Advancing DNA Research: The Synergy of Cohesin Mechanics and Nanotracker Innovations

Deciphering Cohesin's Function in DNA Organization

Cohesin, a vital protein complex, is instrumental in maintaining the integrity of sister chromatids and facilitating the formation of DNA loops, which are critical for proper gene regulation and cell division. Its structure, characterized by a ring-like shape with extended coiled arms and two ATPase domains, allows it to perform these essential functions. Research primarily focuses on the three-dimensional arrangement and dynamic reorganization of DNA throughout the cellular cycle, underscoring its significance in gene expression, genetic recombination, and DNA repair mechanisms.

Exploring the Mechanical Robustness of Cohesin Using Optical Traps

To ascertain cohesin's capacity to withstand the forces exerted by the mitotic spindle, researchers developed an experimental setup mirroring the interaction between two DNA molecules secured by cohesin. This approach involves immobilizing a single DNA molecule on a glass slide, subsequently binding it to a cohesin molecule. The precision of single-molecule analysis is paramount, as multiple molecules can complicate data interpretation. By attaching fluorescent tags to both cohesin and a second DNA molecule, researchers can confirm the presence of individual molecules and their successful binding. An optical trap, employing a highly focused laser beam, is then utilized to apply tension to the DNA-cohesin complex. This sophisticated technique permits precise manipulation and accurate force measurement, enabling scientists to observe cohesin's behavior under stress. The fundamental objective is to apply force to the cohesin-bound DNA until rupture, thereby revealing the mechanical limits of the complex. The Bruker JPK NanoTracker 2, integrated with a turf elimination system, facilitates the visualization of individual cohesin molecules, enabling detailed observation of their interaction with DNA under varying salt concentrations. As salt levels increase, cohesin exhibits diffusive movement along the DNA, which can be monitored via climography. This step also confirms the presence of a single cohesin molecule through photobleaching. The introduction of a second fluorophore-tagged DNA molecule allows for the visualization of correlated movement between cohesin and DNA, further validating single-molecule integrity. During the stretching of DNA with the glass bead, cohesin maintains its grip until a critical force is reached, causing it to rupture while the DNA remains intact. This experimental design allows for the precise determination of the force required to break cohesin's hold, with single cohesin molecules typically rupturing at approximately 20 piconewtons, as evidenced by detachment rupture force histograms.

Identifying Structural Vulnerabilities in Cohesin

To comprehend the dynamics of cohesin's interaction with DNA, the simplest hypothesis suggests that cohesin encircles the DNA in a ring, and any applied force would cause it to break at its weakest point. A crucial point of interest is the kleisin gate, which allows DNA to escape under normal physiological conditions. Researchers investigated whether DNA also escapes through this gate under external force by covalently cross-linking this interface. The experimental results indicated that the rupture force remained unchanged, suggesting that DNA escapes from an alternative site. Another potential weak point is the hinge region, where DNA coils interact. A similar experiment was conducted, where cohesins were loaded onto DNA and their hinges cross-linked, though only about half of the cohesins underwent cross-linking. The rupture force histograms from this experiment exhibited two distinct peaks, indicating two populations of cohesins. Approximately half of the population ruptured at forces similar to the initial experiment, likely representing the uncross-linked complexes. The other half demonstrated significantly higher rupture forces, suggesting that these cross-linked molecules possess a much stronger interface, preventing easy DNA escape due to strong covalent bonds. Furthermore, repeating the experiment with two DNA molecules revealed similar rupture forces, with a slight distribution shift. It was observed that stretching two DNA strands prolonged the breaking process, leading to rupture at a lower force over an extended period. These findings propose that cohesin physically entraps sister chromatids, and this physical linkage can be overcome by forces around 20 piconewtons. This mechanical disengagement of the cohesin ring, driven by this force, may represent a crucial regulatory mechanism during processes like mitosis or nuclear envelope breakdown, where separase cleaves cohesin to allow chromosome separation. The reversible nature of this process and the implications for DNA breathing during replication, where cohesin may mechanically unload and reload, highlight the significance of mechanical regulation in DNA dynamics.

