Biophysical reports最新文献

{"title":"Correlating disordered activation domain ensembles with gene expression levels.","authors":"Eduardo Flores, Aleah R Camacho, Estefania Cuevas-Zepeda, Mary B McCoy, Feng Yu, Max V Staller, Shahar Sukenik","doi":"10.1016/j.bpr.2024.100195","DOIUrl":"10.1016/j.bpr.2024.100195","url":null,"abstract":"<p><p>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.</p>","PeriodicalId":72402,"journal":{"name":"Biophysical reports","volume":" ","pages":"100195"},"PeriodicalIF":2.4,"publicationDate":"2025-03-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11791265/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142928673","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"DiffMAP-GP: Continuous 2D diffusion maps from particle trajectories without data binning using Gaussian processes.","authors":"Vishesh Kumar, J Shepard Bryan, Alex Rojewski, Carlo Manzo, Steve Pressé","doi":"10.1016/j.bpr.2024.100194","DOIUrl":"10.1016/j.bpr.2024.100194","url":null,"abstract":"<p><p>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.</p>","PeriodicalId":72402,"journal":{"name":"Biophysical reports","volume":" ","pages":"100194"},"PeriodicalIF":2.4,"publicationDate":"2025-03-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11959874/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142866556","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Toward measurements of absolute membrane potential in Bacillus subtilis using fluorescence lifetime.","authors":"Debjit Roy, Xavier Michalet, Evan W Miller, Kiran Bharadwaj, Shimon Weiss","doi":"10.1016/j.bpr.2025.100196","DOIUrl":"10.1016/j.bpr.2025.100196","url":null,"abstract":"<p><p>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.</p>","PeriodicalId":72402,"journal":{"name":"Biophysical reports","volume":" ","pages":"100196"},"PeriodicalIF":2.4,"publicationDate":"2025-03-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11835658/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142973653","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Robust quantification of cellular mechanics using optical tweezers.","authors":"Wessel S Rodenburg, Sven F A Ebben, Jorine M Eeftens","doi":"10.1016/j.bpr.2025.100199","DOIUrl":"10.1016/j.bpr.2025.100199","url":null,"abstract":"<p><p>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.</p>","PeriodicalId":72402,"journal":{"name":"Biophysical reports","volume":" ","pages":"100199"},"PeriodicalIF":2.4,"publicationDate":"2025-03-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11910095/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143416442","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Fluorescent labeling strategies for molecular bioimaging.","authors":"Marcel Streit, Made Budiarta, Marvin Jungblut, Gerti Beliu","doi":"10.1016/j.bpr.2025.100200","DOIUrl":"10.1016/j.bpr.2025.100200","url":null,"abstract":"<p><p>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.</p>","PeriodicalId":72402,"journal":{"name":"Biophysical reports","volume":" ","pages":"100200"},"PeriodicalIF":2.4,"publicationDate":"2025-03-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11914189/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143416423","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Deep learning-driven automated high-content dSTORM imaging with a scalable open-source toolkit.","authors":"Janis T Linke, Luise Appeltshauser, Kathrin Doppler, Katrin G Heinze","doi":"10.1016/j.bpr.2025.100201","DOIUrl":"10.1016/j.bpr.2025.100201","url":null,"abstract":"<p><p>Super-resolution microscopy offers the ability to visualize molecular structures in biological samples with unprecedented detail. However, the full potential of these techniques is often hindered by a lack of automated, user-independent workflows. Here, we present an open-source toolkit that automates dSTORM super-resolution microscopy using deep learning for segmentation and object detection. This standalone program enables reliable segmentation of diverse biomedical images, even in low-contrast samples, surpassing existing solutions. Integrated into the imaging pipeline, it rapidly processes high-content data in minutes, reducing manual labor. Demonstrated by biological examples, such as microtubules in cell culture and the βII-spectrin in nerve fibers, our approach makes super-resolution imaging faster, more robust, and easy to use, even by nonexperts. This broadens its potential applications in biomedicine, including high-throughput experimentation.