Biophysical journal最新文献

{"title":"Heterogenous condensates of transcription factors in embryonic stem cells: Molecular simulations.","authors":"Azuki Mizutani, Cheng Tan, Yuji Sugita, Shoji Takada","doi":"10.1016/j.bpj.2025.04.001","DOIUrl":"https://doi.org/10.1016/j.bpj.2025.04.001","url":null,"abstract":"<p><p>Biomolecular condensates formed via liquid-liquid phase separation are ubiquitous in cells, especially in the nucleus. While condensates containing one or two kinds of biomolecules have been relatively well characterized, those with more heterogenous biomolecular components and interactions between biomolecules inside are largely unknown. This study used residue-resolution molecular dynamics (MD) simulations to investigate heterogeneous protein assemblies that include four master transcription factors in mammalian embryonic stem cells: Oct4, Sox2, Klf4, and Nanog. MD simulations of the mixture systems showed highly heterogeneous and dynamic behaviors; protein condensates mainly contain Sox2, Klf4, and Nanog, while most Oct4 are dissolved into the dilute phase. The condensate forms loosely interacting clusters where Klf4 is the most abundant, suggesting that Klf4 serves as a scaffold of the condensate, and Sox2 and Nanog are bound to Klf4 for stabilizing the condensate. Oct4 is moderately recruited to the condensate, serving as a client mainly via its interaction with Sox2. This study highlights the importance of inter-molecular interaction between different transcription factors on the condensate formations with heterogeneous biomolecular components.</p>","PeriodicalId":8922,"journal":{"name":"Biophysical journal","volume":" ","pages":""},"PeriodicalIF":3.2,"publicationDate":"2025-04-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143802272","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":"Biophysical characterization of anion channels in Mitochondrion-Endoplasmic-Reticulum Contact sites (MERCs).","authors":"Shridhar Kiran Sanghvi, Denis Gabrilovich, Satish K Raut, Ajay Gopalan, Aryan Singh, Harmeet Rireika Bhachu, Mayukha Dyta, Veronica Loyo-Celis, Jenna Thuma, Devasena Ponnalagu, Jonathan Davis, Shubha Gururaja Rao, Harpreet Singh","doi":"10.1016/j.bpj.2025.04.002","DOIUrl":"https://doi.org/10.1016/j.bpj.2025.04.002","url":null,"abstract":"<p><p>The mitochondrion-endoplasmic reticulum (ER) contact sites (MERCs, also known as mitochondrial-associated membranes (MAMs)) are specialized regions of the endoplasmic reticulum that are in close proximity to the mitochondrion. These organelle structures play essential roles in a variety of processes, such as calcium signaling, lipid metabolism, renin-angiotensin-aldosterone system control, the unfolded protein response, and autophagy. MERCs are known to actively participate in ion transport between the ER and mitochondria. Although active calcium channels in MERCs have been detected, limited studies have been carried out to identify or characterize functional anion channels. Here, we tested whether functional anion channels are present in MERCs. We isolated MERCs from mouse organs (heart and brain) and reconstituted them in planar bilayers. The single-channel properties were recorded in the presence of various anion channel blockers or antagonists (IAA-94, DIDS, A9C, and NPPB). We corroborated the presence of anion channels targeted by these drugs using immunoblotting and immunocytochemistry. Biochemical analysis and immunocytochemistry corroborate that CLIC4, CLIC3, and VDACs are present in MERCs. Our results indicate that anion channels are active in MERCs, which could play a pertinent role in intracellular organelle communication.</p>","PeriodicalId":8922,"journal":{"name":"Biophysical journal","volume":" ","pages":""},"PeriodicalIF":3.2,"publicationDate":"2025-04-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143794664","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":"Several common methods of making vesicles (except an emulsion method) capture intended lipid ratios.","authors":"Heidi M J Weakly, Kent J Wilson, Gunnar J Goetz, Emily L Pruitt, Amy Li, Libin Xu, Sarah L Keller","doi":"10.1016/j.bpj.2025.03.029","DOIUrl":"https://doi.org/10.1016/j.bpj.2025.03.029","url":null,"abstract":"","PeriodicalId":8922,"journal":{"name":"Biophysical journal","volume":" ","pages":""},"PeriodicalIF":3.2,"publicationDate":"2025-04-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143787689","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":"Chromatin unfolding via loops can drive clustered transposon insertion.","authors":"Roshan Prizak, Aaron Gadzekpo, Lennart Hilbert","doi":"10.1016/j.bpj.2025.03.038","DOIUrl":"https://doi.org/10.1016/j.bpj.2025.03.038","url":null,"abstract":"<p><p>Transposons, DNA sequences capable of relocating within the genome, make up a significant portion of eukaryotic genomes and are often found in clusters. Within the cell nucleus, the genome is organized into chromatin, a structure with varying degrees of compaction due to three-dimensional folding. Transposon insertion or activation can lead to chromatin decompaction, increasing accessibility and potentially facilitating further