The bacterial SOS response promotes DNA repair, survival, and mutagenesis under genotoxic stress, especially during sub-lethal antibiotic exposure. This response is regulated by LexA, a transcriptional repressor controlling SOS gene expression, but how LexA coordinates this network as antibiotic stress alters nucleoid structure is unclear. Using ciprofloxacin-induced DNA damage, we investigated how increasing antibiotic stress affects the spatial relationship between LexA and the nucleoid in single E. coli cells. Through 3D single-molecule imaging and functional assays, we found that sublethal ciprofloxacin doses activated SOS, expanded nucleoids, and maintained LexA association. In contrast, higher stress caused severe DNA damage, compacted nucleoids, and LexA exclusion, yet some cells remained viable and recovered after drug removal. These results demonstrate that the SOS response involves both temporal and spatial regulation, with LexA and nucleoid organization adapting to damage severity to modulate SOS functions.

Lumacaftor and Ivacaftor are two FDA-approved medications currently used to treat cystic fibrosis (CF), a genetic disease caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR), a chloride ion channel located in epithelial cell membranes; however, the detailed mechanism(s) of their action remains to be elucidated. Both drugs, termed modulators, bind CFTR at a protein-lipid interface, yet Lumacaftor acts at the endoplasmic reticulum (ER), while Ivacaftor acts at the plasma membrane (PM). A major difference among biological membranes is their level of cholesterol (viz., the ER, 5% cholesterol; the Golgi apparatus, 12.5%; and the PM, 30%). Therefore, we investigated the ability of each molecule to interact with membranes of the corresponding cholesterol content to determine if lipid cholesterol content provides a physical basis for their observed localized activity. Using differential scanning calorimetry and a terbium-based liposome disruption assay, we show that both Lumacaftor (a corrector) and Ivacaftor (a potentiator) penetrate/diffuse through membranes containing high cholesterol concentrations, such as in Golgi and the PM. The results further suggest that (1) Lumacaftor resides within membranes containing 5% cholesterol, supporting the proposition that Lumacaftor acts as a corrector of the CFTR channel at the ER level where the nascent protein is in its initial folding stage; and (2) Ivacaftor is well-suited to penetrate the PM and reach its binding pocket on CFTR. Our findings provide evidence that membrane cholesterol levels significantly modulate CFTR corrector/potentiator activity and consequently may affect sensitivity to clinical therapeutics in CF patients.

Ribonucleoprotein (RNP) granules have been linked to translation regulation and disease, but their assembly and regulatory mechanisms are not well understood. Here, we show that the RNA-binding protein G3BP1 preferentially interacts with unfolded RNA, driving the assembly of RNP granule-like condensates that establish RNA-RNA interactions. These RNA-RNA interactions limit the mobility and translatability of sequestered mRNAs and stabilize the condensates. The DEAD-box RNA helicase DDX3X attenuates RNA-RNA interactions inside RNP granule-like condensates, rendering the condensates dynamic and enabling mRNA translation. Importantly, disease-associated and catalytically inactive DDX3X variants fail to resolve such RNA-RNA interactions. Inhibiting DDX3X in cultured cells accelerates RNP granule assembly and delays their disassembly, indicating that RNA-RNA interactions contribute to RNP granule stability in cells. Our findings reveal how RNP granules generate inhibitory RNA-RNA interactions that are modulated by DEAD-box RNA helicases to ensure RNA availability and translatability.

