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The size of coarse sedimentary particles in fluvial systems is key for quantifying sedimentation and transport conditions in both active and ancient fluvial systems. In particular, the grain size of the bed load in gravel-bed rivers allows inferring information on sediment entrainment or deposition mechanisms, and on the hydraulic conditions controlling them. However, collecting data on such coarse-grained sedimentary particles traditionally involved time-intensive and costly fieldwork, leading to the development of image-based techniques for grain size estimation over the last two decades. Nevertheless, despite much progress and the recent deployment of deep learning methods that were trained on large datasets (i.e., > 100 000 manually annotated grains; Lang et al., 2021; Chen et al., 2022), image-based grain size data is limited to single percentile values, often due to a systematic bias and/or a low accuracy (e.g., Chardon et al., 2020; Mair et al., 2022). Specifically, the core challenge for most existing methods is the accurate segmentation, i.e., the identification and delineation of individual grains, across distinctly different types of data. Here we present a new approach designated to improve the segmentation in images, which is based on the capability of transfer learning of deep learning models. Such a strategy allows us to re-train existing models for new tasks that are similar to their original purpose. In particular, we use the python-based and open-source tool cellpose (Stringer et al., 2021), which is a state-of-the-art machine-learning model based on neural networks and designed to segment cells in biomedical images. We retrained such a cellpose model on several image datasets of fluvial gravel. The rationale for our approach is based on an inferred geometric similarity between cell nuclei and rock pebbles. Our re-trained models outperform existing methods designed for the segmentation of fluvial pebbles in all datasets, despite an order of magnitude smaller number of training data than currently used in machine learning models. Furthermore, our results show that models trained on specialized datasets for a specific sediment setting yield significantly better results than models trained on larger and more diverse datasets. Fortunately, the model’s flexibility, accessibility, and ability for easy and fast training (Pachitariu and Stringer, 2022) enable the training of task- or image-type-specific models. To facilitate the segmentation power of such models, we built an open-source software tool, ImageGrains. This tool allows for easy use of the models we trained, or of other custom models, as well as streamlined grain size and shape measurements. This allows for fast and nearly automated measurements of large numbers of coarse sedimentary particles with high precision and across vastly different image settings. ReferencesChardon, V., et al., 2022: River Res. Appl., 38, 358–367, https://doi.org/10.1002/rra.3910.Chen, X., et al., 2022: Earth Surf. Dyn., 10, 349–366, https://doi.org/10.5194/esurf-10-349-2022.Lang, N., et al. 2021: Hydrol. Earth Syst. Sci., 25, 2567–2597, https://doi.org/10.5194/hess-25-2567-2021.Mair, D., et al. 2022: Earth Surf. Dyn., 10, 953–973, https://doi.org/10.5194/esurf-10-953-2022.Pachitariu, M. and Stringer, C. 2022: Nat. Methods, 19, 1634–1641, https://doi.org/10.1038/s41592-022-01663-4.Stringer, C., et al. 2021: Nat. Methods, 18, 100–106, https://doi.org/10.1038/s41592-020-01018-x.

Pairing of homologous chromosomes represents a critical step of meiosis in nearly all sexually reproducing species. While in some organisms meiotic pairing requires programmed DNA breakage and recombination, in many others it engages homologous chromosomes that remain apparently intact. The mechanistic nature of such recombination-independent pairing represents a fundamental question in molecular genetics. Using ‘meiotic silencing by unpaired DNA’ (MSUD) in Neurospora crassa as a model process, we demonstrate the existence of a cardinally different approach to DNA homology recognition in meiosis. The main advantage of MSUD over other experimental systems lies in its ability to identify any relatively short DNA fragment lacking a homologous allelic partner. Here we show that MSUD does not rely on the canonical mechanism of meiotic recombination, yet it is promoted by REC8, a conserved component of the meiotic cohesin complex. We also show that certain patterns of interspersed homology are recognized as pairable during MSUD. Such patterns need to be co-linear and must contain short tracts of sequence identity spaced apart with a periodicity of 21 or 22 base-pairs. By using these values as a guiding parameter in all-atom molecular modeling, we discover that homologous double-stranded DNA molecules can associate by forming quadruplex-based contacts with an interval of 2.5 helical turns, which requires right-handed plectonemic coiling and additional conformational changes in the intervening double-helical segments. These results (i) reconcile genetic and biophysical lines of evidence for the existence of direct homologous dsDNA-dsDNA pairing, (ii) identify a role for this process in initiating post-transcriptional silencing, and (iii) suggest that chromosomes are cross-matched in meiosis by a precise mechanism that operates on intact double-stranded DNA molecules.

