In the past three decades, the ability to optically manipulate biomolecules has spurred a new era of medical and biophysical research. Optical tweezers (OT) have enabled experimenters to trap, sort, ...and probe cells, as well as discern the structural dynamics of proteins and nucleic acids at single molecule level. The steady improvement in OT's resolving power has progressively pushed the envelope of their applications; there are, however, some inherent limitations that are prompting researchers to look for alternatives to the conventional techniques. To begin with, OT are restricted by their one-dimensional approach, which makes it difficult to conjure an exhaustive three-dimensional picture of biological systems. The high-intensity trapping laser can damage biological samples, a fact that restricts the feasibility of in vivo applications. Finally, direct manipulation of biological matter at nanometer scale remains a significant challenge for conventional OT. A significant amount of literature has been dedicated in the last 10 years to address the aforementioned shortcomings. Innovations in laser technology and advances in various other spheres of applied physics have been capitalized upon to evolve the next generation OT systems. In this review, we elucidate a few of these developments, with particular focus on their biological applications. The manipulation of nanoscopic objects has been achieved by means of plasmonic optical tweezers (POT), which utilize localized surface plasmons to generate optical traps with enhanced trapping potential, and photonic crystal optical tweezers (PhC OT), which attain the same goal by employing different photonic crystal geometries. Femtosecond optical tweezers (fs OT), constructed by replacing the continuous wave (cw) laser source with a femtosecond laser, promise to greatly reduce the damage to living samples. Finally, one way to transcend the one-dimensional nature of the data gained by OT is to couple them to the other large family of single molecule tools, i.e., fluorescence-based imaging techniques. We discuss the distinct advantages of the aforementioned techniques as well as the alternative experimental perspective they provide in comparison to conventional OT.
Liquid–liquid phase separation of multivalent intrinsically disordered protein–RNA complexes is ubiquitous in both natural and biomimetic systems. So far, isotropic liquid droplets are the most ...commonly observed topology of RNA–protein condensates in experiments and simulations. Here, by systematically studying the phase behavior of RNA–protein complexes across varied mixture compositions, we report a hollow vesicle-like condensate phase of nucleoprotein assemblies that is distinct from RNA–protein droplets. We show that these vesicular condensates are stable at specific mixture compositions and concentration regimes within the phase diagram and are formed through the phase separation of anisotropic protein–RNA complexes. Similar to membranes composed of amphiphilic lipids, these nucleoprotein−RNA vesicular membranes exhibit local ordering, size-dependent permeability, and selective encapsulation capacity without sacrificing their dynamic formation and dissolution in response to physicochemical stimuli. Our findings suggest that protein−RNA complexes can robustly create lipid-free vesicle-like enclosures by phase separation.
Mechanosensing drives acuity of αβ T-cell recognition Feng, Yinnian; Brazin, Kristine N.; Kobayashi, Eiji ...
Proceedings of the National Academy of Sciences - PNAS,
09/2017, Letnik:
114, Številka:
39
Journal Article
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T lymphocytes use surface αβ T-cell receptors (TCRs) to recognize peptides bound to MHC molecules (pMHCs) on antigen-presenting cells (APCs). How the exquisite specificity of high-avidity T cells is ...achieved is unknown but essential, given the paucity of foreign pMHC ligands relative to the ubiquitous self-pMHC array on an APC. Using optical traps, we determine physicochemical triggering thresholds based on load and force direction. Strikingly, chemical thresholds in the absence of external load require orders of magnitude higher pMHC numbers than observed physiologically. In contrast, force applied in the shear direction (∼10 pN per TCR molecule) triggers T-cell Ca2+ flux with as few as two pMHC molecules at the interacting surface interface with rapid positional relaxation associated with similarly directed motor-dependent transport via ∼8-nm steps, behaviors inconsistent with serial engagement during initial TCR triggering. These synergistic directional forces generated during cell motility are essential for adaptive T-cell immunity against infectious pathogens and cancers.
