Condensin extrudes DNA loops in steps up to hundreds of base pairs that are generated by ATP binding events.

The condensin SMC protein complex organizes chromosomal structure by extruding loops of DNA. Its ATP-dependent motor mechanism remains unclear but likely involves steps associated with large conformational changes within the ∼50 nm protein complex. Here, using high-resolution magnetic tweezers, we resolve single steps in the loop extrusion process by individual yeast condensins. The measured median step sizes range between 20-40 nm at forces of 1.0-0.2 pN, respectively, comparable with the holocomplex size. These large steps show that, strikingly, condensin typically reels in DNA in very sizeable amounts with ∼200 bp on average per single extrusion step at low force, and occasionally even much larger, exceeding 500 bp per step. Using Molecular Dynamics simulations, we demonstrate that this is due to the structural flexibility of the DNA polymer at these low forces. Using ATP-binding-impaired and ATP-hydrolysis-deficient mutants, we find that ATP binding is the primary step-generating stage underlying DNA loop extrusion. We discuss our findings in terms of a scrunching model where a stepwise DNA loop extrusion is generated by an ATP-binding-induced engagement of the hinge and the globular domain of the SMC complex.

The progression of replication forks at natural replication barriers in live bacteria.

Protein-DNA complexes are one of the principal barriers the replisome encounters during replication. One such barrier is the Tus-ter complex, which is a direction dependent barrier for replication fork progression. The details concerning the dynamics of the replisome when encountering these Tus-ter barriers in the cell are poorly understood. By performing quantitative fluorescence microscopy with microfuidics, we investigate the effect on the replisome when encountering these barriers in live Escherichia coli cells. We make use of an E. coli variant that includes only an ectopic origin of replication that is positioned such that one of the two replisomes encounters a Tus-ter barrier before the other replisome. This enables us to single out the effect of encountering a Tus-ter roadblock on an individual replisome. We demonstrate that the replisome remains stably bound after encountering a Tus-ter complex from the non-permissive direction. Furthermore, the replisome is only transiently blocked, and continues replication beyond the barrier. Additionally, we demonstrate that these barriers affect sister chromosome segregation by visualizing specific chromosomal loci in the presence and absence of the Tus protein. These observations demonstrate the resilience of the replication fork to natural barriers and the sensitivity of chromosome alignment to fork progression.

Quantitative Analysis of Intracellular Fluorescent Foci in Live Bacteria.

Fluorescence microscopy has revolutionized in vivo cellular biology. Through the specific labeling of a protein of interest with a fluorescent protein, one is able to study movement and colocalization, and even count individual proteins in a live cell. Different algorithms exist to quantify the total intensity and position of a fluorescent focus. Although these algorithms have been rigorously studied for in vitro conditions, which are greatly different than the in-homogenous and variable cellular environments, their exact limits and applicability in the context of a live cell have not been thoroughly and systematically evaluated. In this study, we quantitatively characterize the influence of different background subtraction algorithms on several focus analysis algorithms. We use, to our knowledge, a novel approach to assess the sensitivity of the focus analysis algorithms to background removal, in which simulated and experimental data are combined to maintain full control over the sensitivity of a focus within a realistic background of cellular fluorescence. We demonstrate that the choice of algorithm and the corresponding error are dependent on both the brightness of the focus, and the cellular context. Expectedly, focus intensity estimation and localization accuracy suffer in all algorithms at low focus to background ratios, with the bacteroidal background subtraction in combination with the median excess algorithm, and the region of interest background subtraction in combination with a two-dimensional Gaussian fit algorithm, performing the best. We furthermore show that the choice of background subtraction algorithm is dependent on the expression level of the protein under investigation, and that the localization error is dependent on the distance of a focus from the bacterial edge and pole. Our results establish a set of guidelines for what signals can be analyzed to give a targeted spatial and intensity accuracy within a bacterial cell.

A simple self-calibrating method to measure the height of fluorescent molecules and beads at nanoscale resolution.