The pairing of homologous chromosomes represents a critical step of meiosis in nearly all sexually reproducing species. In many organisms, pairing involves chromosomes that remain apparently intact. The mechanistic nature of homology recognition at the basis of such pairing is unknown. Using "meiotic silencing by unpaired DNA" (MSUD) as a model process, we demonstrate the existence of a cardinally different approach to DNA homology recognition in meiosis. The main advantage of MSUD over other experimental systems lies in its ability to identify any relatively short DNA fragment lacking a homologous allelic partner. Here, we show that MSUD does not rely on the canonical mechanism of meiotic recombination, yet it is promoted by REC8, a conserved component of the meiotic cohesion complex. We also show that certain patterns of interspersed homology are recognized as pairable during MSUD. Such patterns need to be colinear and must contain short tracts of sequence identity spaced apart at 21 or 22 base pairs. By using these periodicity values as a guiding parameter in all-atom molecular modeling, we discover that homologous DNA molecules can pair by forming quadruplex-based contacts with an interval of 2.5 helical turns. This process requires right-handed plectonemic coiling and additional conformational changes in the intervening double-helical segments. Our results 1) reconcile genetic and biophysical evidence for the existence of direct homologous double-stranded DNA (dsDNA)-dsDNA pairing, 2) identify a role for this process in initiating RNA interference, and 3) suggest that chromosomes can be cross-matched by a precise mechanism that operates on intact dsDNA molecules.

The migration of activated T cells across the blood–brain barrier (BBB) is a critical step in central nervous system (CNS) immune surveillance and inflammation. Whereas T cell diapedesis across the intact BBB seems to occur preferentially through the BBB cellular junctions, impaired BBB integrity during neuroinflammation is accompanied by increased transcellular T cell diapedesis. The underlying mechanisms directing T cells to paracellular versus transcellular sites of diapedesis across the BBB remain to be explored. By combining in vitro live-cell imaging of T cell migration across primary mouse brain microvascular endothelial cells (pMBMECs) under physiological flow with serial block-face scanning electron microscopy (SBF-SEM), we have identified BBB tricellular junctions as novel sites for T cell diapedesis across the BBB. Downregulated expression of tricellular junctional proteins or protein-based targeting of their interactions in pMBMEC monolayers correlated with enhanced transcellular T cell diapedesis, and abluminal presence of chemokines increased T cell diapedesis through tricellular junctions. Our observations assign an entirely novel role to BBB tricellular junctions in regulating T cell entry into the CNS. This article has an associated First Person interview with the first author of the paper., Highlighted Article: Ultrastructural analysis of T cell migration across the blood–brain barrier (BBB) under physiological flow identifies BBB tricellular junctions as sites of T cell diapedesis.

Last year we published an article (Witz et al., 2019) in which we used time-lapse microscopy in combination with microfluidics to measure growth, division and replication in single E. coli cells on the one hand, and developed a new statistical analysis method to calculate the ability of different cell cycle models to capture the correlation structure observed in the data on the other hand. This led us to propose a new model of cell cycle control in E. coli which we called the double-adder model.Recently Le Treut et al. published a comment (Le Treut et al., 2020) on our article which made a number of highly critical claims, including allegations that our own data support a different model than the one we proposed, and that our model cannot reproduce the ‘adder phenotype’ observed in the data. We here show that all these allegations are false and based on basic analysis errors. Although our focus is on explaining the errors in the analysis of Le Treut et al, we have attempted to make the presentation of interest to a broader scientific audience by discussing the issues in the context of what our current understanding is of the bacterial cell cycle, and to what extent recent data either support or reject various proposed models.

