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FRETsael: Nanometer localization of biomolecular interactions using fluorescence lifetime imaging.
IF 3.2 3区 生物学 Q2 BIOPHYSICS Pub Date : 2025-02-19 DOI: 10.1016/j.bpj.2025.02.017
Yair Razvag, Paz Drori, Shalhevet Klemfner, Eran Meshorer, Eitan Lerner

Super-resolution light microscopy techniques facilitate the observation of nm-sized biomolecules, which are 1-2 orders of magnitude smaller than the diffraction limit of light. Using super-resolution microscopy techniques, it is possible to observe fluorescence from two biomolecules in close proximity; however, not necessarily in direct interaction. Using FRETsael, we localize biomolecular interactions exhibiting FRET with nanometer accuracy, from two-color fluorescence lifetime imaging data. The concepts of FRETsael were tested first against simulations, in which the recovered localization accuracy is 20-30 nm for true-positive detections of FRET pairs. Further analysis of the simulation results reports the conditions in which true-positive rates are maximal. We then show the capabilities of FRETsael on simulated samples of actin-vinculin and ER-ribosome interactions, as well as experimental samples of actin-myosin two-color confocal imaging. Overall, the FRETsael approach paves the way toward studying biomolecular interactions with improved spatial resolution from laser scanning confocal two-color fluorescence lifetime imaging.

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引用次数: 0
Channel rectification made simple.
IF 3.2 3区 生物学 Q2 BIOPHYSICS Pub Date : 2025-02-18 Epub Date: 2025-01-24 DOI: 10.1016/j.bpj.2025.01.013
Harley T Kurata, Christopher N Rowley
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引用次数: 0
Polarity sorting of actin filaments by motor-driven cargo transport. 电动货物运输中肌动蛋白丝的极性分选。
IF 3.2 3区 生物学 Q2 BIOPHYSICS Pub Date : 2025-02-18 Epub Date: 2025-01-17 DOI: 10.1016/j.bpj.2025.01.007
Oghosa H Akenuwa, Steven M Abel

During the active transport of cellular cargo, forces generated by cargo-associated molecular motors propel the cargo along cytoskeletal tracks. However, the forces impact not only the cargo, but also the underlying cytoskeletal filaments. To better understand the interplay between cargo transport and the organization of cytoskeletal filaments, we employ coarse-grained computer simulations to study actin filaments interacting with cargo-anchored myosin motors in a confined domain. We show that cargo transport can lead to the segregation of filaments into domains of preferred filament polarity separated by clusters of aggregated cargoes. The formation of polarity-sorted filament domains is enhanced by larger numbers of cargoes, more motors per cargo, and longer filaments. Analysis of individual trajectories reveals dynamic and heterogeneous behavior, including locally stable aggregates of cargoes that undergo rapid coalescence into larger clusters when sufficiently close. Our results provide insight into the impact of motor-driven organelle transport on actin filaments, which is relevant both in cells and in synthetic environments.

在细胞货物的主动运输过程中,由与货物相关的分子马达产生的力推动货物沿着细胞骨架轨道前进。然而,这些力不仅影响货物,而且影响底层的细胞骨架细丝。为了更好地理解货物运输和细胞骨架细丝组织之间的相互作用,我们采用粗粒度的计算机模拟来研究肌动蛋白细丝与货物锚定的肌球蛋白马达在一个有限区域内的相互作用。我们表明,货物运输可以导致长丝的隔离进入优选的长丝极性的区域,由聚集的货物簇分开。极性分选的细丝结构域的形成被大量的货物、每货物更多的马达和更长的细丝所增强。对单个轨迹的分析揭示了动态和异质行为,包括局部稳定的货物聚集,当足够接近时,它们会迅速合并成更大的集群。我们的研究结果提供了对运动驱动细胞器运输对肌动蛋白丝的影响的见解,这在细胞和合成环境中都是相关的。
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引用次数: 0
Binuclear ruthenium complex linker length tunes DNA threading intercalation kinetics. 双核钌络合物连接体长度调节DNA穿线插入动力学。
IF 3.2 3区 生物学 Q2 BIOPHYSICS Pub Date : 2025-02-18 Epub Date: 2025-01-10 DOI: 10.1016/j.bpj.2025.01.002
Ali A Almaqwashi, Micah J McCauley, Johanna Andersson, Ioulia Rouzina, Fredrik Westerlund, Per Lincoln, Mark C Williams