nearby insertions. This positive feedback between chromatin unfolding and transposon insertion may result in transposon clustering. Here, we combine bioinformatics with polymer modeling to explore possible mechanisms and conditions that promote clustered transposon insertions. Our analysis of human cell line genomic repeat data reveals extensive clustering of heterochromatic LINE-1 elements and euchromatic Alu elements. For Alu elements, this clustering correlates with increased chromatin accessibility. Both Alu and LINE-1 deviate in their sequence-inherent flexibility from the overall genome, with above-average flexibility for Alu and below-average flexibility for most LINE-1 sequences. Flexibility was highest in young transposons, so that young Alu and LINE-1 exceed overall genome flexibility. We developed an according polymer model of transposon insertion, consisting of a self-attracting chromatin domain. Transposon insertions locally disrupt self-attraction, leading to unfolding of the domain as more transposons are inserted. In simulations where transposons are inserted adjacent to existing ones, we observed gradual unfolding through loop extensions from a folded core. Including transposases as explicit particles, our model shows that adjacent transposon insertion occurs when densely packed chromatin excludes transposases or when insertion rates exceed the thermal equilibration rate of polymer configurations. We conclude that (i) dense chromatin packing that hinders transposase access as well as (ii) a local loss of compaction upon transposon insertion favor clustered transposon insertion via loop formation. This biophysical mechanism of clustered insertion site preference would act in combination with selective pressures shaping transposon distribution over evolutionary time scales.</p>","PeriodicalId":8922,"journal":{"name":"Biophysical journal","volume":" ","pages":""},"PeriodicalIF":3.2,"publicationDate":"2025-04-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143794665","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":"Bacteriorhodopsin proton-pumping mechanism: successes and challenges in computational approaches.","authors":"Ana-Nicoleta Bondar, Jeremy C Smith","doi":"10.1016/j.bpj.2025.03.035","DOIUrl":"https://doi.org/10.1016/j.bpj.2025.03.035","url":null,"abstract":"<p><p>Bacteriorhodopsin (bR) is perhaps the best-studied proton pump. Over about four decades, research on this fascinating photocyclic light-driven protein inspired the development of key experimental and computational methodologies that are now widely used in membrane protein studies. We review here failures and successes in computational approaches that have been applied to study the bR proton-transfer steps. Conflict between experimental results pertaining to the proton transfer mechanisms in the early photocycle intermediates was resolved by detailed quantum mechanical/molecular mechanical (QM/MM) computation, the results of which were confirmed more than a decade later. Key to this approach was the realization that, to understand how the pump works and achieves directional transfer of protons, the individual reaction steps-proton transfer and reorganization of the internal hydrogen-bond network- needed to be considered within the context of the energy landscape of the complete reaction cycle.</p>","PeriodicalId":8922,"journal":{"name":"Biophysical journal","volume":" ","pages":""},"PeriodicalIF":3.2,"publicationDate":"2025-04-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143787684","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":"In Silico Design of Foldable Lasso Peptides.","authors":"John D M Nguyen, Gabriel C A da Hora, Marcus C Mifflin, Andrew G Roberts, Jessica M J Swanson","doi":"10.1016/j.bpj.2025.03.036","DOIUrl":"https://doi.org/10.1016/j.bpj.2025.03.036","url":null,"abstract":"<p><p>Lasso peptides are a unique class of natural products with distinctively threaded structures, conferring exceptional stability against thermal and proteolytic degradation. Despite their promising biotechnological and pharmaceutical applications, reported attempts to prepare them by chemical synthesis result in forming the nonthreaded branched-cyclic isomer, rather than the desired lassoed structure. This is likely due to the entropic challenge of folding a short, threaded motif prior to chemically mediated cyclization. Accordingly, this study aims to better understand and enhance the relative stability of pre-lasso conformations-the essential precursor to lasso peptide formation-through sequence optimization, chemical modification, and disulfide incorporation. Using Rosetta fixed backbone design, optimal sequences for several class II lasso peptides are identified. Enhanced sampling with well-tempered metadynamics confirmed that designed sequences derived from the lasso structures of rubrivinodin and microcin J25 exhibit a notable improvement in pre-lasso stability relative to the competing nonthreaded conformations. Chemical modifications to the isopeptide bond-forming residues of microcin J25 further increase the probability of pre-lasso formation, highlighting the beneficial role of non-canonical amino acid residues. Counterintuitively, the introduction of a disulfide cross-link decreased pre-lasso stability. Although cross-linking inherently constrains the peptide structure, decreasing the entropic dominance of unfolded phase space, it hinders the requisite wrapping of the N-terminal end around the tail to adopt the pre-lasso conformation. However, combining chemical modifications with the disulfide cross-link results in further pre-lasso stabilization, indicating that the ring modifications counteract the constraints and provide a cooperative benefit with cross-linking. These findings lay the groundwork for further design efforts to enable synthetic access to the lasso peptide scaffold.