We describe a simple self-calibrating technique, incident-beam interference sweeping, for measuring the height of fluorescent labels. Using a tilted back-reflecting mirror and a scanning laser beam, a modulated fluorescence emission allows height determination of a label from a surface with a resolution of ∼ 3 nm. In addition, we show that the absolute distance of a label from the top-mounted mirror can be determined with a resolution of a few tens of nanometers over a micrometer range.

Magnetic torque tweezers: measuring torsional stiffness in DNA and RecA-DNA filaments.

We introduce magnetic torque tweezers, which enable direct single-molecule measurements of torque. Our measurements of the effective torsional stiffness C of dsDNA indicated a substantial force dependence, with C = approximately 40 nm at low forces up to C = approximately 100 nm at high forces. The initial torsional stiffness of RecA filaments was nearly twofold larger than that for dsDNA, yet at moderate torques further build-up of torsional strain was prevented.

Electron beam fabrication of a microfluidic device for studying submicron-scale bacteria.

BACKGROUND: Controlled restriction of cellular movement using microfluidics allows one to study individual cells to gain insight into aspects of their physiology and behaviour. For example, the use of micron-sized growth channels that confine individual Escherichia coli has yielded novel insights into cell growth and death. To extend this approach to other species of bacteria, many of whom have dimensions in the sub-micron range, or to a larger range of growth conditions, a readily-fabricated device containing sub-micron features is required. RESULTS: Here we detail the fabrication of a versatile device with growth channels whose widths range from 0.3 µm to 0.8 µm. The device is fabricated using electron beam lithography, which provides excellent control over the shape and size of different growth channels and facilitates the rapid-prototyping of new designs. Features are successfully transferred first into silicon, and subsequently into the polydimethylsiloxane that forms the basis of the working microfluidic device. We demonstrate that the growth of sub-micron scale bacteria such as Lactococcus lactis or Escherichia coli cultured in minimal medium can be followed in such a device over several generations. CONCLUSIONS: We have presented a detailed protocol based on electron beam fabrication together with specific dry etching procedures for the fabrication of a microfluidic device suited to study submicron-sized bacteria. We have demonstrated that both Gram-positive and Gram-negative bacteria can be successfully loaded and imaged over a number of generations in this device. Similar devices could potentially be used to study other submicron-sized organisms under conditions in which the height and shape of the growth channels are crucial to the experimental design.

MukBEF-dependent chromosomal organization in widened Escherichia coli.

The bacterial chromosome is spatially organized through protein-mediated compaction, supercoiling, and cell-boundary confinement. Structural Maintenance of Chromosomes (SMC) complexes are a major class of chromosome-organizing proteins present throughout all domains of life. Here, we study the role of the Escherichia coli SMC complex MukBEF in chromosome architecture and segregation. Using quantitative live-cell imaging of shape-manipulated cells, we show that MukBEF is crucial to preserve the toroidal topology of the Escherichia coli chromosome and that it is non-uniformly distributed along the chromosome: it prefers locations toward the origin and away from the terminus of replication, and it is unevenly distributed over the origin of replication along the two chromosome arms. Using an ATP hydrolysis-deficient MukB mutant, we confirm that MukBEF translocation along the chromosome is ATP-dependent, in contrast to its loading onto DNA. MukBEF and MatP are furthermore found to be essential for sister chromosome decatenation. We propose a model that explains how MukBEF, MatP, and their interacting partners organize the chromosome and contribute to sister segregation. The combination of bacterial cell-shape modification and quantitative fluorescence microscopy paves way to investigating chromosome-organization factors in vivo.

Non-bias-limited tracking of spherical particles, enabling nanometer resolution at low magnification.

We present a three-dimensional tracking routine for nondiffraction-limited particles, which significantly reduces pixel bias. Our technique allows for increased resolution compared to that of previous methods, especially at low magnification or at high signal/noise ratio. This enables tracking with nanometer accuracy in a wide field of view and tracking of many particles. To reduce bias induced by pixelation, the tracking algorithm uses interpolation of the image on a circular grid to determine the x-, y-, and z-positions. We evaluate the proposed algorithm by tracking simulated images and compare it to well-known center-of-mass and cross-correlation methods. The final resolution of the described method improves up to an order of magnitude in three dimensions compared to conventional tracking methods. We show that errors in x,y-tracking can seriously affect z-tracking if interpolation is not used. We validate our results with experimental data obtained for conditions matching those used in the simulations. Finally, we show that the increased performance of the proposed algorithm uniquely enables it to extract accurate data for the persistence length and end-to-end distance of 107 DNA tethers in a single experiment.

