For experiments on chiral self-assembly, we used a two-component mixture consisting of 880 nm long rod-like fd viruses and the non-adsorbing polymer Dextran. In aqueous suspension, fd viruses alone exhibit purely repulsive interactions 13. Adding non-adsorbing polymer to a dilute isotropic suspension of fd rods induces attractive interactions via the depletion mechanism and leads to their condensation into colloidal membranes, equilibrium structures consisting of one-rod-length thick liquid-like monolayers of aligned rods (Fig. 1a) 11. Despite having different structures on molecular lengthscales, the longwavelength coarse-grained properties of colloidal membranes are identical to those of conventional lipid bilayers. However, unlike their amphiphilic counterparts, colloidal membranes do not form vesicles and are instead observed as freely suspended disks with exposed edges. Here, we investigate the behavior of these exposed edges in a manner analogous to previously studied liquid-liquid domains embedded in lipid bilayers [14][15][16] . For our experiments, it is essential that fd viruses are chiral, i.e. a pair of aligned viruses minimizes their interaction energy when they are slightly twisted in a preferred direction with respect to each other. The strength of chiral interactions can be continuously tuned to zero through either genetic or physical methods ( Supplementary Fig. 1) 13,17 .Before investigating chiral membranes, we determined the structure of a membrane's edge composed of simpler achiral rods using three complimentary imaging techniques, namely 2D and 3D polarization microscopy and electron microscopy. The local tilting of the rods within a membrane was determined using 2D LC-PolScope, which produces images in which the intensity of each pixel represents the local retardance of the membrane (Fig. 1d) 18. Such images can be quantitatively related to the tilting of the rods away from the layer normal, the z-axis 19. Rods in the bulk of a membrane are aligned along the zaxis, so that 2D LC-PolScope images appear black in that region (Fig. 1e). In contrast, the bright birefringent ring along the membrane's periphery reveals local tilting of the rods (Fig. 1e, Supplementary Fig. 2). For achiral rods, this indicates that a membrane has a hemi-toroidal curved edge (Fig. 1b, c). In comparison to an untilted edge, a curved edge structure lowers the area of the rod/polymer interface, thus reducing interfacial tension, at the cost of increasing the elastic energy due to twist distortion. This hypothesis is confirmed by visualizing the 3D membrane structure using electron tomography, whichshows that the viruses' long axis transitions from being parallel to the z-axis in the membrane bulk to perpendicular to the z-axis and tangent to the edge along the membrane periphery ( When viewed with optical microscopy, a membrane's edge exhibits significant thermal fluctuations, the analysis of which yields the line tension γ eff , the energetic cost required to move rods from the membrane interior to the periphe...
Any macroscopic deformation of a filamentous bundle is necessarily accompanied by local sliding and/or stretching of the constituent filaments1,2. Yet the nature of the sliding friction between two aligned filaments interacting through multiple contacts remains largely unexplored. Here, by directly measuring the sliding forces between two bundled F-actin filaments, we show that these frictional forces are unexpectedly large, scale logarithmically with sliding velocity as in solid-like friction, and exhibit complex dependence on the filaments’ overlap length. We also show that a reduction of the frictional force by orders of magnitude, associated with a transition from solid-like friction to Stokes’s drag, can be induced by coating F-actin with polymeric brushes. Furthermore, we observe similar transitions in filamentous microtubules and bacterial flagella. Our findings demonstrate how altering a filament’s elasticity, structure and interactions can be used to engineer interfilament friction and thus tune the properties of fibrous composite materials.
We present a miniature centrifuge force microscope (CFM) that repurposes a benchtop centrifuge for high-throughput single-molecule experiments with high-resolution particle tracking, a large force range, temperature control and simple push-button operation. Incorporating DNA nanoswitches to enable repeated interrogation by force of single molecular pairs, we demonstrate increased throughput, reliability and the ability to characterize population heterogeneity. We perform spatiotemporally multiplexed experiments to collect 1,863 bond rupture statistics from 538 traceable molecular pairs in a single experiment, and show that 2 populations of DNA zippers can be distinguished using per-molecule statistics to reduce noise.
Liquid-liquid phase separation is ubiquitous in suspensions of nanoparticles, proteins and colloids. It plays an important role in gel formation, protein crystallization and perhaps even as an organizing principle in cellular biology 1,2 .With a few notable exceptions 3,4 , surface-tension-minimizing liquid droplets in bulk suspensions continuously coalesce, increasing in size without bound until achieving macroscale phase separation. In comparison, the phase behavior of colloids, nanoparticles or proteins confined to interfaces, surfaces or membranes is significantly more complex 5-11 . Inclusions distort the local interface structure leading to interactions that are fundamentally different from the well-studied interactions mediated by isotropic solvents 12,13 . Here, we investigate liquid-liquid phase separation in monolayer membranes composed of dissimilar chiral colloidal rods. We demonstrate that colloidal rafts are a ubiquitous feature of binary colloidal membranes. We measure the raft free energy landscape by visualizing its assembly kinetics. Subsequently, we quantify repulsive raft-raft interactions and relate them to directly imaged raft-induced membrane distortions, demonstrating that particle chirality plays a key role in this microphase separation. At high densities, rafts assemble into cluster crystals which constantly exchange monomeric rods with the background reservoir to maintain a self-limited size. Lastly, we 2 demonstrate that rafts can form bonds to assemble into higher-order suprastructures. Our work demonstrates that membrane-mediated liquid-liquid phase separation can be fundamentally different from the well-characterized behavior of bulk liquids. It outlines a robust membrane-based pathway for assembly of monodisperse liquid clusters which is complementary to existing methods which take place in bulk suspensions [14][15][16] . Finally, it reveals that chiral inclusions in membranes acquire long-ranged repulsive interactions, which might play a role in stabilizing assemblages of finite size 11,17 .In the presence of non-adsorbing polymer, mono-disperse rod-like viruses experience effective depletion attractions that drive their lateral association. These interactions can lead to assembly of one-rod-length-thick colloidal monolayer membranes that are held together by the osmotic pressure of the enveloping polymer suspension 18 . We allow membranes to sediment to the bottom of the sample chambers in which case the constituent rods point in the z direction, while all images are taken in the x-y plane.Although they differ on molecular scales, the long-wavelength fluctuations of colloidal monolayers and lipid bilayers are described by the same free energy. In this work, we investigated the behavior of colloidal membranes containing a mixture of two rods: 880 nm long rod-like fd-Y21M virus and 1200 nm long M13KO7 virus 19 . Membranes were prepared by adding a depletant to a dilute isotropic fd-Y21M/M13KO7 mixture. For all parameters investigated, both rods co-assembled into binary membranes. ...
