Tuesday, October 10, 2017

Ionic effects on the temperature–force phase diagram of DNA

Sitichoke Amnuanpol

Double-stranded DNA (dsDNA) undergoes a structural transition to single-stranded DNA (ssDNA) in many biologically important processes such as replication and transcription. This strand separation arises in response either to thermal fluctuations or to external forces. The roles of ions are twofold, shortening the range of the interstrand potential and renormalizing the DNA elastic modulus. The dsDNA-to-ssDNA transition is studied on the basis that dsDNA is regarded as a bound state while ssDNA is regarded as an unbound state. The ground state energy of DNA is obtained by mapping the statistical mechanics problem to the imaginary time quantum mechanics problem. In the temperature–force phase diagram the critical force Fc(T) increases logarithmically with the Na+ concentration in the range from 32 to 110 mM. Discussing this logarithmic dependence of Fc(T) within the framework of polyelectrolyte theory, it inevitably suggests a constraint on the difference between the interstrand separation and the length per unit charge during the dsDNA-to-ssDNA transition.


Actin and microtubule networks contribute differently to cell response for small and large strains

H Kubitschke, J Schnauss, K D Nnetu, E Warmt, R Stange and J Kaes

Cytoskeletal filaments provide cells with mechanical stability and organization. The main key players are actin filaments and microtubules governing a cell's response to mechanical stimuli. We investigated the specific influences of these crucial components by deforming MCF-7 epithelial cells at small (≤5% deformation) and large strains (>5% deformation). To understand specific contributions of actin filaments and microtubules, we systematically studied cellular responses after treatment with cytoskeleton influencing drugs. Quantification with the microfluidic optical stretcher allowed capturing the relative deformation and relaxation of cells under different conditions. We separated distinctive deformational and relaxational contributions to cell mechanics for actin and microtubule networks for two orders of magnitude of drug dosages. Disrupting actin filaments via latrunculin A, for instance, revealed a strain-independent softening. Stabilizing these filaments by treatment with jasplakinolide yielded cell softening for small strains but showed no significant change at large strains. In contrast, cells treated with nocodazole to disrupt microtubules displayed a softening at large strains but remained unchanged at small strains. Stabilizing microtubules within the cells via paclitaxel revealed no significant changes for deformations at small strains, but concentration-dependent impact at large strains. This suggests that for suspended cells, the actin cortex is probed at small strains, while at larger strains; the whole cell is probed with a significant contribution from the microtubules.


Integrated Method to Attach DNA Handles and Functionally Select Proteins to Study Folding and Protein-Ligand Interactions with Optical Tweezers

Yuxin Hao, Clare Canavan, Susan S. Taylor & Rodrigo A. Maillard

Optical tweezers has emerged as a powerful tool to study folding, ligand binding, and motor enzymes. The manipulation of proteins with optical tweezers requires attaching molecular handles to the protein of interest. Here, we describe a novel method that integrates the covalent attachment of DNA handles to target proteins with a selection step for functional and properly folded molecules. In addition, this method enables obtaining protein molecules in different liganded states and can be used with handles of different lengths. We apply this method to study the cAMP binding domain A (CBD-A) of Protein kinase A. We find that the functional selection step drastically improves the reproducibility and homogeneity of the single molecule data. In contrast, without a functional selection step, proteins often display misfolded conformations. cAMP binding stabilizes the CBD-A against a denaturing force, and increases the folded state lifetime. Data obtained with handles of 370 and 70 base pairs are indistinguishable, but at low forces short handles provide a higher spatial resolution. Altogether, this method is flexible, selects for properly folded molecules in different liganded states, and can be readily applicable to study protein folding or protein-ligand interactions with force spectroscopy that require molecular handles.


Submicrometer-sized nonspherical particle separation by laser beam

Jaromír Petržala, Miroslav Kocifaj, Ladislav Kómar, and Alexandre Simoneau

The radiation pressure exerted on sub-micrometer-size particles is shown to be an important factor predetermining the impact coordinates of the particles after being illuminated by a laser beam. Unlike spherical particles, the nonspherical ones can be deflected perpendicularly to the beam direction if the momentum transfer from the laser beam to a particle is large enough. Such an optical sorting is a useful technology, which can be used to isolate spherules of a specific size from a population of particles of random sizes and shapes. The system of ideal spheres has a wide range of applications in industry and also in the development of targeted optical devices, and so the methods for fast contact-less particle separation are expected to lead to considerable progress in the field. The theoretical model we have developed is demonstrated in a set of numerical experiments on metallic and nonmetallic particles.


Protein Folding Mediated by Trigger Factor and Hsp70: New Insights from Single-Molecule Approaches

Florian Wruck, Mario J.Avellaneda, Eline J.Koers, David P. Minde, Matthias P. Mayer, Günter Kramer, Alireza Mashaghi, Sander J.Tans

Chaperones assist in protein folding, but what this common phrase means in concrete terms has remained surprisingly poorly understood. We can readily measure chaperone binding to unfolded proteins, but how they bind and affect proteins along folding trajectories has remained obscure. Here we review recent efforts by our labs and others that are beginning to pry into this issue, with a focus on the chaperones trigger factor and Hsp70. Single-molecule methods are central, as they allow the stepwise process of folding to be followed directly. First results have already revealed contrasts with long-standing paradigms: rather than acting only “early” by stabilizing unfolded chain segments, these chaperones can bind and stabilize partially folded structures as they grow to their native state. The findings suggest a fundamental redefinition of the protein folding problem and a more extensive functional repertoire of chaperones than previously assumed.


