Biophysical journal最新文献

Single molecule localization microscopy (SMLM) has revolutionized the understanding of cellular organization by reconstructing informative images with quantifiable spatial distributions of molecules far beyond the optical diffraction limit. Much effort has been devoted to optimizing localization accuracy. One such approach is the assessment of SMLM data quality in real-time, rather than after lengthy post-acquisition analysis, which nevertheless represents a computational challenge. We overcame this difficulty by implementing an innovative mathematical approach we designed to drastically reduce the computational analysis of particle localization. Our Quality Control Maps (QCM) workflow enables a much higher rate of data processing compared to that limited by the frequency required by current cameras. Accordingly, by using an innovative computational approach for the detection step and an estimator based on a Gaussian model of the point spread function (PSF), sub-pixel particle locations and their accuracy can be determined through a straightforward analytical calculation, without the need for iterations. As a true parameter-free algorithm, QCM is robust and adaptable to all types of SMLM data, with high speed enabling the real-time calculation of quantitative quality control indicators. Such features are compatible with smart microscopy, the concept of which depends on the adjustment of acquisition parameters in real-time according to analytical results. Finally, the offline QCM mode can be used as a tool to evaluate synthetic or previously acquired data, as well as to teach the basic concepts of SMLM.

Phosphatidylinositides (PIs), constitute only 1 to 3% of plasma membranes, but play vital roles in cellular signaling. In particular, phosphatidylinositol 4,5-bisphosphate (PIP₂) is involved in processes such as cytoskeleton organization and ion channel regulation. Pleckstrin homology (PH) domains, are modular domains found in many proteins, and are known for their strong affinity for PIP₂ headgroups. The role of lipid composition in PH domain binding to PIP₂, particularly the inclusion of phosphatidylserine (PS), is not well understood. This study explores the mechanisms of PH domain binding to PIP₂, using fluorescence spectroscopy, FTIR, two-dimensional IR (2D IR) spectroscopy, and molecular dynamics (MD) simulations. We find that anionic PIP₂ and PS alter the interfacial environment compared to phosphatidylcholines (PC). Additionally, the PH domain promotes the localization of anionic lipid domains upon binding. Our results highlight the role of PS in lipid domain formation within membranes and its potential influence on protein binding affinities and lipid geometries. Specifically, we discovered a strong interaction between PIP₂ and PS, where hydrogen bonding within these anionic lipids drives localization in the membrane. This interaction also regulates protein binding at the membrane interface. Our findings suggest that cooperativity between PIP₂ and PS is key to the formation of localized lipid domains and the recruitment of proteins like the PH domain of PLC-δ1.

The molecular interactions between a fungal membrane model and SJGAP, a 32-amino-acid antimicrobial peptide (AMP) derived from skipjack tuna GAPDH, as well as four analogs, were investigated using molecular dynamics simulations and Fourier-transform infrared (FTIR) spectroscopy. In a previous study, Analog 7, modified by replacing three alanine residues with leucine residues, exhibited unique antifungal activity without any antibacterial effect. This contrasts with other analogs, which showed both antifungal and antibacterial effects. In the present study, Analog 7 displayed the strongest interactions with the membrane's hydrophobic core, inserting more deeply and causing significantly greater membrane deformation and thinning compared to the other analogs. Its presence caused significant membrane deformation, evident from the displacement of both the phosphate groups and terminal methyls of the lipids. Notably, Analog 7 was the only analog to induce a marked depletion of ergosterol around the peptide insertion site. Fourier-transform infrared (FTIR) spectroscopy experiments further confirmed the distinctive impact of Analog 7 on a fungal membrane model. The combined results from molecular dynamics simulations and spectroscopy emphasize the critical role of leucine substitutions in Analog 7, particularly at residues 18 and 19 within the central α helix, in promoting membrane thinning and inducing ergosterol depletion, suggesting increased membrane permeabilization, which could explain its previously reported antifungal specificity. This study provides the first insights into the molecular interactions between a GAPDH-derived AMP and a fungal membrane model, offering valuable information about its antifungal mechanism of action.

The human genome consists of about 2 m of DNA packed inside the cell nucleus barely 10 μm in diameter. DNA is complexed with histones, forming chromatin fiber, which folds inside the nucleus into loops, TADs, A/B compartments and chromosome territories. This organization is knot-free and self-similar across length scales, leading to a hypothesis that the genome presents a fractal globule, which was corroborated by chromosome conformation capture experiments. In addition, many microscopy techniques have been used to obtain the fractal dimension of the genome's spatial distribution from its images. However, different techniques often required that different definitions of fractal dimension be adapted, making the comparison of these results not trivial. In this study, we use spinning disc confocal microscopy to collect high-resolution images of nuclei in live human cells during the cell cycle. We then systematically compare existing image-based fractal analyses - including mass-scaling, box-counting, lacunarity and multifractal spectrum - by applying them to images of human cell nuclei and investigate changes in the genome's spatial organization during the cell cycle. Our data reveal that different image-based fractal measurements offer distinct metrics, highlighting different features of the genome's spatial organization. Yet, all these metrics consistently indicate the following trend for the changes in the genome's organization during the cell cycle: the genome being compactly packed in early G1 phase, followed by a decondensation throughout the G1 phase, and a subsequent condensation in the S and G2 phases. Our comprehensive comparison of image-based fractal analyses reconciles the perceived discrepancies between different methods. Moreover, our results offer new insights into the physical principles underlying the genome's organization and its changes during the cell cycle.

