Synergistic Coordination of Chromatin Torsional Mechanics and Topoisomerase Activity.

DNA replication in eukaryotes generates DNA supercoiling, which may intertwine (braid) daughter chromatin fibers to form precatenanes, posing topological challenges during chromosome segregation. The mechanisms that limit precatenane formation remain unclear. By making direct torque measurements, we demonstrate that the intrinsic mechanical properties of chromatin play a fundamental role in dictating precatenane formation and regulating chromatin topology. Whereas a single chromatin fiber is torsionally soft, a braided fiber is torsionally stiff, indicating that supercoiling on chromatin substrates is preferentially directed in front of the fork during replication. We further show that topoisomerase II relaxation displays a strong preference for a single chromatin fiber over a braided fiber. These results suggest a synergistic coordination-the mechanical properties of chromatin inherently suppress precatenane formation during replication elongation by driving DNA supercoiling ahead of the fork, where supercoiling is more efficiently removed by topoisomerase II. VIDEO ABSTRACT.

Optical Tweezers: A Force to Be Reckoned With.

The 2018 Nobel Prize in Physics has been awarded jointly to Arthur Ashkin for the discovery and development of optical tweezers and their applications to biological systems and to Gérard Mourou and Donna Strickland for the invention of laser chirped pulse amplification. Here we focus on Arthur Ashkin and how his revolutionary work opened a window into the world of molecular mechanics and spurred the rise of single-molecule biophysics.

Biocompatible and High Stiffness Nanophotonic Trap Array for Precise and Versatile Manipulation.

The advent of nanophotonic evanescent field trapping and transport platforms has permitted increasingly complex single molecule and single cell studies on-chip. Here, we present the next generation of nanophotonic Standing Wave Array Traps (nSWATs) representing a streamlined CMOS fabrication process and compact biocompatible design. These devices utilize silicon nitride (Si3N4) waveguides, operate with a biofriendly 1064 nm laser, allow for several watts of input power with minimal absorption and heating, and are protected by an anticorrosive layer for sustained on-chip microelectronics in aqueous salt buffers. In addition, due to Si3N4's negligible nonlinear effects, these devices can generate high stiffness traps while resolving subnanometer displacements for each trapped particle. In contrast to traditional table-top counterparts, the stiffness of each trap in an nSWAT device scales linearly with input power and is independent of the number of trapping centers. Through a unique integration of microcircuitry and photonics, the nSWAT can robustly trap, and controllably position, a large number of nanoparticles along the waveguide surface, operating in an all-optical, constant-force mode without need for active feedback. By reducing device fabrication cost, minimizing trapping laser specimen heating, increasing trapping force, and implementing commonly used trapping techniques, this new generation of nSWATs significantly advances the development of a high performance, low cost optical tweezers array laboratory on-chip.

Dextran-coated iron oxide nanoparticle-induced nanotoxicity in neuron cultures.

Recent technological advances have introduced diverse engineered nanoparticles (ENPs) into our air, water, medicine, cosmetics, clothing, and food. However, the health and environmental effects of these increasingly common ENPs are still not well understood. In particular, potential neurological effects are one of the most poorly understood areas of nanoparticle toxicology (nanotoxicology), in that low-to-moderate neurotoxicity can be subtle and difficult to measure. Culturing primary neuron explants on planar microelectrode arrays (MEAs) has emerged as one of the most promising in vitro techniques with which to study neuro-nanotoxicology, as MEAs enable the fluorescent tracking of nanoparticles together with neuronal electrical activity recording at the submillisecond time scale, enabling the resolution of individual action potentials. Here we examine the dose-dependent neurotoxicity of dextran-coated iron oxide nanoparticles (dIONPs), a common type of functionalized ENP used in biomedical applications, on cultured primary neurons harvested from postnatal day 0-1 mouse brains. A range of dIONP concentrations (5-40 µg/ml) were added to neuron cultures, and cells were plated either onto well plates for live cell, fluorescent reactive oxidative species (ROS) and viability observations, or onto planar microelectrode arrays (MEAs) for electrophysiological measurements. Below 10 µg/ml, there were no dose-dependent cellular ROS increases or effects in MEA bursting behavior at sub-lethal dosages. However, above 20 µg/ml, cell death was obvious and widespread. Our findings demonstrate a significant dIONP toxicity in cultured neurons at concentrations previously reported to be safe for stem cells and other non-neuronal cell types.

High-Performance Image-Based Measurements of Biological Forces and Interactions in a Dual Optical Trap.

Optical traps enable the nanoscale manipulation of individual biomolecules while measuring molecular forces and lengths. This ability relies on the sensitive detection of optically trapped particles, typically accomplished using laser-based interferometric methods. Recently, image-based particle tracking techniques have garnered increased interest as a potential alternative to laser-based detection; however, successful integration of image-based methods into optical trapping instruments for biophysical applications and force measurements has remained elusive. Here, we develop a camera-based detection platform that enables accurate and precise measurements of biological forces and interactions in a dual optical trap. In demonstration, we stretch and unzip DNA molecules while measuring the relative distances of trapped particles from their trapping centers with sub-nanometer accuracy and precision. We then use the DNA unzipping technique to localize bound proteins with sub-base-pair precision, revealing how thermal DNA "breathing" fluctuations allow an unzipping fork to detect and respond to the presence of a protein bound downstream. This work advances the capabilities of image tracking in optical traps, providing a state-of-the-art detection method that is accessible, highly flexible, and broadly compatible with diverse experimental substrates and other nanometric techniques.

Recent advances in single molecule studies of nucleosomes.

As the fundamental packing units of DNA in eukaryotes, nucleosomes play a central role in governing DNA accessibility in a variety of cellular processes. Our understanding of the mechanisms underlying this complex regulation has been aided by unique structural and dynamic perspectives offered by single molecule techniques. Recent years have witnessed remarkable advances achieved using these techniques, including the generation of a detailed histone-DNA energy landscape, elucidation of nucleosome disassembly processes, and real-time monitoring of molecular motors interacting with nucleosomes. These and other highlights of single molecule nucleosome studies will be discussed in this review.