Biophysical reports最新文献

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.

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.

All living systems display remarkable spatial and temporal precision, despite operating in intrinsically fluctuating environments. It is even more surprising given that biological phenomena are regulated by multiple chemical reactions that are also random. Although the underlying molecular mechanisms of surprisingly high precision in biology remain not well understood, a novel theoretical picture that relies on the coupling of relevant stochastic processes has recently been proposed and applied to explain different phenomena. To illustrate this approach, in this review, we discuss two systems that exhibit precision control: spatial regulation in bacterial cell size and temporal regulation in the timing of cell lysis by λ bacteriophage. In cell-size regulation, it is argued that a balance between stochastic cell growth and cell division processes leads to a narrow distribution of cell sizes. In cell lysis, it is shown that precise timing is due to the coupling of holin protein accumulation and the breakage of the cellular membrane. The stochastic coupling framework also allows us to explicitly evaluate dynamic properties for both biological systems, eliminating the need to utilize the phenomenological concept of thresholds. Excellent agreement with experimental observations is observed, supporting the proposed theoretical ideas. These observations also suggest that the stochastic coupling method captures the important aspects of molecular mechanisms of precise cellular regulation, providing a powerful new tool for more advanced investigations of complex biological phenomena.

The B-DNA of the genome contains numerous sequences that can form various noncanonical structures including G-quadruplex (G4), formed by two or more stacks of four guanine residues in a plane, and intercalating motif (i-motif [iM]) formed by alternately arranged C-C⁺ pairs. One of the easy yet sensitive methods to study G4s and iMs is circular dichroism (CD) spectroscopy, which generates characteristic G4 and iM peaks. We have analyzed and compared the effects of various environmental factors including pH, buffer composition, temperature, flanking sequences, complimentary DNA strands, and single-stranded DNA binding protein (SSB) on the CD patterns of G4s and iMs generated by two groups of DNA molecules, one containing tandem repeats of GGGGCC and CCCCGG from the C9ORF72 gene associated with amyotrophic lateral sclerosis and frontotemporal dementia, and the second containing polyG/polyC clusters from oncogene promoter-proximal regions without such tandem repeats. Changes in pH caused drastic changes in CCCCGG-iM and GGGGCC-G4 and the changes were dependent on repeat numbers and G-C basepairing. In contrast, with the DNA sequences from the promoter-proximal regions of oncogenes, iMs disassembled upon pH changes with the peak slowly shifting to lower wavelength but the G4s did not show significant change. Complementary DNA strands and flanking DNA sequences also regulate G4 and iM formation. The SSB disassembled both G4s and iMs formed by almost all sequences suggesting an in vivo role for SSBs in the disassembly of G4s and iMs during DNA replication and other DNA transactions.

Fibroblast growth factor 21 (FGF21) is an endocrine FGF that plays a vital role in regulating essential metabolic pathways. FGF21 increases glucose uptake by cells, promotes fatty acid oxidation, reduces blood glucose levels, and alleviates metabolic diseases. However, detailed studies on its stability and biophysical characteristics have not been reported. Herein, we present the overexpression, biophysical characterization, and metabolic activity of a soluble recombinant FGF21 (rFGF21). The far-UV circular dichroism spectra of rFGF21 show a negative trough at 215 nm, indicating that the protein's backbone predominantly adopts a β sheet conformation. rFGF21 shows intrinsic tyrosine fluorescence at 305 nm. Thermal denaturation using differential scanning calorimetry reveals that rFGF21 is relatively thermally unstable, with a melting temperature of 46.8°C (±0.1°C). The urea-induced unfolding of rFGF21 is rapid, with a chemical transition midpoint of 0.4 M. rFGF21 is readily cleaved by trypsin in limited trypsin digestion assays. Isothermal titration calorimetry experiments show that rFGF21 does not bind to heparin. Interestingly, rFGF21 demonstrates proliferative activity in NIH/3T3 fibroblasts and enhances mitochondrial oxidative phosphorylation and fatty acid oxidation in 3T3-L1 adipocytes. These findings provide a crucial framework for the engineering of novel structure-based variants of FGF21 with improved stability and biological activity to treat metabolic disorders.

