Biophysical reports最新文献

While the transduction process has been well-studied in hair cells, the possible presence of mechanical perturbations in the hair cell soma has not been explored in non-mammalian species. Hair cell mechanotransduction involves rapid biophysical events that remain difficult to observe in intact tissue. We developed a label-free optical method to image active motility within the soma during both spontaneous and mechanically driven hair bundle motion. Localized light-intensity fluctuations were detected at distinct focal planes, particularly near the periphery and basal pole of the soma. These optical signals exhibited spectral components that matched those of the hair bundle and were substantially reduced when mechanotransduction channel gating was disrupted, indicating that the somatic activity reflects physiological processes linked to mechanotransduction. Activity hotspots consistently aligned with regions of ionic channels and synaptic contacts, and strong stimulation produced phase-locked somatic responses that diminished after tip-link disruption. These findings parallel reports of mechanical correlates of neuronal activity and support the presence of an optical signature of transduction within the soma. Our results demonstrate that wide-field, label-free imaging can resolve intrinsic optical events in semi-intact sensory epithelia, offering a promising approach for non-invasive studies of hair cell and afferent-neuron signaling.

The human eye lens plays an essential role in vision by focusing light onto the retina. This transparent tissue consists of densely packed crystallin proteins that exhibit remarkable solu- bility despite minimal protein turnover. Post-translational modifications that accumulate over a lifetime can reduce crystallin solubility, resulting in the precipitation or phase-separation of pro- tein aggregates. Oxidation is a common type of modification that can cause such opacification of the lens, particularly in age-related cataract. Here, we study the oxidation of W163 in γS- crystallin, a structural lens protein that is particularly vulnerable to oxidative stress. We were motivated by previous findings, which report the oxidation of this residue in diseased as well as UV- and γ-irradiated samples. Using genetic code expansion (GCE), we incorporated an oxi- dation mimic, 5-hydroxytryptophan (5HTP), at position 163 of γS-crystallin (γS-W163(5HTP)). This subtle change in the structural and electronic properties of its side-chain is hypothesized to destabilize the hydrophobic core of the C-terminal domain. γS-W163(5HTP) was characterized and compared to the wild-type (γS-WT). Although the overall fold and stability of the two proteins were comparable, the aggregation of γS-W163(5HTP) was triggered at notably lower temperatures compared to γS-WT. Subsequent investigation of this observation using both simulations andexperiments suggests a potential mechanism for polymerization as well as oxidation-induced conformational changes that may cause susceptibility to thermal aggregation. Our findings high- light the utility of GCE platforms for systematically evaluating the impact of post-translational modifications on disease-related proteins.

Vibrational frequency profiling (VFP) using optical tweezers (OT) has emerged as a promising technique to characterize single cell behavior for diagnostic applications. These applications include cancer screening, identifying bacterial resistance to antibiotics, and assessing cellular response to metabolic treatments. Previously, we demonstrated that vibrational profiling can successfully differentiate samples in real time using single-cell vibrational signatures. However, the sensitivity of this approach, and its ability to be applied across variable experimental conditions and cell lines has not been well identified. This study builds on previous work, demonstrating the feasibility of our established vibrational analysis to quantitively assess whether changes in experimental design can improve model separation of different cell types. U251 human glioblastoma cells and A549 human lung carcinoma cells were used as our model system. We illustrate this capability through three examples: (i) comparing open Petri-dish to closed microfluidic chambers, (ii) synchronizing cells in the cell cycle, and (iii) altering medium viscosity. These factors were selected for their potential to influence signal quality and model accuracy. Vibrations were collected using OT, Fourier Transformed to produce power spectra and were then parameterized using peak detection, extracting area-under-curve values. Peaks were aggregated across samples and classified using Partial Least Squares Discriminant Analysis. PLS-DA classification was performed with 70/30 train-test splits of data and 10-fold CV. Petri-dishes yielded a more well classified sample than microfluidics chambers (F1 score 0.89 vs 0.79). Synchronized cells reduced vibrational variability and improved classification (F1 score 0.83 vs 0.79). Increased viscosity produced slight classifier improvements (F1 score 0.81 vs 0.79). These findings demonstrate that VFPs are sensitive to the mechanical and environmental context of cell measurement, highlighting that careful standardization of experimental conditions is crucial for developing reproducible and clinically translatable VFP-based diagnostic platforms.

