Single-Molecule Assay for Proteolytic Susceptibility: Force-Induced Collagen Destabilization

Force plays a key role in regulating dynamics of biomolecular structure and interactions, yet techniques are lacking to manipulate and continuously read out this response with high throughput. We present an enzymatic assay for force-dependent accessibility of structure that makes use of a wireless mini-radio centrifuge force microscope to provide a real-time readout of kinetics. The microscope is designed for ease of use, fits in a standard centrifuge bucket, and offers high-throughput, video-rate readout of individual proteolytic cleavage events. Proteolysis measurements on thousands of tethered collagen molecules show a load-enhanced trypsin sensitivity, indicating destabilization of the triple helix.     

Single-Molecule Light-Sheet Imaging of Suspended T Cells

Adaptive immune responses are initiated by triggering of the T cell receptor. Single-molecule imaging based on total internal reflection fluorescence microscopy at coverslip/basal cell interfaces is commonly used to study this process. These experiments have suggested, unexpectedly, that the diffusional behavior and organization of signaling proteins and receptors may be constrained before activation. However, it is unclear to what extent the molecular behavior and cell state is affected by the imaging conditions, i.e., by the presence of a supporting surface. In this study, we implemented single-molecule light-sheet microscopy, which enables single receptors to be directly visualized at any plane in a cell to study protein dynamics and organization in live, resting T cells. The light sheet enabled the acquisition of high-quality single-molecule fluorescence images that were comparable to those of total internal reflection fluorescence microscopy. By comparing the apical and basal surfaces of surface-contacting T cells using single-molecule light-sheet microscopy, we found that most coated-glass surfaces and supported lipid bilayers profoundly affected the diffusion of membrane proteins (T cell receptor and CD45) and that all the surfaces induced calcium influx to various degrees. Our results suggest that, when studying resting T cells, surfaces are best avoided, which we achieve here by suspending cells in agarose.     

Resolving Cytosolic Diffusive States in Bacteria by Single-Molecule Tracking

The trajectory of a single protein in the cytosol of a living cell contains information about its molecular interactions in its native environment. However, it has remained challenging to accurately resolve and characterize the diffusive states that can manifest in the cytosol using analytical approaches based on simplifying assumptions. Here, we show that multiple intracellular diffusive states can be successfully resolved if sufficient single-molecule trajectory information is available to generate well-sampled distributions of experimental measurements and if experimental biases are taken into account during data analysis. To address the inherent experimental biases in camera-based and MINFLUX-based single-molecule tracking, we use an empirical data analysis framework based on Monte Carlo simulations of confined Brownian motion. This framework is general and adaptable to arbitrary cell geometries and data acquisition parameters employed in two-dimensional or three-dimensional single-molecule tracking. We show that, in addition to determining the diffusion coefficients and populations of prevalent diffusive states, the timescales of diffusive state switching can be determined by stepwise increasing the time window of averaging over subsequent single-molecule displacements. Time-averaged diffusion analysis of single-molecule tracking data may thus provide quantitative insights into binding and unbinding reactions among rapidly diffusing molecules that are integral for cellular functions.     

Probing Position-Dependent Diffusion in Folding Reactions Using Single-Molecule Force Spectroscopy

Folding of proteins and nucleic acids involves a diffusive search over a multidimensional conformational energy landscape for the minimal-energy structure. When examining the projection of conformational motions onto a one-dimensional reaction coordinate, as done in most experiments, the diffusion coefficient D is generally position dependent. However, it has proven challenging to measure such position-dependence experimentally. We investigated the position-dependence of D in the folding of DNA hairpins as a simple model system in two ways: first, by analyzing the round-trip time to return to a given extension in constant-force extension trajectories measured by force spectroscopy, and second, by analyzing the fall time required to reach a given extension in force jump measurements. These methods yielded conflicting results: the fall time implied a fairly constant D, but the round-trip time implied variations of over an order of magnitude. Comparison of experiments with computational simulations revealed that both methods were strongly affected by experimental artifacts inherent to force spectroscopy measurements, which obscured the intrinsic position-dependence of D. Lastly, we applied Kramers’s theory to the kinetics of hairpins with energy barriers located at different positions along the hairpin stem, as a crude probe of D at different stem positions, and we found that D did not vary much as the barrier was moved along the reaction coordinate. This work underlines the difficulties faced when trying to deduce position-dependent diffusion coefficients from experimental folding trajectories.     