Force spectroscopy gives access to the underlying free energy landscape of protein folding. Proteins exhibit folding rates between microseconds and hours. To access slow folding rates, magnetic tweezers have shown to be a promising tool, yet it remained unclear if magnetic tweezers capture kinetics of ultra-fast folding proteins. Here, we study the folding mechanics and kinetics of λ6-85; a five-helix bundle protein with fast 20 µs folding time in the thermal denaturation midpoint. We observed two-state folding of λ6-85 at 6.2 pN and 250 ms folding time in the mechanical midpoint. With optical tweezers, we found that λ6-85 folds at the mechanical midpoint with 15 ms, 16-fold faster than in magnetic tweezers. To resolve the discrepancy between magnetic and optical tweezers, we developed a physics-based model taking into account the constant force condition of magnetic tweezers and the spacer mechanics. Using this model, we reach agreement between magnetic tweezers, optical tweezers and thermal denaturation experiments. In summary, we show that magnetic tweezers capture kinetics of ultrafast conformational changes, even at low forces. The model for extrapolation of the kinetics to force-free conditions provides opportunities of comparability for the growing community of magnetic tweezers force spectroscopy. Magnetic tweezers can access stability and dynamics of proteins. Here, the authors show that magnetic tweezers resolve also dynamics of a fast-folding protein, with 16-fold slower transition rates compared to optical tweezers, and provide a comprehensive view of the kinetics from different force spectroscopy techniques.

Single-molecule localization microscopy (SMLM) advanced biological discoveries beyond the diffraction limit. Various implementations enable 3D SMLM to reconstruct volumetric cell images. Yet, the inherent anisotropic point spread function of optical microscopes often limits the localization precision in the axial direction compared to the lateral precision. Such localization anisotropy could also expand spherical cellular structures to ellipsoidal cellular structures. Structure identification, however, is often performed using DBSCAN cluster algorithms, considering an isotropic search volume. Here, we show that an anisotropic DBSCAN search volume identifies anisotropic clusters more reliably using simulated ground truth data sets. Given experimental localization precisions, we suggest optimized search parameters based on an expanded computational grid search and show an enhanced performance of anisotropic DBSCAN amidst variations in localization precision. We demonstrate the capability of anisotropic DBSCAN on experimental data and anticipate that the algorithm allows for a more rigorous identification of clusters in cells, considering the anisotropic localization precisions of astigmatism-based 3D SMLM.

Multiple antibiotic resistances are a major global health threat. The predominant tool for adaptation in Gram-negative bacteria is the integron. Under stress, it rearranges gene cassettes to offer an escape using the tyrosine recombinase IntI, recognizing folded DNA hairpins, the attC sites. Four recombinases and two attC sites form the synaptic complex. Yet, for unclear reasons, the recombination efficiency varies greatly. Here, we established an optical tweezers force spectroscopy assay to probe the synaptic complex stability and revealed, for seven combinations of attC sites, significant variability in the mechanical stability. We found a strong correlation between mechanical stability and recombination efficiency of attC sites in vivo, indicating a regulatory mechanism from the DNA structure to the macromolecular complex stability. Taking into account known forces during DNA metabolism, we propose that the variation of the integron in vivo recombination efficiency is mediated by the synaptic complex stability. We anticipate that further recombination processes are also affected by their corresponding mechanical stability.

The predominant tool for adaptation in Gram-negative bacteria is a genetic system called integron. Under conditions of stress, it rearranges gene cassettes, ensuring their sampling through expression, to offer a solution for overcoming the initial stress. Integrons are a major actor of multiple antibiotic resistances, a recognized major global health threat. Cassettes are recombined by a unique recombination process involving a tyrosine recombinase – the IntI integrase – and folded single-stranded DNA hairpins – the attC sites which terminate each cassette. Four recombinases and two attC sites form a macromolecular synaptic complex, which is key to the recombination process and the focus of our study. The bottom strand of all attC sites shows highest recombination efficiency in vivo than the top one, however, the efficiency still varies several orders of magnitude and the underlying reason remains unclear. Here, we established an optical tweezers force-spectroscopy assay that allows us to probe the synaptic complex stability. We found for seven combinations of attC sites great variability in the mechanical stability. Two protein variants also showed a strong influence on the mechanical stability. We then determined the in vivo recombination efficiencies of the different attC site combinations and protein variants and discovered a strong correlation between recombination efficiency and mechanical stability of the synaptic complex, indicating a regulatory mechanism from the DNA sequence to the macromolecular complex stability. Taking into account known forces during DNA metabolism, we suggest that the variation of the in vivo recombination efficiency is mediated strongly by the synaptic complex stability.### Competing Interest StatementThe authors have declared no competing interest.