Super-resolution light microscopy techniques facilitate the observation of nm-sized biomolecules, which are 1-2 orders of magnitude smaller than the diffraction limit of light. Using super-resolution microscopy techniques, it is possible to observe fluorescence from two biomolecules in close proximity; however, not necessarily in direct interaction. Using FRETsael, we localize biomolecular interactions exhibiting FRET with nanometer accuracy, from two-color fluorescence lifetime imaging data. The concepts of FRETsael were tested first against simulations, in which the recovered localization accuracy is 20-30 nm for true-positive detections of FRET pairs. Further analysis of the simulation results reports the conditions in which true-positive rates are maximal. We then show the capabilities of FRETsael on simulated samples of actin-vinculin and ER-ribosome interactions, as well as experimental samples of actin-myosin two-color confocal imaging. Overall, the FRETsael approach paves the way toward studying biomolecular interactions with improved spatial resolution from laser scanning confocal two-color fluorescence lifetime imaging.

During the active transport of cellular cargo, forces generated by cargo-associated molecular motors propel the cargo along cytoskeletal tracks. However, the forces impact not only the cargo, but also the underlying cytoskeletal filaments. To better understand the interplay between cargo transport and the organization of cytoskeletal filaments, we employ coarse-grained computer simulations to study actin filaments interacting with cargo-anchored myosin motors in a confined domain. We show that cargo transport can lead to the segregation of filaments into domains of preferred filament polarity separated by clusters of aggregated cargoes. The formation of polarity-sorted filament domains is enhanced by larger numbers of cargoes, more motors per cargo, and longer filaments. Analysis of individual trajectories reveals dynamic and heterogeneous behavior, including locally stable aggregates of cargoes that undergo rapid coalescence into larger clusters when sufficiently close. Our results provide insight into the impact of motor-driven organelle transport on actin filaments, which is relevant both in cells and in synthetic environments.

Binuclear ruthenium complexes have been investigated for potential DNA-targeted therapeutic and diagnostic applications. Studies of DNA threading intercalation, in which DNA basepairs must be broken for intercalation, have revealed means of optimizing a model binuclear ruthenium complex to obtain reversible DNA-ligand assemblies with the desired properties of high affinity and slow kinetics. Here, we used single-molecule force spectroscopy to study a binuclear ruthenium complex with a longer semirigid linker relative to the model complex. Equilibrium results suggest a DNA affinity that is an order of magnitude higher than the parent binuclear ruthenium complex, likely due to a sterically relieved DNA threading intercalation mechanism. Notably, kinetics analysis shows that less DNA elongation is required for threading intercalation compared to the parent complex, and the association rate is two orders of magnitude faster. The ruthenium complex elongates the DNA duplex by ∼0.3 nm per bound ligand to reach the equilibrium intercalated state, with a significantly different energy landscape relative to the parent complex. Mechanical properties of the ligand-saturated DNA duplex show a higher persistence length, indicating that the longer semirigid linker provides enough molecular spacing to allow a single monomer to fully stack with basepairs, comparable to the monomeric parent ruthenium complex. The DNA basepairs in the equilibrium threading intercalated state are likely intact, and the ruthenium complex is shielded from the polar solution, providing measurable single-molecule confocal fluorescence signals. The obtained confocal fluorescence imaging of the bound dye confirms mostly uniform intercalation along the tethered DNA, consistent with other intercalators. The results of this study, along with previously examined ruthenium complex variants, illustrate tunable intercalation mechanisms guided by the rational design of therapeutic and diagnostic small molecules to target and modify the DNA duplex.

Humans have three known ATP-binding cassette (ABC) transporters in the inner mitochondrial membrane (ABCB7, ABCB8, and ABCB10). ABCB10, the most studied of them thus far, is essential for normal red blood cell development and protection against oxidative stress, and it was recently found to export biliverdin, a heme degradation product with antioxidant properties. The molecular mechanism underlying the function of ABC transporters remains controversial. Their nucleotide binding domains (NBDs) must dimerize to hydrolyze ATP, but capturing the transporters in such conformation for structural studies has been experimentally difficult, especially for ABCB10 and related eukaryotic transporters. Purified transporters are commonly studied in detergent micelles, or after their reconstitution in nanodiscs, usually at nonphysiological temperature and using nonhydrolyzable ATP analogs or mutations that prevent ATP hydrolysis. Here, we have used luminescence resonance energy transfer to evaluate the effect of experimental conditions on the NBD dimerization of ABCB10. Our results indicate that all conditions used for determination of currently available ABCB10 structures have failed to induce NBD dimerization. ABCB10 in detergent responded only to MgATP at 37°C, whereas reconstituted protein shifted toward dimeric NBDs more easily, including in response to MgAMP-PNP and even present NBD dimerization with MgATP at room temperature. The nanodisc's size affects the nucleotide-free conformational equilibrium of ABCB10 and the response to ATP in the absence of magnesium, but for all analyzed sizes (scaffold proteins MSP1D1, MSP1E3D1, and MSP2N2), a conformation with dimeric NBDs is clearly preferred during active ATP hydrolysis (MgATP, 37°C). These results highlight the sensitivity of this human ABC transporter to experimental conditions and the need for a more cautious interpretation of structural models obtained under far from physiological conditions. A dimeric NBD conformation that has been elusive in previous studies seems to be dominant during MgATP hydrolysis at physiological temperature.