Wednesday, October 4, 2017

Assessment of Local Heterogeneity in Mechanical Properties of Nanostructured Hydrogel Networks

Zhaokai Meng, Teena Thakur, Chandani Chitrakar, Manish K. Jaiswal, Akhilesh K. Gaharwar, and Vladislav V. Yakovlev

Our current understanding of the mechanical properties of nanostructured biomaterials is rather limited to invasive/destructive and low-throughput techniques such as atomic force microscopy, optical tweezers, and shear rheology. In this report, we demonstrate the capabilities of recently developed dual Brillouin/Raman spectroscopy to interrogate the mechanical and chemical properties of nanostructured hydrogel networks. The results obtained from Brillouin spectroscopy show an excellent correlation with the conventional uniaxial and shear mechanical testing. Moreover, it is confirmed that, unlike the macroscopic conventional mechanical measurement techniques, Brillouin spectroscopy can provide the elasticity characteristic of biomaterials at a mesoscale length, which is remarkably important for understanding complex cell–biomaterial interactions. The proposed technique experimentally demonstrated the capability of studying biomaterials in their natural environment and may facilitate future fabrication and inspection of biomaterials for various biomedical and biotechnological applications.


Polarization-Induced Chirality in Metamaterials via Optomechanical Interaction

Mingkai Liu, David A. Powell, Rui Guo, Ilya V. Shadrivov and Yuri S. Kivshar

A novel type of metamaterial is introduced, where the structural symmetry can be controlled by optical forces. Since symmetry sets fundamental bounds on the optical response, symmetry breaking changes the properties of metamaterials qualitatively over the entire resonant frequency band. This is achieved by a polarized pump beam, exerting optical forces which are not constrained by the structural symmetry. This new concept is illustrated for a metasurface composed of zig-zag chains of dipole meta-atoms, in which a highly asymmetric optical force exists for an appropriate incident polarization. The effect is employed to transform a planar achiral metasurface into a stereoscopic chiral structure. Importantly, the handedness of the induced chirality can be actively switched by changing the incident polarization. The proposed concept can be employed to achieve dynamic spatial control of metamaterials and metasurfaces at infrared and optical frequencies with subwavelength resolution.


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.


Repulsion–attraction switching of nematic colloids formed by liquid crystal dispersions of polygonal prisms

B. Senyuk, Q. Liu, P. D. Nystrom and I. I. Smalyukh

Self-assembly of colloidal particles due to elastic interactions in nematic liquid crystals promises tunable composite materials and can be guided by exploiting surface functionalization, geometric shape and topology, though these means of controlling self-assembly remain limited. Here, we realize low-symmetry achiral and chiral elastic colloids in the nematic liquid crystals using colloidal polygonal concave and convex prisms. We show that the controlled pinning of disclinations at the prism edges alters the symmetry of director distortions around the prisms and their orientation with respect to the far-field director. The controlled localization of the disclinations at the prism's edges significantly influences the anisotropy of the diffusion properties of prisms dispersed in liquid crystals and allows one to modify their self-assembly. We show that elastic interactions between polygonal prisms can be switched between repulsive and attractive just by controlled re-pinning the disclinations at different edges using laser tweezers. Our findings demonstrate that elastic interactions between colloidal particles dispersed in nematic liquid crystals are sensitive to the topologically equivalent but geometrically rich controlled configurations of the particle-induced defects.


Freezing shortens the lifetime of DNA molecules under tension

Wei-Ju Chung, Yujia Cui, Chi-Shuo Chen, Wesley H. Wei, Rong-Shing Chang, Wun-Yi Shu, Ian C. Hsu

DNA samples are commonly frozen for storage. However, freezing can compromise the integrity of DNA molecules. Considering the wide applications of DNA molecules in nanotechnology, changes to DNA integrity at the molecular level may cause undesirable outcomes. However, the effects of freezing on DNA integrity have not been fully explored. To investigate the impact of freezing on DNA integrity, samples of frozen and non-frozen bacteriophage lambda DNA were studied using optical tweezers. Tension (5–35 pN) was applied to DNA molecules to mimic mechanical interactions between DNA and other biomolecules. The integrity of the DNA molecules was evaluated by measuring the time taken for single DNA molecules to break under tension. Mean lifetimes were determined by maximum likelihood estimates and variances were obtained through bootstrapping simulations. Under 5 pN of force, the mean lifetime of frozen samples is 44.3 min with 95% confidence interval (CI) between 36.7 min and 53.6 min while the mean lifetime of non-frozen samples is 133.2 min (95% CI: 97.8–190.1 min). Under 15 pN of force, the mean lifetimes are 10.8 min (95% CI: 7.6–12.6 min) and 78.5 min (95% CI: 58.1–108.9 min). The lifetimes of frozen DNA molecules are significantly reduced, implying that freezing compromises DNA integrity. Moreover, we found that the reduced DNA structural integrity cannot be restored using regular ligation process. These results indicate that freezing can alter the structural integrity of the DNA molecules.