32 research outputs found
Response to comment on textquoteleftInitiation of chromosome replication controls both division and replication cycles in E. coli through a double-adder mechanismtextquoteright
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
Conformation of Circular DNA in 2 Dimensions
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 is topologically invariant and
equal to the SAW value (in the present case ), and that the topology
and dimensionality of the system strongly influences the cross-over between the
rigid regime and the self-avoiding regime at a scale .
Additionally, the bond correlation function scales with the molecular length
as predicted. For molecular lengths , circular DNA behaves
like a stiff molecule with approximately elliptic shape.Comment: 4 pages, 5 figure
Organization and Dynamics of the Living E. Coli Nucleoid at High Resolution in Space and Time
Cooperative kinking at distant sites in mechanically stressed DNA
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° 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 DN
Persistence length and scaling properties of single-stranded DNA adsorbed on modified graphite
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
Conformation of Ring Polymers in 2D Constrained Environments
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
Interplay of DNA supercoiling and catenation during the segregation of sister duplexes
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 viv
Immune cell extravasation in an organ-on-chip to model lung inflammation.
Acute respiratory distress syndrome (ARDS) is a severe lung condition with high mortality and various causes, including lung infection. No specific treatment is currently available and more research aimed at better understanding the pathophysiology of ARDS is needed. Most lung-on-chip models that aim at mimicking the air-blood barrier are designed with a horizontal barrier through which immune cells can migrate vertically, making it challenging to visualize and investigate their migration. In addition, these models often lack a barrier of natural protein-derived extracellular matrix (ECM) suitable for live cell imaging to investigate ECM-dependent migration of immune cells as seen in ARDS. This study reports a novel inflammation-on-chip model with live cell imaging of immune cell extravasation and migration during lung inflammation. The three-channel perfusable inflammation-on-chip system mimics the lung endothelial barrier, the ECM environment and the (inflamed) lung epithelial barrier. A chemotactic gradient was established across the ECM hydrogel, leading to the migration of immune cells through the endothelial barrier. We found that immune cell extravasation depends on the presence of an endothelial barrier, on the ECM density and stiffness, and on the flow profile. In particular, bidirectional flow, broadly used in association with rocking platforms, was found to importantly delay extravasation of immune cells in contrast to unidirectional flow. Extravasation was increased in the presence of lung epithelial tissue. This model is currently used to study inflammation-induced immune cell migration but can be used to study infection-induced immune cell migration under different conditions, such as ECM composition, density and stiffness, type of infectious agents used, and the presence of organ-specific cell types
Fractal Dimension and Localization of DNA Knots
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 gyration
radius of the adsorbed DNA knot scales with the 3D Flory exponent within error. In (ii), we find , a value between the 3D
and 2D () exponents, indicating an incomplete 2D relaxation or a
different polymer universality class. Compelling evidence is also presented for
the localization of the knot crossings in 2D.Comment: 4 pages, 3 figure
Brain endothelial tricellular junctions as novel sites for T cell diapedesis across the blood–brain barrier
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