Force generation by dynamic microtubules.

The assembly and disassembly of microtubules can generate pushing and pulling forces that, together with motor proteins, contribute to the correct positioning of chromosomes, mitotic spindles and nuclei in cells. In vitro experiments combined with modeling have shed light on the intrinsic capability of dynamic microtubules to generate force, and various observations of positioning processes in cells and model systems have shown how pushing and pulling forces are used in different situations. A sophisticated set of microtubule-end-binding proteins is responsible for steering dynamic microtubules toward their cellular target and regulating the pushing and/or pulling forces that are generated once contact is established.

Assembly dynamics of microtubules at molecular resolution.

Microtubules are highly dynamic protein polymers that form a crucial part of the cytoskeleton in all eukaryotic cells. Although microtubules are known to self-assemble from tubulin dimers, information on the assembly dynamics of microtubules has been limited, both in vitro and in vivo, to measurements of average growth and shrinkage rates over several thousands of tubulin subunits. As a result there is a lack of information on the sequence of molecular events that leads to the growth and shrinkage of microtubule ends. Here we use optical tweezers to observe the assembly dynamics of individual microtubules at molecular resolution. We find that microtubules can increase their overall length almost instantaneously by amounts exceeding the size of individual dimers (8 nm). When the microtubule-associated protein XMAP215 (ref. 6) is added, this effect is markedly enhanced and fast increases in length of about 40-60 nm are observed. These observations suggest that small tubulin oligomers are able to add directly to growing microtubules and that XMAP215 speeds up microtubule growth by facilitating the addition of long oligomers. The achievement of molecular resolution on the microtubule assembly process opens the way to direct studies of the molecular mechanism by which the many recently discovered microtubule end-binding proteins regulate microtubule dynamics in living cells.

Highly parallel magnetic tweezers by targeted DNA tethering.

Single-molecule force-spectroscopy methods such as magnetic and optical tweezers have emerged as powerful tools for the detailed study of biomechanical aspects of DNA-enzyme interactions. As typically only a single molecule of DNA is addressed in an individual experiment, these methods suffer from a low data throughput. Here, we report a novel method for targeted, nonrandom immobilization of DNA-tethered magnetic beads in regular arrays through microcontact printing of DNA end-binding labels. We show that the increase in density due to the arrangement of DNA-bead tethers in regular arrays can give rise to a one-order-of-magnitude improvement in data-throughput in magnetic tweezers experiments. We demonstrate the applicability of this technique in tweezers experiments where up to 450 beads are simultaneously tracked in parallel, yielding statistical data on the mechanics of DNA for 357 molecules from a single experimental run. Our technique paves the way for kilo-molecule force spectroscopy experiments, enabling the study of rare events in DNA-protein interactions and the acquisition of large statistical data sets from individual experimental runs.

Force-generation and dynamic instability of microtubule bundles.

Individual dynamic microtubules can generate pushing or pulling forces when their growing or shrinking ends are in contact with cellular objects such as the cortex or chromosomes. These microtubules can operate in parallel bundles, for example when interacting with mitotic chromosomes. Here, we investigate the force-generating capabilities of a bundle of growing microtubules and study the effect that force has on the cooperative dynamics of such a bundle. We used an optical tweezers setup to study microtubule bundles growing against a microfabricated rigid barrier in vitro. We show that multiple microtubules can generate a pushing force that increases linearly with the number of microtubules present. In addition, the bundle can cooperatively switch to a shrinking state, due to a force-induced coupling of the dynamic instability of single microtubules. In the presence of GMPCPP, bundle catastrophes no longer occur, and high bundle forces are reached more effectively. We reproduce the observed behavior with a simple simulation of microtubule bundle dynamics that takes into account previously measured force effects on single microtubules. Using this simulation, we also show that a constant compressive force on a growing bundle leads to oscillations in bundle length that are of potential relevance for chromosome oscillations observed in living cells.

