Biophysical reports最新文献

{"title":"Diffusion and interaction dynamics of the cytosolic peroxisomal import receptor PEX5.","authors":"S Galiani, K Reglinski, P Carravilla, A Barbotin, I Urbančič, J Ott, J Sehr, E Sezgin, F Schneider, D Waithe, P Hublitz, W Schliebs, R Erdmann, C Eggeling","doi":"10.1016/j.bpr.2022.100055","DOIUrl":"10.1016/j.bpr.2022.100055","url":null,"abstract":"<p><p>Cellular functions rely on proper actions of organelles such as peroxisomes. These organelles rely on the import of proteins from the cytosol. The peroxisomal import receptor PEX5 takes up target proteins in the cytosol and transports them to the peroxisomal matrix. However, its cytosolic molecular interactions have so far not directly been disclosed. Here, we combined advanced optical microscopy and spectroscopy techniques such as fluorescence correlation spectroscopy and stimulated emission depletion microscopy with biochemical tools to present a detailed characterization of the cytosolic diffusion and interaction dynamics of PEX5. Among other features, we highlight a slow diffusion of PEX5, independent of aggregation or target binding, but associated with cytosolic interaction partners via its N-terminal domain. This sheds new light on the functionality of the receptor in the cytosol as well as highlighting the potential of using complementary microscopy tools to decipher molecular interactions in the cytosol by studying their diffusion dynamics.</p>","PeriodicalId":72402,"journal":{"name":"Biophysical reports","volume":"2 2","pages":"None"},"PeriodicalIF":0.0,"publicationDate":"2022-06-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://ftp.ncbi.nlm.nih.gov/pub/pmc/oa_pdf/8f/8d/main.PMC9586885.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"10853977","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":"Identifying vaccine escape sites via statistical comparisons of short-term molecular dynamics","authors":"Madhusudan Rajendran, M. C. Ferran, G. Babbitt","doi":"10.1016/j.bpr.2022.100056","DOIUrl":"https://doi.org/10.1016/j.bpr.2022.100056","url":null,"abstract":"","PeriodicalId":72402,"journal":{"name":"Biophysical reports","volume":"36 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2022-04-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"80751517","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":"Chromatin as self-returning walks: From population to single cell and back.","authors":"Anne R Shim,&nbsp;Kai Huang,&nbsp;Vadim Backman,&nbsp;Igal Szleifer","doi":"10.1016/j.bpr.2021.100042","DOIUrl":"https://doi.org/10.1016/j.bpr.2021.100042","url":null,"abstract":"<p><p>With a growing understanding of the chromatin structure, many efforts remain focused on bridging the gap between what is suggested by population-averaged data and what is visualized for single cells. A popular approach to traversing these scales is to fit a polymer model to Hi-C contact data. However, Hi-C is an average of millions to billions of cells, and each cell may not contain all population-averaged contacts. Therefore, we employ a novel approach of summing individual chromosome trajectories-determined by our Self-Returning Random Walk model-to create populations of cells. We allow single cells to consist of disparate structures and reproduce a variety of experimentally relevant contact maps. We show that the amount of shared topology between cells, and their mechanism of formation, changes the population-averaged structure. Therefore, we present a modeling technique that, with few constraints and little oversight, can be used to understand which single-cell chromatin structures underlie population-averaged behavior.</p>","PeriodicalId":72402,"journal":{"name":"Biophysical reports","volume":"2 1","pages":"100042"},"PeriodicalIF":0.0,"publicationDate":"2022-03-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://ftp.ncbi.nlm.nih.gov/pub/pmc/oa_pdf/24/c6/main.PMC9680733.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"9252291","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":"Long-term STED imaging of membrane packing and dynamics by exchangeable polarity-sensitive dyes.","authors":"Pablo Carravilla, Anindita Dasgupta, Gaukhar Zhurgenbayeva, Dmytro I Danylchuk, Andrey S Klymchenko, Erdinc Sezgin, Christian Eggeling","doi":"10.1016/j.bpr.2021.100023","DOIUrl":"10.1016/j.bpr.2021.100023","url":null,"abstract":"<p><p>Understanding the plasma membrane nanoscale organization and dynamics in living cells requires microscopy techniques with high spatial and temporal resolution that permit for long acquisition times and allow for the quantification of membrane biophysical properties, such as lipid ordering. Among the most popular super-resolution techniques, stimulated emission depletion (STED) microscopy offers one of the highest temporal resolutions, ultimately defined by the scanning speed. However, monitoring live processes using STED microscopy is significantly limited by photobleaching, which recently has been circumvented by exchangeable membrane dyes that only temporarily reside in the membrane. Here, we show that NR4A, a polarity-sensitive exchangeable plasma membrane probe based on Nile red, permits the super-resolved quantification of membrane biophysical parameters in real time with high temporal and spatial resolution as well as long acquisition times. The potential of this polarity-sensitive exchangeable dye is showcased by live-cell real-time three-dimensional STED recordings of bleb formation and lipid exchange during membrane fusion as well as by STED-fluorescence correlation spectroscopy experiments for the simultaneous quantification of membrane dynamics and lipid packing that correlate in model and live-cell membranes.