Clostridium perfringens β-toxin (CPB) is a highly active β-pore-forming toxin (β-PFT) and the essential virulence factor for fatal, necro-hemorrhagic enteritis in animals and humans. The molecular mechanisms involved in CPB's action on its target, the endothelium of small intestinal vessels, are poorly understood. Here, we identify platelet endothelial cell adhesion molecule-1 (CD31 or PECAM-1) as the specific membrane receptor for CPB on endothelial cells. CD31 expression corresponds with the cell-type specificity of CPB, and it is essential for toxicity in cultured cells and mice. Ectopic CD31 expression renders resistant cells and liposomes susceptible to CPB-induced membrane damage. Moreover, the extracellular Ig6 domain of mouse, human, and porcine CD31 is essential for the interaction with CPB. Hence, our results explain the cell-type specificity of CPB in vitro and in the natural disease caused by C. perfringens type C.

Living cells proliferate by completing and coordinating two essential cycles, a division cycle that controls cell size, and a DNA replication cycle that controls the number of chromosomal copies in the cell. Despite lacking dedicated cell cycle control regulators such as cyclins in eukaryotes, bacteria such as E. coli manage to tightly coordinate those two cycles across a wide range of growth conditions, including situations where multiple nested rounds of replication progress simultaneously. Various cell cycle models have been proposed to explain this feat, but it has been impossible to validate them so far due to a lack of experimental tools for systematically testing their different predictions. Recently new insights have been gained on the division cycle through the study of the structure of fluctuations in growth, size, and division in individual cells. In particular, it was found that cell size appears to be controlled by an adder mechanism, i.e. the added volume between divisions is held approximately constant and fluctuates independently of growth rate and cell size at birth. However, how replication initiation is regulated and coupled to cell size control remains unclear, mainly due to scarcity of experimental measurements on replication initiation at the single-cell level. Here, we used time-lapse microscopy in combination with microfluidics to directly measure growth, division and replication in thousands of single E. coli cells growing in both slow and fast growth conditions. In order to compare different phenomenological models of the cell cycle, we introduce a statistical framework which assess their ability to capture the correlation structure observed in the experimental data. Using this in combination with stochastic simulations, our data indicate that, instead of thinking of the cell cycle as running from birth to division, the cell cycle is controlled by two adder mechanisms starting at the initiation of replication: the added volume since the last initiation event controls the timing of both the next division event and the next replication initiation event. Interestingly the double-adder mechanism identified in this study has recently been found to explain the more complex cell cycle of mycobacteria, suggesting shared control strategies across species.

SUMMARYClostridium perfringensβ-toxin (CPB) is a highly active hemolysin β-pore forming toxin and the essential virulence factor for a severe, necro-hemorrhagic enteritis in animals and humans.In vivoandin vitroit exerts a remarkable cell type specificity towards endothelial cells, platelets and some leucocytic cell lines. The target cell specificity of CPB is, however, poorly understood and a receptor explaining this selective toxicity has not been identified. This has hampered further research into the pathogenesis ofC. perfringenstype C induced enteritis. Here we identify Platelet Endothelial Cell Adhesion Molecule-1 (CD31 or PECAM-1) as the specific membrane receptor for CPB on endothelial cells. CD31 expression is essential for CPB toxicity in endothelial cells and lethality in mice and sufficient to render previously resistant cells highly susceptible to the toxin. We further demonstrate, that the extracellular membrane proximal Ig6 domain of CD31 is required for the interaction with CPB and that expression of CD31 corresponds with the specificity of the toxin towards cultured cell lines. Our results thus provide an explanation for the cell type specificity of CPB and can be linked to the characteristic lesions observed a devastating enteric disease in animals and humans.

International audience; Mammalian mitotic chromosome morphogenesis was analyzed by 4D live-cell and snapshot deconvolution fluorescence imaging. Prophase chromosomes, whose organization was previously unknown, are revealed to comprise co-oriented sister linear loop arrays displayed along a single, peripheral, regularly kinked topoisomerase II/cohesin/condensin II axis. Thereafter, rather than smooth, progressive compaction as generally envisioned, progression to metaphase is a discontinuous process involving chromosome expansion as well as compaction. At late prophase, dependent on topoisomerase II and with concomitant cohesin release, chromosomes expand, axes split and straighten, and chromatin loops transit to a radial disposition around now-central axes. Finally, chromosomes globally compact, giving the metaphase state. These patterns are consistent with the hypothesis that the molecular events of chromosome morphogenesis are governed by accumulation and release of chromosome stress, created by chromatin compaction and expansion. Chromosome state could evolve analogously throughout the cell cycle.