Single-Molecule Unbinding Forces between the Polysaccharide Hyaluronan and Its Binding Proteins

The extracellular polysaccharide hyaluronan (HA) is ubiquitous in all vertebrate tissues, where its various functions are encoded in the supramolecular complexes and matrices that it forms with HA-binding proteins (hyaladherins). In tissues, these supramolecular architectures are frequently subjected to mechanical stress, yet how this affects the intermolecular bonding is largely unknown. Here, we used a recently developed single-molecule force spectroscopy platform to analyze and compare the mechanical strength of bonds between HA and a panel of hyaladherins from the Link module superfamily, namely the complex of the proteoglycan aggrecan and cartilage link protein, the proteoglycan versican, the inflammation-associated protein TSG-6, the HA receptor for endocytosis (stabilin-2/HARE), and the HA receptor CD44. We find that the resistance to tensile stress for these hyaladherins correlates with the size of the HA-binding domain. The lowest mean rupture forces are observed for members of the type A subgroup (i.e., with the shortest HA-binding domains; TSG-6 and HARE). In contrast, the mechanical stability of the bond formed by aggrecan in complex with cartilage link protein (two members of the type C subgroup, i.e., with the longest HA-binding domains) and HA is equal or even superior to the high affinity streptavidin?biotin bond. Implications for the molecular mechanism of unbinding of HA?hyaladherin bonds under force are discussed, which underpin the mechanical properties of HA?hyaladherin complexes and HA-rich extracellular matrices.     

Nuclear Transport and Accumulation of Smad Proteins Studied by Single-Molecule Microscopy

Nuclear translocation of stimulated Smad heterocomplexes is a critical step in the signal transduction of transforming growth factor β (TGF-β) from transmembrane receptors into the nucleus. Specifically, normal nuclear accumulation of Smad2/Smad4 heterocomplexes induced by TGF-β1 is involved in carcinogenesis. However, the relationship between nuclear accumulation and the nucleocytoplasmic transport kinetics of Smad proteins in the presence of TGF-β1 remains obscure. By combining a high-speed single-molecule tracking microscopy and Förster resonance energy transfer technique, we tracked the entire TGF-β1-induced process of Smad2/Smad4 heterocomplex formation, as well as their transport through nuclear pore complexes in live cells, with a high single-molecule localization precision of 2 ms and <20 nm. Our single-molecule Förster resonance energy transfer data have revealed that in TGF-β1-treated cells, Smad2/Smad4 heterocomplexes formed in the cytoplasm, imported through the nuclear pore complexes as entireties, and finally dissociated in the nucleus. Moreover, we found that basal-state Smad2 or Smad4 cannot accumulate in the nucleus without the presence of TGF-β1, mainly because both of them have an approximately twofold higher nuclear export efficiency compared to their nuclear import. Remarkably and reversely, heterocomplexes of Smad2/Smad4 induced by TGF-β1 can rapidly concentrate in the nucleus because of their almost fourfold higher nuclear import rate in comparison with their nuclear export rate. Thus, we believe that the determined TGF-β1-dependent transport configurations and efficiencies for the basal-state Smad or stimulated Smad heterocomplexes elucidate the basic molecular mechanism to understand their nuclear transport and accumulation.     

Quantifying the Precision of Single-Molecule Torque and Twist Measurements Using Allan Variance

Single-molecule manipulation techniques have provided unprecedented insights into the structure, function, interactions, and mechanical properties of biological macromolecules. Recently, the single-molecule toolbox has been expanded by techniques that enable measurements of rotation and torque, such as the optical torque wrench (OTW) and several different implementations of magnetic (torque) tweezers. Although systematic analyses of the position and force precision of single-molecule techniques have attracted considerable attention, their angle and torque precision have been treated in much less detail. Here, we propose Allan deviation as a tool to systematically quantitate angle and torque precision in single-molecule measurements. We apply the Allan variance method to experimental data from our implementations of (electro)magnetic torque tweezers and an OTW and find that both approaches can achieve a torque precision better than 1 pN · nm. The OTW, capable of measuring torque on (sub)millisecond timescales, provides the best torque precision for measurement times$\mathrm{?}$10 s, after which drift becomes a limiting factor. For longer measurement times, magnetic torque tweezers with their superior stability provide the best torque precision. Use of the Allan deviation enables critical assessments of the torque precision as a function of measurement time across different measurement modalities and provides a tool to optimize measurement protocols for a given instrument and application.     

