Myosin II dynamics and the influence of S100A4

细胞包含成千上万的成分，这些成分以协调的方式相互作用，产生生命的特征（如细胞分裂、运动）。肌凝蛋白是一种关键的蛋白质成分，它聚合形成丝状结构，是细胞骨架的一部分，帮助维持和改变细胞形状。这些纤维是动态的，肌凝蛋白成分可能在几秒钟内结合和分离，即使在整个纤维看起来稳定了许多分钟的情况下。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似乎是一个重要的因素。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浓度的变化可导致细胞形态和迁移行为的改变。本提案的目的是研究S100A4、肌球蛋白和其他蛋白在纯状态下结合的详细时间过程，并设计基于显微镜的检测方法，通过这种方法可以在活细胞中跟踪它们的相互作用。申请人的初步工作已经建立了本研究所需的许多工具。已经确定了S100A4的原子结构，并对其在肌球蛋白上的相互作用位点进行了部分表征。已经开发出一种细胞系，其S100A4浓度可以通过特定诱导和使用siRNA（一种阻止该蛋白质表达的特定分子）随意改变。此外，肌凝蛋白可以与绿色荧光蛋白融合表达，以便在显微镜下可以看到肌凝蛋白细丝。一种定制的显微镜已经开发出来，可以观察细胞的薄切片，并且在这个区域内的小区域可以通过短暂的激光照射进行光漂白。虽然这破坏了融合蛋白的荧光，但肌凝蛋白部分不受影响，并继续与细丝结合和分离。漂白区域恢复的时间过程提供了蛋白质的扩散速率及其在丝状结构中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来分解不稳定的Ca2+复合体，该复合体是由先前的孵育加载到细胞中。将在细胞内测定的相互作用速率与在没有其他细胞成分的情况下用纯化蛋白质在溶液中观察到的相互作用速率进行比较，看它们是否一致。如果没有达成一致，则表明涉及到其他组件，需要对其进行定义。由于细胞相互作用的复杂性，需要仔细控制以确定是否由肌球蛋白- s100a4相互作用直接产生特定效应。一种方法是使肌凝蛋白和S100A4发生突变，从而破坏它们的结合位点。了解S100A4的原子结构以及与之相互作用的肌凝蛋白尾部区域将有助于这种方法。进一步的工作是在高分辨率下定义完整的相互作用区域。对活细胞进行定量测量的一个困难是样品之间细胞形状的变化。我们将探索在各种形状（例如X， Y和U）的载玻片上沉积有利底物的图图化技术，这应该鼓励细胞以明确的形状结合。此外，图案的角落将促进粘附复合物的形成，而细胞骨架结构，如肌动球蛋白应力纤维将被定位在末端之间（例如X将诱导一个方形细胞，每个角落连接四组应力纤维）。这种固定的细胞可以更好地再现测量结果。

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.