In cells, DNA is routinely subjected to significant levels of bending and twisting. In some cases, such as under physiological levels of supercoiling, DNA can be so highly strained, that it transitions into non-canonical structural conformations that are capable of relieving mechanical stress within the template. DNA minicircles offer a robust model system to study stress-induced DNA structures. Using DNA minicircles on the order of 100 bp in size, we have been able to control the bending and torsional stresses within a looped DNA construct. Through a combination of cryo-EM image reconstructions, Bal31 sensitivity assays and Brownian dynamics simulations, we have been able to analyze the effects of biologically relevant underwinding-induced kinks in DNA on the overall shape of DNA minicircles. Our results indicate that strongly underwound DNA minicircles, which mimic the physical behavior of small regulatory DNA loops, minimize their free energy by undergoing sequential, cooperative kinking at two sites that are located about 180 degrees apart along the periphery of the minicircle. This novel form of structural cooperativity in DNA demonstrates that bending strain can localize hyperflexible kinks within the DNA template, which in turn reduces the energetic cost to tightly loop DNA.

Using numerical simulations, we compare properties of knotted DNA molecules that are either torsionally relaxed or supercoiled. We observe that DNA supercoiling tightens knotted portions of DNA molecules and accentuates the difference in curvature between knotted and unknotted regions. The increased curvature of knotted regions is expected to make them preferential substrates of type IIA topoisomerases because various earlier experiments have concluded that type IIA DNA topoisomerases preferentially interact with highly curved DNA regions. The supercoiling-induced tightening of DNA knots observed here shows that torsional tension in DNA may serve to expose DNA knots to the unknotting action of type IIA topoisomerases, and thus explains how these topoisomerases could maintain a low knotting equilibrium in vivo, even for long DNA molecules.

Chromosomal and plasmid DNA molecules in bacterial cells are maintained under torsional tension and are therefore supercoiled. With the exception of extreme thermophiles, supercoiling has a negative sign, which means that the torsional tension diminishes the DNA helicity and facilitates strand separation. In consequence, negative supercoiling aids such processes as DNA replication or transcription that require global- or local-strand separation. In extreme thermophiles, DNA is positively supercoiled which protects it from thermal denaturation. While the role of DNA supercoiling connected to the control of DNA stability, is thoroughly researched and subject of many reviews, a less known role of DNA supercoiling emerges and consists of aiding DNA topoisomerases in DNA decatenation and unknotting. Although DNA catenanes are natural intermediates in the process of DNA replication of circular DNA molecules, it is necessary that they become very efficiently decatenated, as otherwise the segregation of freshly replicated DNA molecules would be blocked. DNA knots arise as by-products of topoisomerase-mediated intramolecular passages that are needed to facilitate general DNA metabolism, including DNA replication, transcription or recombination. The formed knots are, however, very harmful for cells if not removed efficiently. Here, we overview the role of DNA supercoiling in DNA unknotting and decatenation.

Recent studies reveal that the bacterial nucleoid has a defined, self-adherent shape and an underlying longitudinal organization and comprises a viscoelastic matrix. Within this shape, mobility is enhanced by ATP-dependent processes and individual loci can undergo ballistic off-equilibrium movements. In Escherichia coli, two global dynamic nucleoid behaviors emerge pointing to nucleoid-wide accumulation and relief of internal stress. Sister segregation begins with local splitting of individual loci, which is delayed at origin, terminus and specialized interstitial snap regions. Globally, as studied in several systems, segregation is a multi-step process in which internal nucleoid state plays critical roles that involve both compaction and expansion. The origin and terminus regions undergo specialized programs partially driven by complex ATP burning mechanisms such as a ParAB Brownian ratchet and a septum-associated FtsK motor. These recent findings reveal strong, direct parallels among events in different systems and between bacterial nucleoids and mammalian chromosomes with respect to physical properties, internal organization and dynamic behaviors.

