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Chromatin is an essential component of nuclear mechanical response and shape that maintains nuclear compartmentalization and function. The biophysical properties of chromatin alter nuclear shape and stability, but little is known about whether or how major genomic functions can impact the integrity of the nucleus. We hypothesized that transcription might affect cell nuclear shape and rupture through its effects on chromatin structure and dynamics. To test this idea, we inhibited transcription with the RNA polymerase II inhibitor alpha-amanitin in wild type cells and perturbed cells that present increased nuclear blebbing. Transcription inhibition suppresses nuclear blebbing for several cell types, nuclear perturbations, and transcription inhibitors. Furthermore, transcription is necessary for robust nuclear bleb formation, bleb stabilization, and bleb-based nuclear ruptures. These morphological effects appear to occur through a novel biophysical pathway, since transcription does not alter either chromatin histone modification state or nuclear rigidity, which typically control nuclear blebbing. We find that active/phosphorylated RNA pol II Ser5, marking transcription initiation, is enriched in nuclear blebs relative to DNA. Thus, transcription initiation is a hallmark of nuclear blebs. Polymer simulations suggest that motor activity within chromatin, such as that of RNA pol II, can generate active forces that deform the nuclear periphery, and that nuclear deformations depend on motor dynamics. Our data provide evidence that the genomic function of transcription impacts nuclear shape stability, and suggests a novel mechanism, separate and distinct from chromatin rigidity, for regulating large-scale nuclear shape and function.

Cohesin and CCCTC-binding factor (CTCF) are key regulatory proteins of three-dimensional (3D) genome organization. Cohesin extrudes DNA loops that are anchored by CTCF in a polar orientation. Here, we present direct evidence that CTCF binding polarity controls cohesin-mediated DNA looping. Using single-molecule imaging of CTCF-cohesin collisions, we demonstrate that a critical N-terminal motif of CTCF blocks cohesin translocation and DNA looping. The cryo-electron microscopy structure of the intact cohesin-CTCF complex reveals that this CTCF motif ahead of zinc-fingers can only reach its binding site on the STAG1 cohesin subunit when the N-terminus of CTCF faces cohesin. Remarkably, a C-terminally oriented CTCF accelerates DNA compaction by cohesin. DNA-bound Cas9 and Cas12a ribonucleoproteins are also polar cohesin barriers, indicating that stalling is intrinsic to cohesin itself, and other proteins can substitute for CTCF in fruit flies and other eukaryotes. Finally, we show that RNA-DNA hybrids (R-loops) block cohesin-mediated DNA compaction in vitro and are enriched with cohesin subunits in vivo, likely forming TAD boundaries. Our results provide direct evidence that CTCF orientation and R-loops shape the 3D genome by directly regulating cohesin.

Objective. To develop a metaphase chromosome model representing the complete genome of a human lymphocyte cell to support microscopic Monte Carlo (MMC) simulation-based radiation-induced DNA damage studies. Approach. We first employed coarse-grained polymer physics simulation to obtain a rod-shaped chromatid segment of 730 nm in diameter and 460 nm in height to match Hi–C data. We then voxelized the segment with a voxel size of 11 nm per side and connected the chromatid with 30 types of pre-constructed nucleosomes and 6 types of linker DNAs in base pair (bp) resolutions. Afterward, we piled different numbers of voxelized chromatid segments to create 23 pairs of chromosomes of 1–5 μm long. Finally, we arranged the chromosomes at the cell metaphase plate of 5.5 μm in radius to create the complete set of metaphase chromosomes. We implemented the model in gMicroMC simulation by denoting the DNA structure in a four-level hierarchical tree: nucleotide pairs, nucleosomes and linker DNAs, chromatid segments, and chromosomes. We applied the model to compute DNA damage under different radiation conditions and compared the results to those obtained with G0/G1 model and experimental measurements. We also performed uncertainty analysis for relevant simulation parameters. Main results. The chromatid segment was successfully voxelized and connected in bps resolution, containing 26.8 mega bps (Mbps) of DNA. With 466 segments, we obtained the metaphase chromosome containing 12.5 Gbps of DNA. Applying it to compute the radiation-induced DNA damage, the obtained results were self-consistent and agreed with experimental measurements. Through the parameter uncertainty study, we found that the DNA damage ratio between metaphase and G0/G1 phase models was not sensitive to the chemical simulation time. The damage was also not sensitive to the specific parameter settings in the polymer physics simulation, as long as the produced metaphase model followed a similar contact map distribution. Significance. Experimental data reveal that ionizing radiation induced DNA damage is cell cycle dependent. Yet, DNA chromosome models, except for the G0/G1 phase, are not available in the state-of-the-art MMC simulation. For the first time, we successfully built a metaphase chromosome model and implemented it into MMC simulation for radiation-induced DNA damage computation.

