Biophysical reports最新文献

{"title":"Characteristic energy scales of active fluctuations in adherent cells.","authors":"Avraham Moriel, Haguy Wolfenson, Eran Bouchbinder","doi":"10.1016/j.bpr.2022.100099","DOIUrl":"10.1016/j.bpr.2022.100099","url":null,"abstract":"<p><p>Cell-matrix and cell-cell adhesion play important roles in a wide variety of physiological processes, from the single-cell level to the large scale, multicellular organization of tissues. Cells actively apply forces to their environment, either extracellular matrix or neighboring cells, as well as sense its biophysical properties. The fluctuations associated with these active processes occur on an energy scale much larger than that of ordinary thermal equilibrium fluctuations, yet their statistical properties and characteristic scales are not fully understood. Here, we compare measurements of the energy scale of active cellular fluctuations-an effective cellular temperature-in four different biophysical settings, involving both single-cell and cell-aggregate experiments under various control conditions, different cell types, and various biophysical observables. The results indicate that a similar energy scale of active fluctuations might characterize the same cell type in different settings, though it may vary among different cell types, being approximately six to eight orders of magnitude larger than the ordinary thermal energy at room temperature. These findings call for extracting the energy scale of active fluctuations over a broader range of cell types, experimental settings, and biophysical observables and for understanding the biophysical origin and significance of such cellular energy scales.</p>","PeriodicalId":72402,"journal":{"name":"Biophysical reports","volume":"3 1","pages":"100099"},"PeriodicalIF":2.4,"publicationDate":"2022-12-29","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://ftp.ncbi.nlm.nih.gov/pub/pmc/oa_pdf/c3/23/main.PMC9867956.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"10620867","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":"The space between us: Modeling spatial heterogeneity in synthetic microbial consortia dynamics.","authors":"Ryan Godin,&nbsp;Bhargav R Karamched,&nbsp;Shawn D Ryan","doi":"10.1016/j.bpr.2022.100085","DOIUrl":"https://doi.org/10.1016/j.bpr.2022.100085","url":null,"abstract":"<p><p>A central endeavor in bioengineering concerns the construction of multistrain microbial consortia with desired properties. Typically, a gene network is partitioned between strains, and strains communicate via quorum sensing, allowing for complex behaviors. Yet a fundamental question of how emergent spatiotemporal patterning in multistrain microbial consortia affects consortial dynamics is not understood well. Here, we propose a computationally tractable and straightforward modeling framework that explicitly allows linking spatiotemporal patterning to consortial dynamics. We validate our model against previously published results and make predictions of how spatial heterogeneity impacts interstrain communication. By enabling the investigation of spatial patterns effects on microbial dynamics, our modeling framework informs experimentalists, helps advance the understanding of complex microbial systems, and supports the development of applications involving them.</p>","PeriodicalId":72402,"journal":{"name":"Biophysical reports","volume":"2 4","pages":"100085"},"PeriodicalIF":0.0,"publicationDate":"2022-12-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://ftp.ncbi.nlm.nih.gov/pub/pmc/oa_pdf/dd/bc/main.PMC9720408.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"10740663","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":"A dark intermediate in the fluorogenic reaction between tetrazine fluorophores and <i>trans</i>-cyclooctene.","authors":"Felix Hild,&nbsp;Philipp Werther,&nbsp;Klaus Yserentant,&nbsp;Richard Wombacher,&nbsp;Dirk-Peter Herten","doi":"10.1016/j.bpr.2022.100084","DOIUrl":"https://doi.org/10.1016/j.bpr.2022.100084","url":null,"abstract":"<p><p>Fluorogenic labeling via bioorthogonal tetrazine chemistry has proven to be highly successful in fluorescence microscopy of living cells. To date, <i>trans</i>-cyclooctene (TCO) and bicyclonyne have been found to be the most useful substrates for live-cell labeling owing to their fast labeling kinetics, high biocompatibility, and bioorthogonality. Recent kinetic studies of fluorogenic click reactions with TCO derivatives showed a transient fluorogenic effect but could not explain the reaction sequence and the contributions of different intermediates. More recently, fluorescence quenching by potential intermediates has been investigated, suggesting their occurrence in the reaction sequence. However, in situ studies of the click reaction that directly relate these observations to the known reaction sequence are still missing. In this study, we developed a single-molecule fluorescence detection framework to investigate fluorogenic click reactions. In combination with data from ultra-performance liquid chromatography-tandem mass spectrometry, this explains