Transcription factor proteins bind to specific DNA promoter sequences and initiate gene transcription. These proteins often contain intrinsically disordered activation domains (ADs) that regulate their transcriptional activity. Like other disordered protein regions, ADs do not have a fixed three-dimensional structure and instead exist in an ensemble of conformations. Disordered ensembles contain sequence-encoded structural preferences that are often linked to their function. We hypothesize that this link exists between the structural preferences of AD ensembles and their ability to induce gene expression. To test this, we measured the ensemble dimensions of two ADs, HIF-1α and CITED2, in live cells using fluorescence resonance energy transfer microscopy and correlated this structural information with their transcriptional activity. We find that mutations that expanded the ensemble of HIF-1α increased transcriptional activity, while compacting mutations reduced it, highlighting the critical role of structural plasticity in regulating HIF-1α function. Conversely, CITED2 showed no correlation between ensemble dimensions and activity. Our results highlight a possible link between AD ensemble dimensions and their transcriptional activity, with implications for transcriptional regulation and dysfunction.

Diffusion coefficients often vary across regions, such as cellular membranes, and quantifying their variation can provide valuable insight into local membrane properties such as composition and stiffness. Toward quantifying diffusion coefficient spatial maps and uncertainties from particle tracks, we develop a Bayesian framework (DiffMAP-GP) by placing Gaussian process (GP) priors on the family of candidate maps. For sake of computational efficiency, we leverage inducing point methods on GPs arising from the mathematical structure of the data giving rise to nonconjugate likelihood-prior pairs. We analyze both synthetic data, where ground truth is known, as well as data drawn from live-cell single-molecule imaging of membrane proteins. The resulting tool provides an unsupervised method to rigorously map diffusion coefficients continuously across membranes without data binning.

Membrane potential (MP) changes can provide a simple readout of bacterial functional and metabolic state or stress levels. While several optical methods exist for measuring fast changes in MP in excitable cells, there is a dearth of such methods for absolute and precise measurements of steady-state MPs in bacterial cells. Conventional electrode-based methods for the measurement of MP are not suitable for calibrating optical methods in small bacterial cells. While optical measurement based on Nernstian indicators have been successfully used, they do not provide absolute or precise quantification of MP or its changes. We present a novel, calibrated MP recording approach to address this gap. In this study, we used a fluorescence lifetime-based approach to obtain a single-cell-resolved distribution of the membrane potential and its changes upon extracellular chemical perturbation in a population of bacterial cells for the first time. Our method is based on 1) a unique VoltageFluor (VF) optical transducer, whose fluorescence lifetime varies as a function of MP via photoinduced electron transfer and 2) a quantitative phasor-FLIM analysis for high-throughput readout. This method allows MP changes to be easily visualized, recorded and quantified. By artificially modulating potassium concentration gradients across the membrane using an ionophore, we have obtained a Bacillus subtilis-specific MP versus VF lifetime calibration and estimated the MP for unperturbed B. subtilis cells to be -65 mV (in minimal salts glycerol glutamate [MSgg]), -127 mV (in M9), and that for chemically depolarized cells as -14 mV (in MSgg). We observed a population-level MP heterogeneity of ∼6-10 mV indicating a considerable degree of diversity of physiological and metabolic states among individual cells. Our work paves the way for deeper insights into bacterial electrophysiology and bioelectricity research.

The mechanical properties of cells are closely related to function and play a crucial role in many cellular processes, including migration, differentiation, and cell fate determination. Numerous methods have been developed to assess cell mechanics under various conditions, but they often lack accuracy on biologically relevant piconewton-range forces or have limited control over the applied force. Here, we present a straightforward approach for using optically trapped polystyrene beads to accurately apply piconewton-range forces to adherent and suspended cells. We precisely apply a constant force to cells by means of a force-feedback system, allowing for quantification of deformation, cell stiffness, and creep response from a single measurement. Using drug-induced perturbations of the cytoskeleton, we show that this approach is sensitive to detecting changes in cellular mechanical properties. Collectively, we provide a framework for using optical tweezers to apply highly accurate forces to adherent and suspended cells and describe straightforward metrics to quantify cellular mechanical properties.

Super-resolution microscopy (SRM) has transformed biological imaging by circumventing the diffraction limit of light and enabling the visualization of cellular structures and processes at the molecular level. Central to the capabilities of SRM is fluorescent labeling, which ensures the precise attachment of fluorophores to biomolecules and has direct impact on the accuracy and resolution of imaging. Continuous innovation and optimization in fluorescent labeling are essential for the successful application of SRM in cutting-edge biological research. In this review, we discuss recent advances in fluorescent labeling strategies for molecular bioimaging, with a special focus on protein labeling. We compare different approaches, highlight technological breakthroughs, and address challenges such as linkage error and labeling density. By evaluating both established and emerging methods, we aim to guide researchers through all aspects that should be considered before opting for any labeling technique.