International audience; Our understanding of the physical principles organizing the genome in the nucleus is limited by the lack of tools to directly exert and measure forces on interphase chromosomes in vivo and probe their material nature. Here, we introduce an approach to actively manipulate a genomic locus using controlled magnetic forces inside the nucleus of a living human cell. We observed viscoelastic displacements over micrometers within minutes in response to near-piconewton forces, which are consistent with a Rouse polymer model. Our results highlight the fluidity of chromatin, with a moderate contribution of the surrounding material, revealing minor roles for cross-links and topological effects and challenging the view that interphase chromatin is a gel-like material. Our technology opens avenues for future research in areas from chromosome mechanics to genome functions.

Cohesin folds mammalian interphase chromosomes by extruding the chromatin fiber into numerous loops. “Loop extrusion” can be impeded by chromatin-bound factors, such as CTCF, which generates characteristic and functional chromatin organization patterns. It has been proposed that transcription relocalizes or interferes with cohesin, and that active promoters are cohesin loading sites. However, the effects of transcription on cohesin have not been reconciled with observations of active extrusion by cohesin. To determine how transcription modulates extrusion, we studied mouse cells in which we could alter cohesin abundance, dynamics, and localization by genetic ‘knockouts’ of the cohesin regulators CTCF and Wapl. Through Hi-C experiments, we discovered intricate, cohesin-dependent contact patterns near active genes. Chromatin organization around active genes exhibited hallmarks of interactions between transcribing RNA polymerases (RNAPs) and extruding cohesins. These observations could be reproduced by polymer simulations in which RNAPs were “moving barriers” to extrusion that obstructed, slowed, and pushed cohesins. The simulations predicted that preferential loading of cohesin at promoters is inconsistent with our experimental data. Additional ChIP-seq experiments showed that the putative cohesin loader Nipbl is not predominantly enriched at promoters. Therefore, we propose that cohesin is not preferentially loaded at promoters and that the barrier function of RNAP accounts for cohesin accumulation at active promoters. Altogether, we find that RNAP is a new type of extrusion barrier that is not stationary, but rather, translocates and relocalizes cohesin. Loop extrusion and transcription might interact to dynamically generate and maintain gene interactions with regulatory elements and shape functional genomic organization.Significance StatementLoop extrusion by cohesin is critical to folding the mammalian genome into loops. Extrusion can be halted by CTCF proteins bound at specific genomic loci, which generates chromosomal domains and can regulate gene expression. However, the process of transcription itself can modulate cohesin, thus refolding chromosomes near active genes. Through experiments and simulations, we show that transcribing RNA polymerases (RNAPs) act as “moving barriers” to loop-extruding cohesins. Unlike stationary CTCF barriers, RNAPs actively relocalize cohesins, which generates characteristic patterns of spatial organization around active genes. Our model predicts that the barrier function of RNAP can explain why cohesin accumulates at active promoters and provides a mechanism for clustering active promoters. Through transcription-extrusion interactions, cells might dynamically regulate functional genomic contacts.

Cellular structures are continually subjected to forces, which may serve as mechanical signals for cells through their effects on biomolecule interaction kinetics. Typically, molecular complexes interact via “slip bonds,” so applied forces accelerate off rates by reducing transition energy barriers. However, biomolecules with multiple dissociation pathways may have considerably more complicated force dependencies. This is the case for DNA-binding proteins that undergo “facilitated dissociation,” in which competitor biomolecules from solution enhance molecular dissociation in a concentration-dependent manner. Using simulations and theory, we develop a generic model that shows that proteins undergoing facilitated dissociation can form an alternative type of molecular bond, known as a “catch bond,” for which applied forces suppress protein dissociation. This occurs because the binding by protein competitors responsible for the facilitated dissociation pathway can be inhibited by applied forces. Within the model, we explore how the force dependence of dissociation is regulated by intrinsic factors, including molecular sensitivity to force and binding geometry, and the extrinsic factor of competitor protein concentration. We find that catch bonds generically emerge when the force dependence of the facilitated unbinding pathway is stronger than that of the spontaneous unbinding pathway. The sharpness of the transition between slip- and catch-bond kinetics depends on the degree to which the protein bends its DNA substrate. These force-dependent kinetics are broadly regulated by the concentration of competitor biomolecules in solution. Thus, the observed catch bond is mechanistically distinct from other known physiological catch bonds because it requires an extrinsic factor – competitor proteins – rather than a specific intrinsic molecular structure. We hypothesize that this mechanism for regulating force-dependent protein dissociation may be used by cells to modulate protein exchange, regulate transcription, and facilitate diffusive search processes.Statement of significanceMechanotransduction regulates chromatin structure and gene transcription. Forces may be transduced via biomolecular interaction kinetics, particularly, how molecular complexes dissociate under stress. Typically, molecules form “slip bonds” that dissociate more rapidly under tension, but some form “catch bonds” that dissociate more slowly under tension due to their internal structure. We develop a model for a distinct type of catch bond that emerges via an extrinsic factor: protein concentration in solution. Underlying this extrinsic catch bond is “facilitated dissociation,” whereby competing proteins from solution accelerate protein-DNA unbinding by invading the DNA binding site. Forces may suppress invasion, inhibiting dissociation, as for catch bonds. Force-dependent facilitated dissociation can thus govern the kinetics of proteins sensitive to local DNA conformation and mechanical state.

