Biophysical reports最新文献

Since the advent of stochastic localization microscopy approaches in 2006, the number of studies employing this strategy to investigate the subdiffraction limit features of fluorescently labeled structures in biology, biophysics and solid state samples has increased exponentially. Underpinning all these approaches is the notion that the position of single molecules can be determined to high precision, provided enough photons are collected. The determination of exactly how precisely, has been demanded to formulas that try to approximate the so-called Cramer-Rao lower bound based on input parameters such as the number of photons collected from the molecules, or the size of the camera pixel. These estimates should, however, be matched to the experimental localization precision, which can be easily determined if, instead of looking at single beads, we study the distance between a pair. We revisit here a few key works, observing how these theoretical determinations tend to routinely underestimate the experimental localization precision of the order of a factor 2. A software-independent metric to determine, based on each individual setup, the appropriate value to set on the localization error of individual emitters is provided.

Myopia is a prevalent refractive eye disorder closely associated with alterations in corneal biomechanical properties. As fundamental units of corneal tissue, corneal cells significantly influence myopia progression through their nanomechanical characteristics. However, the biophysical mechanisms underlying this process, particularly in human corneal cells, remain unclear. This study investigates the coupling between mechanical properties and cytoskeletal morphology in human corneal cells across varying myopia severity levels. Utilizing atomic force microscopy (AFM), the Young's modulus and adhesion properties of corneal cells obtained from patients with low, moderate, and high myopia were assessed. Additionally, the cytoskeletal morphological variations were quantified by calculating the fractal dimension from AFM topography images. Experimental results reveal that with increasing myopia severity, corneal cells exhibit decreased stiffness, increased adhesion, and reduced regularity and stability of the cytoskeletal network. This evidence highlights a coupling relationship between biomechanical properties and cytoskeletal morphology in human corneal cells during myopia development at the cellular scale, offering significant insights into the pathogenesis of myopia and potential avenues for innovative preventive strategies. VIDEO ABSTRACT.

Assessing protein insertion and association with membranes is often a critical step that follows protein synthesis for both fundamental studies on protein folding and structure as well as translational applications that harness proteins for their activity. Traditionally, membrane protein association with membranes involves ultracentrifugation, which can be time-consuming and inaccessible in low-resource scientific environments. In this study, we develop an accessible method to purify vesicle-integrated cell-free expressed proteins from unincorporated protein or lysed membranes. We use a table-top microcentrifuge, capable of reaching speeds up to 21,130 × g, and a sucrose gradient to effectively separate the bulk of the cell-free expression components from proteoliposomes. We validate our approach can be used to measure membrane association of a variety of proteins, such as peripheral and transmembrane proteins as well as lipid-specific proteins, and that our method can be extended to membrane proteins derived from cellular membranes. Our approach provides a more accessible, cost-effective, and low-volume alternative for isolating proteoliposomes from misfolded and unassociated membrane proteins that should be applicable for fundamental biophysical studies and applications involving cell-free expressed membrane proteins.

The In Vitro Motility Assay (IVMA) is a widely used experimental system to study the chemical and mechanical activity of myosin and other cytoskeletal motor proteins. In the IVMA, myosin molecules are bound to a glass surface and propel fluorescently labeled actin filaments across the surface, which are recorded using video fluorescence microscopy. The length and velocity of the actin filaments offer a measurement of the chemomechanical activity of the myosin motor proteins. Although the assay itself is well suited for high-throughput application, current video analysis approaches are slow, labor intensive, and subject to human bias. To address this shortfall, we introduce ATLAS, an open-source, platform independent software package that utilizes state-of-the-art machine learning algorithms to identify fluorescently labeled actin filaments and then track and analyze their motion in the IVMA. Utilizing both experimental data and a large array of simulated actomyosin motility movies, we demonstrate that ATLAS accurately and efficiently measures both the velocity and length of actin filaments across a broad range of experimental conditions.

Cross-linked fibrous networks are central to maintaining the structural integrity and functional relevance of many biological and engineered materials. Fibrin networks are the building blocks of blood clots, mediators of tissue injury and repair, and synthetic wound sealants. Cross-linking of fibrin fibers is catalyzed by the activated form of transglutaminase enzyme FXIIIa, which becomes available in plasma but is also readily presented on the surface of activated platelets and macrophages. The contribution of surface-bound FXIIIa to fibrin structure has not been well understood. In this work, we investigated the role of surface-bound FXIIIa on the formation and structure of fibrin fibers from FXIII-deficient plasma by confining the cross-linking reactions to the surface of microspheres. Quantitative microscopy revealed that cross-linking on FXIIIa-coated surfaces facilitates fibrin deposition following a sigmoidal kinetics, and that these fibers were straighter, longer, and more numerous compared with uncross-linked fibers bound to surfaces coated with anti-fibrin antibody. Our results suggest that, by modifying local fibrin density and structure, surface-bound FXIIIa may play a significant role in the mechanobiology of hemostasis and inflammation.

