Probing an active role of titin in modulating actin-myosin interactions

这些实验的目的是探索肌联蛋白丝如何调节肌动蛋白-肌球蛋白的相互作用，特别是在心肌中。肌联蛋白是一种多功能多肽，它在多个位点与其他肌节蛋白如肌球蛋白或肌动蛋白相互作用。虽然肌联蛋白在肌肉收缩中的“被动”作用已被充分证明，但肌联蛋白更积极参与的暗示却很丰富，但分散且不系统。我们希望更有系统地跟进和探讨这些暗示。为了研究肌联蛋白的假定的“活性”机械性质，我们将利用独特的方法：单肌原纤维制备，结合荧光团标记的抗肌联蛋白抗体，提供了一个理想的工具来研究肌联蛋白的作用。我们将对单个心脏肌原纤维进行力和刚度测量，以测试肌联蛋白丝在被Ca²+激活后是否变硬，在真正意义上是“收缩的”。我们还将确定肌联蛋白对心肌肌原纤维的活性肌节长度-张力曲线的影响，特别是在高拉伸下。此外，将采用体外运动试验来检验肌联蛋白对细丝滑动速度具有特异性影响的假设。将使用各种重组肌联蛋白构建体，并研究肌联蛋白对肌动蛋白滑动速度的影响受[Ca²+]影响的可能性。如果发现构建体的特定效应，我们将检查是否也可以在单个肌原纤维中检测到类似的效应，以确认在肌节的高度有序环境中观察到的功能相关性。

The goal of these experiments is to explore how titin filaments can modulate actin-myosin interaction, particularly in cardiac muscle. Titin is a multifunctional polypeptide which interacts at multiple sites with other sarcomeric proteins, such as myosin or actin. Wheras the 'passive' roles of titin in muscle contraction are well documented, hints at a more active involvement of titin are abundant but scattered and unsystematic. We wish to follow up and probe these hints in a more systematic way. To study the putative 'active' mechanical properties of titin, we will make use of unique methodology: the single myofibril preparation, combined with fluorophore-labelled anti-titin antibodies, provides an ideal tool to investigate the role of titin. We will perform force and stiffness measurements on single cardiac myofibrils to test whether the titin filament stiffens upon activation with Ca²+, being 'contractile' in a true sense. We will also determine the effect titin has on the active sarcomere length-tension curve of cardiac myofibrils, in particular on high stretch. Further, the in-vitro motility assay will be employed to test the hypothesis that titin has a specific effect on thin-filament sliding velocity. A variety of recombinant titin constructs will be used and the possibility studied that the effect of titin on actin sliding speed is influenced by [Ca²+]. If a specific effect of a construct is found, we will examine whether a comparable effect can also be detected in the single myofibril, to confirm a functional relevance of the observations in the highly ordered enviroment of the sarcomere.