The nucleus houses, organizes, and protects chromatin to ensure genome integrity and proper gene expression, but how the nucleus adapts mechanically to changes in the extracellular environment is poorly understood. Recent studies have revealed that extracellular physical stresses induce chromatin compaction via mechanotransductive processes. We report that increased extracellular multivalent cations lead to increased heterochromatin levels through activation of mechanosensitive ion channels, without large-scale cell stretching. In cells with perturbed chromatin or lamins, this increase in heterochromatin suppresses nuclear blebbing associated with nuclear rupture and DNA damage. Through micromanipulation force measurements, we show that this increase in heterochromatin increases chromatin-based nuclear rigidity, which protects nuclear morphology and function. In addition, transduction of elevated extracellular cations rescues nuclear morphology in model and patient cells of human diseases, including progeria and the breast cancer model cell line MDA-MB-231. We conclude that nuclear mechanics, morphology, and function can be modulated by cell sensing of the extracellular environment through mechanosensitive ion channels and consequent changes to histone modification state and chromatin-based nuclear rigidity.

Chromatin, which consists of DNA and associated proteins, contains genetic information and is a mechanical component of the nucleus. Heterochromatic histone methylation controls nucleus and chromosome stiffness, but the contribution of heterochromatin protein HP1α (CBX5) is unknown. We used a novel HP1α auxin-inducible degron human cell line to rapidly degrade HP1α. Degradation did not alter transcription, local chromatin compaction, or histone methylation, but did decrease chromatin stiffness. Single-nucleus micromanipulation reveals that HP1α is essential to chromatin-based mechanics and maintains nuclear morphology, separate from histone methylation. Further experiments with dimerization-deficient HP1αI165Eindicate that chromatin crosslinking via HP1α dimerization is critical, while polymer simulations demonstrate the importance of chromatin-chromatin crosslinkers in mechanics. In mitotic chromosomes, HP1α similarly bolsters stiffness while aiding in mitotic alignment and faithful segregation. HP1α is therefore a critical chromatin-crosslinking protein that provides mechanical strength to chromosomes and the nucleus throughout the cell cycle and supports cellular functions.

Our understanding of the physical principles organizing the genome in the nucleus is limited by the lack of tools to directly exert and measure forces on interphase chromosomes in vivo and probe their material nature. Here, we present a novel approach to actively manipulate a genomic locus using controlled magnetic forces inside the nucleus of a living human cell. We observe viscoelastic displacements over microns within minutes in response to near-picoNewton forces, which are well captured by a Rouse polymer model. Our results highlight the fluidity of chromatin, with a moderate contribution of the surrounding material, revealing the minor role of crosslinks and topological effects and challenging the view that interphase chromatin is a gel-like material. Our new technology opens avenues for future research, from chromosome mechanics to genome functions.

Compaction of chromosomes is essential for reliable transmission of genetic information. Experiments suggest that this ∼ 1000-fold compaction is driven by condensin complexes that extrude chromatin loops, i.e., progressively collect chromatin fiber from one or both sides of the complex to form a growing loop. Theory indicates that symmetric two-sided loop extrusion can achieve such compaction, but recent single-molecule studies observed diverse dynamics of condensins that perform one-sided, symmetric two-sided, and asymmetric two-sided extrusion.We use simulations and theory to determine how these molecular properties lead to chromosome compaction. High compaction can be achieved if even a small fraction of condensins have two essential properties: a long residence time and the ability to perform two-sided (not necessarily symmetric) extrusion. In mixtures of condensins I and II, coupling of two-sided extrusion and stable chromatin binding by condensin II promotes compaction. These results provide missing connections between single-molecule observations and chromosome-scale organization.