Binuclear ruthenium complexes have been investigated for potential DNA-targeted therapeutic and diagnostic applications. Studies of DNA threading intercalation, in which DNA basepairs must be broken for intercalation, have revealed means of optimizing a model binuclear ruthenium complex to obtain reversible DNA-ligand assemblies with the desired properties of high affinity and slow kinetics. Here, we used single-molecule force spectroscopy to study a binuclear ruthenium complex with a longer semirigid linker relative to the model complex. Equilibrium results suggest a DNA affinity that is an order of magnitude higher than the parent binuclear ruthenium complex, likely due to a sterically relieved DNA threading intercalation mechanism. Notably, kinetics analysis shows that less DNA elongation is required for threading intercalation compared to the parent complex, and the association rate is two orders of magnitude faster. The ruthenium complex elongates the DNA duplex by ∼0.3 nm per bound ligand to reach the equilibrium intercalated state, with a significantly different energy landscape relative to the parent complex. Mechanical properties of the ligand-saturated DNA duplex show a higher persistence length, indicating that the longer semirigid linker provides enough molecular spacing to allow a single monomer to fully stack with basepairs, comparable to the monomeric parent ruthenium complex. The DNA basepairs in the equilibrium threading intercalated state are likely intact, and the ruthenium complex is shielded from the polar solution, providing measurable single-molecule confocal fluorescence signals. The obtained confocal fluorescence imaging of the bound dye confirms mostly uniform intercalation along the tethered DNA, consistent with other intercalators. The results of this study, along with previously examined ruthenium complex variants, illustrate tunable intercalation mechanisms guided by the rational design of therapeutic and diagnostic small molecules to target and modify the DNA duplex.

双核钌配合物已被研究用于潜在的dna靶向治疗和诊断应用。DNA穿线嵌入的研究,其中DNA碱基对必须被破坏插入,揭示了优化模型双核钌配合物的方法,以获得具有高亲和力和慢动力学特性的可逆DNA配体组装。在这里,我们使用单分子力光谱研究了一个双核钌配合物,它具有相对于模型配合物更长的半刚性连接。平衡结果表明,DNA亲和力比母体双核钌配合物高一个数量级,可能是由于空间释放的DNA线插入机制。值得注意的是,动力学分析表明,与母体复合物相比,螺纹插入所需的DNA伸长更少,并且结合速率快了两个数量级。钌配合物将每个结合配体的DNA双链拉长约0.3 nm,以达到平衡插层状态,相对于母体配合物具有明显不同的能量景观。配体饱和DNA双链的机械性能显示出更高的持续长度,这表明较长的半刚性连接体提供了足够的分子间距,允许单个单体与碱基对完全堆叠,可与单体母体钌配合物相媲美。处于平衡线插层状态的DNA碱基对可能是完整的,钌配合物被极性溶液屏蔽,提供可测量的单分子共聚焦荧光信号。所获得的结合染料的共聚焦荧光成像证实了沿系留DNA的大部分均匀嵌入,与其他嵌入物一致。这项研究的结果,以及之前研究的钌复合物变体,说明了可调节的插入机制,通过合理设计治疗和诊断小分子来靶向和修饰DNA双工。
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引用次数: 0
Conformational equilibrium of an ABC transporter analyzed by luminescence resonance energy transfer.
IF 3.2 3区 生物学 Q2 BIOPHYSICS Pub Date : 2025-02-18 DOI: 10.1016/j.bpj.2025.02.016
Maria E Zoghbi, Annabella Nouel Barreto, Alex L Hernandez

