Wednesday, August 15, 2018

Single-Molecule Mechanical Folding and Unfolding of RNA Hairpins: Effects of Single A-U to A·C Pair Substitutions and Single Proton Binding and Implications for mRNA Structure-Induced −1 Ribosomal Frameshifting

Lixia Yang, Zhensheng Zhong, Cailing Tong, Huan Jia, Yiran Liu, and Gang Chen

A wobble A·C pair can be protonated at near physiological pH to form a more stable wobble A+·C pair. Here, we constructed an RNA hairpin (rHP) and three mutants with one A-U base pair substituted with an A·C mismatch on the top (near the loop, U22C), middle (U25C), and bottom (U29C) positions of the stem, respectively. Our results on single-molecule mechanical (un)folding using optical tweezers reveal the destabilization effect of A-U to A·C pair substitution and protonation-dependent enhancement of mechanical stability facilitated through an increased folding rate, or decreased unfolding rate, or both. Our data show that protonation may occur rapidly upon the formation of an apparent mechanical folding transition state. Furthermore, we measured the bulk −1 ribosomal frameshifting efficiencies of the hairpins by a cell-free translation assay. For the mRNA hairpins studied, −1 frameshifting efficiency correlates with mechanical unfolding force at equilibrium and folding rate at around 15 pN. U29C has a frameshifting efficiency similar to that of rHP (∼2%). Accordingly, the bottom 2–4 base pairs of U29C may not form under a stretching force at pH 7.3, which is consistent with the fact that the bottom base pairs of the hairpins may be disrupted by ribosome at the slippery site. U22C and U25C have a similar frameshifting efficiency (∼1%), indicating that both unfolding and folding rates of an mRNA hairpin in a crowded environment may affect frameshifting. Our data indicate that mechanical (un)folding of RNA hairpins may mimic how mRNAs unfold and fold in the presence of translating ribosomes.


Experimental characterization and modeling of optical tweezer particle handling dynamics

Michael D. Porter, Brian Giera, Robert M. Panas, Lucas A. Shaw, Maxim Shusteff, and Jonathan B. Hopkins

We report a new framework for a quantitative understanding of optical trapping (OT) particle handling dynamics. We present a novel three-dimensional particle-based model that includes optical, hydrodynamic, and inter-particle forces. This semi-empirical colloid model is based on an open-source simulation code known as LAMMPS (large-scale atomic/molecular massively parallel simulator) and properly recapitulates the full OT force profile beyond the typical linear approximations valid near the trap center. Simulations are carried out with typical system parameters relevant for our experimental holographic optical trapping (HOT) system, including varied particle sizes, trap movement speeds, and beam powers. Furthermore, we present a new experimental method for measuring both the stable and metastable boundaries of the optical force profile to inform or validate the model’s underlying force profile. We show that our framework is a powerful tool for accurately predicting particle behavior in a practical experimental OT setup and can be used to characterize and predict particle handling dynamics within any arbitrary OT force profile.


Optical trapping and manipulation of plasmonic nanoparticles: fundamentals, applications, and perspectives

Alexander S. Urban, Sol Carretero-Palacios, Andrey A. Lutich, Theobald Lohmüller, Jochen Feldman and Frank Jäckel

This feature article discusses the optical trapping and manipulation of plasmonic nanoparticles, an area of current interest with potential applications in nanofabrication, sensing, analytics, biology and medicine. We give an overview over the basic theoretical concepts relating to optical forces, plasmon resonances and plasmonic heating. We discuss fundamental studies of plasmonic particles in optical traps and the temperature profiles around them. We place a particular emphasis on our own work employing optically trapped plasmonic nanoparticles towards nanofabrication, manipulation of biomimetic objects and sensing.


Impact of Lanthanide Nanomaterials on Photonic Devices and Smart Applications

Jiajia Zhou,  Julius L. Leaño Jr.,  Zhenyu Liu,  Dayong Jin,  Ka‐Leung Wong,  Ru‐Shi Liu, Jean‐Claude G. Bünzli

Half a century after its initial emergence, lanthanide photonics is facing a profound remodeling induced by the upsurge of nanomaterials. Lanthanide‐doped nanomaterials hold promise for bioapplications and photonic devices because they ally the unmatched advantages of lanthanide photophysical properties with those arising from large surface‐to‐volume ratios and quantum confinement that are typical of nanoobjects. Cutting‐edge technologies and devices have recently arisen from this association and are in turn promoting nanophotonic materials as essential tools for a deeper understanding of biological mechanisms and related medical diagnosis and therapy, and as crucial building blocks for next‐generation photonic devices. Here, the recent progress in the development of nanomaterials, nanotechnologies, and nanodevices for clinical uses and commercial exploitation is reviewed. The candidate nanomaterials with mature synthesis protocols and compelling optical uniqueness are surveyed. The specific fields that are directly driven by lanthanide doped nanomaterials are emphasized, spanning from in vivo imaging and theranostics, micro‐/nanoscopic techniques, point‐of‐care medical testing, forensic fingerprints detection, to micro‐LED devices.


A review on phospholipid vesicles flowing through channels

Fikret Aydin, Xiaolei Chu, Joseph Greenstein and Meenakshi Dutt

The flow of particles through confined volumes has appeared under different contexts in nature and technology. Some examples include the flow of red blood cells or drug delivery vehicles through capillaries, or surfactant-based particles in nano- or microfluidic cells. The molecular composition of the particles along with external conditions and the characteristics of the confined volume impact the response of the particle to flow. This review focuses on the problem of phospholipid vesicles constrained to flowing in channels. The review examines how experimental and computational approaches have been harnessed to study the response of these particles to the flow.


