Biophysical reports最新文献

Fungi and bacteria are found living in a wide variety of environments, and their interactions are important in many processes including soil health, human and animal physiology, and in biotechnological applications. Here, we investigate a single morphological feature of cocultures of planktonic bacterial growth within biofilm-forming liquid cultures of mycelium, namely, the attachment of bacterial ectobionts of species Bacillus subtilis to fungal hyphae of species Hericium erinaceus. The bacteria-in-mycelial-biofilm method was developed and utilized to allow for attachment of bacteria to hyphae via containment within exopolymeric substances (EPS) and the overall extracellular matrix of the mycelium. A graded dehydration protocol was used to selectively remove extraneous biofilm components and reveal intact bacteria and surface-interfacing features. The dehydration methods allowed for identification of specific interactions and differentiated these cultures from trivial stochastic mixing of bacteria and mycelium in liquid media. Attachment structures appear to be produced primarily by the mycelium and enveloped the bacterial ectobiont. Nanoscale surface-interfacing EPS constituents were preserved, providing a biophysical basis for a range of contact-dependent modulating properties of the bacteria on this fungal host. The mean biofilm area across triplicates was 3.90μm²±0.72μm², and the mean percentage coverage was 18.33%±5.52%. The bacterial biofilm components could not be ruled out as co-contributing to formation of attachment structures due to the structures being present connecting individual bacteria as well as to hyphae.

Crystallins are highly stable, soluble proteins that refract light and maintain transparency in the vertebrate eye lens. They are not replaced after early development, making them an excellent system for studying protein stability and solubility in crowded environments. To better understand the effects of deamidation on these ubiquitous vertebrate crystallins, we investigated a particularly extreme example, a lens protein from the long-lived Antarctic toothfish (Dissostichus mawsoni), γS1 crystallin (DmγS1). This protein remains soluble in the crowded fish lens, maintaining its transparency even at -2°C and at concentrations more than twofold that of humans (nearly 1000 mg/mL) and over a comparable timescale. As the organism ages, crystallins accumulate oxidative damage such as deamidation of Asn and Gln side chains, leading to aggregation and cataract. Previous studies of human γS crystallin (HγS) have shown that extensive deamidation reduces stability and increases aggregation propensity. Here, we present the biophysical characterization of wild-type DmγS1 and variants with three, five, and seven deamidation sites. In sharp contrast to results for human γS-crystallin, increasing the number of deamidations does not significantly change the thermal stability of DmγS1. These proteins are startlingly resistant to thermal denaturation; despite their psychrophilic origin, they have midpoint unfolding temperatures between 56°C and 63°C. Extensive deamidation does make the protein more vulnerable to chemical denaturation as well as aggregation below the unfolding temperature; however, all the variants resist aggregation well above the fish's physiological temperature. These proteins present a useful model system for aggregation resistance in extreme environments; most studies of protein solubility focus on unusually aggregation-prone proteins, but understanding the underlying biophysics also requires studying extremely soluble proteins.

The degree of over-/underwinding of the DNA double helix, quantified by the superhelical density, is a key feature modulating critical biological processes such as gene expression and regulation. In fact, DNA molecules are able to channel the excess levels of mechanical stress into local defective and denatured states that are promptly detected by, e.g., transcription factors and nuclease enzymes. The occurrence and stability of these motifs are dictated by a complex interplay between topological and sequence-dependent effects, ultimately affecting the global conformational dynamics of the DNA molecule itself. Here, we characterize the impact of the sequence and of the superhelical density on the structural evolution of a 672-bp DNA minicircle via classical molecular dynamics simulations employing the coarse-grained oxDNA force field. We observe that moderately-to-highly undercoiled regimes are associated with the occurrence of stable, somewhat broad denaturation bubbles, typically co-localizing with flexible nucleotide sequences on the DNA minicircle. These defects are hardly reabsorbed by the system, thereby pinning the subsequent dynamics of the molecule. In fact, a similar behavior was recapitulated by enforcing "synthetic,"adjoining DNA mismatches, regardless of the underlying nucleotide sequence, suggesting an effective manner of DNA manipulation.

Measuring dipole orientations of fluorescent probes offers unique local structural insights of labeled biomolecules and has seen expanding applications in structural biology studies. Here, we propose an alternative imaging geometry, "dual-view" microscopy, for single-molecule dipole orientation measurements. We develop a protocol capable of simultaneously measuring absorption and emission dipole orientations of single emitters. Further, through simulation, we demonstrate that absorption dipole orientation can be accurately measured with high and uniform precision in three dimensions, significantly outperforming epifluorescence microscopy. Meanwhile the emission dipole is independently narrowed down to four possible orientations and can be uniquely determined with the co-estimated absorption dipole. Dual-view microscopy represents a new paradigm in single-molecule orientation sensing and could have applications in imaging under cryogenic temperatures.