Unveiling Cohesin's Conformational Changes as a Molecular Engine

Cohesin is widely recognized as a molecular machine capable of extruding DNA loops, yet the precise mechanism underlying this function remains largely elusive. A fundamental characteristic of such molecular machines is their ability to couple conformational transformations with the hydrolysis cycle and directional movement along the DNA strand. Investigations aimed to determine whether specific conformational changes within cohesin could generate force, thereby elucidating how these alterations propel cohesin along the DNA. Cohesin exhibits two primary conformational shifts: the "head-to-head" movement, involving the reciprocal displacement of the head domains, and the "hinge-to-head" movement. To study these dynamics, common cohesin molecules were tagged with two markers: one to immobilize cohesin on a surface and another, a passive coil, to detach cohesin from the bead and the optical trap. By attaching a bead held in an optical trap to cohesin, researchers could precisely monitor how cohesin's conformational changes respond to applied force. For instance, if a hinge bends towards the head, the bead would move in conjunction, and this displacement could be detected. Data corresponding to both bent and unbent cohesin hinges were collected, and the inferred distances aligned with structural data, confirming the real-time observation of head-hinge bending. Regarding the influence of external force on these bends, cohesin remained largely unbent at forces around 1.5 piconewtons but exhibited dynamic bending and unbending at forces around 1 piconewton and lower. This dynamic behavior mirrored Brownian motion, suggesting that thermal fluctuations, rather than chemical transitions, primarily drive this movement. A simple three-state model, incorporating fully bent, half-bent, and fully unbent states, accurately described the data, reinforcing the role of thermal Brownian fluctuations in this movement. The subsequent inquiry focused on the role of ATP hydrolysis and the type of movement it drives. By examining head-to-head movement, immobilizing one head and applying force to the other, a starkly different picture emerged. Head-to-head movement occurred at significantly higher forces (e.g., 5-10 piconewtons) and involved displacements of approximately 10 nanometers, consistent with structural data and AFM measurements. Importantly, the rate of opening and closing of this molecule's domains remained largely independent of external force within a specific range (up to 15 piconewtons), unlike the exponential force dependence expected in thermal Brownian ratchet systems. These findings suggest that head-to-head movement is chemically driven, likely by the ATP cycle, distinguishing it from the thermal fluctuation-driven hinge-to-head movement. In summary, cohesin possesses two distinct force-generating mechanisms: head-hinge bending, primarily driven by thermal fluctuations, and head-to-head movement, chemically driven and capable of generating much higher forces. While the precise reasons for these two mechanisms are unknown, one hypothesis posits that they contribute to different phases of loop extrusion: one for initial DNA bending and the other for subsequent loop elongation. These insights pave the way for a deeper understanding of cohesin's multifaceted role as a molecular motor in cellular processes.

The Bruker NanoTracker: A Leap in Optical Tweezers Technology

The NanoTracker represents a sophisticated, fully automated optical tweezers system, offering unparalleled capabilities in high-resolution force and position measurements. As the second iteration of its kind, the NanoTracker is built upon an inverted microscope platform, allowing seamless integration with diverse light microscopy techniques, including EP fluorescence, confocal, TIF, and TIC. Its compatibility extends to various microscope manufacturers, such as JICE, Nikon, Olympus, and Leica, ensuring broad applicability. The NanoTracker features an intuitive software interface that incorporates automation features, including script-writing for spectrometry modes and both CMOS and CCD cameras for clear sample visualization. The system’s high degree of automation streamlines experimental workflows; users merely need to introduce a sample, and the software manages all subsequent processes. Capable of measuring forces up to 100 or 200 piconewtons, depending on laser power, the NanoTracker boasts a remarkable resolution of 0.1 piconewtons. Beyond force measurement, it can track particle positions with sub-nanometer precision. The system supports high data acquisition rates in megahertz and offers extensive bandwidth. It accommodates single, dual, and multiple traps, managing the latter through a time-sharing method where short time allocations to each trap prevent noticeable interruptions. The number of trapped particles is limited only by the available laser power, allowing for the trapping of up to 255 particles with a single trap. As a Class 1 laser instrument, it eliminates the need for additional laser safety goggles or specialized laser-safe laboratory environments. The NanoTracker is available with either a 3-watt or 5-watt laser, utilizing a 1064-nanometer wavelength. An optional Petri dish heater accommodates 35-millimeter Petri dishes, enabling sample heating up to 45°C, complemented by gas perfusion functionality ideal for long-term live-cell experiments. Another valuable accessory is the magnetic twister, perfect for trapping magnetic particles and studying the effects of toxins. These features collectively enhance the versatility and precision of optical tweezers experiments, pushing the boundaries of single-molecule research.