The cell nucleus houses the chromosomes, which are linked to a soft shell of lamin filaments. Experiments indicate that correlated chromosome dynamics and nuclear shape fluctuations arise from motor activity. To identify the physical mechanisms, we develop a model of an active, crosslinked Rouse chain bound to a polymeric shell. System-sized correlated motions occur but require both motor activity {\it and} crosslinks. Contractile motors, in particular, enhance chromosome dynamics by driving anomalous density fluctuations. Nuclear shape fluctuations depend on motor strength, crosslinking, and chromosome-lamina binding. Therefore, complex chromatin dynamics and nuclear shape emerge from a minimal, active chromosome-lamina system., 18 pages, 21 figures

SMC complexes, such as condensin or cohesin, organize chromatin throughout the cell cycle by a process known as loop extrusion. SMC complexes reel in DNA, extruding and progressively growing DNA loops. Modeling assuming two-sided loop extrusion reproduces key features of chromatin organization across different organisms. In vitro single-molecule experiments confirmed that yeast condensins extrude loops, however, they remain anchored to their loading sites and extrude loops in a ‘one-sided’ manner. We therefore simulate one-sided loop extrusion to investigate whether ‘one-sided’ complexes can compact mitotic chromosomes, organize interphase domains, and juxtapose bacterial chromosomal arms, as can be done by ‘two-sided’ loop extruders. While one-sided loop extrusion cannot reproduce these phenomena, variants can recapitulate in vivo observations. We predict that SMC complexes in vivo constitute effectively two-sided motors or exhibit biased loading and propose relevant experiments. Our work suggests that loop extrusion is a viable general mechanism of chromatin organization., eLife digest The different molecules of DNA in a cell are called chromosomes, and they change shape dramatically when cells divide. Ordinarily, chromosomes are packaged by proteins called histones to make thick fibres called chromatin. Chromatin fibres are further folded into a sparse collection of loops. These loops are important not only to make genetic material fit inside a cell, but also to make distant regions of the chromosomes interact with each other, which is important to regulate gene activities. The fibres compact to prepare for cell division: they fold into a much denser series of loops. This is a remarkable physical feat in which tiny protein machines wrangle lengthy strands of DNA. A process called loop extrusion could explain how chromatin folding works. In this process, ring-like protein complexes known as SMC complexes would act as motors that can form loops. SMC complexes could bind a chromatin fibre and reel it in to form the loops, with the density of loops increasing before cell division to further compact the chromosomes. Looping by SMC complexes has been observed in a variety of cell types, including mammalian and bacterial cells. From these studies, loop extrusion is generally assumed to be ‘two-sided’. This means that each SMC complex reels in the chromatin on both sides of it, thus growing the chromatin loop. However, imaging individual SMC complexes bound to single molecules of DNA showed that extrusion can be asymmetric, or ‘one-sided’. These observations show the SMC complex remains anchored in place and the chromatin is reeled in and extruded by only one side of the complex. So Banigan, van den Berg, Brandão et al. created a computer model to test whether the mechanism of one-sided extrusion could produce chromosomes that are organised, compact, and ready for cell division, like two-sided extrusion can. To answer this question, Banigan, van den Berg, Brandão et al. analysed imaging experiments and data that had been collected using a technique that captures how chromatin fibres are arranged inside cells. This was paired with computer simulations of chromosomes bound by SMC protein complexes. The simulations and analysis found that the simplest one-sided loop extrusion complexes generally cannot reproduce the same patterns of chromatin loops as two-sided complexes. However, a few specific variations of one-sided extrusion can actually recapitulate correct chromatin folding and organisation. These results show that some aspects of chromosome organization can be attained by one-sided extrusion, but many require two-sided extrusion. Banigan, van den Berg, Brandão et al. explain how the simulated mechanisms of loop extrusion could be consistent with seemingly contradictory observations from different sets of experiments. Altogether, they demonstrate that loop extrusion is a viable general mechanism to explain chromatin organisation, and that it likely possesses physical capabilities that have yet to be observed experimentally.

Chromosomes are organized as chromatin loops that promote segregation, enhancer-promoter interactions, and other genomic functions. Loops were hypothesized to form by ‘loop extrusion,’ by which structural maintenance of chromosomes (SMC) complexes, such as condensin and cohesin, bind to chromatin, reel it in, and extrude it as a loop. However, such exotic motor activity had never been observed. Following an explosion of indirect evidence, recent single-molecule experiments directly imaged DNA loop extrusion by condensin and cohesin in vitro. These experiments observe rapid (kb/s) extrusion that requires ATP hydrolysis and stalls under pN forces. Surprisingly, condensin extrudes loops asymmetrically, challenging previous models. Extrusion by cohesin is symmetric but requires the protein Nipbl. We discuss how SMC complexes may perform their functions on chromatin in vivo.