We introduce a nanoscale experimental platform that enables kinetic and equilibrium measurements of a wide range of molecular interactions by expanding the functionality of gel electrophoresis. Programmable, self-assembled DNA nanoswitches serve both as templates for positioning molecules, and as sensitive, quantitative reporters of molecular association and dissociation. We demonstrate this low cost, versatile, “lab-on-a-molecule” system by characterizing 10 different interactions, including a complex 4-body interaction with 5 discernable states.
In the presence of nonadsorbing polymers, colloidal particles experience ubiquitous attractive interactions induced by depletion forces. Here, we measure the depletion interaction between a pair of microtubule filaments using a method that combines single filament imaging with optical trapping. By quantifying the dependence of filament cohesion on both polymer concentration and solution ionic strength, we demonstrate that the minimal model of depletion, based on the Asakura-Oosawa theory, fails to quantitatively describe the experimental data. By measuring the cohesion strength in two- and three- filament bundles, we verify pairwise additivity of depletion interactions for the specific experimental conditions. The described experimental technique can be used to measure pairwise interactions between various biological or synthetic filaments and complements information extracted from bulk osmotic stress experiments.
In the presence of a nonadsorbing polymer, monodisperse rod-like particles assemble into colloidal membranes, which are one-rodlength-thick liquid-like monolayers of aligned rods. Unlike 3D edgeless bilayer vesicles, colloidal monolayer membranes form open structures with an exposed edge, thus presenting an opportunity to study elasticity of fluid sheets. Membranes assembled from single-component chiral rods form flat disks with uniform edge twist. In comparison, membranes composed of a mixture of rods with opposite chiralities can have the edge twist of either handedness. In this limit, disk-shaped membranes become unstable, instead forming structures with scalloped edges, where two adjacent lobes with opposite handedness are separated by a cuspshaped point defect. Such membranes adopt a 3D configuration, with cusp defects alternatively located above and below the membrane plane. In the achiral regime, the cusp defects have repulsive interactions, but away from this limit we measure effective longranged attractive binding. A phenomenological model shows that the increase in the edge energy of scalloped membranes is compensated by concomitant decrease in the deformation energy due to Gaussian curvature associated with scalloped edges, demonstrating that colloidal membranes have positive Gaussian modulus. A simple excluded volume argument predicts the sign and magnitude of the Gaussian curvature modulus that is in agreement with experimental measurements. Our results provide insight into how the interplay between membrane elasticity, geometrical frustration, and achiral symmetry breaking can be used to fold colloidal membranes into 3D shapes.self-assembly | membranes | liquid crystals | Gaussian curvature | chirality T he possible configurations and shapes of 2D fluid membranes can be described by a continuum energy expression that accounts for the membrane's out-of-plane deformations as well as the line tension associated with the membrane's exposed edge (1, 2). Because an arbitrary deformation of a thin layer can have either mean and/or Gaussian curvature, the full theoretical description of membranes, in principle, requires two parameters, the bending and Gaussian curvature moduli. However, lipid bilayers almost always appear as edgeless 3D vesicles, which further simplify theoretical modeling. In particular, integrating Gaussian curvature over any simply closed surface yields a constant (3). Thus, the shape fluctuations of a closed vesicle only depend on the membrane-bending modulus. Consequently, experiments that interrogated mechanics or shape fluctuations of vesicles provided extensive information about the membrane curvature modulus and how it depends on the structure of the constituent particles (4-6). In comparison, significantly less is known about the Gaussian modulus, despite the significant role it plays in fundamental biological and technological processes such as pore formation as well as vesicle fusion and fission (7)(8)(9)(10)(11).Recent experiments have demonstrated that, in the presence of a d...
Decoding the identity of biomolecules from trace samples is a longstanding goal in the field of biotechnology. Advances in DNA analysis have significantly impacted clinical practice and basic research, but corresponding developments for proteins face challenges due to their relative complexity and our inability to amplify them. Despite progress in methods such as mass spectrometry and mass cytometry, single-molecule protein identification remains a highly challenging objective. Toward this end, we combine DNA nanotechnology with single-molecule force spectroscopy to create a mechanically reconfigurable DNA Nanoswitch Caliper (DNC) capable of measuring multiple coordinates on single biomolecules with atomic resolution. Using optical tweezers, we demonstrate absolute distance measurements with angstrom-level precision for both DNA and peptides, and using multiplexed magnetic tweezers, we demonstrate quantification of relative abundance in mixed samples. Measuring distances between DNA-labeled residues, we perform single-molecule fingerprinting of synthetic and natural peptides, and show discrimination, within a heterogeneous population, between different post-translational modifications. DNA Nanoswitch Calipers are a powerful and accessible tool for characterizing distances within nanoscale complexes that will enable new applications in fields such as single-molecule proteomics.
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