</p>","PeriodicalId":8922,"journal":{"name":"Biophysical journal","volume":" ","pages":""},"PeriodicalIF":3.2,"publicationDate":"2025-04-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143778885","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":"Glycolysis model shows that allostery maintains high ATP and limits accumulation of intermediates.","authors":"Mangyu Choe, Tal Einav, Rob Phillips, Denis V Titov","doi":"10.1016/j.bpj.2025.03.037","DOIUrl":"https://doi.org/10.1016/j.bpj.2025.03.037","url":null,"abstract":"<p><p>Glycolysis is a conserved metabolic pathway that produces ATP and biosynthetic precursors. It is not well understood how the control of mammalian glycolytic enzymes through allosteric feedback and mass action accomplishes various tasks of ATP homeostasis, such as controlling the rate of ATP production, maintaining high and stable ATP levels, ensuring that ATP hydrolysis generates a net excess of energy, and maintaining glycolytic intermediate concentrations within physiological levels. To investigate these questions, we developed a biophysical model of glycolysis based on enzyme rate equations derived from in vitro kinetic data. This is the first biophysical model of human glycolysis that successfully recapitulates the above tasks of ATP homeostasis and predicts absolute concentrations of glycolytic intermediates and isotope tracing kinetics that align with experimental measurements in human cells. We use the model to show that mass action alone is sufficient to control the ATP production rate and maintain the high energy of ATP hydrolysis. Meanwhile, allosteric regulation of hexokinase (HK) and phosphofructokinase (PFK) by ATP, ADP, inorganic phosphate, and glucose-6-phosphate is required to maintain high ATP levels and to prevent uncontrolled accumulation of phosphorylated intermediates of glycolysis. Allosteric feedback achieves the latter by maintaining HK and PFK enzyme activity at one-half of ATP demand and, thus, inhibiting the reaction of Harden and Young, which otherwise converts glucose to supraphysiological levels of phosphorylated glycolytic intermediates at the expense of ATP. Our methodology provides a roadmap for a quantitative understanding of how metabolic homeostasis emerges from the activities of individual enzymes.</p>","PeriodicalId":8922,"journal":{"name":"Biophysical journal","volume":" ","pages":""},"PeriodicalIF":3.2,"publicationDate":"2025-04-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143787687","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":"Comparison of BH3-dependent and BH3-independent membrane interactions of pro-apoptotic factor BAX.","authors":"Mykola V Rodnin, Victor Vasquez-Montes, Pierce T O'Neil, Alexander Kyrychenko, Alexey S Ladokhin","doi":"10.1016/j.bpj.2025.03.034","DOIUrl":"https://doi.org/10.1016/j.bpj.2025.03.034","url":null,"abstract":"<p><p>The pro-apoptotic factor BAX is a key member of the Bcl-2 family of apoptotic regulators. BAX functions by permeating the outer mitochondrial membrane, a process that begins with the targeting of soluble BAX to the membrane. Once associated, BAX refolds, inserts into the bilayer, and ultimately assembles into a multimeric pore of unknown structure. BAX targeting is initiated by an activation signal that can arise from two pathways: (a) a BH3-dependent one in which BAX is activated by one of the BH3-only effectors such as tBid, or (b) a recently discovered BH3-independent pathway, where BAX activity is modulated by changes in lipid composition. In this study, we gain further insight into how these two pathways function, and how their function is impacted by anti-apoptotic factor Bcl-xL. We use fluorescence spectroscopy to compare the BH3-dependent and BH3-independent interactions of BAX with model membranes of varying lipid compositions. We investigate membrane association using FRET between Donor-labeled BAX and Acceptor-labeled vesicles. We monitor membrane insertion by observing changes in the spectral properties of the environment-sensitive probe NBD, which we selectively attached to a series of single-cysteine BAX mutants. Finally, we study membrane permeation through BAX-induced leakage of soluble markers loaded into vesicles. Our results show that BAX-induced permeabilization of zwitterionic vesicles is more efficient for the BH3-dependent pathway compared to the BH3-independent pathway; however, permeabilization of cardiolipin-containing vesicles is equally efficient for both the BH3-dependent and BH3-independent pathways. Interestingly, while anionic lipids are not necessary for the initial BH3-independent membrane association of BAX, they are critical for subsequent stages of membrane insertion and pore assembly. The spectroscopic response of NBD-labeled BAX is comparable for both interaction modes, indicating a similar structure for the final inserted state. We found that the Bcl-xL factor inhibits vesicle permeabilization by preventing BAX from interacting with the bilayer.