The cell nucleus houses, protects, and arranges the genome within the cell. Therefore, nuclear mechanics and morphology are important for dictating gene regulation, and these properties are perturbed in many human diseases, such as cancers and progerias. The field of nuclear mechanics has long been dominated by studies of the nuclear lamina, the intermediate filament shell residing just beneath the nuclear membrane. However, a growing body of work shows that chromatin and chromatin-related factors within the nucleus are an essential part of the mechanical response of the cell nucleus to forces. Recently, our group demonstrated that chromatin and the lamina provide distinct mechanical contributions to nuclear mechanical response. The lamina is indeed important for robust response to large, whole-nucleus stresses, but chromatin dominates the short-extension response. These findings offer a clarifying perspective on varied nuclear mechanics measurements and observations, and they suggest several new exciting possibilities for understanding nuclear morphology, organization, and mechanics.

We study a Brownian dynamics simulation model of a biopolymeric shell deformed by axial forces exerted at opposing poles. The model exhibits two distinct linear force-extension regimes, with the response to small tensions governed by linear elasticity and the response to large tensions governed by an effective spring constant that scales with radius as R−0.25. When extended beyond the initial linear elastic regime, the shell undergoes a hysteretic, temperature-dependent buckling transition. We experimentally observe this buckling transition by stretching and imaging the lamina of isolated cell nuclei. Furthermore, the interior contents of the shell can alter mechanical response and buckling, which we show by simulating a model for the nucleus that quantitatively agrees with our micromanipulation experiments stretching individual nuclei.

Micromanipulation force measurements of single isolated nuclei at physiological strains and strain rates reveal two distinct cell nuclear mechanical regimes differentially governed by chromatin and lamin A. Chromatin and its histone-modification compaction govern short extension, and the lamin A amount dictates long-extension strain stiffening., The cell nucleus must continually resist and respond to intercellular and intracellular mechanical forces to transduce mechanical signals and maintain proper genome organization and expression. Altered nuclear mechanics is associated with many human diseases, including heart disease, progeria, and cancer. Chromatin and nuclear envelope A-type lamin proteins are known to be key nuclear mechanical components perturbed in these diseases, but their distinct mechanical contributions are not known. Here we directly establish the separate roles of chromatin and lamin A/C and show that they determine two distinct mechanical regimes via micromanipulation of single isolated nuclei. Chromatin governs response to small extensions (

The cell nucleus encloses, organizes, and protects the genome. Chromatin maintains nuclear mechanical stability and shape in coordination with lamins and the cytoskeleton. Abnormal nuclear shape is a diagnostic marker for human diseases, and it can cause nuclear dysfunction. Chromatin mechanics underlies this link, as alterations to chromatin and its physical properties can disrupt or rescue nuclear shape. The cell can regulate nuclear shape through mechanotransduction pathways that sense and respond to extracellular cues, thus modulating chromatin compaction and rigidity. These findings reveal how chromatin's physical properties can regulate cellular function and drive abnormal nuclear morphology and dysfunction in disease.

Chromosome segregation during cell division depends on interactions of kinetochores with dynamic microtubules (MTs). In many eukaryotes, each kinetochore binds multiple MTs, but the collective behavior of these coupled MTs is not well understood. We present a minimal model for collective kinetochore-MT dynamics, based on in vitro measurements of individual MTs and their dependence on force and kinetochore phosphorylation by Aurora B kinase. For a system of multiple MTs connected to the same kinetochore, the force-velocity relation has a bistable regime with two possible steady-state velocities: rapid shortening or slow growth. Bistability, combined with the difference between the growing and shrinking speeds, leads to center-of-mass and breathing oscillations in bioriented sister kinetochore pairs. Kinetochore phosphorylation shifts the bistable region to higher tensions, so that only the rapidly shortening state is stable at low tension. Thus, phosphorylation leads to error correction for kinetochores that are not under tension. We challenged the model with new experiments, using chemically induced dimerization to enhance Aurora B activity at metaphase kinetochores. The model suggests that the experimentally observed disordering of the metaphase plate occurs because phosphorylation increases kinetochore speeds by biasing MTs to shrink. Our minimal model qualitatively captures certain characteristic features of kinetochore dynamics, illustrates how biochemical signals such as phosphorylation may regulate the dynamics, and provides a theoretical framework for understanding other factors that control the dynamics in vivo.