</p>","PeriodicalId":72402,"journal":{"name":"Biophysical reports","volume":"1 2","pages":"None"},"PeriodicalIF":0.0,"publicationDate":"2021-12-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8651516/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"39837845","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":"Recharging your fats: CHARMM36 parameters for neutral lipids triacylglycerol and diacylglycerol.","authors":"Pablo Campomanes,&nbsp;Janak Prabhu,&nbsp;Valeria Zoni,&nbsp;Stefano Vanni","doi":"10.1016/j.bpr.2021.100034","DOIUrl":"https://doi.org/10.1016/j.bpr.2021.100034","url":null,"abstract":"<p><p>Neutral lipids (NLs) are an abundant class of cellular lipids. They are characterized by the total lack of charged chemical groups in their structure, and, as a consequence, they play a major role in intracellular lipid storage. NLs that carry a glycerol backbone, such as triacylglycerols (TGs) and diacylglycerols (DGs), are also involved in the biosynthetic pathway of cellular phospholipids, and they have recently been the subject of numerous structural investigations by means of atomistic molecular dynamics simulations. However, conflicting results on the physicochemical behavior of NLs were observed depending on the nature of the atomistic force field used. Here, we show that current phospholipid-derived CHARMM36 parameters for DGs and TGs cannot adequately reproduce interfacial properties of these NLs because of excessive hydrophilicity at the glycerol-ester region. By following a CHARMM36-consistent parameterization strategy, we develop improved parameters for both TGs and DGs that are compatible with both cutoff-based and particle mesh Ewald schemes for the treatment of Lennard-Jones interactions. We show that our improved parameters can reproduce interfacial properties of NLs and their behavior in more complex lipid assemblies. We discuss the implications of our findings in the context of intracellular lipid storage and NLs' cellular activity.</p>","PeriodicalId":72402,"journal":{"name":"Biophysical reports","volume":"1 2","pages":"None"},"PeriodicalIF":0.0,"publicationDate":"2021-12-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8651513/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"39837842","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":"Cooled SPAD array detector for low light-dose fluorescence laser scanning microscopy.","authors":"Eli Slenders,&nbsp;Eleonora Perego,&nbsp;Mauro Buttafava,&nbsp;Giorgio Tortarolo,&nbsp;Enrico Conca,&nbsp;Sabrina Zappone,&nbsp;Agnieszka Pierzynska-Mach,&nbsp;Federica Villa,&nbsp;Enrica Maria Petrini,&nbsp;Andrea Barberis,&nbsp;Alberto Tosi,&nbsp;Giuseppe Vicidomini","doi":"10.1016/j.bpr.2021.100025","DOIUrl":"https://doi.org/10.1016/j.bpr.2021.100025","url":null,"abstract":"<p><p>The single-photon timing and sensitivity performance and the imaging ability of asynchronous-readout single-photon avalanche diode (SPAD) array detectors have opened up enormous perspectives in fluorescence (lifetime) laser scanning microscopy (FLSM), such as super-resolution image scanning microscopy and high-information content fluorescence fluctuation spectroscopy. However, the strengths of these FLSM techniques depend on the many different characteristics of the detector, such as dark noise, photon-detection efficiency, after-pulsing probability, and optical cross talk, whose overall optimization is typically a trade-off between these characteristics. To mitigate this trade-off, we present, to our knowledge, a novel SPAD array detector with an active cooling system that substantially reduces the dark noise without significantly deteriorating any other detector characteristics. In particular, we show that lowering the temperature of the sensor to -15°C significantly improves the signal/noise ratio due to a 10-fold decrease in the dark count rate compared with room temperature. As a result, for imaging, the laser power can be decreased by more than a factor of three, which is particularly beneficial for live-cell super-resolution imaging, as demonstrated in fixed and living cells expressing green-fluorescent-protein-tagged proteins. For fluorescence fluctuation spectroscopy, together with the benefit of the reduced laser power, we show that cooling the detector is necessary to remove artifacts in the correlation function, such as spurious negative correlations observed in the hot elements of the detector, i.e., elements for which dark noise is substantially higher than the median value. Overall, this detector represents a further step toward the integration of SPAD array detectors in any FLSM system.</p>","PeriodicalId":72402,"journal":{"name":"Biophysical reports","volume":"1 2","pages":"None"},"PeriodicalIF":0.0,"publicationDate":"2021-12-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://ftp.ncbi.nlm.nih.gov/pub/pmc/oa_pdf/e5/bd/main.PMC8651514.