Protein condensation is the basis for formation of membrane-less organelles in the cell. Most famously, weak, polyvalent interactions, often including RNA, may lead to a liquid-liquid phase separation. This effect greatly enhances local concentrations and is thought to promote interactions that would remain rare in dilute solution. Synthetic systems provide a means to clarify the underlying biophysical mechanisms at play, both in vitro and in the cell via exogenous expression. In this regard, ferritin is a useful substrate, as its composition of 24 subunits with octahedral symmetry supports self-assembly by close packing in 3D. The conventional diagnostic tool for protein condensation is fluorescence imaging. In this work, we explore the use of refractive index mapping to detect states of condensation and decondensation. Using two related ferritin-based self-assembly systems, we find that refractive index is a sensitive indicator for reversible condensation. Surprisingly, refractive index indicates a rapid decondensation even when molecular dispersal kinetics are slow according to fluorescence. Conversely, in a photoactivated condensation where long activation results in slow decondensation kinetics, the refractive index provides reliable evidence for the physical state independent of fluorescence. The observations suggest a distinction between condensation to a sparse biomolecular network or to a material continuum that supports an optical polarizability distinct from that of the dilute phase in solution.

Skeletal muscle alpha actin (ACTA1) is important for muscle contraction and relaxation, with historical studies focused on ACTA1 mutations in muscle dysfunction. Proteomics reports have consistently observed that actin, including ACTA1, is acetylated at multiple lysine sites. However, few reports have studied the effects of actin acetylation on cellular function, and fewer have examined ACTA1 acetylation on skeletal muscle function. Here, we aimed to examine how ACTA1 acetylation affected actomyosin interactions by determining actin sliding velocity, myosin binding, and calcium sensitivity. In this study, ACTA1 was chemically acetylated via acetic anhydride (AA) to increasing levels of acetylation: low-level acetylation (using 0.1 mM AA), mid-level acetylation (0.3 mM AA), and high-level acetylation (1 mM AA). We report that ACTA1 acetylation significantly decreased actin sliding velocity and actin filament length. Further analysis showed that ACTA1 acetylation significantly increased calcium sensitivity, with a loss of tropomyosin regulation noted with high-level ACTA1 acetylation. Lastly, ACTA1 acetylation enhanced skeletal myosin half maximal binding to actin. These data highlight acetylation as an additional posttranslational modification, outside of phosphorylation, in the regulation of muscle contraction and skeletal muscle alpha actin function.

The presented study aimed to investigate the antibacterial activity of menthol-the main component of one of the widespread plants of the Lamiaceae family-Mentha arvensis. To investigate the mode of action of menthol, we studied its influence on kanamycin-resistant E. coli pARG-25 and wild-type E. coli BW25113 strains. For this, the effect of menthol on ATPase activity, proton and potassium fluxes, and intracellular pH was investigated under aerobic and anaerobic conditions. The results showed that menthol influences these parameters in a concentration- and condition-dependent way. It likely interacts with FoF₁-ATPase and other systems involved in energy-generating processes and ion transport, disrupting the bacterial metabolism of both antibiotic-resistant and -susceptible strains.

Coarse-grained (CG) models are widely used to study membrane proteins at physiologically relevant scales. However, simulating long-range bilayer deformations induced by membrane-embedded proteins at submicrometer scales remains challenging. Here, we assess a generic solvent-free CG lipid model, previously applied to membrane proteins, for large-scale molecular dynamics simulations. We find that beyond a critical membrane size, the model becomes unstable due to membrane poration and unphysical undulations. To overcome this limitation, we systematically optimize this lipid model, significantly extending its stability for larger membrane systems. Using this improved model, we simulate membrane deformation induced by the mechanosensitive ion channel PIEZO in bilayers with varying mechanical properties. This optimized CG model with tunable mechanical properties provides a timely tool for investigating bilayer-mediated membrane protein interactions and bridging the gap between continuum elasticity theory and atomistic simulations.

Controlling cancer cell fate through membrane depolarization, reactive oxygen species (ROS) dynamics, and voltage-gated ion channel (VGIC) activation represents a rapidly advancing paradigm in bioelectronic oncology. Electrically excitable cancer cells-including glioblastoma, retinoblastoma, SH-SY5Y neuroblastoma, MCF-7, and MDA-MB-231-exhibit distinct electrophysiological and redox sensitivities that govern their responsiveness to photoelectrical stimulation. Here, we develop an integrated theoretical and transcriptomic framework describing how photocapacitive and photofaradaic stimulation modulates intracellular calcium signaling, mitochondrial membrane potential (ΔΨm), and ROS homeostasis to determine apoptotic, autophagic, or proliferative outcomes. Experimental data sets from GSE59612, GSE103224 (glioblastoma), GSE97508 (retinoblastoma), and GSE45827 (breast cancer) parameterize VGIC families, antioxidant pathways, and cell death modules. New simulations using a measured 20-Hz photovoltaic waveform show that photocapacitive depolarization elevates glioblastoma ROS levels to ∼150-160 μM over 30 min, placing cells within a proliferative-to-autophagic transition region, with a small apoptotic component. Mapping these ROS trajectories to a bifurcation-based cell fate model reveals glioblastoma-specific redox thresholds that align with transcriptomic VGIC and antioxidant signatures. By unifying stimulation physics, bioelectrical modeling, and omics-based parameterization, this work provides a predictive foundation for designing photovoltaic cancer therapies tuned to cell-type-specific electrophysiological and redox landscapes. Moreover, in MDA-MB-231 cells the same stimulation induces a controlled, early-stage autophagy response, providing an intrinsic antiinflammatory benefit that can suppress early tumorigenic signaling.