Chromatin decompaction via increasing euchromatin or decreasing heterochromatin results in a softer nucleus and abnormal nuclear blebbing, independent of lamin perturbations. Conversely, increasing heterochromatin stiffens the nucleus and rescues nuclear morphology in lamin-perturbed cells that present abnormal nuclear morphology., Nuclear shape and architecture influence gene localization, mechanotransduction, transcription, and cell function. Abnormal nuclear morphology and protrusions termed “blebs” are diagnostic markers for many human afflictions including heart disease, aging, progeria, and cancer. Nuclear blebs are associated with both lamin and chromatin alterations. A number of prior studies suggest that lamins dictate nuclear morphology, but the contributions of altered chromatin compaction remain unclear. We show that chromatin histone modification state dictates nuclear rigidity, and modulating it is sufficient to both induce and suppress nuclear blebs. Treatment of mammalian cells with histone deacetylase inhibitors to increase euchromatin or histone methyltransferase inhibitors to decrease heterochromatin results in a softer nucleus and nuclear blebbing, without perturbing lamins. Conversely, treatment with histone demethylase inhibitors increases heterochromatin and chromatin nuclear rigidity, which results in reduced nuclear blebbing in lamin B1 null nuclei. Notably, increased heterochromatin also rescues nuclear morphology in a model cell line for the accelerated aging disease Hutchinson–Gilford progeria syndrome caused by mutant lamin A, as well as cells from patients with the disease. Thus, chromatin histone modification state is a major determinant of nuclear blebbing and morphology via its contribution to nuclear rigidity.

The binding of transcription factors (TFs) to DNA controls most aspects of cellular function, making the understanding of their binding kinetics imperative. The standard description of bimolecular interactions posits TF off-rates are independent of TF concentration in solution. However, recent observations have revealed that proteins in solution can accelerate the dissociation of DNA-bound proteins. To study the molecular basis of facilitated dissociation (FD), we have used single-molecule imaging to measure dissociation kinetics of Fis, a key E. coli TF and major bacterial nucleoid protein, from single dsDNA binding sites. We observe a strong FD effect characterized by an exchange rate ∽1 × 104 M-1s-1, establishing that FD of Fis occurs at the single-binding-site level, and we find that the off-rate saturates at large Fis concentrations in solution. While spontaneous (i.e., competitor-free) dissociation shows a strong salt dependence, we find that facilitated dissociation depends only weakly on salt. These results are quantitatively explained by a model in which partially dissociated bound proteins are susceptible to invasion by competitor proteins in solution. We also report FD of NHP6A, a yeast TF whose structure differs significantly from Fis. We further perform molecular dynamics simulations, which indicate that FD can occur for molecules that interact far more weakly than those we have studied. Taken together, our results indicate that FD is a general mechanism assisting in the local removal of TFs from their binding sites and does not necessarily require cooperativity, clustering, or binding site overlap.SIGNIFICANCE STATEMENTTranscription factors (TFs) control biological processes by binding and unbinding to DNA. Therefore it is crucial to understand the mechanisms that affect TF binding kinetics. Recent studies challenge the standard picture of TF binding kinetics by demonstrating cases of proteins in solution accelerating TF dissociation rates through a facilitated dissociation (FD) process. Our study shows that FD can occur at the level of single binding sites, without the action of large protein clusters or long DNA segments. Our results quantitatively support a model of FD in which competitor proteins invade partially dissociated states of DNA-bound TFs. FD is expected to be a general mechanism for modulating gene expression by altering the occupancy of TFs on the genome.Author ContributionsRamsey I. Kamardesigned research, performed research, contributed new reagents/analytic tools, analyzed data, wrote the paperEdward J. Banigandesigned research, performed research, contributed new reagents/analytic tools, analyzed data, wrote the paperAykut Erbasdesigned research, performed research, contributed new reagents/analytic tools, analyzed data, wrote the paperRebecca D. Giuntolidesigned research, performed research, contributed new reagents/analytic tools, analyzed data, wrote the paperMonica Olvera de la Cruzdesigned research, performed research, wrote the paperReid C. Johnsondesigned research, performed research, contributed new reagents/analytic tools, analyzed data, wrote the paperJohn F. Markodesigned research, performed research, contributed new reagents/analytic tools, analyzed data, wrote the paper

Free-flowing granular media can quickly become jammed above a critical density. Nonlinear dynamical systems analysis now suggests that jamming arises from the interaction between the density of instabilities and the propagation of disturbances throughout the material.

We introduce a short correlation length for torque in twisting-stiff biomolecules, which is necessary for the physical property that torque fluctuations be finite in amplitude. We develop a nonequilibrium theory of dynamics of DNA twisting which predicts two crossover time scales for temporal torque correlations in single-molecule experiments. Bending fluctuations can be included, and at linear order we find that they do not affect the twist dynamics. However, twist fluctuations affect bending, and we predict the spatial inhomogeneity of twist, torque, and buckling arising in nonequilibrium “rotor-bead” experiments.