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"39837843","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":"Optical probing of local membrane potential with fluorescent polystyrene beads.","authors":"Zehavit Shapira,&nbsp;Nurit Degani-Katzav,&nbsp;Shimon Yudovich,&nbsp;Asaf Grupi,&nbsp;Shimon Weiss","doi":"10.1016/j.bpr.2021.100030","DOIUrl":"https://doi.org/10.1016/j.bpr.2021.100030","url":null,"abstract":"<p><p>The study of electrical activity in single cells and local circuits of excitable cells, such as neurons, requires an easy-to-use, high-throughput methodology that allows for the measurement of membrane potential. Investigating the electrical properties in specific subcompartments of neurons, or in a specific type of neurons, introduces additional complexity. An optical voltage-imaging technique that allows high spatial and temporal resolution could be an ideal solution. However, most valid voltage-imaging techniques are nonspecific. Those that are more site-directed require a lot of preliminary work and specific adaptations, among other drawbacks. Here, we explore a new method for membrane voltage imaging, based on Förster resonance energy transfer between fluorescent polystyrene (FPS) beads and dipicrylamine. Not only has it been shown that fluorescence intensity correlates with membrane potential, but more importantly, the membrane potential from individual particles can be detected. Among other advantages, FPS beads can be synthesized with surface functional groups and can be targeted to specific proteins by conjugation of recognition molecules. Therefore, in the presence of dipicrylamine, FPS beads represent single-particle detectors of membrane potential that can be localized to specific membrane compartments. This new and easily accessible platform for targeted optical voltage imaging can further elucidate the mechanisms of neuronal electrical activity.</p>","PeriodicalId":72402,"journal":{"name":"Biophysical reports","volume":"1 2","pages":"None"},"PeriodicalIF":0.0,"publicationDate":"2021-12-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://ftp.ncbi.nlm.nih.gov/pub/pmc/oa_pdf/ab/d9/main.PMC8651512.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"39749775","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":"From single-molecule to genome-wide mapping of DNA lesions: repair-assisted damage detection sequencing.","authors":"Noa Gilat,&nbsp;Dena Fridman,&nbsp;Hila Sharim,&nbsp;Sapir Margalit,&nbsp;Natalie R Gassman,&nbsp;Yael Michaeli,&nbsp;Yuval Ebenstein","doi":"10.1016/j.bpr.2021.100017","DOIUrl":"https://doi.org/10.1016/j.bpr.2021.100017","url":null,"abstract":"<p><p>Mapping DNA damage and its repair has immense potential in understanding environmental exposures, their genotoxicity, and their impact on human health. Monitoring changes in genomic stability also aids in the diagnosis of numerous DNA-related diseases, such as cancer, and assists in monitoring their progression and prognosis. Developments in recent years have enabled unprecedented sensitivity in quantifying the global DNA damage dose in cells via fluorescence-based analysis down to the single-molecule level. However, genome-wide maps of DNA damage distribution are challenging to produce. Here, we describe the localization of DNA damage and repair loci by repair-assisted damage detection sequencing (RADD-seq). Based on the enrichment of damage lesions coupled with a pull-down assay and followed by next-generation sequencing, this method is easy to perform and can produce compelling results with minimal coverage. RADD-seq enables the localization of both DNA damage and repair sites for a wide range of single-strand damage types. Using this technique, we created a genome-wide map of the oxidation DNA damage lesion 8-oxo-7,8-dihydroguanine before and after repair. Oxidation lesions were heterogeneously distributed along the human genome, with less damage occurring in tight chromatin regions. Furthermore, we showed repair is prioritized for highly expressed, essential genes and in open chromatin regions. RADD-seq sheds light on cellular repair mechanisms and is capable of identifying genomic hotspots prone to mutation.</p>","PeriodicalId":72402,"journal":{"name":"Biophysical reports","volume":"1 2","pages":"None"},"PeriodicalIF":0.0,"publicationDate":"2021-12-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1016/j.bpr.2021.100017","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"39837844","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":"Anisotropic presentation of ligands on cargos modulates degradative function of phagosomes","authors":"Menghi Jiao, Wenqian Li, Yanqi Yu, Yanqi Yu","doi":"10.1016/j.bpr.2021.100041","DOIUrl":"https://doi.org/10.1016/j.bpr.2021.100041","url":null,"abstract":"","PeriodicalId":72402,"journal":{"name":"Biophysical reports","volume":"30 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2021-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"74214090","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":"An economic, square-shaped flat-field illumination module for TIRF-based super-resolution microscopy","authors":"Jeff Y. L. Lam, Yunzhao Wu, Eleni Dimou, Ziwei Zhang, M. Cheetham, Markus Körbel, Zengjie Xia, D. Klenerman, J. S. Danial","doi":"10.1016/j.bpr.2022.100044","DOIUrl":"https://doi.org/10.1016/j.bpr.2022.100044","url":null,"abstract":"","PeriodicalId":72402,"journal":{"name":"Biophysical reports","volume":"6 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2021-11-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"72724550","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}