Aim: Atomic force microscopy nanoindentation of myofibers was used to assess and quantitatively diagnose muscular dystrophies from human patients. Materials & methods: Myofibers were probed from fresh or frozen muscle biopsies from human dystrophic patients and healthy volunteers, as well as mice models, and Young’s modulus stiffness values were determined. Results: Fibers displaying abnormally low mechanical stability were detected in biopsies from patients affected by 11 distinct muscle diseases, and Young’s modulus values were commensurate to the severity of the disease. Abnormal myofiber resistance was also observed from consulting patients whose muscle condition could not be detected or unambiguously diagnosed otherwise. Discussion & conclusion: This study provides a proof-of-concept that atomic force microscopy yields a quantitative read-out of human muscle function from clinical biopsies, and that it may thereby complement current muscular dystrophy diagnosis.

Mitotic chromosomes are among the most recognizable structures in the cell, yet for over a century their internal organization remains largely unsolved. We applied chromosome conformation capture methods, 5C and Hi-C, across the cell cycle and revealed two alternative three-dimensional folding states of the human genome. We show that the highly compartmentalized and cell-type-specific organization described previously for non-synchronous cells is restricted to interphase. In metaphase, we identify a homogenous folding state, which is locus-independent, common to all chromosomes, and consistent among cell types, suggesting a general principle of metaphase chromosome organization. Using polymer simulations, we find that metaphase Hi-C data is inconsistent with classic hierarchical models, and is instead best described by a linearly-organized longitudinally compressed array of consecutive chromatin loops.

Visualization of living E. coli nucleoids, defined by HupA-mCherry, reveals a discrete, dynamic helical ellipsoid. Three basic features emerge. (i) Nucleoid density efficiently coalesces into longitudinal bundles, giving a stiff, low DNA density ellipsoid. (ii) This ellipsoid is radially confined within the cell cylinder. Radial confinement gives helical shape and drives and directs global nucleoid dynamics, including sister segregation. (iii) Longitudinal density waves flux back and forth along the nucleoid, with 5–10% of density shifting within 5s, enhancing internal nucleoid mobility. Furthermore, sisters separate end-to-end in sequential discontinuous pulses, each elongating the nucleoid by 5–15%. Pulses occur at 20min intervals, at defined cell cycle times. This progression is mediated by sequential installation and release of programmed tethers, implying cyclic accumulation and relief of intra-nucleoid mechanical stress. These effects could comprise a chromosome-based cell cycle engine. Overall, the presented results suggest a general conceptual framework for bacterial nucleoid morphogenesis and dynamics.

Circular DNA molecules in vivo form catenanes and knots during such processes as replication or recombination. In addition, DNA molecules are often subjected to a torsional tension, which results in their supercoiling. The interplay between catenation, knotting and supercoiling leads to unexpected conformational changes of entire DNA molecules, and this has interesting physical and biological consequences. We show in this chapter how modelling DNA as a semi-flexible ribbon can be applied to get information about the molecular conformation of DNA molecules with complex topologies. In particular, our results highlight the importance of the chirality of knots and catenanes in the structural changes induced by DNA supercoiling. For example, strongly linked right-handed toroidal DNA catenanes undergo a specific folding that can be reversed by the introduction of negative supercoiling in each chain, or the shape of negatively supercoiled DNA trefoil knots depends on their chirality. In each case, we perform dynamical simulations including hydrodynamics, to investigate the consequences of these structural changes on the sedimentation and gel electrophoretic behaviour of the modelled knotted and catenated DNA molecules.

Living fluorescently-labeled E.coli nucleoids were imaged at ∼75nm and >2sec resolution. Non-replicating single-chromosome nucleoids are fat helicoidal ellipsoids. They are denser centrally than peripherally and have a strong intrinsic tendency to split longitudinally into two, sometimes more, subellipsoids. Nucleoid density distributions and splitting patterns fluctuate on

The combination of ring closure and spatial constraints has a fundamental effect on the statistics of semiflexible polymers such as DNA. However, studies of the interplay between circularity and constraints are scarce and single-molecule experimental data concerning polymer conformations are missing. By means of atomic force microscopy we probe the conformation of circular DNA molecules in two dimensions and in the concentrated regime (above the overlap concentration c*). Molecules in this regime experience a collapse, and their statistical properties agree very well with those of simulated vesicles under pressure. Some circular molecules also create confining regions in which other molecules are trapped. Thus we show further that spatially confined molecules fold into specific conformations close to those found for linear chains, and strongly dependent on the size of the confining box.