Humans have three known ATP-binding cassette (ABC) transporters in the inner mitochondrial membrane (ABCB7, ABCB8, and ABCB10). ABCB10, the most studied of them thus far, is essential for normal red blood cell development and protection against oxidative stress, and it was recently found to export biliverdin, a heme degradation product with antioxidant properties. The molecular mechanism underlying the function of ABC transporters remains controversial. Their nucleotide binding domains (NBDs) must dimerize to hydrolyze ATP, but capturing the transporters in such conformation for structural studies has been experimentally difficult, especially for ABCB10 and related eukaryotic transporters. Purified transporters are commonly studied in detergent micelles, or after their reconstitution in nanodiscs, usually at nonphysiological temperature and using nonhydrolyzable ATP analogs or mutations that prevent ATP hydrolysis. Here, we have used luminescence resonance energy transfer to evaluate the effect of experimental conditions on the NBD dimerization of ABCB10. Our results indicate that all conditions used for determination of currently available ABCB10 structures have failed to induce NBD dimerization. ABCB10 in detergent responded only to MgATP at 37°C, whereas reconstituted protein shifted toward dimeric NBDs more easily, including in response to MgAMP-PNP and even present NBD dimerization with MgATP at room temperature. The nanodisc's size affects the nucleotide-free conformational equilibrium of ABCB10 and the response to ATP in the absence of magnesium, but for all analyzed sizes (scaffold proteins MSP1D1, MSP1E3D1, and MSP2N2), a conformation with dimeric NBDs is clearly preferred during active ATP hydrolysis (MgATP, 37°C). These results highlight the sensitivity of this human ABC transporter to experimental conditions and the need for a more cautious interpretation of structural models obtained under far from physiological conditions. A dimeric NBD conformation that has been elusive in previous studies seems to be dominant during MgATP hydrolysis at physiological temperature.

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引用次数: 0
Migrasome formation is initiated preferentially in tubular junctions by membrane tension. 迁移小体的形成是由膜张力在管状连接处优先开始的。
IF 3.2 3区 生物学 Q2 BIOPHYSICS Pub Date : 2025-02-18 Epub Date: 2025-01-03 DOI: 10.1016/j.bpj.2024.12.029
Ben Zucker, Raviv Dharan, Dongju Wang, Li Yu, Raya Sorkin, Michael M Kozlov

Migrasomes, the vesicle-like membrane microstructures, arise on the retraction fibers (RFs), the branched nanotubules pulled out of cell plasma membranes during cell migration and shaped by membrane tension. Migrasomes form in two steps: a local RF bulging is followed by a protein-dependent stabilization of the emerging spherical bulge. Here, we addressed theoretically and experimentally the previously unexplored mechanism of bulging of membrane tubular systems. We assumed that the bulging could be driven by increases in membrane tension and experimentally verified this hypothesis in live-cell and biomimetic systems. We exposed RF-generating live cells to a hypotonic medium, which produced water flows into the cells and a related increase in the membrane tension. We observed the formation of migrasome-like bulges with a preferential location in the RF branching sites. Next, we developed a biomimetic system of three membrane tubules pulled out of a giant plasma membrane vesicle (GPMV), connected by a junction, and subjected to pulling forces controlled by the GPMV membrane tension. An abrupt increase in the GPMV tension resulted in the generation of migrasome-like bulges mainly in the junctions. To understand the physical forces behind these observations, we considered theoretically the mechanical energy of a membrane system consisting of a three-way tubular junction with emerging tubular arms subjected to membrane tension. Substantiating our experimental observations, the energy minimization predicted a tension increase to drive the formation of membrane bulges, preferably in the junction site, independently of the way of the tension application. We generalized the model to derive universal criteria of bulging in branched membrane tubules.

