Biophysical journal最新文献

{"title":"Nanoscale Clustering and Dynamics of Phosphatidylinositol 4,5-Bisphosphate in an Immune Cell Model.","authors":"Brandon M Aho,Dylan J Wagner,Julie A Gosse,Samuel T Hess","doi":"10.1016/j.bpj.2025.07.004","DOIUrl":"https://doi.org/10.1016/j.bpj.2025.07.004","url":null,"abstract":"Mast cells mediate their immuno- and neuro-modulatory effects by releasing granules containing bioactive substances. Phosphatidylinositol 4,5-bisphosphate (PIP2), enriched at the plasma membrane (PM), is a key signaling lipid involved in numerous physiological functions including the calcium entry needed for antigen stimulated mast cell degranulation. However, functional nanoscale PIP2 clustering and dynamics have not been previously investigated in immune cells. Using the pleckstrin homology domain from PLCδ (PH) tagged with photoswitchable fluorescent protein Dendra2, clustering was revealed in the mast cell model RBL-2H3, both fixed and live. We also discovered that live RBL-2H3 cells have PH clusters which evolve over timescales of ∼100s. Additionally, the distribution of PIP2, and specifically PIP2 clusters themselves, are disrupted upon addition of the cationic, lipidic drug cetylpyridinium chloride (CPC). CPC led to smaller, less dense, and more circular clusters. Furthermore, PH molecular mobility increased after the addition of CPC, suggesting interference of this drug with PH binding to PIP2. In addition to this pharmacological relevance, the physiology of PIP2 clusters during functional stimulation by antigen was investigated. Antigen stimulation led to increased cluster size, which was counteracted by CPC. In live cells, PH density outside clusters was altered by CPC but not by antigen. CPC increased the proportion of regions of high-density PH compared to all other regions of the PM. While PH diffusion was, interestingly, not affected by antigen, it was increased by CPC, particularly in lower density regions. Under all live cell dynamics observed, PH demonstrated confinement which were consistent with simulated diffusion within potential wells with an elliptical shape. These findings illuminate the nanoscale behavior of PIP2 in immune cells and the correlation of that behavior with cell function.","PeriodicalId":8922,"journal":{"name":"Biophysical journal","volume":"50 1","pages":""},"PeriodicalIF":3.4,"publicationDate":"2025-07-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144565959","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"On the curvature and relaxation of microtubule plus-end tips.","authors":"Tomasz Skóra, Jiangbo Wu, Daniel Beckett, Weizhi Xue, Gregory A Voth, Tamara C Bidone","doi":"10.1016/j.bpj.2025.07.003","DOIUrl":"10.1016/j.bpj.2025.07.003","url":null,"abstract":"<p><p>Microtubules are essential cytoskeletal components with a broad range of functions in which the structure and dynamics of their plus-end tips play critical roles. Existing mechanistic models explain the tips curving dynamics in different ways: the allosteric model suggests that GTP hydrolysis induces conformational changes in tubulin subunits that destabilize the lattice, leading to protofilament curving and depolymerization, while the lattice model posits that GTP hydrolysis directly destabilizes the microtubule lattice. However, the effect of GTP hydrolysis on the curving dynamics of microtubule tips remains incompletely understood. In this study, we employed a multiscale modeling approach, combining all-atom molecular dynamics simulations with Brownian dynamics simulations, to investigate the relaxation of microtubule plus-end tips into curved configurations. Our results show that both GDP- and GTP-bound tips exhibit an outward bending of protofilaments into curved, ram's horn-like structures, characterized by a linear relationship between curvature and distance from the plus-end tip. These observations align with experimental cryo-ET images of microtubule plus-end tips in different nucleotide states. Collectively, our findings suggest that the outward bending of protofilaments at the plus-end tip is an intrinsic feature of microtubules, independent of the nucleotide state.</p>","PeriodicalId":8922,"journal":{"name":"Biophysical journal","volume":" ","pages":""},"PeriodicalIF":3.2,"publicationDate":"2025-07-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144567030","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Deciphering the molecular mechanisms of BPTF interactions with nucleosomes via molecular simulations.","authors":"Ryan Hebert, Jeff Wereszczynski","doi":"10.1016/j.bpj.2025.06.042","DOIUrl":"10.1016/j.bpj.2025.06.042","url":null,"abstract":"<p><p>Many transcription factors regulate DNA accessibility and gene expression by recognizing post-translational modifications on histone tails within nucleosomes. These interactions are often studied in vitro using short peptide mimics of histone tails, which may overlook conformational changes that occur in the full nucleosomal context. Here, we employ molecular dynamics simulations to investigate the binding dynamics of the plant homeodomain (PHD) finger and bromodomain of bromodomain PHD-finger transcription factor (BPTF), both in solution and bound to either a histone H3 peptide or a full nucleosome. Our results show that BPTF adopts distinct conformational states depending on its binding context, with nucleosome engagement inducing compaction of the multidomain structure. PHD-finger binding displaces the H3 tail from DNA, increasing H3 tail flexibility and promoting compensatory binding of the H4 tail to nucleosomal DNA. This redistribution of histone-DNA contacts weakens overall hydrogen bonding with DNA, suggesting localized destabilization of the nucleosome core. Despite electrostatic repulsion limiting direct reader-DNA contacts, strong van der Waals interactions with the H3 tail stabilize binding. Our results provide atomistic insight into how BPTF engagement modulates nucleosome structure and may facilitate chromatin remodeling.</p>","PeriodicalId":8922,"journal":{"name":"Biophysical journal","volume":" ","pages":""},"PeriodicalIF":3.2,"publicationDate":"2025-07-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144567028","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"The Effects of DMSO on DNA Conformations and Mechanics.","authors":"Koen R Storm, Caroline Körösy, Enrico Skoruppa, Stefanie D Pritzl, Pauline J Kolbeck, Willem Vanderlinden, Helmut Schiessel, Jan Lipfert","doi":"10.1016/j.bpj.2025.06.041","DOIUrl":"https://doi.org/10.1016/j.bpj.2025.06.041","url":null,"abstract":"<p><p>Dimethyl sulfoxide (DMSO) is a polar aprotic solvent used in a wide range of applications, including uses as a drug and in drug delivery, as a solvent for fluorescence dyes, and in enzymatic reactions that process DNA. Consequently, many assays contain low concentrations (≤ 10%) of DMSO. While it is well known that DMSO lowers the melting temperature of DNA, its effects on DNA conformations and mechanical properties below the melting temperature are unclear. Here we use complementary single-molecule techniques to probe DNA in the presence of 0-60% DMSO. Magnetic tweezers force-extension measurements find that the bending persistence length of DNA decreases moderately and linearly with DMSO concentrations up to 20 vol%, by (0.43 ± 0.02)% per %-DMSO. Magnetic tweezers twist measurements demonstrate a reduction in melting torque in the presence of DMSO and find that the helical twist of DNA remains largely unchanged up to 20% DMSO, while even higher concentrations slightly unwind the helix. Using AFM imaging, we find a moderate compaction of DNA conformations by DMSO and observe a systematic decrease of the mean squared end-to-end distance by 1.2% per %-DMSO. We use coarse grained Monte Carlo simulations of DNA as a semi-flexible polymer with a variable density of flexible segments, representing DMSO-induced local defects or melting, to rationalize the observed behavior. The model quantitates the effects of introducing locally flexible regions into DNA and gives trends in line with the magnetic tweezers and AFM imaging experiments. Our results show that addition of up to 50% DMSO has a gradual effect on DNA structure and mechanics and that for low concentrations (≤ 20%) the induced changes are relatively minor. Our work provides a baseline to understand and model the effects of DMSO on DNA in a range of biophysical and biochemical assays.</p>","PeriodicalId":8922,"journal":{"name":"Biophysical journal","volume":" ","pages":""},"PeriodicalIF":3.2,"publicationDate":"2025-07-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144551851","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Characterization and structural basis for the brightness of mCLIFY: A novel monomeric and circularly permuted bright yellow fluorescent protein.","authors":"Him Shweta, Kushol Gupta, Yufeng Zhou, Xiaonan Cui, Selene Li, Zhe Lu, Yale E Goldman, Jody A Dantzig","doi":"10.1016/j.bpj.2025.05.012","DOIUrl":"10.1016/j.bpj.2025.05.012","url":null,"abstract":"<p><p>Ongoing improvements of genetically encoded fluorescent proteins have enhanced cellular localization studies and performance of biosensors, such as environmentally or mechanically sensitive fluorescence resonance energy transfer pairs, in cell biological and biophysical research. The brightest yellow fluorescent protein, widely used in these studies is YPet, derived from the jellyfish Aequorea victoria via the GFP derivative Venus. YPet dimerizes at concentrations used in cellular studies (K_D^1-2 = 3.4 μM) which impacts quantitative interpretation of emission intensity, rotational freedom, energy transfer, and lifetime. Although YPet is nearly 30% brighter than Venus, no atomic structures of YPet have been reported to ascertain the structural differences leading to the higher brightness, possibly due to the tendency to dimerize or oligomerize. Here, we report properties of a new YPet derivative, mCLIFY, a monomeric, bright, yellow, and long-lived fluorescent protein created by circular permutation of YPet and substitution of the amino acid residues thought to mediate dimerization. mCLIFY retains the advantageous photophysical properties of YPet but does not dimerize at least up to 40 μM concentration. We determined the atomic structure of mCLIFY at 1.57-Å resolution. Extensive characterization of the photophysical and structural properties of YPet and mCLIFY allowed us to elucidate the bases of their long lifetimes, enhanced brightness, and the difference in propensity to dimerize.