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IF 2.3 4区医学 Q3 BIOPHYSICS

Cellular and molecular bioengineering Pub Date : 2024-12-04 eCollection Date: 2025-02-01 DOI:10.1007/s12195-024-00835-w

Emily M Kerivan, Victoria N Amari, William B Weeks, Leigh H Hardin, Lyle Tobin, Omayma Y Al Azzam, Dana N Reinemann

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引用次数: 0

摘要

目的：细胞骨架蛋白质集合体表现出突发性力学，其团队行为并不一定是各组分单分子特性的总和。此外，细丝可能充当力传感器，分配反馈并影响运动蛋白的行为。为了了解这种突发性力学的设计原理，我们开发了一种方法，利用 QCM-D 测量肌动蛋白束如何对改变组成肌球蛋白 II 运动行为的环境变量做出机械响应：我们首次利用 QCM-D 探测骨骼肌肌球蛋白 II 浓度和运动核苷酸状态变化导致的肌动蛋白-肌球蛋白束粘弹性变化。使用微流体装置在金 QCM-D 传感器上构建肌动蛋白束，并记录每种添加成分的频率和耗散变化测量值，以破译哪些检测成分会导致肌动蛋白束结构顺应性发生变化：结果：肌球蛋白浓度降低时，频率和耗散的变化较小，而添加第一种和第二种肌动蛋白时，频率和耗散变化的相对变化相对相似。引人注目的是，用不同的核苷酸（ATP 与 ADP）冲洗缓冲液会产生独特的频率和耗散位移特征。当肌球蛋白 II 的 ADP 结合状态与肌动蛋白丝紧密结合时，我们观察到频率增加而耗散变化减少，这表明粘弹性降低，这可能是由于肌球蛋白对肌动蛋白的亲和力增加，从活性马达转变为静态交联剂，并能从表面招募更多的肌动蛋白丝，使传感器涂层整体更加坚硬。然而，降低 ADP 浓度会增加系统的顺从性，这表明瞬时交联和保持马达活性的平衡可能会产生一个合作性更强、生产力更高的发力系统：结论：QCM-D 可检测分子水平变化（如运动浓度和核苷酸状态）导致的肌动蛋白粘弹性变化。这些结果为肌动蛋白作为机械力反馈传感器的作用提供了支持，并展示了一种新方法，可用于破译驱动新出现的细胞骨架集合串联和细胞内机械传感的反馈机制。这种方法可用于研究环境对更复杂的细胞骨架集合力学的影响，包括添加其他马达、交联剂和丝状物类型：在线版本包含补充材料，可查阅 10.1007/s12195-024-00835-w。

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Deciphering Mechanochemical Influences of Emergent Actomyosin Crosstalk Using QCM-D.

Purpose: Cytoskeletal protein ensembles exhibit emergent mechanics where behavior in teams is not necessarily the sum of the components' single molecule properties. In addition, filaments may act as force sensors that distribute feedback and influence motor protein behavior. To understand the design principles of such emergent mechanics, we developed an approach utilizing QCM-D to measure how actomyosin bundles respond mechanically to environmental variables that alter constituent myosin II motor behavior.

Methods: QCM-D is used for the first time to probe alterations in actin-myosin bundle viscoelasticity due to changes in skeletal myosin II concentration and motor nucleotide state. Actomyosin bundles were constructed on a gold QCM-D sensor using a microfluidic setup, and frequency and dissipation change measurements were recorded for each component addition to decipher which assay constituents lead to changes in bundle structural compliancy.

Results: Lowering myosin concentration is detected as lower shifts in frequency and dissipation, while the relative changes in frequency and dissipation shifts for both the first and second actin additions are relatively similar. Strikingly, buffer washes with different nucleotides (ATP vs. ADP) yielded unique signatures in frequency and dissipation shifts. As myosin II's ADP-bound state tightly binds actin filaments, we observe an increase in frequency and decrease in dissipation change, indicating a decrease in viscoelasticity, likely due to myosin's increased affinity for actin, conversion from an active motor to a static crosslinker, and ability to recruit additional actin filaments from the surface, making an overall more rigid sensor coating. However, lowering the ADP concentration results in increased system compliancy, indicating that transient crosslinking and retaining a balance of motor activity perhaps results in a more cooperative and productive force generating system.

Conclusions: QCM-D can detect changes in actomyosin viscoelasticity due to molecular-level alterations, such as motor concentration and nucleotide state. These results provide support for actin's role as a mechanical force-feedback sensor and demonstrate a new approach for deciphering the feedback mechanisms that drive emergent cytoskeletal ensemble crosstalk and intracellular mechanosensing. This approach can be adapted to investigate environmental influences on more complex cytoskeletal ensemble mechanics, including addition of other motors, crosslinkers, and filament types.

Supplementary information: The online version contains supplementary material available at 10.1007/s12195-024-00835-w.

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来源期刊

Cellular and molecular bioengineering 生物-生物物理

CiteScore

5.60

自引率

3.60%

发文量

审稿时长

>12 weeks

期刊介绍： The field of cellular and molecular bioengineering seeks to understand, so that we may ultimately control, the mechanical, chemical, and electrical processes of the cell. A key challenge in improving human health is to understand how cellular behavior arises from molecular-level interactions. CMBE, an official journal of the Biomedical Engineering Society, publishes original research and review papers in the following seven general areas: Molecular: DNA-protein/RNA-protein interactions, protein folding and function, protein-protein and receptor-ligand interactions, lipids, polysaccharides, molecular motors, and the biophysics of macromolecules that function as therapeutics or engineered matrices, for example. Cellular: Studies of how cells sense physicochemical events surrounding and within cells, and how cells transduce these events into biological responses. Specific cell processes of interest include cell growth, differentiation, migration, signal transduction, protein secretion and transport, gene expression and regulation, and cell-matrix interactions. Mechanobiology: The mechanical properties of cells and biomolecules, cellular/molecular force generation and adhesion, the response of cells to their mechanical microenvironment, and mechanotransduction in response to various physical forces such as fluid shear stress. Nanomedicine: The engineering of nanoparticles for advanced drug delivery and molecular imaging applications, with particular focus on the interaction of such particles with living cells. Also, the application of nanostructured materials to control the behavior of cells and biomolecules.