Scheid Lisa-Mareike, Weber Cornelia, Bopp Nasrin, Mosqueira Matias, Fink Rainer H A
Medical Biophysics Unit, Medical Faculty, Institute of Physiology and Pathophysiology, University of HeidelbergHeidelberg, Germany.
Front Physiol. 2017 May 31;8:367. doi: 10.3389/fphys.2017.00367. eCollection 2017.
The motility assay (IVMA) is a technique that enables the measurement of the interaction between actin and myosin providing a relatively simple model to understand the mechanical muscle function. For actin-myosin IVMA, myosin is immobilized in a measurement chamber, where it converts chemical energy provided by ATP hydrolysis into mechanical energy. The result is the movement of fluorescently labeled actin filaments that can be recorded microscopically and analyzed quantitatively. Resulting sliding speeds and patterns help to characterize the underlying actin-myosin interaction that can be affected by different factors such as mutations or active compounds. Additionally, modulatory actions of the regulatory proteins tropomyosin and troponin in the presence of calcium on actin-myosin interaction can be studied with the IVMA. Zebrafish is considered a suitable model organism for cardiovascular and skeletal muscle research. In this context, straightforward protocols for the isolation and use of zebrafish muscle proteins in the IVMA would provide a useful tool in molecular studies. Currently, there are no protocols available for the mentioned purpose. Therefore, we developed fast and easy protocols for characterization of zebrafish proteins in the IVMA. Our protocols enable the interested researcher to (i) isolate actin from zebrafish skeletal muscle and (ii) extract functionally intact myosin from cardiac and skeletal muscle of individual adult zebrafish. Zebrafish tail muscle actin is isolated after acetone powder preparation, polymerized, and labeled with Rhodamine-Phalloidin. Myosin from ventricles of adult zebrafish is extracted directly into IVMA flow-cells. The same extraction protocol is applicable for comparably small tissue pieces as from zebrafish tail, mouse and frog muscle. After addition of the fluorescently labeled F-actin from zebrafish-or other origin-and ATP, sliding movement can be visualized using a fluorescence microscope and an intensified CCD camera. Taken together, we introduce a method for functional analysis in zebrafish cardiac and skeletal muscle research to study mutations at the molecular level of thick or thin filament proteins. Additionally, preliminary data indicate the usefulness of the presented method to perform the IVMA with myosin extracted from muscles of other animal models.
运动性分析（IVMA）是一种能够测量肌动蛋白和肌球蛋白之间相互作用的技术，它提供了一个相对简单的模型来理解肌肉的机械功能。对于肌动蛋白 - 肌球蛋白IVMA，肌球蛋白固定在测量室中，在那里它将ATP水解提供的化学能转化为机械能。结果是荧光标记的肌动蛋白丝的运动，这可以通过显微镜记录并进行定量分析。由此产生的滑动速度和模式有助于表征潜在的肌动蛋白 - 肌球蛋白相互作用，这种相互作用可能会受到不同因素的影响，如突变或活性化合物。此外，在有钙存在的情况下，调节蛋白原肌球蛋白和肌钙蛋白对肌动蛋白 - 肌球蛋白相互作用的调节作用可以通过IVMA进行研究。斑马鱼被认为是心血管和骨骼肌研究的合适模式生物。在这种情况下，用于在IVMA中分离和使用斑马鱼肌肉蛋白的简单方案将为分子研究提供一个有用的工具。目前，没有用于上述目的的方案。因此，我们开发了快速简便的方案来在IVMA中表征斑马鱼蛋白。我们的方案使感兴趣的研究人员能够：（i）从斑马鱼骨骼肌中分离肌动蛋白，以及（ii）从成年斑马鱼个体的心脏和骨骼肌中提取功能完整的肌球蛋白。斑马鱼尾部肌肉肌动蛋白在制备丙酮粉后分离、聚合，并用罗丹明 - 鬼笔环肽标记。成年斑马鱼心室中的肌球蛋白直接提取到IVMA流动池中。相同的提取方案适用于来自斑马鱼尾巴、小鼠和青蛙肌肉等相对较小的组织块。加入来自斑马鱼或其他来源的荧光标记的F - 肌动蛋白和ATP后，可以使用荧光显微镜和增强型CCD相机观察滑动运动。综上所述，我们介绍了一种用于斑马鱼心脏和骨骼肌研究功能分析的方法，以研究粗细肌丝蛋白分子水平的突变。此外，初步数据表明所提出的方法对于用从其他动物模型肌肉中提取的肌球蛋白进行IVMA是有用的。