Enzymatic “machines,” such as catalytic rods or colloids, can self-propel and interact by generating gradients of their substrates. We theoretically investigate the behaviors of such machines in a chemically active environment where their catalytic substrates are continuously synthesized and destroyed, as occurs in living cells. We show how the kinetic properties of the medium modulate self-propulsion and pairwise interactions between machines, with the latter controlled by a tunable characteristic interaction range analogous to the Debye screening length in an electrolytic solution. Finally, we discuss the effective force arising between interacting machines and possible biological applications, such as partitioning of bacterial plasmids.

Chemokines have a central role in regulating processes essential to the immune function of T cells, such as their migration within lymphoid tissues and targeting of pathogens in sites of inflammation. Here we track T cells using multi-photon microscopy to demonstrate that the chemokine CXCL10 enhances the ability of CD8+ T cells to control the pathogen Toxoplasma gondii in the brains of chronically infected mice. This chemokine boosts T-cell function in two different ways: it maintains the effector T-cell population in the brain and speeds up the average migration speed without changing the nature of the walk statistics. Notably, these statistics are not Brownian; rather, CD8+ T-cell motility in the brain is well described by a generalized Lévy walk. According to our model, this unexpected feature enables T cells to find rare targets with more than an order of magnitude more efficiency than Brownian random walkers. Thus, CD8+ T-cell behaviour is similar to Lévy strategies reported in organisms ranging from mussels to marine predators and monkeys, and CXCL10 aids T cells in shortening the average time taken to find rare targets.

We determine the elastic constants and the Raman spectra of spinel under pressure using density functional perturbation theory. Like other spinels, MgAl 2 O 4 shows an increasing bulk modulus and a weakly varying shear modulus under pressure. The Raman spectrum is dominated by two mains peaks whose intensities decrease under pressure. The theoretical Raman spectra of disordered spinel shows a better agreement with measurements on natural samples than the ordered one. We also investigate the high-pressure stability of different structures and find the calcium titanate type to be the stable phase beyond 45 GPa.

The rate of dissociation of a DNA-protein complex is often considered to be a property of that complex, without dependence on other nearby molecules in solution. We study the kinetics of dissociation of the abundant Escherichia coli nucleoid protein Fis from DNA, using a single-molecule mechanics assay. The rate of Fis dissociation from DNA is strongly dependent on the solution concentration of DNA. The off-rate (k(off)) of Fis from DNA shows an initially linear dependence on solution DNA concentration, characterized by an exchange rate of k(ex)≈9×10(-4) (ng/μl)(-1) s(-1) for 100 mM univalent salt buffer, with a very small off-rate at zero DNA concentration. The off-rate saturates at approximately k(off,max)≈8×10(-3) s(-1) for DNA concentrations above ≈20 ng/μl. This exchange reaction depends mainly on DNA concentration with little dependence on the length of the DNA molecules in solution or on binding affinity, but this does increase with increasing salt concentration. We also show data for the yeast HMGB protein NHP6A showing a similar DNA-concentration-dependent dissociation effect, with faster rates suggesting generally weaker DNA binding by NHP6A relative to Fis. Our results are well described by a model with an intermediate partially dissociated state where the protein is susceptible to being captured by a second DNA segment, in the manner of "direct transfer" reactions studied for other DNA-binding proteins. This type of dissociation pathway may be important to protein-DNA binding kinetics in vivo where DNA concentrations are large.

The three-dimensional positions of immune cells can be tracked in live tissues precisely as a function of time using two-photon microscopy. However, standard methods of analysis used in the field and experimental artifacts can bias interpretations and obscure important aspects of cell migration such as directional migration and non-Brownian walk statistics. Therefore, methods were developed for minimizing drift artifacts, identifying directional and anisotropic (asymmetric) migration, and classifying cell migration statistics. These methods were applied to describe the migration statistics of CD8+ T cells in uninflamed lymph nodes. Contrary to current models, CD8+ T cell statistics are not well described by a straightforward persistent random walk model. Instead, a model in which one population of cells moves via Brownian-like motion and another population follows variable persistent random walks with noise reproduces multiple statistical measures of CD8+ T cell migration in the lymph node in the absence of inflammation., Author Summary Migration is fundamental to immune cell function, and accurate quantitative methods are crucial for analyzing and interpreting migration statistics. However, existing methods of analysis cannot uniquely describe cell behavior and suffer from various limitations. This complicates efforts to address questions such as to what extent chemotactic signals direct cellular behaviors and how random migration of many cells leads to coordinated immune response. We therefore develop methods that provide a complete description of migration with a minimum of assumptions and describe specific quantities for characterizing directional motion. Using numerical simulations and experimental data, we evaluate these measures and discuss methods to minimize the effects of experimental artifacts. These methodologies may be applied to various migrating cells or organisms. We apply our approach to an important model system, T cells migrating in lymph node. Surprisingly, we find that the canonical Brownian-walker-like model does not accurately describe migration. Instead, we find that T cells move heterogeneously and are described by a two-population model of persistent and diffusive random walkers. This model is completely different from the generalized Lévy walk model that describes activated T cells in brains infected with Toxoplasma gondii, indicating that T cells exhibit distinct migration statistics in different tissues.

