A conserved ATP- and Scc2/4-dependent activity for cohesin in tethering DNA molecules

Pilar Gutierrez-Escribano, Matthew D. Newton, Aida Llauró, Jonas Huber, Loredana Tanasie, Joseph Davy, Isabel Aly, Ricardo Aramayo, Alex Montoya, Holger Kramer, Johannes Stigler, David S. Rueda, and Luis Aragon

Sister chromatid cohesion requires cohesin to act as a protein linker to hold chromatids together. How cohesin tethers chromatids remains poorly understood. We have used optical tweezers to visualize cohesin as it holds DNA molecules. We show that cohesin complexes tether DNAs in the presence of Scc2/Scc4 and ATP demonstrating a conserved activity from yeast to humans. Cohesin forms two classes of tethers: a “permanent bridge” resisting forces over 80 pN and a force-sensitive “reversible bridge.” The establishment of bridges requires physical proximity of dsDNA segments and occurs in a single step. “Permanent” cohesin bridges slide when they occur in trans, but cannot be removed when in cis. Therefore, DNAs occupy separate physical compartments in cohesin molecules. We finally demonstrate that cohesin tetramers can compact linear DNA molecules stretched by very low force (below 1 pN), consistent with the possibility that, like condensin, cohesin is also capable of loop extrusion.

DOI

The post-PAM interaction of RNA-guided spCas9 with DNA dictates its target binding and dissociation

Qian Zhang, Fengcai Wen, Siqi Zhang, Jiachuan Jin, Lulu Bi, Ying Lu, Ming Li, Xu-Guang Xi, Xingxu Huang, Bin Shen and Bo Sun

Cas9 is an RNA-guided endonuclease that targets complementary DNA for cleavage and has been repurposed for many biological usages. Cas9 activities are governed by its direct interactions with DNA. However, information about this interplay and the mechanism involved in its direction of Cas9 activity remain obscure. Using a single-molecule approach, we probed Cas9/sgRNA/DNA interactions along the DNA sequence and found two stable interactions flanking the protospacer adjacent motif (PAM). Unexpectedly, one of them is located approximately 14 base pairs downstream of the PAM (post-PAM interaction), which is beyond the apparent footprint of Cas9 on DNA. Loss or occupation of this interaction site on DNA impairs Cas9 binding and cleavage. Consistently, a downstream helicase could readily displace DNA-bound Cas9 by disrupting this relatively weak post-PAM interaction. Our work identifies a critical interaction of Cas9 with DNA that dictates its binding and dissociation, which may suggest distinct strategies to modulate Cas9 activity.

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Optical pulling at macroscopic distances

Xiao Li, Jun Chen, Zhifang Lin and Jack Ng

Optical tractor beams, proposed in 2011 and experimentally demonstrated soon after, offer the ability to pull particles against light propagation. It has attracted much research and public interest. Yet, its limited microscopic-scale range severely restricts its applicability. The dilemma is that a long-range Bessel beam, the most accessible beam for optical traction, has a small half-cone angle, θ0, making pulling difficult. Here, by simultaneously using several novel and compatible mechanisms, including transverse isotropy, Snell’s law, antireflection coatings (or impedance-matched metamaterials), and light interference, we overcome this dilemma and achieve long-range optical pulling at θ0 ≈ 1°. The range is estimated to be 14 cm when using ~1 W of laser power. Thus, macroscopic optical pulling can be realized in a medium or in a vacuum, with good tolerance of the half-cone angle and the frequency of the light.

