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Titin细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。肌动蛋白是一种多功能多肽，可与肌球蛋白或肌动蛋白等其他肌瘤蛋白在多个部位相互作用。虽然肌动蛋白在肌肉收缩中的“被动”作用已经得到了很好的证明，但有大量迹象表明，肌动蛋白参与了更积极的活动，但却是分散的和不系统的。我们希望对这些提示进行更系统的跟踪和探讨。为了研究肌动蛋白的机械性能，我们将使用独特的方法：单一的肌原纤维制备与荧光团标记的抗肌动蛋白抗体相结合，为研究肌动蛋白的作用提供了理想的工具。我们将对单个心肌肌原纤维进行力和硬度测量，以测试肌动蛋白细丝在被钙离子激活后是否变硬，真正意义上的“收缩”。我们还将确定肌动蛋白对心肌原纤维活性肌节长度-张力曲线的影响，特别是在高伸展时。此外，体外运动试验将被用来检验Titin对细丝滑动速度有特定影响的假设。将使用多种重组肌动蛋白构建物，并研究肌动蛋白对肌动蛋白滑动速度的影响是否受[钙离子]的影响。如果发现了结构的特定效应，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，以确认在高度有序的肌节环境中观察到的功能相关性。