</p>","PeriodicalId":8922,"journal":{"name":"Biophysical journal","volume":" ","pages":"2133-2149"},"PeriodicalIF":3.2,"publicationDate":"2025-07-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12256888/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144126511","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Single-particle tracking of genetically encoded nanoparticles: Optimizing expression for cytoplasmic diffusion studies.","authors":"Elizaveta Korunova, Vitali Sikirzhytski, Jeffery L Twiss, Paula Vasquez, Michael Shtutman","doi":"10.1016/j.bpj.2025.05.025","DOIUrl":"10.1016/j.bpj.2025.05.025","url":null,"abstract":"<p><p>Single-particle tracking (SPT) is a powerful technique for probing the diverse physical properties of the cytoplasm. Genetically encoded nanoparticles provide an especially convenient tool for such investigations, as they can be expressed and tracked in cells via fluorescence. Among these, 40-nm genetically encoded multimerics (GEMs) provide a unique opportunity to explore the cytoplasm. Their size corresponds to that of ribosomes and big protein complexes, allowing us to investigate the effects of the cytoplasm on the diffusivity of these objects while excluding the influence of chemical interactions during stressful events and pathological conditions. However, the effects of GEM expression levels on the measured cytoplasmic diffusivity remain largely uncharacterized in mammalian cells. To optimize the GEMs tracking and assess expression level effects, we developed a doxycycline-inducible GEM expression system and compared it with a previously reported constitutive expression system. The inducible GEM expression system reduced the number of GEM particles from 2000 to as low as 5-500 per average 2D cell cytoplasmic area, depending on doxycycline concentration and incubation time. This optimization enabled adjustment of particle density for imaging and improved homogeneity across the cell population. Moreover, we enhanced the analysis of GEM diffusivity by incorporating an effective diffusion coefficient that accounts for the type of motion and by quantifying motion heterogeneity through standard deviations of particle displacements within and between cells.</p>","PeriodicalId":8922,"journal":{"name":"Biophysical journal","volume":" ","pages":"2222-2235"},"PeriodicalIF":3.2,"publicationDate":"2025-07-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12256916/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144191434","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Coarse-grained chromatin dynamics by tracking multiple similarly labeled gene loci.","authors":"Alexander Mader, Andrew I Rodriguez, Tianyu Yuan, Ivan Surovtsev, Megan C King, Simon G J Mochrie","doi":"10.1016/j.bpj.2025.05.008","DOIUrl":"10.1016/j.bpj.2025.05.008","url":null,"abstract":"<p><p>The \"holy grail\" of chromatin research would be to follow the chromatin configuration in individual live cells over time. One way to achieve this goal would be to track the positions of multiple loci arranged along the chromatin polymer with fluorescent labels. Using distinguishable labels would define each locus uniquely in a microscopic image but would restrict the number of loci that could be observed simultaneously due to experimental limits to the number of distinguishable labels. Using the same label for all loci circumvents this limitation but requires a (currently lacking) framework for how to establish each observed locus identity, i.e., to which genomic position it corresponds. Here, we analyze theoretically, using simulations of Rouse model polymers, how single-particle tracking of multiple identically labeled loci enables the determination of loci identity. We show that the probability of correctly assigning observed loci to genomic positions converges exponentially to unity as the number of observed loci configurations increases. The convergence rate depends only weakly on the number of labeled loci, so that even large numbers of loci can be identified with high fidelity by tracking them across about eight independent chromatin configurations. In the case of two distinct labels that alternate along the chromatin polymer, we find that the probability of the correct assignment converges faster than for same-labeled loci, requiring observation of fewer independent chromatin configurations to establish loci identities. Finally, for a modified Rouse model polymer, which realizes a population of dynamic loops, we find that the success probability also converges to unity exponentially as the number of observed loci configurations increases, albeit slightly more slowly than for a classical Rouse model polymer. Altogether, these results establish particle tracking of multiple identically or alternately labeled loci over time as a feasible way to infer temporal dynamics of the coarse-grained configuration of the chromatin polymer in individual living cells.