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Single-molecule polarization microscopy of DNA intercalators sheds light on the structure of S-DNA

Adam S. Backer, Andreas S. Biebricher, Graeme A. King, Gijs J. L. Wuite, Iddo Heller and Erwin J. G. Peterman

DNA structural transitions facilitate genomic processes, mediate drug-DNA interactions, and inform the development of emerging DNA-based biotechnology such as programmable materials and DNA origami. While some features of DNA conformational changes are well characterized, fundamental information such as the orientations of the DNA base pairs is unknown. Here, we use concurrent fluorescence polarization imaging and DNA manipulation experiments to probe the structure of S-DNA, an elusive, elongated conformation that can be accessed by mechanical overstretching. To this end, we directly quantify the orientations and rotational dynamics of fluorescent DNA-intercalated dyes. At extensions beyond the DNA overstretching transition, intercalators adopt a tilted (θ ~ 54°) orientation relative to the DNA axis, distinct from the nearly perpendicular orientation (θ ~ 90°) normally assumed at lower extensions. These results provide the first experimental evidence that S-DNA has substantially inclined base pairs relative to those of the standard (Watson-Crick) B-DNA conformation.

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Viscoelastic properties of vimentin originate from nonequilibrium conformational changes

Johanna Block, Hannes Witt, Andrea Candelli, Jordi Cabanas Danes, Erwin J. G. Peterman, Gijs J. L. Wuite, Andreas Janshoff and Sarah Köster

Structure and dynamics of living matter rely on design principles fundamentally different from concepts of traditional material science. Specialized intracellular filaments in the cytoskeleton permit living systems to divide, migrate, and grow with a high degree of variability and durability. Among the three filament systems, microfilaments, microtubules, and intermediate filaments (IFs), the physical properties of IFs and their role in cellular mechanics are the least well understood. We use optical trapping of individual vimentin filaments to investigate energy dissipation, strain history dependence, and creep behavior of stretched filaments. By stochastic and numerical modeling, we link our experimental observations to the peculiar molecular architecture of IFs. We find that individual vimentin filaments display tensile memory and are able to dissipate more than 70% of the input energy. We attribute these phenomena to distinct nonequilibrium folding and unfolding of α helices in the vimentin monomers constituting the filaments.

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Nanometer-precision linear sorting with synchronized optofluidic dual barriers

Yuzhi Shi, Sha Xiong, Lip Ket Chin, Jingbo Zhang, Wee Ser, Jiuhui Wu, Tianning Chen, Zhenchuan Yang, Yilong Hao, Bo Liedberg, Peng Huat Yap, Din Ping Tsai, Cheng-Wei Qiu and Ai Qun Liu

The past two decades have witnessed the revolutionary development of optical trapping of nanoparticles, most of which deal with trapping stiffness larger than 10−8 N/m. In this conventional regime, however, it remains a formidable challenge to sort out sub–50-nm nanoparticles with single-nanometer precision, isolating us from a rich flatland with advanced applications of micromanipulation. With an insightfully established roadmap of damping, the synchronization between optical force and flow drag force can be coordinated to attempt the loosely overdamped realm (stiffness, 10−10 to 10−8 N/m), which has been challenging. This paper intuitively demonstrates the remarkable functionality to sort out single gold nanoparticles with radii ranging from 30 to 50 nm, as well as 100- and 150-nm polystyrene nanoparticles, with single nanometer precision. The quasi-Bessel optical profile and the loosely overdamped potential wells in the microchannel enable those aforementioned nanoparticles to be separated, positioned, and microscopically oscillated. This work reveals an unprecedentedly meaningful damping scenario that enriches our fundamental understanding of particle kinetics in intriguing optical systems, and offers new opportunities for tumor targeting, intracellular imaging, and sorting small particles such as viruses and DNA.