</p>","PeriodicalId":72402,"journal":{"name":"Biophysical reports","volume":" ","pages":"100201"},"PeriodicalIF":2.4,"publicationDate":"2025-02-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11986538/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143537880","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"TC10 differently controls the dynamics of Exo70 in growth cones of cortical and hippocampal neurons.","authors":"Hiteshika Gosain, Karin B Busch","doi":"10.1016/j.bpr.2024.100186","DOIUrl":"10.1016/j.bpr.2024.100186","url":null,"abstract":"<p><p>The exocyst is an octameric protein complex that acts as a tether for GOLGI-derived vesicles at the plasma membrane during exocytosis. It is involved in membrane expansion during axonal outgrowth. Exo70 is a major subunit of the exocyst complex and is controlled by TC10, a Rho family GTPase. How TC10 affects the dynamics of Exo70 at the plasma membrane is not well understood. There is also evidence that TC10 controls Exo70 dynamics differently in nonpolar cells and axons. To address this, we used super-resolution microscopy to study the spatially resolved effects of TC10 on Exo70 dynamics in HeLa cells and the growth cone of cortical and hippocampal neurons. We generated single-particle localization and trajectory maps and extracted mean square displacements, diffusion coefficients, and alpha coefficients to characterize Exo70 diffusion. We found that the diffusivity of Exo70 was different in nonpolar cells and the growth cone of neurons. TC10 stimulated the mobility of Exo70 in HeLa cells but decreased the diffusion of Exo70 in the growth cone of cortical neurons. In contrast to cortical neurons, TC10 overexpression did not affect the mobility of Exo70 in the axonal growth cone of hippocampal neurons. These data suggest that mainly exocyst tethering in cortical neurons was under the control of TC10.</p>","PeriodicalId":72402,"journal":{"name":"Biophysical reports","volume":" ","pages":"100186"},"PeriodicalIF":2.4,"publicationDate":"2024-12-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11617994/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142633883","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Growing bacterial colonies harness emergent genealogical demixing to regulate organizational entropy.","authors":"Garima Rani, Anupam Sengupta","doi":"10.1016/j.bpr.2024.100175","DOIUrl":"10.1016/j.bpr.2024.100175","url":null,"abstract":"<p><p>Spatiotemporal organization of individuals within growing bacterial colonies is a key determinant of intraspecific interactions and colony-scale heterogeneities. The evolving cellular distribution, in relation to the genealogical lineage, is thus central to our understanding of bacterial fate across scales. Yet, how bacteria self-organize genealogically as a colony expands has remained unknown. Here, by developing a custom-built label-free algorithm, we track and study the genesis and evolution of emergent self-similar genealogical enclaves, whose dynamics are governed by biological activity. Topological defects at enclave boundaries tune finger-like morphologies of the active interfaces. The Shannon entropy of cell arrangements reduce over time; with faster-dividing cells possessing higher spatial affinity to genealogical relatives, at the cost of a well-mixed, entropically favorable state. Our coarse-grained lattice model demonstrates that genealogical enclaves emerge due to an interplay of division-mediated dispersal, stochasticity of division events, and cell-cell interactions. The study reports so-far hidden emergent self-organizing features arising due to entropic suppression, ultimately modulating intraspecific genealogical distances within bacterial colonies.