We have characterized the polymer physics of single-stranded DNA (ssDNA) using atomic force microscopy. The persistence length l(p) of circular ssDNA adsorbed on a modified graphite surface was determined independently of secondary structure. At a very low ionic strength we obtained l(p)=9.1 nm from the bond correlation function. Increasing the salt concentration lead to a decrease in l(p); at 1 mM NaCl we found l(p)=6.7 nm, while at 10 mM NaCl a value l(p)=4.6 nm was obtained. The persistence length was also extracted from the root-mean-square end-to-end distance and the end-to-end distance distribution function. Finally, we have investigated the scaling behavior using the two latter quantities, and found that on long length scales ssDNA behaves as a two-dimensional self-avoiding walk.

12 páginas, 8 figuras -- PAGS nros. 3845-3853, The discrete regulation of supercoiling, catenation and knotting by DNA topoisomerases is well documented both in vivo and in vitro, but the interplay between them is still poorly understood. Here we studied DNA catenanes of bacterial plasmids arising as a result of DNA replication in Escherichia coli cells whose topoisomerase IV activity was inhibited. We combined high-resolution two-dimensional agarose gel electrophoresis with numerical simulations in order to better understand the relationship between the negative supercoiling of DNA generated by DNA gyrase and the DNA interlinking resulting from replication of circular DNA molecules. We showed that in those replication intermediates formed in vivo, catenation and negative supercoiling compete with each other. In interlinked molecules with high catenation numbers negative supercoiling is greatly limited. However, when interlinking decreases, as required for the segregation of newly replicated sister duplexes, their negative supercoiling increases. This observation indicates that negative supercoiling plays an active role during progressive decatenation of newly replicated DNA molecules in vivo., Spanish Ministerio de Ciencia e Innovacion[BIO2005-02224, BFU2008-00408/BMC, BFU2007-62670]; Swiss National Science Foundation[3100A0-116275]

The conformation of circular DNA molecules of various lengths adsorbed in a 2D conformation on a mica surface is studied. The results confirm the conjecture that the critical exponent nu is topologically invariant and equal to the self-avoiding walk value (in the present case nu=3/4), and that the topology and dimensionality of the system strongly influence the crossover between the rigid regime and the self-avoiding regime at a scale L approximately 7l{p}. Additionally, the bond correlation function scales with the molecular length L as predicted. For molecular lengths Lor=5l{p}, circular DNA behaves like a stiff molecule with an approximately elliptic shape.

We study the behavior of single-stranded DNA (ssDNA) in the presence of well-known drugs with either an intercalating binding mode, such as daunorubicin, actinomycin D, and chloroquine, or a minor groove binding mode, such as netropsin and berenil, by atomic force microscopy (AFM). At very low salt conditions, ssDNA molecules adopt an unstructured conformation without secondary structures. We observe that under these conditions additions of drugs that bind to double-stranded DNA ( dsDNA) promote the formation of secondary structures in ssDNA. Furthermore, with an increase of concentration of the drugs, the extension as well as the thermal stabilization of these hairpins was observed.

Atomic force microscopy was used to image single-stranded DNA (ssDNA) adsorbed on mica modified by Mg2+, by 3-aminopropyltriethoxysilane or on modified highly oriented pyrolytic graphite (HOPG). ssDNA molecules on mica have compact structures with lumps, loops and super twisting, while on modified HOPG graphite ssDNA molecules adopt a conformation without secondary structures. We have shown that the immobilization of ssDNA under standard conditions on modified HOPG eliminates intramolecular base-pairing, thus this method could be important for studying certain processes involving ssDNA in more details.

