions, irrespective of cell length at birth. This simple mechanism provides a remarkably robust cell size control without the need of being precise, abating size deviations exponentially within a few generations. This size homeostasis mechanism is broadly applicable for symmetric and asymmetric divisions, as well as for different growth rates. Furthermore, our data suggest that constant size extension is implemented at or close to division. Altogether, our findings provide fundamentally distinct governing principles for cell size and cell-cycle control in bacteria.

Evidence for a DNA-relay mechanism in ParABS-mediated chromosome segregation.

The widely conserved ParABS system plays a major role in bacterial chromosome segregation. How the components of this system work together to generate translocation force and directional motion remains uncertain. Here, we combine biochemical approaches, quantitative imaging and mathematical modeling to examine the mechanism by which ParA drives the translocation of the ParB/parS partition complex in Caulobacter crescentus. Our experiments, together with simulations grounded on experimentally-determined biochemical and cellular parameters, suggest a novel 'DNA-relay' mechanism in which the chromosome plays a mechanical function. In this model, DNA-bound ParA-ATP dimers serve as transient tethers that harness the elastic dynamics of the chromosome to relay the partition complex from one DNA region to another across a ParA-ATP dimer gradient. Since ParA-like proteins are implicated in the partitioning of various cytoplasmic cargos, the conservation of their DNA-binding activity suggests that the DNA-relay mechanism may be a general form of intracellular transport in bacteria.DOI: http://dx.doi.org/10.7554/eLife.02758.001.

The bacterial cytoplasm has glass-like properties and is fluidized by metabolic activity.

The physical nature of the bacterial cytoplasm is poorly understood even though it determines cytoplasmic dynamics and hence cellular physiology and behavior. Through single-particle tracking of protein filaments, plasmids, storage granules, and foreign particles of different sizes, we find that the bacterial cytoplasm displays properties that are characteristic of glass-forming liquids and changes from liquid-like to solid-like in a component size-dependent fashion. As a result, the motion of cytoplasmic components becomes disproportionally constrained with increasing size. Remarkably, cellular metabolism fluidizes the cytoplasm, allowing larger components to escape their local environment and explore larger regions of the cytoplasm. Consequently, cytoplasmic fluidity and dynamics dramatically change as cells shift between metabolically active and dormant states in response to fluctuating environments. Our findings provide insight into bacterial dormancy and have broad implications to our understanding of bacterial physiology, as the glassy behavior of the cytoplasm impacts all intracellular processes involving large components.

Spatial organization of the flow of genetic information in bacteria.

Eukaryotic cells spatially organize mRNA processes such as translation and mRNA decay. Much less is clear in bacterial cells where the spatial distribution of mature mRNA remains ambiguous. Using a sensitive method based on quantitative fluorescence in situ hybridization, we show here that in Caulobacter crescentus and Escherichia coli, chromosomally expressed mRNAs largely display limited dispersion from their site of transcription during their lifetime. We estimate apparent diffusion coefficients at least two orders of magnitude lower than expected for freely diffusing mRNA, and provide evidence in C. crescentus that this mRNA localization restricts ribosomal mobility. Furthermore, C. crescentus RNase E appears associated with the DNA independently of its mRNA substrates. Collectively, our findings show that bacteria can spatially organize translation and, potentially, mRNA decay by using the chromosome layout as a template. This chromosome-centric organization has important implications for cellular physiology and for our understanding of gene expression in bacteria.

Mathematical modeling of a minimal protocell with coordinated growth and division.

Self-replication is an essential attribute of life but the molecular-level mechanisms involved are not well understood. Cellular self-replication requires not only duplicating all cellular components and doubling volume and membrane area, but also replicating cellular geometry. A whole-cell modeling framework is presented in which an assumed reaction network determines both concentration changes of cellular components and cell geometry. Cell shape is calculated by minimizing membrane-bending energy. Using this framework, simultaneous doubling of volume, surface area, and all components was found to be insufficient to provide mid-cell "pinching" of the parental cell to form two daughter cells. This prompted the design of a minimal protocell that includes a growing shell, a cell-cycle engine, and a contractile ring to enforce cytokinesis. Kinetic parameters were found such that the system exhibited periodic behavior with fundamental aspects of self-replication. This involved simultaneous doubling of all cellular components during a cell cycle, doubling cell volume and membrane area, achieving periodic changes in surface/volume ratio, and forming daughter cells that were geometrically equivalent to each other and to the "newborn" parental cell. The results presented here impact the design of laboratory protocells and the development of a modular strategy for constructing a comprehensive in silico whole-cell model.

Kinetic modeling of the assembly, dynamic steady state, and contraction of the FtsZ ring in prokaryotic cytokinesis.