In adult cardiomyocytes, the type 2a sarco/endoplasmic reticulum Ca²⁺-ATPase (SERCA2a) plays a vital role in intracellular Ca²⁺ regulation. Reduced SERCA2a function has been associated with decreased myocardial contraction and cardiac output in several heart diseases. Consequently, increasing SERCA2a activity is a high-priority target for treating cardiac pathologies associated with abnormal Ca²⁺ homeostasis. In our previous SERCA ATPase-based screening study, we identified several small molecules as potential activators of SERCA2a function, including Compound 9, a piperidinyl amide. With porcine cardiac sarcoplasmic reticulum (SR) preparations, we confirmed activation of both SERCA2a ATPase and Ca²⁺-uptake activities. In the current study, we analyzed the effect of Compound 9 on SERCA2a activity on intracellular Ca²⁺ dynamics in ventricular myocytes. Using FRET with human SERCA2a overexpressed in mammalian cells, we confirm that Compound 9 binds and alters SERCA structural dynamics independent of peptide regulators, including phospholamban (PLB). Confocal microscopy and in-cell Ca²⁺ imaging revealed that Compound 9 enhanced Ca²⁺ dynamics in mouse ventricular myocytes. Compound 9 (10 μM) increased the action potential-induced Ca²⁺ transients by 65% and SR Ca²⁺ load by 29%. Moreover, Compound 9 increased Ca²⁺ dynamics during adrenergic receptor stimulation and in PLB knockout cardiomyocytes, suggesting the stimulatory effect of Compound 9 is PLB independent. Overall, Compound 9 displays characteristics that can be beneficial to enhance cardiac intracellular Ca²⁺ dynamics by increasing SERCA2a function.

E-hooks, or the C-terminal tails of tubulin, mediate interactions between microtubules and associated proteins. Despite their functional importance in cellular and physiological processes, their structural variability and mechanistic roles remain poorly understood. E-hooks are thought to be intrinsically disordered to some degree, making crystallographic studies difficult and necessitating the use of computational tools to study their structures and how they change E-hook function. This review synthesizes recent computational efforts to elucidate E-hook structure, dynamics, and functional differentiation across tubulin isotypes. We examine studies probing E-hooks in isolation or with globular cores, which have revealed subunit-specific features influencing microtubule behavior. We also evaluate the role of E-hooks in modulating binding affinity and conformational states of motor proteins and microtubule-associated proteins (MAPs). Finally, we highlight adjacent technological and methodological advances that have implications for both the interpretation of past findings and the design of future studies, offering new directions for the investigation of E-hook-mediated microtubule regulation.

Hemostasis prevents bleeding by forming clots, although disruptions can cause insufficient or excessive clotting. Shear stress is a critical factor influencing the normal function of the blood and can stimulate the formation of unnecessary clots. Stenosed vessels significantly contribute to this phenomenon by increasing shear stress on the vessel walls. This study aims to quantify the effects of shear stress and stenosis severity on thrombus formation in patient-specific left coronary bifurcations. Three-dimensional patient-specific geometries were reconstructed from angiographic data. Blood flow was modeled using the Brinkman equation, while transport and interactions of coagulation factors were simulated through the convection-diffusion-reaction equation. Model predictions were validated using literature data. The findings showed that all patients experienced significant shear stress at the stenosed regions, which are highly susceptible to clot formation. Shear stress was found to be inversely related to vessel diameter and directly related to the stenosis degree. Furthermore, in bifurcated vessels, blood flow may reach zero before complete occlusion by the clot, emphasizing the role of hydrodynamic resistance in redirecting blood flow. Among these factors, the stenosis degree emerges as the most significant predictor of blood flow cessation time. A relationship was developed allowing physicians to accurately and rapidly monitor a patient's condition using angiographic images, providing new insights into the hemodynamic mechanisms of coronary thrombosis and supporting improved diagnosis and treatment of cardiovascular diseases.

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.

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.

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.

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.