The scaling properties of DNA knots of different complexities were studied by atomic force microscope. Following two different protocols DNA knots are adsorbed onto a mica surface in regimes of (i) strong binding, that induces a kinetic trapping of the three-dimensional (3D) configuration, and of (ii) weak binding, that permits (partial) relaxation on the surface. In (i) the radius of gyration of the adsorbed DNA knot scales with the 3D Flory exponent ν 0.60 within error. In (ii), we find ν 0.66, a value between the 3D and 2D (ν=3/4) exponents. Evidence is also presented for the localization of knot crossings in 2D under weak adsorption conditions. © 2007 The American Physical Society., We thank the Swiss National Science Foundation Grant No. 200021-101851, NSERC and the Canada Research Chairs program, and the Spanish Plan Nacional de I+D+I for support

The organization and morphogenesis of chromosomes as they prepare to segregate during cell division has fascinated researchers since their first microscopic visualizations. On page XXX of this issue, Naumova et al. (1) combine chromosome conformation capture with polymer physics simulations to see inside metaphase chromosomes and elucidate their principles of organization. A new, yet satisfyingly familiar, view emerges. Electron microscopic visualizations provide compelling evidence that chromosomes are organized as loops (see the figure). Naumova et al.’s analysis suggests that chromosome organization is a highly probabilistic process that, without much specification, yields a robust longitudinal assembly of loop bases running along the length of the chromosome. The model reproduces known properties of mitotic chromosomes, including variable positioning of loci and spatial mixing of regions of intermediate (megabase) size. Although average loop size cannot be determined from chromosome capture data, the loop size necessary for the simulations to match experimental results was 80 kilobases, very similar to experimental estimates. The proposed model is highly attractive in its simplicity and robustness. A linear array of chromatin loops forms, at consecutive but apparently random positions, and then is longitudinally compressed. These few rules ensure that chromosomes globally always fold into the same shape, without requiring precise specification of either highly conserved genetic loci or specific length scales. Figure 1 Meiotic and mitotic chromosomes The model of Naumova et al. supports the seminal description of Ulrich Laemmli, formulated nearly 40 years ago (2). Laemmli envisioned a constraining protein network located centrally within the chromosome. This “scaffolding” brings segments together into loops, thereby shaping the chromatin to give rod-like chromosomal bodies rather than spherical balls (3). The underlying organization comprises a linearly folded assembly of scaffold associated regions (SARs), rich in the bases adenine (A) and thymine (T), yielding an “AT queue” (3–5). Topoisomerase II, condensin and AT-hook architectural proteins synergistically collaborate with SARs to give shape (3–5). Compellingly, the AT queue–scaffold model explains classic cytological G/R banding patterns (5). Importantly, in the model of Naumova et al., positioning of loop bases appears random. However, chromosome capture analysis provides a population average description. Different cells may stochastically choose different SARs from a larger array of possibilities. [The common textbook model of highly ordered scaffold and loops was never Laemmli’s view.] In Naumova et al.’s model, the compressed loop array could run along the chromosome in a straight path or a meandering path. Experimental data define a topoisomerase II–condensin core lying radially within the center of the chromosome and running along its entire length (4, 6). However, the substructure of this scaffold may well be complex (with folds or coils) (4, 5). Laemmli postulated that loops emanate outward radially from the scaffold (7). Naumova et al.’s model does not specify a radial array but, similarly, envisions loops emanating out from the central axial feature. Metaphase chromosome organization has also been proposed to comprise a hierarchical folding of larger domains (8) or connectivity spread uniformly through the chromatin (9). Naumova et al. exclude these two models. At prophase of meiosis, chromosomes comprise linear arrays of loops whose bases are decorated with proteins, including topoisomerase II and condensin (10). In yeast, loop-base sequences are locally AT-rich regions (11). In mammals, loop sizes for meiotic prophase chromosomes are the same as for mitotic metaphase chromosomes (~100 kb). The two cases are obviously related. Remarkably, when a mammalian chromosome segment is introduced into yeast, it acquires the same meiotic prophase organization (loop size and spacing) as the endogenous yeast genome (12). Loop organization is thus clearly determined by probabilistic selection of a subset of available sequences. Also, alterations in structural axis components can alter, and increase variability of, loop size (13). Maybe loop size is determined in part by “chromatin fiber” persistence length: loops would form down to the minimum size compatible with fiber stiffness. In the model of Naumova et al., a linear array of continuous chromatin loops forms and then undergoes longitudinal compression. It will not be surprising if the first stage corresponds to prophase (mitotic and meiotic), while the second stage corresponds to evolution to the metaphase state. The findings of Naumova et al. will reinvigorate discussions of late-stage mitotic chromosome organization and morphogenesis. What is the molecular nature of the final axial compressed loop array? It may comprise a DNA-protein meshwork, as meiotic prophase chromosome axes (10), rather than a continuous protein-protein structure. Primary components of the metaphase scaffold all mediate DNA-DNA linkages. Most strikingly, if newt metaphase chromosomes are treated with deoxyribonuclease, they simply disappear, rather than leaving behind a discrete core (9). Also of interest is how mature mitotic chromosomes can be forcibly extended, allowed to retract, and then extended again for multiple successive cycles. Are protein links metastable? Do they act as “slip-rings”? Does an open DNA-protein meshwork allows reversible deformation without disruption of protein-mediated DNA-DNA links? It is also unclear how sister chromatids acquire their side-by-side metaphase relationship. Perhaps loosely connected sister chromatids become longitudinally organized in parallel, and then further compact, with stress on linkages between sisters promoting their further removal (5). However, classically, mitotic and meiotic prophase chromosomes appear as morphologically single units, hinting at a more complex situation. Finally, by comparing chromosome capture data for interphase and metaphase, Naumova et al. show that mitotic chromosomes lose major features observed previously for interphase stages, e.g. cell-type–specific spatial segregation of open domains and closed domains. How epigenetic information for cell-type–specific gene expression is retained during the passage through mitosis is unknown. Are relevant elements locally marked? Or are some cell-type–specific interactions maintained but invisible at the current level of resolution? These answers to these questions remain for the future, with imaging, conformation capture, polymer modeling, and genetic analysis all destined to play key roles.