Tuesday, August 14, 2018

Dynamic Clustering of Dyneins on Axonal Endosomes: Evidence from High-Speed Darkfield Imaging

Praveen D. Chowdary, Luke Kaplan, Daphne L. Che, Bianxiao Cui

One of the fundamental features that govern the cooperativity of multiple dyneins during cargo trafficking in cells is the spatial distribution of these dyneins on the cargo. Geometric considerations and recent experiments indicate that clustered distributions of dyneins are required for effective cooperation on micron-sized cargos. However, very little is known about the spatial distribution of dyneins and their cooperativity on smaller cargos, such as vesicles or endosomes <200 nm in size, which are not amenable to conventional immunostaining and optical trapping methods. In this work, we present evidence that dyneins can dynamically be clustered on endosomes in response to load. Using a darkfield imaging assay, we measured the repeated stalls and detachments of retrograde axonal endosomes under load with <10 nm localization accuracy at imaging rates up to 1 kHz for over a timescale of minutes. A three-dimensional stochastic model was used to simulate the endosome motility under load to gain insights on the mechanochemical properties and spatial distribution of dyneins on axonal endosomes. Our results indicate that 1) the distribution of dyneins on endosomes is fluid enough to support dynamic clustering under load and 2) the detachment kinetics of dynein on endosomes differs significantly from the in vitro measurements possibly due to an increase in the unitary stall force of dynein on endosomes.


Optical lattices and optical vortex arrays in clustered speckles

Changwei He, Li Ma, Ruirui Zhang, Xing Li, Yuqin Zhang, and Chuanfu Cheng

Clustered speckle, optical lattices, and their optical vortex array are subjects of interest in optical wave manipulation. In this study, disordered optical lattices and vortex arrays with different unit structures were found in the clustered speckles generated by a circularly-distributed multi-pinhole scattering screen when it was illuminated by coherent light. These structures included hexagonal lattices, kagome lattices, and honeycomb lattices. Moreover, optical lattices with asymmetric units generated by modulation of phases with non-integer multiples of 2π were discussed. Theoretical analysis and numerical calculations demonstrated that optical lattices in clustered speckles in the observation plane were generated by the phase modulations of the random scattering screen. The lattice type depended on the number of 2π multiples of the summed phase difference between the pinholes. Additionally, the conditions for the formation of periodical optical lattices and their vortex arrays were given. Different optical lattices and their vortex arrays appearing simultaneously in the clustered speckle were difficult to generate using the common multi-beam interference system. This phenomenon is of great significance in the study of the orbital angular momentum of photons and other fields.


Using single molecule force spectroscopy to facilitate a rational design of Ca2+-responsive β-roll peptide-based hydrogels

Lichao Liu, Han Wang, Yueying Han, Shanshan Lv and Jianfeng Chen

This study demonstrated that incorporation of Ca2+-responsive β-roll peptides arising from repeat-in-toxin (RTX) into elastomeric proteins provided an approach to construct hydrogels that exhibit Ca2+-responsive mechanical properties through a force analysis-based approach. Use of circular dichroism spectroscopy confirmed that there was a Ca2+-induced conformational change of RTX-based recombinant polyproteins. The polyproteins could be crosslinked into solid hydrogels. Shrinking/swelling measurements showed a Ca2+-responsive dimensional change of the RTX-based hydrogels. Mechanical measurements at constant pulling speed and at constant extension suggested that the mechanical properties of the RTX-based hydrogels were Ca2+-responsive. Experimental single molecule force spectroscopies were used to investigate the nano-mechanical stability of the RTX domains. Single molecule atomic force microscopy and optical tweezers provided evidence that the Ca2+-dependent refolding of the intrinsically disordered RTX led to the force increase. The results indicated that the unique Ca2+-responsive mechanical properties of the RTX-based hydrogels at the macroscopic level could be attributed to the nano-mechanical properties of the RTX domains engineered into individual polyproteins at the single molecule level.


Generation of reconfigurable optical traps for microparticles spatial manipulation through dynamic split lens inspired light structures

Angel Lizana, Haolin Zhang, Alex Turpin, Albert Van Eeckhout, Fabian A. Torres-Ruiz, Asticio Vargas, Claudio Ramirez, Francesc Pi & Juan Campos

We present an experimental method, based on the use of dynamic split-lens configurations, useful for the trapping and spatial control of microparticles through the photophoretic force. In particular, the concept of split-lens configurations is exploited to experimentally create customized and reconfigurable three-dimensional light structures, in which carbon coated glass microspheres, with sizes in a range of 63–75 μm, can be captured. The generation of light spatial structures is performed by properly addressing phase distributions corresponding to different split-lens configurations onto a spatial light modulator (SLM). The use of an SLM allows a dynamic variation of the light structures geometry just by modifying few control parameters of easy physical interpretation. We provide some examples in video format of particle trapping processes. What is more, we also perform further spatial manipulation, by controlling the spatial position of the particles in the axial direction, demonstrating the generation of reconfigurable three-dimensional photophoretic traps for microscopic manipulation of absorbing particles.


Pseudopolymorph Control of l-Phenylalanine Achieved by Laser Trapping

Chi-Shiun Wu, Pei-Yun Hsieh, Ken-ichi Yuyama, Hiroshi Masuhara, and Teruki Sugiyama

The pseudopolymorphism of l-phenylalanine, monohydrate and anhydrous crystals, was arbitrarily controlled by laser trapping with a focused continuous-wave near-infrared laser beam. Crystallization was realized even from unsaturated solution, when laser trapping always produced the anhydrous crystal. Contrarily, the monohydrate crystal was constantly formed from supersaturated solution. In saturated solution, the pseudopolymorphism strongly depended on laser power and polarization.