Dynamics of Membrane-Bound G12V-KRAS from Simulations and Single-Molecule FRET in Native Nanodiscs

Recent studies have shown that the small GTPase KRAS adopts multiple orientations with respect to the plane of anionic model membranes, whereby either the three C-terminal helices or the three N-terminal β-strands of the catalytic domain face the membrane. This has functional implications because, in the latter, the membrane occludes the effector-interacting surface. However, it remained unclear how membrane reorientation occurs and, critically, whether it occurs in the cell in which KRAS operates as a molecular switch in signaling pathways. Herein, using data from a 20 μs-long atomistic molecular dynamics simulation of the oncogenic G12V-KRAS mutant in a phosphatidylcholine/phosphatidylserine bilayer, we first show that internal conformational fluctuations of flexible regions in KRAS result in three distinct membrane orientations. We then show, using single-molecule fluorescence resonance energy transfer measurements in native lipid nanodiscs derived from baby hamster kidney cells, that G12V-KRAS samples three conformational states that correspond to the predicted orientations. The combined results suggest that relatively small energy barriers separate orientation states and that signaling-competent conformations dominate the overall population.     

Single-Molecule Resolution of Antimicrobial Peptide Interactions with Supported Lipid A Bilayers

The molecular interactions between antimicrobial peptides (AMPs) and lipid A-containing supported lipid bilayers were probed using single-molecule total internal reflection fluorescence microscopy. Hybrid supported lipid bilayers with lipid A outer leaflets and phospholipid (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE)) inner leaflets were prepared and characterized, and the spatiotemporal trajectories of individual fluorescently labeled LL37 and Melittin AMPs were determined as they interacted with the bilayer surfaces comprising either monophosphoryl or diphosphoryl lipid A (from Escherichia coli) to determine the impact of electrostatic interactions. Large numbers of trajectories were obtained and analyzed to obtain the distributions of surface residence times and the statistics of the spatial trajectories. Interestingly, the AMP species were sensitive to subtle differences in the charge of the lipid, with both peptides diffusing more slowly and residing longer on the diphosphoryl lipid A. Furthermore, the single-molecule dynamics indicated a qualitative difference between the behavior of AMPs on hybrid Lipid A bilayers and on those composed entirely of DOPE. Whereas AMPs interacting with a DOPE bilayer exhibited two-dimensional Brownian diffusion with a diffusion coefficient of ～1.7 μm²/s, AMPs adsorbed to the lipid A surface exhibited much slower apparent diffusion (on the order of ～0.1 μm²/s) and executed intermittent trajectories that alternated between two-dimensional Brownian diffusion and desorption-mediated three-dimensional flights. Overall, these findings suggested that bilayers with lipid A in the outer leaflet, as it is in bacterial outer membranes, are valuable model systems for the study of the initial stage of AMP-bacterium interactions. Furthermore, single-molecule dynamics was sensitive to subtle differences in electrostatic interactions between cationic AMPs and monovalent or divalent anionic lipid A moieties.     

Compaction of Single-Molecule Megabase-Long Chromatin under the Influence of Macromolecular Crowding

The megabase-sized length of chromatin is highly relevant to the state of chromatin in vivo, where it is subject to a highly crowded environment and is organized in topologically associating domains of similar dimension. We developed an in vitro experimental chromatin model system reconstituted from T4 DNA (approximately 166 kbp) and histone octamers and studied the monomolecular compaction of this megabase-sized chromatin fiber under the influence of macromolecular crowding. We used single-molecule fluorescence microscopy and observed compaction in aqueous solutions containing poly(ethylene glycol) in the presence of monovalent (Na⁺ and K⁺) and divalent (Mg²⁺) cations. Both DNA and chromatin demonstrated compaction under comparable conditions in the presence of poly(ethylene glycol) and Na⁺ or Mg²⁺ salt. However, the mechanism of the compaction changed from a first-order phase transition for DNA to a continuous folding for megabase-sized chromatin fibers. A more efficient and pronounced chromatin compaction was observed in the presence of Na⁺ compared to K⁺. A flow-stretching technique to unfold DNA and chromatin coils was used to gain further insight into the morphology of partially folded chromatin fibers. The results revealed a distribution of partially folded chromatin fibers. This variability is likely the result of the heterogeneous distribution of nucleosomes on the DNA chain. The packaging of DNA in the form of chromatin in the crowded nuclear environment appears essential to ensure gradual conformational changes of DNA.     

