Membrane bending by protein phase separation.
Membrane bending by protein phase separation.
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通过蛋白质相分离使膜弯曲。
DOI：
10.1073/pnas.2017435118
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发表时间：
2021
影响因子：
11.1
通讯作者：
Stachowiak,JeanneC
中科院分区：
文献类型：
--
作者：
Yuan,Feng;Alimohamadi,Haleh;Bakka,Brandon;Trementozzi,AndreaN;Day,KaseyJ;Fawzi,NicolasL;Rangamani,Padmini;Stachowiak,JeanneC
美国密西西比大学。单分子生物物理学领域的一个挑战是重建反映生理功能的运动丝环境。许多光学捕获马达蛋白的研究采用了一个单一的马达与一个单一的灯丝相互作用的还原几何。这些构象不能准确地代表具有交联能力的马达（如肌球蛋白或有丝分裂驱动蛋白）发挥功能的结构体系。细胞骨架丝，结合蛋白和电机的网络已经组装和研究，以帮助这个问题，然而，一种方法，结合光学捕获与积极组装的细胞骨架层次在体外将揭示未开发的理解本地，molecularlevel生物力学，需要完成大规模的细胞任务，如细胞分裂和肌肉收缩。我们已经设计了“纳米细胞”，使用光镊以高分辨率探测分层细胞骨架力学。纳米细胞利用重组和分离细胞骨架丝和蛋白质的能力，通过微流体、分阶段引入和主动操纵来主动定制更高阶的结构，以研究分子马达力学。使用这种方法，我们询问actomyosin力学使用光镊，以阐明肌球蛋白II合奏协同作用和力调节内捆绑肌动蛋白组装。26-亚克的N-末端叶的肌球蛋白调控轻链是一个肌节长度传感器的一部分，在心肌托马斯Kampourakis。英国伦敦国王学院。心肌的正常功能取决于它在较长时间内收缩得更强的能力。当心肌细胞在两次心跳之间放松时，静脉充盈的增加会拉伸心肌细胞，从而在下一次心跳中触发更强的收缩--Frank-Starling关系。相反，当心肌细胞在血液喷射过程中缩短时，它们会被灭活，加速放松，以便于在下一次跳动之前重新充盈。这两种效应对于心脏响应身体不同需求的有效功能都是必不可少的，并且通常在心脏病中受损。然而，尽管进行了大量的研究工作，但其潜在的分子机制仍然未知。在这里，我们使用双功能荧光探针来监测肌球蛋白的调节轻链（RLC）的构象，因为我们改变了收缩心肌细胞中肌节的长度。我们发现，构象的N-末端叶的RLC（NRLC）的轨迹的肌节长度的变化几乎是瞬间的。相反，RLC（CRLC）的C端叶对肌球蛋白马达与肌动蛋白的连接和马达的工作行程敏感。这些结果表明，心脏收缩强度和速度的长度依赖性是由粗丝的调节状态介导的，并且肌细胞长度的变化是由NRLC作为肌球蛋白马达的不同调节域来感知的。
University of Mississippi, University, MS, USA. A challenge within the single molecule biophysics field is reconstituting a motor-filament environment that reflects physiological function. Many optical trapping studies of motor proteins employ a reductionist geometry of a single motor interacting with a single filament. These conformations do not accurately represent the structural architecture in which motors with crosslinking ability, such as myosins or mitotic kinesins, function. Networks of cytoskeletal filaments, binding proteins, and motors have been assembled and studied to aid this issue; yet, an approach that combines optical trapping with active assembly of cytoskeletal hierarchy in vitro would reveal untapped understanding of the native, molecularlevel biomechanics that are required to accomplish large-scale cellular tasks, such as cell division and muscle contraction. We have engineered ‘‘nanocells’’to probe hierarchical cytoskeletal mechanics with high resolution using optical tweezers. Nanocells take advantage of the ability to reconstitute and isolate cytoskeletal filaments and proteins to actively custom-build higher order architectures through microfluidics, staged introduction, and active manipulation in which to study molecular motor mechanics. Using this approach, we interrogate actomyosin mechanics using optical tweezers to elucidate myosin II ensemble synergy and force regulation within a bundled actin assembly.26-Subg The N-Terminal Lobe of The Myosin Regulatory Light Chain is Part of a Sarcomeric Length Sensor in Cardiac Muscle Thomas Kampourakis. King’s College London, London, United Kingdom. The normal function of heart muscle depends on its ability to contract more strongly at longer length. increased venous filling stretches heart muscle cells when they are relaxed between beats, triggering a stronger contraction in the next beat-the Frank-Starling relation. Conversely, heart muscle cells are deactivated when they shorten during the ejection of blood, accelerating relaxation to facilitate refilling before the next beat. Both effects are essential for the efficient function of the heart in response to the varying needs of the body, and are commonly impaired in heart disease. However, despite intense research effort, the underlying molecular mechanisms remained unknown. Here we used bifunctional fluorescent probes to monitor the conformation of the regulatory light chain (RLC) of myosin as we changed the length of the sarcomere in contracting heart muscle cells. We show that the conformation of the N-terminal lobe of the RLC (NRLC) tracks changes in sarcomere length almost instantaneously. The C-terminal lobe of the RLC (CRLC), in contrast, is sensitive to attachment of myosin motors to actin and the working stroke of the motor. These results show that lengthdependence of the strength and speed of contraction in the heart is mediated by the regulatory state of the thick filament, and that changes in muscle cell length are sensed by NRLC acting as a distinct regulatory domain of the myosin motor.