We introduce a short correlation length for torque in twisting-stiff biomolecules, which is necessary for the physical property that torque fluctuations be finite in amplitude. We develop a nonequilibrium theory of dynamics of DNA twisting which predicts two crossover time scales for temporal torque correlations in single-molecule experiments. Bending fluctuations can be included, and at linear order we find that they do not affect the twist dynamics. However, twist fluctuations affect bending, and we predict the spatial inhomogeneity of twist, torque, and buckling arising in nonequilibrium "rotor-bead" experiments.

A wide variety of cell biological and biomimetic systems use actin polymerization to drive motility. It has been suggested that an object such as a bacterium can propel itself by self-assembling a high concentration of actin behind it, if it is repelled by actin. However, it is also known that it is essential for the moving object to bind actin. Therefore, a key question is how the actin tail can propel an object when it both binds and repels the object. We present a physically consistent Brownian dynamics model for actin-based motility that includes the minimal components of the dendritic nucleation model and allows for both attractive and repulsive interactions between actin and a moveable disc. We find that the concentration gradient of filamentous actin generated by polymerization is sufficient to propel the object, even with moderately strong binding interactions. Additionally, actin binding can act as a biophysical cap, and may directly control motility through modulation of network growth. Overall, this mechanism is robust in that it can drive motility against a load up to a stall pressure that depends on the Young's modulus of the actin network and can explain several aspects of actin-based motility.

We present a theoretical model describing metaphase chromosome oscillations, microtubule (MT) attachment error correction, and anaphase chromosome separation. During metaphase, chromosome pairs align near the center of a bipolar MT spindle and oscillate as the MTs attaching them to the cell poles polymerize and depolymerize. Simultaneously, the cell fixes misaligned chromosome pairs by some tension-dependent mechanism. In anaphase, chromosome pairs separate as depolymerizing MTs pull each chromosome toward its respective cell pole. Instead of including all known components to develop a comprehensive, species-specific description, we introduce a minimal model based on fundamental properties of MT kinetics. We use the tension-dependence of single MT polymerization/depolymerization kinetics measured by Akiyoshi et al. [1] and assume the same functional dependence for compressed MTs. We apply these to a many MT model, and solve this stochastic model numerically and by a master equation approach. We find that the tension dependence of rates enhances the speed of single chromosome pulling by MTs during anaphase- or error-correction-like behavior. Additionally, the force-velocity curve for a single chromosome attached to dynamic MTs exhibits bi-stability: at high loads, large tension fluctuations induce MTs to spontaneously switch from a depolymerizing state into a polymerizing state. The system is hysteretic; to recover depolymerization from the polymerizing state, the load must be decreased to a far smaller value than that required to initially induce polymerization. This behavior leads to the chromosome oscillations we observe in the two-chromosome system. Interestingly, we observe breathing oscillations, which are not captured by any other chromosome oscillation model. Our minimal model reflects general features of the underlying mechanisms of these phenomena, and reveals how different components control chromosome dynamics through the rate constants.[1] Akiyoshi et al. (2010) Nature 468, 576-579.

Single-molecule DNA experiments have revealed a number of observations about the mechanical properties of DNA, but little is known about the dynamics of twist and bend in these experiments. To understand these experiments, we develop and solve a stochastic and elastic model for twist dynamics in a DNA molecule that is driven at one end and dissipates torsional energy through viscous drag at the other end. To ensure that torque fluctuations remain finite in our model, we include a term depending on the gradient-squared of twist strain. We find that the boundary conditions of our system quantize the spectrum of DNA twisting modes and modulate the dynamics in a striking way. To see this, we derive a nonequilibrium fluctuation relation for twist and construct the spatiotemporal correlation function. We find that a novel elastic correlation length controls spatial correlations between twist fluctuations. In the middle of a long DNA molecule, twist correlations initially decay as t−1/4 and crossover to t−1/2 decay at longer times. In contrast, in a shorter molecule, correlations decay exponentially with a decay time that depends on the magnitude of the viscous drag. For nearly straight DNA molecules, twist dynamics are unaffected by bending fluctuations at linear order, even though writhing dynamics are modulated by twist at this order. Our model for twist dynamics suggests several new single-molecule experiments, which could shed further light on DNA mechanics and dynamics.