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Feedback-tracking microrheology in living cells

Kenji Nishizawa, Marcel Bremerich, Heev Ayade, Christoph F. Schmidt, Takayuki Ariga and Daisuke Mizuno

Living cells are composed of active materials, in which forces are generated by the energy derived from metabolism. Forces and structures self-organize to shape the cell and drive its dynamic functions. Understanding the out-of-equilibrium mechanics is challenging because constituent materials, the cytoskeleton and the cytosol, are extraordinarily heterogeneous, and their physical properties are strongly affected by the internally generated forces. We have analyzed dynamics inside two types of eukaryotic cells, fibroblasts and epithelial-like HeLa cells, with simultaneous active and passive microrheology using laser interferometry and optical trapping technology. We developed a method to track microscopic probes stably in cells in the presence of vigorous cytoplasmic fluctuations, by using smooth three-dimensional (3D) feedback of a piezo-actuated sample stage. To interpret the data, we present a theory that adapts the fluctuation-dissipation theorem (FDT) to out-of-equilibrium systems that are subjected to positional feedback, which introduces an additional nonequilibrium effect. We discuss the interplay between material properties and nonthermal force fluctuations in the living cells that we quantify through the violations of the FDT. In adherent fibroblasts, we observed a well-known polymer network viscoelastic response where the complex shear modulus scales as G* ∝ (−iω)3/4. In the more 3D confluent epithelial cells, we found glassy mechanics with G* ∝ (−iω)1/2 that we attribute to glassy dynamics in the cytosol. The glassy state in living cells shows characteristics that appear distinct from classical glasses and unique to nonequilibrium materials that are activated by molecular motors.

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Opto-thermophoretic assembly of colloidal matter

Linhan Lin, Jianli Zhang, Xiaolei Peng, Zilong Wu, Anna C. H. Coughlan, Zhangming Mao, Michael A. Bevan and Yuebing Zheng

Colloidal matter exhibits unique collective behaviors beyond what occurs at single-nanoparticle and atomic scales. Treating colloidal particles as building blocks, researchers are exploiting new strategies to rationally organize colloidal particles into complex structures for new functions and devices. Despite tremendous progress in directed assembly and self-assembly, a truly versatile assembly technique without specific functionalization of the colloidal particles remains elusive. We develop a new strategy to assemble colloidal matter under a light-controlled temperature field, which can solve challenges in the existing assembly techniques. By adding an anionic surfactant (that is, cetyltrimethylammonium chloride), which serves as a surface charge source, a macro ion, and a micellar depletant, we generate a light-controlled thermoelectric field to manipulate colloidal atoms and a depletion attraction force to assemble the colloidal atoms into two-dimensional (2D) colloidal matter. The general applicability of this opto-thermophoretic assembly (OTA) strategy allows us to build colloidal matter of diverse colloidal sizes (from subwavelength scale to micrometer scale) and materials (polymeric, dielectric, and metallic colloids) with versatile configurations and tunable bonding strengths and lengths. We further demonstrate that the incorporation of the thermoelectric field into the optical radiation force can achieve 3D reconfiguration of the colloidal matter. The OTA strategy releases the rigorous design rules required in the existing assembly techniques and enriches the structural complexity in colloidal matter, which will open a new window of opportunities for basic research on matter organization, advanced material design, and applications.

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Mechanical deformation induces depolarization of neutrophils

Andrew E. Ekpenyong, Nicole Toepfner, Christine Fiddler, Maik Herbig, Wenhong Li, Gheorghe Cojoc, Charlotte Summers, Jochen Guck and Edwin R. Chilvers

The transition of neutrophils from a resting state to a primed state is an essential requirement for their function as competent immune cells. This transition can be caused not only by chemical signals but also by mechanical perturbation. After cessation of either, these cells gradually revert to a quiescent state over 40 to 120 min. We use two biophysical tools, an optical stretcher and a novel microcirculation mimetic, to effect physiologically relevant mechanical deformations of single nonadherent human neutrophils. We establish quantitative morphological analysis and mechanical phenotyping as label-free markers of neutrophil priming. We show that continued mechanical deformation of primed cells can cause active depolarization, which occurs two orders of magnitude faster than by spontaneous depriming. This work provides a cellular-level mechanism that potentially explains recent clinical studies demonstrating the potential importance, and physiological role, of neutrophil depriming in vivo and the pathophysiological implications when this deactivation is impaired, especially in disorders such as acute lung injury.

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