</p>","PeriodicalId":72402,"journal":{"name":"Biophysical reports","volume":" ","pages":"100175"},"PeriodicalIF":2.4,"publicationDate":"2024-12-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11416667/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142094260","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Structural studies of the human α₁ glycine receptor via site-specific chemical cross-linking coupled with mass spectrometry.","authors":"Rathna J Veeramachaneni, Chelsee A Donelan, Kayce A Tomcho, Shaili Aggarwal, David J Lapinsky, Michael Cascio","doi":"10.1016/j.bpr.2024.100184","DOIUrl":"10.1016/j.bpr.2024.100184","url":null,"abstract":"<p><p>By identifying distance constraints, chemical cross-linking coupled with mass spectrometry (CX-MS) can be a powerful complementary technique to other structural methods by interrogating macromolecular protein complexes under native-like conditions. In this study, we developed a CX-MS approach to identify the sites of chemical cross-linking from a single targeted location within the human α1 glycine receptor (α1 GlyR) in its apo state. The human α1 GlyR belongs to the family of pentameric ligand-gated ion channel receptors that function in fast neurotransmission. A single chemically reactive cysteine was reintroduced into a Cys null α1 GlyR construct at position 41 within the extracellular domain of human α1 homomeric GlyR overexpressed in a baculoviral system. After purification and reconstitution into vesicles, methanethiosulfonate-benzophenone-alkyne, a heterotrifunctional cross-linker, was site specifically attached to Cys41 via disulfide bond formation. The resting receptor was then subjected to UV photocross-linking. Afterward, monomeric and oligomeric α1 GlyR bands from SDS-PAGE gels were trypsinized and analyzed by tandem MS in bottom-up studies. Dozens of intrasubunit and intersubunit sites of α1 GlyR cross-linking were differentiated and identified from single gel bands of purified protein, showing the utility of this experimental approach to identify a diverse array of distance constraints of the α1 GlyR in its resting state. These studies highlight CX-MS as an experimental approach to identify chemical cross-links within full-length integral membrane protein assemblies in a native-like lipid environment.</p>","PeriodicalId":72402,"journal":{"name":"Biophysical reports","volume":" ","pages":"100184"},"PeriodicalIF":2.4,"publicationDate":"2024-12-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11550363/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142407301","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Expression level of cardiac ryanodine receptors dictates properties of Ca²⁺-induced Ca²⁺ release.","authors":"Roman Nikolaienko, Elisa Bovo, Aleksey V Zima","doi":"10.1016/j.bpr.2024.100183","DOIUrl":"10.1016/j.bpr.2024.100183","url":null,"abstract":"<p><p>The type 2 ryanodine receptor (RyR2) is the major Ca²⁺ release channel required for Ca²⁺-induced Ca²⁺ release (CICR) and cardiac excitation-contraction coupling. The cluster organization of RyR2 at the dyad is critical for efficient CICR. Despite its central role in cardiac Ca²⁺ signaling, the mechanisms that control CICR are not fully understood. As a single RyR2 Ca²⁺ flux dictates local CICR that underlies Ca²⁺ sparks, RyR2 density in a cluster, and therefore the distance between RyR2s, should have a profound impact on local CICR. Here, we studied the effect of the RyR2 expression level ([RyR2]) on CICR activation, termination, and amplitude. The endoplasmic reticulum (ER)-targeted Ca²⁺ sensor RCEPIA-1er was used to directly measure the ER [Ca²⁺] (Ca²⁺]_ER) in the T-Rex-293 the sarco/endoplasmic reticulum Ca²⁺-ATPase (SERCA2a) stable cell line expressing human RyR2. Cells coexpressing RyR2 and SERCA2a produced periodic [Ca²⁺]_ER depletions in the form of spontaneous Ca²⁺ waves due to propagating CICR. For each studied cell, the [Ca²⁺]_ER at which Ca²⁺ waves are activated and terminated was analyzed as a function of [RyR2]. CICR parameters, such as [Ca²⁺]_ER activation, termination, and amplitude, were inversely proportional to [RyR2] at low-intermediate levels. Increasing the sensitivity of RyR2 to cytosolic Ca²⁺ lowered the [Ca²⁺]_ER at which CICR is activated and terminated. Decreasing the sensitivity of RyR2 to cytosolic Ca²⁺ had the opposite effect on CICR. These results suggest that RyR2 density in the release cluster should have a significant impact on local CICR activation and termination. Since SR Ca²⁺ load is evenly distributed throughout the SR network, clusters with higher RyR2 density would have a higher probability of initiating spontaneous CICR.</p>","PeriodicalId":72402,"journal":{"name":"Biophysical reports","volume":" ","pages":"100183"},"PeriodicalIF":2.4,"publicationDate":"2024-12-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11532243/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142333654","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}