Laminar Flow Setup for DNA Stretching Experiments

The NanoTracker's laminar flow cells are ingeniously designed with five input channels and one output channel, enabling the introduction of distinct fluids without intermixing. This architecture facilitates diverse experiments within different channels. For instance, streptavidin-coated beads can be introduced via one channel, while biotinylated DNA molecules flow through another. This setup is particularly effective for fluorescence in situ hybridization (FISH) to DNA molecules, enabling detailed DNA stretching experiments. Such experiments typically involve two traps, positioning a DNA molecule between them. One trap remains static, while the other moves, recording the forces exerted on the DNA as it is stretched. The system's control unit manages all electronics and continuously communicates with the computer for data recording and storage. Above the controller, the laser power supply and steering unit regulate various optical elements to create, steer, and modulate multiple traps. Light is directed along an optical path to the inverted Zeiss microscope head, guided by a dichroic mirror positioned at a 45° angle, and then passed through a high numerical aperture objective. This configuration precisely focuses laser light to trap particles, utilizing a second detection objective and a quadrant photodiode to determine particle position or applied forces through back focal plane interferometry. The NanoTracker software allows precise adjustment of laser power distribution among traps, and an attenuation filter prevents detection oversaturation. A dedicated window facilitates switching between detection and trapping objectives for fine focal plane adjustments. The system also boasts motorized sample stages, enabling highly precise nano-positioning in X, Y, and Z directions up to 100 micrometers. The NanoTracker’s force spectrometer functionality is ideal for DNA stretching experiments. Prior to force measurements, optical tweezers are calibrated using a power spectrum method, requiring input of particle diameter, temperature, and the medium's density and viscosity. Calibration involves fitting the power spectrum with a Lorentzian function, yielding crucial values like sensitivity and trap stiffness. In a typical experiment, with one static and one moving trap, the DNA is extended by 12 micrometers at a rate of one micrometer per second, with the data sampling rate adjusted accordingly. Force spectrometry data, accessible via the spectrometer tab, reveals the static trap's signal. DNA rupture is clearly indicated by the force spectroscopy results returning to zero, typically occurring after extending the DNA by approximately 7.8 to 7.9 micrometers.

Ensuring Successful DNA Tethering Between Beads

The system confirms successful DNA capture when the measured force surpasses a predefined threshold as one of the optical traps moves. In a typical laminar flow setup, one channel contains polystyrene beads, 3 to 3.2 micrometers in diameter, coated with streptavidin, while another channel introduces biotinylated lambda DNA. The strong binding affinity between biotin and streptavidin ensures DNA attachment to the beads. Once the traps are activated, these beads can be precisely captured. With one trap fixed, the other trap can be programmed to circle in 0.1-micrometer steps, systematically searching for DNA. A script monitors force measurements, and if the recorded force increases by more than 20 piconewtons above the baseline, it signals successful DNA capture, allowing spectroscopy measurements to commence. Tools such as the NanoTracker's ramp designer are invaluable for setting up these experiments. Furthermore, the system offers robust automation capabilities through its experiment planner or by enabling custom script development in Java or Python, providing researchers with flexible control over complex experimental protocols.

Synergizing NanoTracker with Atomic Force Microscopy

The NanoTracker system can be integrated with Atomic Force Microscopy (AFM) for correlative measurements. The NanoTracker's COMBI stage is designed to accommodate a trapping objective for particle manipulation. By removing the NanoTracker's head, an AFM can be seamlessly mounted on top, enabling simultaneous optical tracking and AFM measurements. This hybrid approach has already been successfully employed by researchers to investigate interactions between various cell types, demonstrating its potential for multifaceted biological studies at the nanoscale.