迁移体是一种囊泡状的膜微结构,出现在收缩纤维(RFs)上,收缩纤维是在细胞迁移过程中从细胞膜上拉出的支状纳米管,由膜张力形成。偏头痛的形成分两个步骤:局部RF膨胀,随后是蛋白质依赖的球形膨胀的稳定。本文从理论上和实验上探讨了膜管系统胀形的机理。我们假设鼓胀可能是由膜张力的增加引起的,并在活细胞和仿生系统中实验验证了这一假设。我们将产生rf的活细胞暴露在低渗介质中,使水流入细胞,并使膜张力相应增加。我们观察到迁移体样凸起的形成,其优先位置在RF分支位点。接下来,我们开发了一个仿生系统,从一个巨大的囊泡(GPMV)中拔出三个膜管,通过一个结连接,并受到由GPMV膜张力控制的拉力。GPMV张力的突然增加导致主要在连接处产生偏头痛样凸起。为了理解这些观察结果背后的物理力,我们从理论上考虑了膜系统的机械能,该系统由三向管状结组成,管状臂受到膜张力的影响。证实了我们的实验观察,能量最小化预测了张力的增加,以驱动膜凸起的形成,最好是在结处,独立于张力的应用方式。对该模型进行了推广,得到了支膜管胀形的通用判据。
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引用次数: 0
A Drosophila cardiac myosin increases jump muscle stretch activation and shortening deactivation. 果蝇心肌肌球蛋白增加跳跃肌拉伸激活和缩短失活。
IF 3.2 3区 生物学 Q2 BIOPHYSICS Pub Date : 2025-02-18 Epub Date: 2025-01-10 DOI: 10.1016/j.bpj.2025.01.001
Kaylyn M Bell, Alon T Brown, Sarah K Van Houten, Anna C Blice-Baum, William A Kronert, Amy K Loya, Jared Rafael T Camillo, Anthony Cammarato, David T Corr, Sanford I Bernstein, Douglas M Swank

Stretch activation (SA), a delayed increase in force production after rapid muscle lengthening, is critical to the function of vertebrate cardiac muscle and insect asynchronous indirect flight muscle. SA enables or increases power generation in muscle types used in a cyclical manner. Recently, myosin isoform expression has been implicated as a mechanism for varying the amplitude of SA in some muscle types. For instance, we found that expressing a larval Drosophila myosin isoform in a muscle type with minimal SA, the Drosophila jump muscle, substantially increased SA amplitude and enabled positive cyclical power generation. To test whether other myosin isoforms could increase SA amplitude and whether the Drosophila heart benefits from SA, we identified two Drosophila cardiac myosin isoforms, CardM1 and CardM2, and expressed them in Drosophila jump muscle. CardM1, CardM2, and control jump muscle fibers all displayed the characteristic phase 3 of SA, with CardM2 SA amplitude ∼60% greater than that of CardM1 and control fibers. Increasing [Pi] from 0 to 16 mM increased CardM2 SA tension amplitude by 74%, yet had minimal or no effect on CardM1 or control muscle SA amplitude. CardM2 displayed the most prominent phase 3 dip when we induced shortening deactivation, a delayed decrease in force after muscle shortening. The magnitude of CardM2 shortening deactivation tension was ∼50% greater than control or CardM1 fibers. This, along with its greater stretch-activated tension, caused CardM2 to be the only isoform to produce positive power when its fiber length was sinusoidally oscillated. The results support our hypotheses that some myosin isoforms enable greater SA tension levels and suggest that the Drosophila heart is benefiting from SA and shortening deactivation in a manner similar to vertebrate hearts.