Single-molecule Förster-resonance energy transfer (smFRET) experiments allow the study of biomolecular structure and dynamics in vitro and in vivo. We performed an international blind study involving 19 laboratories to assess the uncertainty of FRET experiments for proteins with respect to the measured FRET efficiency histograms, determination of distances, and the detection and quantification of structural dynamics. Using two protein systems with distinct conformational changes and dynamics, we obtained an uncertainty of the FRET efficiency ≤0.06, corresponding to an interdye distance precision of ≤2 Å and accuracy of ≤5 Å. We further discuss the limits for detecting fluctuations in this distance range and how to identify dye perturbations. Our work demonstrates the ability of smFRET experiments to simultaneously measure distances and avoid the averaging of conformational dynamics for realistic protein systems, highlighting its importance in the expanding toolbox of integrative structural biology.

Abstract The actin cortex is an active polymer network underneath the plasma membrane at the periphery of mammalian cells. It is a major regulator of cell shape through the generation of active cortical tension. In addition, the cortex constitutes a mechanical shield that protects the cell during mechanical agitation. Cortical mechanics is tightly controlled by the presence of actin cross‐linking proteins that dynamically bind and unbind actin filaments. Cross‐linker actin bonds are weak non‐covalent bonds whose bond lifetime is likely affected by mechanical tension in the actin cortex making cortical composition inherently mechanosensitive. Here, a quantitative study of changes in cortex composition and turnover dynamics upon short‐lived peaks in active and passive mechanical tension in mitotic HeLa cells is presented. These findings dsclose a twofold mechanical reinforcement strategy of the cortex upon tension peaks entailing i) a direct catch‐bond mechanosensitivity of cross‐linkers filamin and α‐actinin and ii) an indirect cortical mechanosensitivity that triggers actin cortex reinforcement via enhanced polymerization of actin. Thereby a “molecular safety belt” mechanism that protects the cortex from injury upon mechanical challenges is disclosed.

Single-molecule FRET (smFRET) has become a versatile tool for probing the structure and functional dynamics of biomolecular systems, and is extensively used to address questions ranging from biomolecular folding to drug discovery. Confocal smFRET measurements are amongst the widely used smFRET assays and are typically performed in a single-well format. Thus, sampling of many experimental parameters is laborious and time consuming. To address this challenge, we extend here the capabilities of confocal smFRET beyond single-well measurements by integrating a multiwell plate functionality to allow for continuous and automated smFRET measurements. We demonstrate the broad applicability of the multiwell plate assay towards DNA hairpin dynamics, protein folding, competitive and cooperative protein-DNA interactions, and drug-discovery, revealing insights that would be very difficult to achieve with conventional single-well format measurements. For the adaptation into existing instrumentations, we provide a detailed guide and open-source acquisition and analysis software. Single-molecule FRET (smFRET) studies often struggle with large parameter spaces. Here, the authors introduce an automated smFRET platform for multiwell plate screening of biomolecular conformations and dynamics.

Upon subjecting molecules to nonequilibrium conditions, many biophysical and biochemical features such as molecular diffusion, protein folding, dissociation constant, as well as enzyme-catalyzed reactions can be characterized in an aqueous solution. However, conducting assays under nonequilibrium conditions in complex self-assembled biomatrices (e.g., extracellular matrices) remains challenging due to the limitations associated with sample handling, reaction design, and optical detection. Herein, we present the investigation of biomolecular thermodiffusion in noncovalently assembled synthetic or naturally derived hydrogels. This approach has been demonstrated with a large variety of analytes of different sizes across the nanoscale, including small molecules, polysaccharides, proteins, DNA, and five DNA origamis of different geometries in various polymer networks. As the aggregation of analytes can be suppressed, the in-biomatrix method has also shown advantages over in-solution measurements. Remarkably, the method provides a unique opportunity to study how a thermophoretic movement of matrix surroundings can impact the thermophoretic movement of analytes, with dimensions from low to high nm and a million-fold variation in mass. Most importantly, the method is capable of measuring binding affinity in biomatrices, allowing for characterizing the protein-ligand interaction within a more biologically relevant context.