Cytokinesis in prokaryotes involves the assembly of a polymeric ring composed of FtsZ protein monomeric units. The Z ring forms at the division plane and is attached to the membrane. After assembly, it maintains a stable yet dynamic steady state. Once induced, the ring contracts and the membrane constricts. In this work, we present a computational deterministic biochemical model exhibiting this behavior. The model is based on biochemical features of FtsZ known from in vitro studies, and it quantitatively reproduces relevant in vitro data. An essential part of the model is a consideration of interfacial reactions involving the cytosol volume, where monomeric FtsZ is dispersed, and the membrane surface in the cell's mid-zone where the ring is assembled. This approach allows the same chemical model to simulate either in vitro or in vivo conditions by adjusting only two geometrical parameters. The model includes minimal reactions, components, and assumptions, yet is able to reproduce sought-after in vivo behavior, including the rapid assembly of the ring via FtsZ-polymerization, the formation of a dynamic steady state in which GTP hydrolysis leads to the exchange of monomeric subunits between cytoplasm and the ring, and finally the induced contraction of the ring. The model gives a quantitative estimate for coupling between the rate of GTP hydrolysis and of FtsZ subunit turnover between the assembled ring and the cytoplasmic pool as observed. Membrane constriction is chemically driven by the strong tendency of GTP-bound FtsZ to self-assembly. The model suggests a possible mechanism of membrane contraction without a motor protein. The portion of the free energy of GTP hydrolysis released in cyclization is indirectly used in this energetically unfavorable process. The model provides a limit to the mechanistic complexity required to mimic ring behavior, and it highlights the importance of parallel in vitro and in vivo modeling.

Nickel-dependent oligomerization of the alpha subunit of acetyl-coenzyme a synthase/carbon monoxide dehydrogenase.

After activation with NiCl2, the recombinant alpha subunit of the Ni-containing alpha2beta2 acetyl-CoA synthase/carbon monoxide dehydrogenase (ACS/CODH) catalyzes the synthesis of acetyl-CoA from CO, CoA, and a methyl group donated from the corrinoid-iron-sulfur protein (CoFeSP). The alpha subunit has two conformations (open and closed), and contains a novel [Fe4S4]-[Nip Nid] active site in which the proximal Nip ion is labile. Prior to Ni activation, recombinant apo-alpha contain only an Fe4S4 cluster. Ni-activated alpha subunits exhibit catalytic, spectroscopic and heterogeneity properties typical of alpha subunits contained in ACS/CODH. Evidence presented here indicates that apo-alpha is a monomer whereas Ni-treated alpha oligomerizes, forming dimers and higher molecular weight species including tetramers. No oligomerization occurred when apo-alpha was treated with Cu(II), Zn(II), or Co(II) ions, but oligomerization occurred when apo-alpha was treated with Pt(II) and Pd(II) ions. The dimer accepted only 0.5 methyl group/alpha and exhibited, upon treatment with CO and under reducing conditions, the NiFeC EPR signal quantifying to 0.4 spin/alpha. Dimers appear to consist of two types of alpha subunits, including one responsible for catalytic activity and one that provides a structural scaffold. Higher molecular weight species may be similarly constituted. It is concluded that Ni binding to the A-cluster induces a conformational change in the alpha subunit, possibly to the open conformation, that promotes oligomerization. These interrelated events demonstrate previously unrealized connections between (a) the conformation of the alpha subunit; (b) the metal which occupies the proximal/distal sites of the A-cluster; and (c) catalytic activity.

Kinetics of CO insertion and acetyl group transfer steps, and a model of the acetyl-CoA synthase catalytic mechanism.

Acetyl-CoA synthase/carbon monoxide dehydrogenase is a Ni-Fe-S-containing enzyme that catalyzes the synthesis of acetyl-CoA from CO, CoA, and a methyl group. The methyl group is transferred onto the enzyme from a corrinoid-iron-sulfur protein (CoFeSP). The kinetics of two steps within the catalytic mechanism were studied using the stopped-flow method, including the insertion of CO into a putative Ni(2+)-CH(3) bond and the transfer of the resulting acetyl group to CoA. Neither step had been studied previously. Reactions were monitored indirectly, starting with the methylated intermediate form of the enzyme. Resulting traces were analyzed by constructing a simple kinetic model describing the catalytic mechanism under reducing conditions. Besides methyl group transfer, CO insertion, and acetyl group transfer, fitting to experimental traces required the inclusion of an inhibitory step in which CO reversibly bound to the form of the enzyme obtained immediately after product release. Global simulation of the reported datasets afforded a consistent set of kinetic parameters. The equilibrium constant for the overall synthesis of acetyl-CoA was estimated and compared to the product of the individual equilibrium constants. Simulations obtained with the model duplicated the essential behavior of the enzyme, in terms of the variation of activity with [CO], and the time-dependent decay of the NiFeC EPR signal upon reaction with CoFeSP. Under standard assay conditions, the model suggests that the vast majority of active enzyme molecules in a population should be in the methylated form, suggesting that the subsequent catalytic step, namely CO insertion, is rate limiting. This conclusion is further supported by a sensitivity analysis showing that the rate is most sensitively affected by a change in the rate coefficient associated with the CO insertion step.