AutoStepfinder: A fast and automated step detection method for single-molecule analysis.

Single-molecule techniques allow the visualization of the molecular dynamics of nucleic acids and proteins with high spatiotemporal resolution. Valuable kinetic information of biomolecules can be obtained when the discrete states within single-molecule time trajectories are determined. Here, we present a fast, automated, and bias-free step detection method, AutoStepfinder, that determines steps in large datasets without requiring prior knowledge on the noise contributions and location of steps. The analysis is based on a series of partition events that minimize the difference between the data and the fit. A dual-pass strategy determines the optimal fit and allows AutoStepfinder to detect steps of a wide variety of sizes. We demonstrate step detection for a broad variety of experimental traces. The user-friendly interface and the automated detection of AutoStepfinder provides a robust analysis procedure that enables anyone without programming knowledge to generate step fits and informative plots in less than an hour.

Quantitative guidelines for force calibration through spectral analysis of magnetic tweezers data.

Single-molecule techniques are powerful tools that can be used to study the kinetics and mechanics of a variety of enzymes and their complexes. Force spectroscopy, for example, can be used to control the force applied to a single molecule and thereby facilitate the investigation of real-time nucleic acid-protein interactions. In magnetic tweezers, which offer straightforward control and compatibility with fluorescence measurements or parallel tracking modes, force-measurement typically relies on the analysis of positional fluctuations through video microscopy. Significant errors in force estimates, however, may arise from incorrect spectral analysis of the Brownian motion in the magnetic tweezers. Here we investigated physical and analytical optimization procedures that can be used to improve the range over which forces can be reliably measured. To systematically probe the limitations of magnetic tweezers spectral analysis, we have developed a magnetic tweezers simulator, whose outcome was validated with experimental data. Using this simulator, we evaluate methods to correctly perform force experiments and provide guidelines for correct force calibration under configurations that can be encountered in typical magnetic tweezers experiments.

Direct observation of independently moving replisomes in Escherichia coli.

The replication and transfer of genomic material from a cell to its progeny are vital processes in all living systems. Here we visualize the process of chromosome replication in widened E. coli cells. Monitoring the replication of single chromosomes yields clear examples of replication bubbles that reveal that the two replisomes move independently from the origin to the terminus of replication along each of the two arms of the circular chromosome, providing direct support for the so-called train-track model, and against a factory model for replisomes. The origin of replication duplicates near midcell, initially splitting to random directions and subsequently towards the poles. The probability of successful segregation of chromosomes significantly decreases with increasing cell width, indicating that chromosome confinement by the cell boundary is an important driver of DNA segregation. Our findings resolve long standing questions in bacterial chromosome organization.

Direct imaging of the circular chromosome in a live bacterium.

Although the physical properties of chromosomes, including their morphology, mechanics, and dynamics are crucial for their biological function, many basic questions remain unresolved. Here we directly image the circular chromosome in live E. coli with a broadened cell shape. We find that it exhibits a torus topology with, on average, a lower-density origin of replication and an ultrathin flexible string of DNA at the terminus of replication. At the single-cell level, the torus is strikingly heterogeneous, with blob-like Mbp-size domains that undergo major dynamic rearrangements, splitting and merging at a minute timescale. Our data show a domain organization underlying the chromosome structure of E. coli, where MatP proteins induce site-specific persistent domain boundaries at Ori/Ter, while transcription regulators HU and Fis induce weaker transient domain boundaries throughout the genome. These findings provide an architectural basis for the understanding of the dynamic spatial organization of bacterial genomes in live cells.

Magnetic tweezers for the measurement of twist and torque.