</p>","PeriodicalId":8922,"journal":{"name":"Biophysical journal","volume":" ","pages":"2120-2132"},"PeriodicalIF":3.2,"publicationDate":"2025-07-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12256843/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144075633","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"RNA kink-turns are highly anisotropic with respect to lateral displacement of the flanking stems.","authors":"Eva Matoušková, Tomáš Dršata, Lucie Pfeiferová, Jiří Šponer, Kamila Réblová, Filip Lankaš","doi":"10.1016/j.bpj.2025.05.024","DOIUrl":"10.1016/j.bpj.2025.05.024","url":null,"abstract":"","PeriodicalId":8922,"journal":{"name":"Biophysical journal","volume":" ","pages":"2251"},"PeriodicalIF":3.2,"publicationDate":"2025-07-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12256884/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144180165","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Modulation of striated-muscle contractility by a high-affinity myosin-targeting peptide.","authors":"Thomas Kampourakis, Negar Aboonasrshiraz, Theodore J Kalogeris, Rohit Singh, Dua'a Quedan, Motamed Qadan, Md Mozammel Hossain, Nasrin Taei, Michael Bih, Alysha Joseph, Kerry S McDonald, Douglas D Root","doi":"10.1016/j.bpj.2025.05.027","DOIUrl":"10.1016/j.bpj.2025.05.027","url":null,"abstract":"<p><p>Myosin-based regulation has emerged as a fundamental new concept governing both cardiac and skeletal muscle contractile function during both health and disease states. Myosin-targeted therapeutics have the potential to treat heart failure with either systolic or diastolic dysfunction based on either activating or inhibiting the function of myosin. In this study, we developed a striated-muscle myosin-specific high-affinity peptide that targeted the proximal subfragment 2 (S2) region of the MYH7 myosin, which has been shown to undergo conformational changes associated with force generation by the myosin head domains. We characterized the peptide called Stabilizer using a wide range of biochemical, biophysical, and physiological methods, creating a multi-scale structure-activity relationship ranging from single-molecule assays to contractile measurements in intact cardiac muscle cells. The Stabilizer binds myosin S2 with low nanomolar affinity and strongly increases its mechanical stability as measured by single-molecule gravitational force spectroscopy and Förster resonance energy transfer measurements. The Stabilizer significantly inhibits myofibrilar contractility and ATPase activity, and it reduces myosin crossbridge kinetics in demembranated cardiac muscle cells. Biochemical modification of the Stabilizer further allowed measurements in intact porcine cardiomyocytes showing decreased contraction and relaxation kinetics in the presence of the peptide. Our results show that myosin S2-targeting peptides are biologicals with potential therapeutic applications for muscle diseases.</p>","PeriodicalId":8922,"journal":{"name":"Biophysical journal","volume":" ","pages":"2236-2250"},"PeriodicalIF":3.2,"publicationDate":"2025-07-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12256866/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144156242","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Microtubule polymerization generates microtentacles important in circulating tumor cell invasion.","authors":"Lucina Kainka, Reza Shaebani, Kathi Kaiser, Jonas Bosche, Ludger Santen, Franziska Lautenschläger","doi":"10.1016/j.bpj.2025.05.018","DOIUrl":"10.1016/j.bpj.2025.05.018","url":null,"abstract":"<p><p>Circulating tumor cells (CTCs) have crucial roles in the spread of tumors during metastasis. A decisive step is the extravasation of CTCs from the blood stream or lymph system, which depends on the ability of cells to attach to vessel walls. Recent work suggests that such adhesion is facilitated by microtubule (MT)-based membrane protrusions called microtentacles (McTNs). However, how McTNs facilitate such adhesion and how MTs can generate protrusions in CTCs remain unclear. By combining fluorescence recovery after photobleaching experiments and simulations we show that polymerization of MTs provides the main driving force for McTN formation, whereas the contribution of MTs sliding with respect to each other is minimal. Further, the forces exerted on the McTN tip result in curvature, as the MTs are anchored at the other end in the MT organizing center. When approaching vessel walls, McTN curvature is additionally influenced by the adhesion strength between the McTN and wall. Moreover, increasing McTN length, reducing its bending rigidity, or strengthening adhesion enhances the cell-wall contact area and, thus, promotes cell attachment to vessel walls. Our results demonstrate a link between the formation and function of McTNs, which may provide new insight into metastatic cancer diagnosis and therapy.</p>","PeriodicalId":8922,"journal":{"name":"Biophysical journal","volume":" ","pages":"2161-2175"},"PeriodicalIF":3.2,"publicationDate":"2025-07-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12256914/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144156204","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}