Using numerical simulations, we compare properties of knotted DNA molecules that are either torsionally relaxed or supercoiled. We observe that DNA supercoiling tightens knotted portions of DNA molecules and accentuates the difference in curvature between knotted and unknotted regions. The increased curvature of knotted regions is expected to make them preferential substrates of type IIA topoisomerases because various earlier experiments have concluded that type IIA DNA topoisomerases preferentially interact with highly curved DNA regions. The supercoiling-induced tightening of DNA knots observed here shows that torsional tension in DNA may serve to expose DNA knots to the unknotting action of type IIA topoisomerases, and thus explains how these topoisomerases could maintain a low knotting equilibrium in vivo, even for long DNA molecules.

The bond orientational correlation function (BCF) of a semiflexible ring polymer on a flat surface is studied theoretically. For a stiff chain, we give an exact analytic form of BCF with perturbation calculations. For a chain sufficiently longer than its persistence length, the conventional exponential decay vanishes and a long-range order along the chain contour appears. We demonstrate that the bond orientational correlation satisfies the scaling properties, and construct an interpolating formula for its universal curve that encompasses the short- and large-distance behaviors. Our analytical findings are confirmed by extensive Langevin dynamics simulations, and are in excellent agreement with recent experimental data obtained from DNA molecules imaged by atomic force microscopy without any fitting parameters.

Two-dimensional semiflexible polymer rings are studied both by imaging circular DNA adsorbed on a mica surface and by Monte Carlo simulations of phantom polymers as well as of polymers with finite thickness. Comparison of size and shape of the different models over the full range of flexibilities shows that excluded volume caused by finite thickness induces an anisotropic increase of the main axes of the conformations, a change of shape, accomplished by an enhanced correlation along the contour., 5 pages, 4 figures, version as published in Nano Letters

Replication of circular DNA proceeds through a stage of multiply interlinked catenanes that have to be rapidly spatially separated. In addition, knotting of circular DNA has also to be avoided. In bacteria, topology simplification requires participation of two type II topoisomerases: gyrase and topo IV. Several simulation approaches were applied to explain the very efficient topology simplification in that system. Mainly two strategies were explored: in the first one, the system follows its free energy gradient influenced by supercoiling, and in the second one, specific geometrical rules are defined for the selection of strand passages (hooking, chirality). The Monte-Carlo methods usually used to estimate the efficiency of these strategies do not allow to follow DNA topology simplification dynamically, to evaluate its speed, for example. To overcome this limitation, we simulated DNA unknotting and decatenation by Brownian dynamics, which allows for a natural integration of the strategies mentioned above. By following the topological state of the simulated DNA chains (see figure), we show that the combination of supercoiling and local geometrical selection rules provides an important drive for unknotting and decatenation, especially at low topological complexity.View Large Image | View Hi-Res Image | Download PowerPoint Slide