Single-molecule techniques make it possible to investigate the behavior of individual biological molecules in solution in real time. These techniques include so-called force spectroscopy approaches such as atomic force microscopy, optical tweezers, flow stretching, and magnetic tweezers. Amongst these approaches, magnetic tweezers have distinguished themselves by their ability to apply torque while maintaining a constant stretching force. Here, it is illustrated how such a "conventional" magnetic tweezers experimental configuration can, through a straightforward modification of its field configuration to minimize the magnitude of the transverse field, be adapted to measure the degree of twist in a biological molecule. The resulting configuration is termed the freely-orbiting magnetic tweezers. Additionally, it is shown how further modification of the field configuration can yield a transverse field with a magnitude intermediate between that of the "conventional" magnetic tweezers and the freely-orbiting magnetic tweezers, which makes it possible to directly measure the torque stored in a biological molecule. This configuration is termed the magnetic torque tweezers. The accompanying video explains in detail how the conversion of conventional magnetic tweezers into freely-orbiting magnetic tweezers and magnetic torque tweezers can be accomplished, and demonstrates the use of these techniques. These adaptations maintain all the strengths of conventional magnetic tweezers while greatly expanding the versatility of this powerful instrument.

Slow unloading leads to DNA-bound ß2-sliding clamp accumulation in live Escherichia coli cells.

The ubiquitous sliding clamp facilitates processivity of the replicative polymerase and acts as a platform to recruit proteins involved in replication, recombination and repair. While the dynamics of the E. coli ß2-sliding clamp have been characterized in vitro, its in vivo stoichiometry and dynamics remain unclear. To probe both ß2-clamp dynamics and stoichiometry in live E. coli cells, we use custom-built microfluidics in combination with single-molecule fluorescence microscopy and photoactivated fluorescence microscopy. We quantify the recruitment, binding and turnover of ß2-sliding clamps on DNA during replication. These quantitative in vivo results demonstrate that numerous ß2-clamps in E. coli remain on the DNA behind the replication fork for a protracted period of time, allowing them to form a docking platform for other enzymes involved in DNA metabolism.

Scanning a DNA molecule for bound proteins using hybrid magnetic and optical tweezers.

The functional state of the genome is determined by its interactions with proteins that bind, modify, and move along the DNA. To determine the positions and binding strength of proteins localized on DNA we have developed a combined magnetic and optical tweezers apparatus that allows for both sensitive and label-free detection. A DNA loop, that acts as a scanning probe, is created by looping an optically trapped DNA tether around a DNA molecule that is held with magnetic tweezers. Upon scanning the loop along the λ-DNA molecule, EcoRI proteins were detected with ~17 nm spatial resolution. An offset of 33 ± 5 nm for the detected protein positions was found between back and forwards scans, corresponding to the size of the DNA loop and in agreement with theoretical estimates. At higher applied stretching forces, the scanning loop was able to remove bound proteins from the DNA, showing that the method is in principle also capable of measuring the binding strength of proteins to DNA with a force resolution of 0.1 pN/[Formula: see text]. The use of magnetic tweezers in this assay allows the facile preparation of many single-molecule tethers, which can be scanned one after the other, while it also allows for direct control of the supercoiling state of the DNA molecule, making it uniquely suitable to address the effects of torque on protein-DNA interactions.

Freely orbiting magnetic tweezers to directly monitor changes in the twist of nucleic acids.

The double-stranded nature of DNA links its replication, transcription and repair to rotational motion and torsional strain. Magnetic tweezers (MT) are a powerful single-molecule technique to apply both forces and torques to individual DNA or RNA molecules. However, conventional MT do not track rotational motion directly and constrain the free rotation of the nucleic acid tether. Here we present freely orbiting MT (FOMT) that allow the measurement of equilibrium fluctuations and changes in the twist of tethered nucleic acid molecules. Using a precisely aligned vertically oriented magnetic field, FOMT enable tracking of the rotation angle from straight forward (x,y)-position tracking and permits the application of calibrated stretching forces, without biasing the tether's free rotation. We utilize FOMT to measure the force-dependent torsional stiffness of DNA from equilibrium rotational fluctuations and to follow the assembly of recombination protein A filaments on DNA.