</p>","PeriodicalId":8922,"journal":{"name":"Biophysical journal","volume":" ","pages":""},"PeriodicalIF":3.2,"publicationDate":"2025-04-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143778883","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":"Super-resolution algorithms for Imaging FCS enhancement: A comparative study.","authors":"Shambhavi Pandey, Nithin Pathoor, Thorsten Wohland","doi":"10.1016/j.bpj.2025.03.031","DOIUrl":"https://doi.org/10.1016/j.bpj.2025.03.031","url":null,"abstract":"<p><p>Understanding the structure and dynamics of biological systems is often limited by the trade-off between spatial and temporal resolution. Imaging fluorescence correlation spectroscopy (ImFCS) is a powerful technique for capturing molecular dynamics with high temporal precision but remains diffraction-limited. This constraint poses challenges for quantifying dynamics of subcellular structures like membrane-proximal cortical actin fibers. Computational super-resolution microscopy (CSRM) presents an accessible strategy for enhancing spatial resolution without specialized instrumentation, enabling compatibility with ImFCS. In this study, we evaluated various CSRM techniques, including super-resolution radial fluctuations (SRRF), mean-shift super-resolution (MSSR), and multiple signal classification imaging (MUSICAL), among others, using TIRF datasets of actin fibers labeled with F-tractin-mApple. By combining structural masks from TIRF and CSRM, we distinguished off-fiber, mixed, and on-fiber regions for region-specific diffusion analyses. Although all CSRM algorithms improve ImFCS data analysis, SRRF demonstrated superior performance in identifying cortical actin fibers, showing minimal variance in on-fiber diffusion coefficients. These findings establish a framework for integrating CSRM with ImFCS to achieve high-resolution spatial and dynamic characterization of subcellular structures from single measurements.</p>","PeriodicalId":8922,"journal":{"name":"Biophysical journal","volume":" ","pages":""},"PeriodicalIF":3.2,"publicationDate":"2025-04-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143778886","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":"Barrier effects on the kinetics of cohesin-mediated loop extrusion.","authors":"Leiyan Chen, Zhenquan Zhang, Zihao Wang, Liu Hong, Haohua Wang, Jiajun Zhang","doi":"10.1016/j.bpj.2025.03.026","DOIUrl":"https://doi.org/10.1016/j.bpj.2025.03.026","url":null,"abstract":"<p><p>Chromosome organization mediated by structural maintenance of chromosome complexes is crucial in many organisms. Cohesin extrudes chromatin into loops that are thought to lengthen until it is obstructed by CTCF proteins. In complex cellular environments, the loop extrusion machinery may encounter other chromatin-binding proteins. How these proteins interfere with the cohesin-meditated extrusion process is largely unexplored, but recent experiments have shown that some proteins serve as physical barriers that block cohesin translocation. Other proteins containing a cohesin-interaction motif serve as chemical barriers to induce cohesin pausing through interactions with it. Here, we develop an analytically solvable approach for the loop extrusion model incorporating barriers to investigate the effect of the barrier on the passive extrusion process. To further quantify the impact of barriers, we calculate the mean looping time it takes for cohesin to translocate to form a stable loop before dissociation. Our finding reveals that the physical barrier can accelerate the loop formation, and the degree of acceleration is closely related to the impedance strength of the physical barrier. In particular, the synergy of the cohesin loading site and the physical barrier site accelerates loop formation more significantly. The proximity of the cohesin loading site to the barrier site facilitates the rapid formation of stable loops in long genomes, which implies loop extrusion and chromatin-binding proteins might shape functional genomic organization. Conversely, chemical barriers consistently impede loop formation, with increasing impedance strength of the chemical barrier leading to longer loop formation time. Our study contributes to a more comprehensive understanding of the complexity of the loop extrusion process, providing a new perspective on the potential mechanisms of gene regulation.</p>","PeriodicalId":8922,"journal":{"name":"Biophysical journal","volume":" ","pages":""},"PeriodicalIF":3.2,"publicationDate":"2025-04-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143778879","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}