拉伸激活(SA)是肌肉快速延长后产生力的延迟增加,对脊椎动物心肌和昆虫异步间接飞行肌(IFM)的功能至关重要。SA能够或增加循环使用的肌肉类型的能量产生。最近,肌球蛋白异构体表达被认为是一些肌肉类型中SA振幅变化的机制。例如,我们发现在具有最小SA的肌肉类型中表达幼虫果蝇肌球蛋白异构体,果蝇跳跃肌,大大增加了SA振幅并使正循环发电成为可能。为了验证其他肌球蛋白异构体是否能增加SA振幅,以及果蝇心脏是否受益于SA,我们鉴定了两种果蝇心脏肌球蛋白异构体CardM1和CardM2,并在果蝇跳跃肌中表达。CardM1、CardM2和对照跳肌纤维均表现出特有的3期SA,其中CardM2的SA振幅比CardM1和对照纤维大60%。将[Pi]从0 mM增加到16 mM,使CardM2 SA张力振幅增加74%,但对CardM1或对照肌SA振幅的影响很小或没有影响。当我们诱导缩短失活(SD)时,CardM2显示出最显著的第3阶段下降,即肌肉缩短后的延迟力下降。CardM2缩短失活张力的大小比对照或CardM1纤维大50%。这与其更大的拉伸激活张力一起导致CardM2成为唯一在其纤维长度正弦振荡时产生正功率的异构体。结果支持了我们的假设,即一些肌球蛋白同型体能够提高SA张力水平,并表明果蝇心脏以类似于脊椎动物心脏的方式受益于SA和SD。
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引用次数: 0
Dissecting current rectification through asymmetric nanopores. 通过不对称纳米孔剖析电流整流。
IF 3.2 3区 生物学 Q2 BIOPHYSICS Pub Date : 2025-02-18 Epub Date: 2024-11-29 DOI: 10.1016/j.bpj.2024.11.3318
Yichun Lin, Jerome J Lacroix, James D Sterling, Yun Lyna Luo

Rectification, the tendency of bidirectional ionic conductors to favor ion flow in a specific direction, is an intrinsic property of many ion channels and synthetic nanopores. Despite its frequent occurrence in ion channels and its phenomenological explanation using Eyring's rate theory, a quantitative relationship between the rectified current and the underlying ion-specific and voltage-dependent free energy profile has been lacking. In this study, we designed nanopores in which potassium and chloride current rectification can be manipulated by altering the electrostatic pore polarity. Using molecular dynamics-based free energy simulations, we quantified voltage-dependent changes of free energy barriers in six ion-nanopore systems. Our results illustrate how the energy barriers for inward and outward fluxes become unequal in the presence of an electromotive driving force, leading to varying degrees of rectification for cation and anion currents. By establishing a direct link between potential of mean force and current rectification rate, we demonstrate that rectification caused by energy barrier asymmetry depends on the nature of the permeating ion, can be tuned by pore polarity, does not require ion binding sites, conformational flexibility, or specific pore geometry, and, as such, may be widespread among ion channels.

整流,即双向离子导体有利于离子向特定方向流动的趋势,是许多离子通道和合成纳米孔的固有特性。尽管它经常出现在离子通道中,并且使用Eyring速率理论对其进行了现象学解释,但一直缺乏整流电流与潜在离子特异性和电压依赖性自由能分布之间的定量关系。在这项研究中,我们设计了可以通过改变静电孔极性来控制钾和氯电流整流的纳米孔。利用基于分子动力学的自由能模拟,我们量化了六个离子纳米孔系统中自由能势垒的电压依赖性变化。我们的研究结果说明了在电动势的存在下,向内和向外通量的能量垒是如何变得不相等的,从而导致正离子和阴离子电流的不同程度的整流。通过建立平均力势和电流整流率之间的直接联系,我们证明了能量势垒不对称引起的整流取决于渗透离子的性质,可以通过孔隙极性来调节,不需要离子结合位点、构象柔韧性或特定的孔隙几何形状,因此,可能在离子通道中广泛存在。
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引用次数: 0
Protein target search diffusion-association/dissociation free energy landscape around DNA binding site with flanking sequences. 蛋白质靶标搜索:DNA结合位点附近的扩散关联/解离自由能景观。
IF 3.2 3区 生物学 Q2 BIOPHYSICS Pub Date : 2025-02-18 Epub Date: 2025-01-16 DOI: 10.1016/j.bpj.2025.01.005
Biao Wan, Jin Yu

