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

探讨肌联蛋白在调节肌动蛋白-肌球蛋白相互作用中的积极作用

• 批准号: 5241032
• 负责人: Professor Dr. Wolfgang Linke, Ph.D.
• 金额: --
• 依托单位: Institut für Physiologie II (Vegetative Physiologie)
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2000
• 资助国家: 德国
• 起止时间: 1999-12-31 至 2011-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/5241032?language=en
• 关键词: Probing; active; role; titin; modulating

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.

这些实验的目的是探索肌动蛋白丝如何调节肌动蛋白-肌球蛋白相互作用，特别是在心肌中。肌动蛋白是一种多功能多肽，它在多个位点与其他肌节蛋白（例如肌球蛋白或肌动蛋白）相互作用。肌动蛋白在肌肉收缩中的作用已有充分记录，关于肌动蛋白更积极参与的线索很多，但分散且不系统，我们希望以更系统的方式跟踪和探索这些线​​索。为了确定肌联蛋白的“活性”机械特性，我们将利用独特的方法：单一肌原纤维制剂与荧光团标记的抗肌联蛋白抗体相结合，为研究肌联蛋白的作用提供了理想的工具。对单个心脏肌原纤维进行测量，以测试肌联蛋白丝是否在 Ca2+ 激活后变硬，真正意义上的“收缩”。我们还将确定肌联蛋白对活性肌节的影响。心肌原纤维的长度-张力曲线，特别是在高拉伸时，将采用体外运动测定来检验肌原纤维对细丝滑动速度具有特定影响的假设。使用并研究了肌动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果，我们将检查是否也可以在单个结构中检测到类似的效果。肌原纤维，以确认肌节高度有序环境中观察结果的功能相关性。