the transient intensity increase by relating fluorescent intermediates to the known reaction sequence of TCO with fluorogenic tetrazine dyes. More specifically, we confirm that the reaction of TCO with tetrazine rapidly forms a fluorescent 4,5-dihydropyridazine species that slowly tautomerizes to a weakly fluorescent 1,4-dihydropyridazine, explaining the observed drop in fluorescence intensity. On a much slower timescale of hours/days, the fluorescence intensity may be recovered by oxidation of the intermediate to a pyridazine. Our findings are of importance for quantitative applications in fluorescence microscopy and spectroscopy as the achieved peak intensity with TCO depends on the specific experimental settings. They clearly indicate the requirement for more robust benchmarking of click reactions with tetrazine dyes and the need for alternative dienophiles with fast reaction kinetics and stable fluorescence emission to further applications in advanced fluorescence microscopy.</p>","PeriodicalId":72402,"journal":{"name":"Biophysical reports","volume":"2 4","pages":"100084"},"PeriodicalIF":0.0,"publicationDate":"2022-12-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9782730/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"10497597","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":"Single-photon smFRET: II. Application to continuous illumination.","authors":"Ayush Saurabh, Matthew Safar, Mohamadreza Fazel, Ioannis Sgouralis, Steve Pressé","doi":"10.1016/j.bpr.2022.100087","DOIUrl":"10.1016/j.bpr.2022.100087","url":null,"abstract":"<p><p>Here we adapt the Bayesian nonparametrics (BNP) framework presented in the first companion article to analyze kinetics from single-photon, single-molecule Förster resonance energy transfer (smFRET) traces generated under continuous illumination. Using our sampler, BNP-FRET, we learn the escape rates and the number of system states given a photon trace. We benchmark our method by analyzing a range of synthetic and experimental data. Particularly, we apply our method to simultaneously learn the number of system states and the corresponding kinetics for intrinsically disordered proteins using two-color FRET under varying chemical conditions. Moreover, using synthetic data, we show that our method can deduce the number of system states even when kinetics occur at timescales of interphoton intervals.</p>","PeriodicalId":72402,"journal":{"name":"Biophysical reports","volume":"3 1","pages":"100087"},"PeriodicalIF":2.4,"publicationDate":"2022-12-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9792399/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"10455672","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":"Single-photon smFRET. I: Theory and conceptual basis.","authors":"Ayush Saurabh, Mohamadreza Fazel, Matthew Safar, Ioannis Sgouralis, Steve Pressé","doi":"10.1016/j.bpr.2022.100089","DOIUrl":"10.1016/j.bpr.2022.100089","url":null,"abstract":"<p><p>We present a unified conceptual framework and the associated software package for single-molecule Förster resonance energy transfer (smFRET) analysis from single-photon arrivals leveraging Bayesian nonparametrics, BNP-FRET. This unified framework addresses the following key physical complexities of a single-photon smFRET experiment, including: 1) fluorophore photophysics; 2) continuous time kinetics of the labeled system with large timescale separations between photophysical phenomena such as excited photophysical state lifetimes and events such as transition between system states; 3) unavoidable detector artefacts; 4) background emissions; 5) unknown number of system states; and 6) both continuous and pulsed illumination. These physical features necessarily demand a novel framework that extends beyond existing tools. In particular, the theory naturally brings us to a hidden Markov model with a second-order structure and Bayesian nonparametrics on account of items 1, 2, and 5 on the list. In the second and third companion articles, we discuss the direct effects of these key complexities on the inference of parameters for continuous and pulsed illumination, respectively.</p>","PeriodicalId":72402,"journal":{"name":"Biophysical reports","volume":"3 1","pages":"100089"},"PeriodicalIF":2.4,"publicationDate":"2022-12-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9793182/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"10455670","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":"Single-photon smFRET. III. Application to pulsed illumination.","authors":"Matthew Safar, Ayush Saurabh, Bidyut Sarkar, Mohamadreza Fazel, Kunihiko Ishii, Tahei Tahara, Ioannis Sgouralis, Steve Pressé","doi":"10.1016/j.bpr.2022.100088","DOIUrl":"10.1016/j.bpr.2022.100088","url":null,"abstract":"<p><p>Förster resonance energy transfer (FRET) using pulsed illumination has been pivotal in leveraging lifetime information in FRET analysis. However, there remain major challenges in quantitative single-photon, single-molecule FRET (smFRET) data analysis under pulsed illumination including 1) simultaneously deducing kinetics and number of system states; 2) providing uncertainties over estimates, particularly uncertainty over the number of system states; and 3) taking into account detector noise sources such as cross talk and the instrument response function contributing to uncertainty; in addition to 4) other experimental noise sources such as background. Here, we implement the Bayesian nonparametric framework described in the first companion article that addresses all aforementioned issues in smFRET data analysis specialized for the case of pulsed illumination. Furthermore, we apply our method to both synthetic as well as experimental data acquired using Holliday junctions.