In this work we present a minimal structure-based model of protein diffusional search along local DNA amid protein binding and unbinding events on the DNA, taking into account protein-DNA electrostatic interactions and hydrogen-bonding (HB) interactions or contacts at the interface. We accordingly constructed the protein diffusion-association/dissociation free energy surface and mapped it to 1D as the protein slides along DNA, maintaining the protein-DNA interfacial HB contacts that presumably dictate the DNA sequence information detection. Upon DNA helical path correction, the protein 1D diffusion rates along local DNA can be physically derived to be consistent with experimental measurements. We also show that the sequence-dependent protein sliding or stepping patterns along DNA are regulated by collective interfacial HB dynamics, which also determines the ruggedness of the protein diffusion free energy landscape on the local DNA. In comparison, protein association or binding with DNA are generically dictated by the protein-DNA electrostatic interactions, with an interaction zone of nanometers around DNA. Extra degrees of freedom (DOFs) of the protein such as rotations and conformational fluctuations can be well accommodated within the protein-DNA electrostatic interaction zone. As such we demonstrate that the protein binding or association free energy profiling along DNA smoothens over the 1D diffusion free energy landscape, which leads to population variations for an order of magnitude upon a marginal free energetic smoothening around the specific or consensus sites. We further show that the protein unbinding or dissociation from a comparatively high-binding affinity DNA site is dominated by lateral diffusion to the flanking low-affinity sites. The results predict that experimental characterizations on the relative protein-DNA binding affinities or population profiling on the DNA are systematically and physically impacted by the extra DOFs of protein motions aside from 1D translation or helical tracking, as well as from flanking DNA sequences due to protein 1D diffusion and nonspecific binding/unbinding.

在这项工作中,我们提出了一个基于最小结构的蛋白质扩散搜索模型,该模型在DNA上的蛋白质结合和解结合事件中沿着局部DNA进行搜索,考虑到蛋白质-DNA静电相互作用和氢键(HB)相互作用或界面上的接触。因此,我们构建了蛋白质扩散-结合/解离自由能表面,并在蛋白质沿着DNA滑动时将其映射为1D,保持蛋白质-DNA界面的HB接触,这可能决定了DNA序列信息的检测。在DNA螺旋路径修正后,蛋白质沿局部DNA的一维扩散速率可以物理推导出与实验测量一致。我们还表明,序列依赖的蛋白质沿着DNA滑动或步进模式受到集体界面HB动力学的调节,这也决定了局部DNA上蛋白质扩散自由能景观的坚固性。相比之下,蛋白质与DNA的结合通常是由蛋白质-DNA静电相互作用决定的,在DNA周围有一个纳米级的相互作用区。蛋白质的额外自由度(DOFs),如旋转和构象波动可以很好地适应在蛋白质- dna静电相互作用区。因此,我们证明沿DNA的蛋白质结合或结合自由能谱在一维扩散自由能景观上平滑,这导致种群在特定或共识位点周围的边缘自由能平滑上一个数量级的变化。我们进一步表明,蛋白质从一个相对高结合亲和力的DNA位点解离或解离主要是侧向扩散到侧翼的低亲和力位点。结果预测,除了1D翻译或螺旋跟踪之外,蛋白质运动的额外自由度以及蛋白质一维扩散和非特异性结合/解结合引起的侧翼DNA序列,系统地和物理地影响了蛋白质-DNA相对结合亲和性或DNA种群分析的实验表征。
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引用次数: 0
BPS2025 - Electrical shock injuries: Clarifying the biophysics
IF 3.4 3区 生物学 Q2 BIOPHYSICS Pub Date : 2025-02-13 DOI: 10.1016/j.bpj.2024.11.3283
Sumavarshini Suma Gangidi, Mukul Govande, Kyley McCollum, Raphael C. Lee
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引用次数: 0
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