We performed single molecule force spectroscopy assays to elucidate the competition between the two binding modes (intercalation and groove binding) that occur between the drug chloroquine (CLQ) and ...DNA in HEPES-based buffers. Force–extension curves were obtained under different drug concentrations and buffer compositions, allowing the determination of the changes on the mechanical parameters of these complexes and the physical chemistry of the interactions. The presence of HEPES in the buffer, when combined with NaCl, can modulate the interaction. The conclusions allowed us to advance in the understanding of the role of the driving forces in the balance between distinct binding modes.
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•Chloroquine is a complex DNA ligand that can interact by different binding modes.•The effective binding can be modulated by changing the buffer ionic strength and composition.•We advance in the understanding of the specific role of the driving forces in the balance between distinct binding modes.
Acoustic tweezers are a versatile set of tools that use sound waves to manipulate bioparticles ranging from nanometer-sized extracellular vesicles to millimeter-sized multicellular organisms. Over ...the past several decades, the capabilities of acoustic tweezers have expanded from simplistic particle trapping to precise rotation and translation of cells and organisms in three dimensions. Recent advances have led to reconfigured acoustic tweezers that are capable of separating, enriching, and patterning bioparticles in complex solutions. Here, we review the history and fundamentals of acoustic-tweezer technology and summarize recent breakthroughs.
Multiphase fluids that contain dispersed oil droplets are fundamental to many industrial processes and formulated products. In the present work, the surface wetting and interaction behaviour between ...an emulsion droplet and a single fibre were quantitatively investigated as a function of surfactant chemistry, pH, and salt concentration using an optical tweezers apparatus. Silicone droplets were emulsified in water and stabilised by surfactants. An individual droplet was then captured using an optical tweezers setup to allow controlled contact with a fibre surface. During droplet approach the surface interaction forces were measured and spreading behaviour was quantified after contact was made between the droplet and fibre. Salt concentration was increased to change the surface interaction behaviour. The pH and ζ-potential of silicone emulsions decreased from 9.5 to 6.0 and from 39mV to -15 mV, respectively, when increasing salt concentration for both of the surfactants studied, SDS and CTAB. The attraction between the fibre and a silicone oil droplet was found to increase as the electrostatic repulsion was suppressed with an increased salt concentration. This was confirmed by an increased number of droplet-fibre adhesion events and in some cases droplet spreading on the fibre. Such observations for adhesion are based on electrostatic attractions exceeding the maximum force that can be exerted by the trapping laser. When adhesion was not be observed, attraction could still be recorded through a thin-film hydrodynamic suction effect during the process of retraction, which becomes more pronounced on the polyester fibre. Our results provide a novel method to directly quantify the surface interaction and wetting of liquid droplets on microscopic fibres simultaneously, which could be valuable when investigating formulated products that involve emulsion droplets.
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Laser cooling and trapping
, and magneto-optical trapping methods in particular
, have enabled groundbreaking advances in science, including Bose-Einstein condensation
, quantum computation with ...neutral atoms
and high-precision optical clocks
. Recently, magneto-optical traps (MOTs) of diatomic molecules have been demonstrated
, providing access to research in quantum simulation
and searches for physics beyond the standard model
. Compared with diatomic molecules, polyatomic molecules have distinct rotational and vibrational degrees of freedom that promise a variety of transformational possibilities. For example, ultracold polyatomic molecules would be uniquely suited to applications in quantum computation and simulation
, ultracold collisions
, quantum chemistry
and beyond-the-standard-model searches
. However, the complexity of these molecules has so far precluded the realization of MOTs for polyatomic species. Here we demonstrate magneto-optical trapping of a polyatomic molecule, calcium monohydroxide (CaOH). After trapping, the molecules are laser cooled in a blue-detuned optical molasses to a temperature of 110 μK, which is below the Doppler cooling limit. The temperatures and densities achieved here make CaOH a viable candidate for a wide variety of quantum science applications, including quantum simulation and computation using optical tweezer arrays
. This work also suggests that laser cooling and magneto-optical trapping of many other polyatomic species
will be both feasible and practical.
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