Myosin II dynamics and the influence of S100A4

肌球蛋白 II 动力学和 S100A4 的影响

• 批准号: BB/F007213/1
• 负责人: Igor Barsukov
• 金额: $ 9.79万
• 依托单位: University of Liverpool
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2008
• 资助国家: 英国
• 起止时间: 2008 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FF007213%2F1
• 关键词: Myosin; II; dynamics; influence; S100A4

Cells contain many thousands of components that interact in a coordinated way to give rise to properties characteristic of life (e.g. cell division, motility). Myosin is a key protein component that polymerises to form filamentous structures that are part of the cell cytoskeleton that help both maintain and change cell shape. These filaments are dynamic and myosin components may associate and dissociate over a period of seconds, even under conditions where the overall filament appears to be stable for many minutes. The cell regulates the assembly of myosin filaments by a number of mechanisms including interactions with other proteins, of which S100A4 appears a significant factor. S100A4 is a small Ca2+ binding protein that attaches to the myosin tail and inhibits polymerisation. Changes in the concentration of S100A4 in cells can lead to changes in morphology and migration behaviour. The objectives of this proposal are to study the detailed time courses of binding between S100A4, myosin and other proteins in pure state and to devise microscope-based assays through which their interactions can be followed in living cells. Preliminary work by the applicants has established many of the tools required for this research. The atomic structure of S100A4 has been determined and its interaction sites on myosin have been partially characterised. A cell line has been developed whose S100A4 concentration can be changed at will through specific induction and the use of siRNA (a specific molecule which blocks the expression of this protein). Furthermore the myosin can be expressed as fusion with a green fluorescent protein so that the myosin filaments can be visualised under the microscope. A custom-built microscope has been developed to allow a thin section of the cell to be observed and within this area as small region can be photobleached by brief exposure to laser light. While this destroys the fluorescence of the fusion protein, the myosin part is unaffected and continues to associate and dissociate from filaments. The time course of recovery of the bleached area provides key information about the diffusion rates of the proteins and their exchange rates into filamentous structures. We will also change rates of interaction by modulating the intracellular Ca2+ concentration. This will be done using a ultraviolet light flash to breakdown an unstable complex of Ca2+ which is loaded into the cell by prior incubation. The interaction rates determined within the cell will be compared with those observed with purified proteins in solution in the absence of other cellular components to see if they agree. Absence of agreement will indicate other components that are involved and that will need to be defined. Because of the complexity of cellular interactions, careful controls will be required to determine if specific effects arise directly from myosin-S100A4 interactions. One approach is to make mutations in the myosin and S100A4 so their binding sites are destroyed. Knowledge of the atomic structure of S100A4 and the region within the myosin tail that it interacts with, will aid this approach. Further work is proposed to define the complete interaction region at high resolution. One difficulty in carrying out quantitative measurements on living cells is the variation in cell shape between samples. We will explore patterning techniques whereby a favourable substrate is deposited on a slide in various shapes (e.g. X, Y and U) which should encourage the cells to bind with a well-defined shape. Furthermore, the corners of the patterns will encourage the formation of adhesion complexes, while cytoskeletal structures such as a actomyosin stress fibres will be positioned between the extremities (e.g. X will induce a square-shaped cell with four sets of stress fibres linking each corner). Such immobilised cells should allow better reproducibility of measurements.