</p>","PeriodicalId":72402,"journal":{"name":"Biophysical reports","volume":"2 4","pages":"100088"},"PeriodicalIF":2.4,"publicationDate":"2022-11-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9747580/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"10404386","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":"Single-photon smFRET. III. Application to pulsed illumination","authors":"Matthew Safar, A. Saurabh, Bidyut Sarkar, M. Fazel, Kunihiko Ishii, T. Tahara, Ioannis Sgouralis, S. Pressé","doi":"10.1101/2022.07.20.500892","DOIUrl":"https://doi.org/10.1101/2022.07.20.500892","url":null,"abstract":"Förster resonance energy transfer (FRET) using pulsed illumination has been pivotal in leveraging lifetime information in FRET analysis. However, there remain major challenges in quantitative single photon, single molecule FRET (smFRET) data analysis under pulsed illumination including: 1) simultaneously deducing kinetics and number of system states; 2) providing uncertainties over estimates, particularly uncertainty over the number of system states; 3) taking into account detector noise sources such as crosstalk, and the instrument response function contributing to uncertainty; in addition to 4) other experimental noise sources such as background. Here, we implement the Bayesian nonparametric framework described in the first companion manuscript that addresses all aforementioned issues in smFRET data analysis specialized for the case of pulsed illumination. Furthermore, we apply our method to both synthetic as well as experimental data acquired using Holliday junctions. Why It Matters In the first companion manuscript of this series, we developed new methods to analyze noisy smFRET data. These methods eliminate the requirement of a priori specifying the dimensionality of the physical model describing a molecular complex’s kinetics. Here, we apply these methods to experimentally obtained datasets with samples illuminated by laser pulses at regular time intervals. In particular, we study conformational dynamics of Holliday junctions.","PeriodicalId":72402,"journal":{"name":"Biophysical reports","volume":"17 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2022-11-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"76290200","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Electrically stimulated droplet injector for reduced sample consumption in serial crystallography.","authors":"Mukul Sonker, Diandra Doppler, Ana Egatz-Gomez, Sahba Zaare, Mohammad T Rabbani, Abhik Manna, Jorvani Cruz Villarreal, Garrett Nelson, Gihan K Ketawala, Konstantinos Karpos, Roberto C Alvarez, Reza Nazari, Darren Thifault, Rebecca Jernigan, Dominik Oberthür, Huijong Han, Raymond Sierra, Mark S Hunter, Alexander Batyuk, Christopher J Kupitz, Robert E Sublett, Frederic Poitevin, Stella Lisova, Valerio Mariani, Alexandra Tolstikova, Sebastien Boutet, Marc Messerschmidt, J Domingo Meza-Aguilar, Raimund Fromme, Jose M Martin-Garcia, Sabine Botha, Petra Fromme, Thomas D Grant, Richard A Kirian, Alexandra Ros","doi":"10.1016/j.bpr.2022.100081","DOIUrl":"10.1016/j.bpr.2022.100081","url":null,"abstract":"<p><p>With advances in X-ray free-electron lasers (XFELs), serial femtosecond crystallography (SFX) has enabled the static and dynamic structure determination for challenging proteins such as membrane protein complexes. In SFX with XFELs, the crystals are typically destroyed after interacting with a single XFEL pulse. Therefore, thousands of new crystals must be sequentially introduced into the X-ray beam to collect full data sets. Because of the serial nature of any SFX experiment, up to 99% of the sample delivered to the X-ray beam during its \"off-time\" between X-ray pulses is wasted due to the intrinsic pulsed nature of all current XFELs. To solve this major problem of large and often limiting sample consumption, we report on improvements of a revolutionary sample-saving method that is compatible with all current XFELs. We previously reported 3D-printed injection devices coupled with gas dynamic virtual nozzles (GDVNs) capable of generating samples containing droplets segmented by an immiscible oil phase for jetting crystal-laden droplets into the path of an XFEL. Here, we have further improved the device design by including metal electrodes inducing electrowetting effects for improved control over droplet generation frequency to stimulate the droplet release to matching the XFEL repetition rate by employing an electrical feedback mechanism. We report the improvements in this electrically triggered segmented flow approach for sample conservation in comparison with a continuous GDVN injection using the microcrystals of lysozyme and 3-deoxy-D-manno-octulosonate 8-phosphate synthase and report the segmented flow approach for sample injection applied at the Macromolecular Femtosecond Crystallography instrument at the Linear Coherent Light Source for the first time.