Synergistic SHAPE/Single-Molecule Deconvolution of RNA Conformation under Physiological Conditions

Structural RNA domains are widely involved in the regulation of biological functions, such as gene expression, gene modification, and gene repair. Activity of these dynamic regions depends sensitively on the global fold of the RNA, in particular, on the binding affinity of individual conformations to effector molecules in solution. Consequently, both the 1) structure and 2) conformational dynamics of noncoding RNAs prove to be essential in understanding the coupling that results in biological function. Toward this end, we recently reported observation of three conformational states in the metal-induced folding pathway of the tRNA-like structure domain of Brome Mosaic Virus, via single-molecule fluorescence resonance energy transfer studies. We report herein selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE)-directed structure predictions as a function of metal ion concentrations ([Mⁿ⁺]) to confirm the three-state folding model, as well as test 2° structure models from the literature. Specifically, SHAPE reactivity data mapped onto literature models agrees well with the secondary structures observed at 0–10 mM [Mg²⁺], with only minor discrepancies in the E hairpin domain at low [Mg²⁺]. SHAPE probing and SHAPE-directed structure predictions further confirm the stepwise unfolding pathway previously observed in our single-molecule studies. Of special relevance, this means that reduction in the metal-ion concentration unfolds the 3′ pseudoknot interaction before unfolding the long-range stem interaction. This work highlights the synergistic power of combining 1) single-molecule Förster resonance energy transfer and 2) SHAPE-directed structure-probing studies for detailed analysis of multiple RNA conformational states. In particular, single-molecule guided deconvolution of the SHAPE reactivities permits 2° structure predictions of isolated RNA conformations, thereby substantially improving on traditional limitations associated with current structure prediction algorithms.     

Measuring Intracellular Viscosity in Conditions of Hypergravity

Gravity-sensitive cellular responses are regularly observed in both specialized and nonspecialized cells. One potential mechanism for this sensitivity is a changing viscosity of the intracellular organelles. Here, we report a novel, to our knowledge, viscosity-sensitive molecular rotor based on mesosubstituted boron-dipyrrin used to investigate the response of viscosity of cellular membranes to hypergravity conditions created at the large diameter centrifuge at the European Space Agency Technology Centre. Mouse osteoblastic (MC3T3-E1) and endothelial (human umbilical vein endothelial cell) cell lines were tested, and an increase in viscosity was found with increasing hypergravity loading. This response is thought to be primarily biologically driven, with the potential for a small, instantaneous physical mechanism also contributing to the observed effect. This work provides the first, to our knowledge, quantitative data for cellular viscosity changes under hypergravity, up to 15 × g.     

Measuring Mobility in Chromatin by Intensity-Sorted FCS

The architectural organization of chromatin can play an important role in genome regulation by affecting the mobility of molecules within its surroundings via binding interactions and molecular crowding. The diffusion of molecules at specific locations in the nucleus can be studied by fluorescence correlation spectroscopy (FCS), a well-established technique based on the analysis of fluorescence intensity fluctuations detected in a confocal observation volume. However, detecting subtle variations of mobility between different chromatin regions remains challenging with currently available FCS methods. Here, we introduce a method that samples multiple positions by slowly scanning the FCS observation volume across the nucleus. Analyzing the data in short time segments, we preserve the high temporal resolution of single-point FCS while probing different nuclear regions in the same cell. Using the intensity level of the probe (or a DNA marker) as a reference, we efficiently sort the FCS segments into different populations and obtain average correlation functions that are associated to different chromatin regions. This sorting and averaging strategy renders the method statistically robust while preserving the observation of intranuclear variations of mobility. Using this approach, we quantified diffusion of monomeric GFP in high versus low chromatin density regions. We found that GFP mobility was reduced in heterochromatin, especially within perinucleolar heterochromatin. Moreover, we found that modulation of chromatin compaction by ATP depletion, or treatment with solutions of different osmolarity, differentially affected the ratio of diffusion in both regions. Then, we used the approach to probe the mobility of estrogen receptor-α in the vicinity of an integrated multicopy prolactin gene array. Finally, we discussed the coupling of this method with stimulated emission depletion FCS for performing FCS at subdiffraction spatial scales.