细胞含有数千种成分，它们以协调的方式相互作用，产生生命特征（例如细胞分裂、运动）。肌球蛋白是一种关键的蛋白质成分，可聚合形成丝状结构，丝状结构是细胞骨架的一部分，有助于维持和改变细胞形状。这些肌丝是动态的，肌球蛋白成分可能会在几秒钟内结合和解离，即使在整个肌丝看起来稳定许多分钟的情况下也是如此。细胞通过多种机制调节肌球蛋白丝的组装，包括与其他蛋白质的相互作用，其中 S100A4 似乎是一个重要因素。 S100A4 是一种小的 Ca2+ 结合蛋白，附着在肌球蛋白尾部并抑制聚合。细胞中 S100A4 浓度的变化可导致形态和迁移行为的变化。该提案的目的是研究纯状态下 S100A4、肌球蛋白和其他蛋白质之间结合的详细时间过程，并设计基于显微镜的测定法，通过该测定法可以在活细胞中追踪它们的相互作用。申请人的初步工作已经建立了本研究所需的许多工具。 S100A4 的原子结构已确定，其在肌球蛋白上的相互作用位点已部分表征。已经开发出一种细胞系，其S100A4浓度可以通过特异性诱导和使用siRNA（一种阻断该蛋白表达的特定分子）随意改变。此外，肌球蛋白可以与绿色荧光蛋白融合表达，以便肌球蛋白丝可以在显微镜下可视化。已经开发出一种定制的显微镜，可以观察细胞的薄切片，并且在该区域内，可以通过短暂暴露于激光来对小区域进行光漂白。虽然这会破坏融合蛋白的荧光，但肌球蛋白部分不受影响，并继续与细丝结合和解离。漂白区域恢复的时间过程提供了有关蛋白质扩散速率及其进入丝状结构的交换速率的关键信息。我们还将通过调节细胞内 Ca2+ 浓度来改变相互作用的速率。这将使用紫外线闪光分解不稳定的 Ca2+ 复合物来完成，该复合物通过预先孵育加载到细胞中。将细胞内确定的相互作用速率与在没有其他细胞成分的情况下在溶液中观察到的纯化蛋白质的相互作用速率进行比较，看看它们是否一致。如果没有达成一致，则表明还涉及其他需要定义的组件。由于细胞相互作用的复杂性，需要仔细控制以确定特定效应是否直接由肌球蛋白-S100A4 相互作用产生。一种方法是使肌球蛋白和 S100A4 发生突变，从而破坏它们的结合位点。了解 S100A4 的原子结构以及与其相互作用的肌球蛋白尾部区域将有助于这种方法。提出了进一步的工作来定义高分辨率的完整交互区域。对活细胞进行定量测量的一大困难是样品之间细胞形状的变化。我们将探索图案化技术，将有利的基质以各种形状（例如 X、Y 和 U）沉积在载玻片上，这应该鼓励细胞以明确的形状结合。此外，图案的角将促进粘附复合物的形成，而肌动球蛋白应力纤维等细胞骨架结构将位于四肢之间（例如，X 将诱导一个方形细胞，其中四组应力纤维连接每个角） 。这种固定化细胞应具有更好的测量重现性。

项目成果

Mechanism of the Ca²+-dependent interaction between S100A4 and tail fragments of nonmuscle myosin heavy chain IIA.

S100A4 和非肌肉肌球蛋白重链 IIA 尾部片段之间 Ca2 依赖性相互作用的机制。

• DOI: http://dx.10.1016/j.jmb.2010.11.036
• 发表时间: 2011-01-28
• 期刊: Journal of molecular biology
• 影响因子: 5.6
• 作者: Badyal SK;Basran J;Bhanji N;Kim JH;Chavda AP;Jung HS;Craig R;Elliott PR;Irvine AF;Barsukov IL;Kriajevska M;Bagshaw CR
• 通讯作者: Bagshaw CR

Asymmetric mode of Ca²?-S100A4 interaction with nonmuscle myosin IIA generates nanomolar affinity required for filament remodeling.

Ca2+-S100A4 与非肌肉肌球蛋白 IIA 相互作用的不对称模式产生肌丝重塑所需的纳摩尔亲和力。

• DOI: http://dx.10.1016/j.str.2012.02.002
• 发表时间: 2012
• 期刊: 1993)
• 作者: Elliott PR

S100A4 regulates the Src-tyrosine kinase dependent differentiation of Th17 cells in rheumatoid arthritis.

S100A4 调节类风湿性关节炎中 Th17 细胞的 Src-酪氨酸激酶依赖性分化。

• DOI: 10.1016/j.bbadis.2014.07.003
• 发表时间: 2014-11-01
• 期刊: Biochimica et biophysica acta
• 作者: M. Brisslert;L. Bian;M. Svensson;Rita F. Santos;I. Jonsson;I. Barsukov;M. Erl;sson;sson;K. Andersson;A. Carmo;M. Bokarewa
• 通讯作者: M. Bokarewa