Functional capacities of lead halide perovskites are strongly dependent on their morphology, crystallographic texture, and internal ultrastructure on the nano- and the meso-scale. In the last decade, significant efforts are directed towards the development of novel synthesis routes that would overcome the morphological constraints provided by the physical and crystallographic properties of these materials. In contrast, various living organisms, such as unicellular algae, have the ability to mold biogenic crystals into a vast variety of intricate nano-architectured shapes while keeping their single crystalline nature. Here, using the cell wall of the dinoflagellate L. granifera as a model, sustainably harvested biogenic calcite is successfully transformed into nano-structured perovskites. Three variants of lead halide perovskites CH3 NH3 PbX3 are generated with X = Cl- , Br- and I- ; exhibiting emission peak-wavelength ranging from blue, to green, to near-infrared, respectively. The approach can be used for the mass production of nano-architectured perovskites with desired morphological, textural and, consequently, physical properties exploiting the numerous templates provided by calcite forming unicellular organisms.

In bacteria, the key mechanism facilitating survival and adaptation upon DNA damage is the SOS response. Antibiotics cause DNA damage and trigger autoproteolytic cleavage of the transcriptional repressor LexA, which controls over 50 SOS genes including drivers of mutation. Efforts to inhibit this response and thereby combat antibiotic resistance rely on a broad understanding of its behavior in vivo. Here, we use a single-molecule localization microscopy assay to directly track LexA dynamics in Escherichia coli under low antibiotic stress.

Recent advances in magnetic tweezers set them as a promising tool to unveil the dynamics of proteins and nucleic acids by means of hours to days-long stable constant force experiments. Archetypal folds play a crucial role in our means to uncover the intricacies of protein folding. One prominent case is the λ-phage cI transcription repressor fragment λ6-85; a five-helix bundle protein were single point mutations shift its folding time from fast 200 μs to ultrafast downhill-like 2.3 μs kinetics. Can we resolve ultrafast kinetics with the milliseconds time-resolution of the magnetic tweezers? Here, we report the observation of ultrafast protein folding in magnetic tweezers. Small pN forces slow down the folding to tens of milliseconds; ∼10-fold bigger compared to optical tweezers measurements. We developed an analytic model to describe folding kinetics in magnetic tweezers and extract folding and unfolding rates at a broad range of dynamics ranging from milliseconds to seconds. We decipher free energy landscapes of three different variants of λ6-85, namely λWt, λHA and λYA, and compare transition state positions and barrier heights as small as few kBT measured with magnetic tweezers and optical tweezers.

Cystic fibrosis (CF) is caused by mutations in the gene that codes for the chloride channel cystic fibrosis transmembrane conductance regulator (CFTR). Recent advances in CF treatment have included use of small-molecule drugs known as modulators, such as Lumacaftor (VX-809), but their detailed mechanism of action and interplay with the surrounding lipid membranes, including cholesterol, remain largely unknown. To examine these phenomena and guide future modulator development, we prepared a set of wild type (WT) and mutant helical hairpin constructs consisting of CFTR transmembrane (TM) segments 3 and 4 and the intervening extracellular loop (termed TM3/4 hairpins) that represent minimal membrane protein tertiary folding units. These hairpin variants, including CF-phenotypic loop mutants E217G and Q220R, and membrane-buried mutant V232D, were reconstituted into large unilamellar phosphatidylcholine (POPC) vesicles, and into corresponding vesicles containing 70 mol% POPC +30 mol% cholesterol, and studied by single-molecule FRET and circular dichroism experiments. We found that the presence of 30 mol% cholesterol induced an increase in helicity of all TM3/4 hairpins, suggesting an increase in bilayer cross-section and hence an increase in the depth of membrane insertion compared to pure POPC vesicles. Importantly, when we added the corrector VX-809, regardless of the presence or absence of cholesterol, all mutants displayed folding and helicity largely indistinguishable from the WT hairpin. Fluorescence spectroscopy measurements suggest that the corrector alters lipid packing and water accessibility. We propose a model whereby VX-809 shields the protein from the lipid environment in a mutant-independent manner such that the WT scaffold prevails. Such 'normalization' to WT conformation is consistent with the action of VX-809 as a protein-folding chaperone.