Super-resolution microscopy has enabled imaging at nanometer-scale resolution. However, achieving this level of detail without introducing artifacts that could mislead data interpretation requires maintaining sample stability throughout the entire imaging acquisition. This process can range from a few seconds to several hours, particularly when combining live-cell imaging with super-resolution techniques. Here, we present a three-dimensional active sample stabilization system based on real-time tracking of fiducial markers. To ensure broad accessibility, the system is designed using readily available off-the-shelf optical and photonic components. Additionally, the accompanying software is open source and written in Python, facilitating adoption and customization by the community. We achieve a standard deviation of the sample movement within 1 nm in both the lateral and axial directions for a duration in the range of hours. Our approach allows easy integration into existing microscopes, not only making prolonged super-resolution microscopy more accessible but also allowing confocal and widefield live-cell imaging experiments spanning hours or even days.

Super-resolution microscopy often entails long acquisition times of minutes to hours. Since drifts during the acquisition adversely affect data quality, active sample stabilization is commonly used for some of these techniques to reach their full potential. Although drifts in the lateral plane can often be corrected after acquisition, this is not always possible or may come with drawbacks. Therefore, it is appealing to stabilize sample position in three dimensions (3D) during acquisition. Various schemes for active sample stabilization have been demonstrated previously, with some reaching sub-nanometer stability in 3D. Here, we present a scheme for active drift correction that delivers the nanometer-scale 3D stability demanded by state-of-the-art super-resolution techniques and is straightforward to implement compared to previous schemes capable of reaching this level of stabilization precision. Using a refined algorithm that can handle various types of reference structure, without sparse signal peaks being mandatory, we stabilized sample position to ∼1 nm in 3D using objective lenses both with high and low numerical aperture. Our implementation requires only the addition of a simple widefield imaging path and we provide an open-source control software with graphical user interface to facilitate easy adoption of the module. Finally, we demonstrate how this has the potential to enhance data collection for diffraction-limited and super-resolution imaging techniques using single-molecule localization microscopy and cryo-confocal imaging as showcases.

Exocytosis, which mediates important functions like synaptic transmission and stress responses, has been postulated to release all transmitter molecules in the vesicle in the "all-or-none" quantal hypothesis. Challenging this hypothesis, amperometric current recordings of catecholamine release propose that sub-quantal or partial transmitter release is dominant in various cell types, particularly chromaffin cells. The sub-quantal hypothesis predicts that fusion pore closure (kiss-and-run fusion), the cause of sub-quantal release, is dominant, and blocking pore closure increases quantal size. We tested these predictions by imaging fusion pore closure and amperometric recording of catecholamine release in chromaffin cells during high potassium application, the most-used stimulation protocol for sub-quantal release study. We found that fusion pore closure is not predominant, and inhibition of the fusion pore closure does not increase the quantal size calculated from the amperometric current charge when a sufficiently long integration time is used. These results suggest that sub-quantal release is not prevalent during high potassium application in adrenal chromaffin cells.

We investigate to what extent yeast complementation assays, which in principle can provide large amounts of training data for machine-learning models, yield quantitative correlations between growth rescue and single-channel recordings. If this were the case, yeast complementation results could be used as surrogate data for machine-learning-based channel design. Therefore, we mutated position L94 at the cavity entry of the model K⁺ channel Kcv_PBCV1 to all proteinogenic amino acids. The function of the wild-type channel and its mutants was investigated by reconstituting them in planar lipid bilayers and by their ability to rescue the growth of a yeast strain deficient in K⁺ uptake. The single-channel data show a distinct effect of mutations in this critical position on unitary conductance and open probability, with no apparent causal relationship between the two functional parameters. We also found that even conservative amino acid replacements can alter the unitary conductance and/or open probability and that most functional changes show no systematic relationship with the physicochemical nature of the amino acids. This emphasizes that the functional influence of an amino acid on channel function cannot be reduced to a single chemical property. Mutual comparison of single-channel data and yeast complementation results exhibit only a partial correlation between their electrical parameters and their potency of rescuing growth. Hence, complementation data alone are not sufficient for enabling functional channel design; they need to be complemented by additional parameters such as the number of channels in the plasma membrane.

This study investigates the use of spectral phasor analysis, hyperspectral imaging, and 6-acetyl-2-dimethylaminonaphthalene (ACDAN) fluorescence to explore key protein transitions: unfolding, amyloid aggregation, and liquid-liquid phase separation. We show that ACDAN fluorescence can sensitively detect subtle conformational changes before the complete protein unfolds, revealing early microenvironmental shifts. During amyloid formation, ACDAN identifies solvent dipolar relaxation events undetectable by conventional thioflavin T, providing critical insight into early aggregation events. Additionally, we map the physicochemical properties of protein biocondensates and highlight distinct microenvironments within these condensates, emphasizing the significance of dipolar relaxation in phase-separated systems. The approach provides a flexible and user-friendly toolkit for studying protein transitions, which can be easily implemented in commercial spectrofluorometers and microscopes.