</p>","PeriodicalId":72402,"journal":{"name":"Biophysical reports","volume":"2 4","pages":"100081"},"PeriodicalIF":2.4,"publicationDate":"2022-09-29","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://ftp.ncbi.nlm.nih.gov/pub/pmc/oa_pdf/48/eb/main.PMC9680787.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"10565696","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":"A comparative study of interfacial environments in lipid nanodiscs and vesicles.","authors":"Xiao You,&nbsp;Naveen Thakur,&nbsp;Arka Prabha Ray,&nbsp;Matthew T Eddy,&nbsp;Carlos R Baiz","doi":"10.1016/j.bpr.2022.100066","DOIUrl":"https://doi.org/10.1016/j.bpr.2022.100066","url":null,"abstract":"<p><p>Membrane protein conformations and dynamics are driven by the protein-lipid interactions occurring within the local environment of the membrane. These environments remain challenging to accurately capture in structural and biophysical experiments using bilayers. Consequently, there is an increasing need for realistic cell-membrane mimetics for <i>in vitro</i> studies. Lipid nanodiscs provide certain advantages over vesicles for membrane protein studies. Nanodiscs are increasingly used for structural and spectroscopic characterization of membrane proteins. Despite the common use of nanodiscs, the interfacial environments of lipids confined to a ~10-nm diameter area have remained relatively underexplored. Here, we use ultrafast two-dimensional infrared spectroscopy and temperature-dependent infrared absorption measurements of the ester carbonyls to compare the interfacial hydrogen bond structure and dynamics in lipid nanodiscs of varying lipid compositions and sizes with ~100-nm vesicles. We examine the effects of lipid composition and nanodisc size. We found that nanodiscs and vesicles share largely similar lipid-water H-bond environments and interfacial dynamics. Differences in measured enthalpies of H-bonding suggest that H-bond dynamics in nanodiscs are modulated by the interaction between the annular lipids and the scaffold protein.</p>","PeriodicalId":72402,"journal":{"name":"Biophysical reports","volume":"2 3","pages":""},"PeriodicalIF":0.0,"publicationDate":"2022-09-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://ftp.ncbi.nlm.nih.gov/pub/pmc/oa_pdf/d4/19/main.PMC9518727.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"9623325","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":"Optimization of cryo-electron microscopy for quantitative analysis of lipid bilayers","authors":"Frederick A. Heberle, Doug Welsch, H. L. Scott, M. Waxham","doi":"10.1101/2022.08.23.505005","DOIUrl":"https://doi.org/10.1101/2022.08.23.505005","url":null,"abstract":"Cryogenic electron microscopy (cryo-EM) is among the most powerful tools available for interrogating nanoscale structure of biological structures. We recently showed that cryo-EM can be used to measure the bilayer thickness of lipid vesicles and biological membranes with sub-angstrom precision, resulting in the direct visualization of nanoscopic domains of different thickness in multicomponent lipid mixtures and giant plasma membrane vesicles. Despite the great potential of cryo-EM for revealing the lateral organization of biomembranes, a large parameter space of experimental conditions remains to be optimized. Here, we systematically investigate the influence of instrument parameters and image post-processing steps on the ability to accurately measure bilayer thickness and discriminate regions of different thickness within unilamellar liposomes. We also demonstrate a spatial autocorrelation analysis to extract additional information about lateral heterogeneity. Significance Raft domains in unstimulated cells have proven difficult to directly visualize owing to their nanoscopic size and fleeting existence. The few techniques capable of nanoscopic spatial resolution typically rely on interpretation of indirect spectroscopic or scattering signals or require stabilizing the membrane on a solid support. In contrast, cryo-EM yields direct images of nanoscale domains in probe-free, unsupported membranes. Here, we systematically optimize key steps in the experimental and analysis workflow for this new and specialized application. Our findings represent an important step toward developing cryo-EM into a robust method for investigating phase behavior of membranes at length scales relevant to lipid rafts.","PeriodicalId":72402,"journal":{"name":"Biophysical reports","volume":"183 12 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2022-08-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"78579660","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}