Actin-based Force Generation in the Vascular Endothelium

血管内皮是一个系统性分布的、高度动态的、严格调节的器官系统，在多种疾病的发病机制中起着核心作用。收缩肌动蛋白复合物介导内皮细胞的屏障功能、机械转导、定向跨细胞转运过程，并且在血管生成过程中对细胞迁移至关重要。尽管它们对血管内皮的完整性和功能做出了不可或缺的贡献，但我们对所涉及的肌动球蛋白结构的异构体组成和功能特性的了解是有限的。使用重组肌动蛋白-原肌球蛋白-肌球蛋白复合物的初步实验显示了细胞骨架原肌球蛋白亚型的作用如何超越了看门人的作用。 运动活动的变化不仅观察到交换肌球蛋白亚型，但相同的肌球蛋白在应变敏感性，持续合成能力，和相关的原肌球蛋白亚型交换后的速度显着的变化。我们将确定血管内皮细胞中存在的主要收缩复合物，以阐明其功能定义的结构特征，推导出第一原理，该原理允许对变构触发事件对其化学机械性质，热稳定性，蛋白质折叠稳定性和动力学的影响进行准确建模。我们将利用我们在细胞骨架蛋白的生产，收缩复合物的重建及其功能和结构表征中的主导作用，绘制变构通信途径，并确定化学机械耦合中观察到的异构体依赖性差异的基础。我们将使用系列晶体学方法来获得有关构象和折叠动力学的补充信息。基于我们对合适的药理学肌球蛋白分子伴侣的表征，我们可以在这些实验中防止不可逆变性并遵循展开和重折叠过程。我们希望我们的研究结果将提供前所未有的丰富的结构和动态数据。他们的整合与分子模拟有望产生可测试的模型，这将导致识别的变构触发位置在不同的蛋白质亚型，有助于肌动球蛋白依赖的力在血管内皮细胞的生产，以及它们如何有助于化学机械耦合和力的产生在不同组合的背景下的亚型。

The vascular endothelium is a systemically distributed, highly dynamic, and tightly regulated organ system that plays a central role in the pathogenesis of a wide range of diseases. Contractile actin complexes mediate barrier function, mechano-transduction, directed transcellular transport processes in endothelial cells and are essential for cell migration during angiogenesis. Despite their indispensable contribution to the integrity and function of the vascular endothelium, our knowledge about the isoform-composition and functional properties of the actomyosin-based structures involved is limited. Preliminary experiments using reconstituted actin-tropomyosin-myosin complexes show how the role of cytoskeletal tropomyosin isoforms extends beyond the role of a gatekeeper. Changes in motor activity are not only observed upon exchange of myosin isoforms, but the same myosin shows pronounced variations in strain-sensitivity, processivity, and velocity upon exchange of the associated tropomyosin isoform. We will identify the major contractile complexes present in vascular endothelial cells, to elucidate their function-defining structural features, to deduce first principles that allow an accurate modelling of the impact of allosteric trigger events on their chemo-mechanical properties, thermal stability, protein folding stability and dynamics. We will exploit our leading role in the production of cytoskeletal proteins, reconstitution of contractile complexes, and their functional and structural characterisation, to map the allosteric communication pathways and determine the basis for the observed isoform-dependent differences in chemo-mechanical coupling. We will use serial crystallography approaches to obtain complementary information about conformational and folding dynamics. Based on our characterisation of suitable pharmacological myosin chaperones, we can prevent irreversible denaturation and follow unfolding and refolding processes in these experiments. We expect that our results will provide an unprecedented wealth of structural and dynamic data. Their integration with molecular simulations promises to produce testable models that will lead to the identification of allosteric trigger positions in the different protein isoforms that contribute to actomyosin-dependent force production in vascular endothelial cells and how they contribute to chemo-mechanical coupling and force generation in the context of different combinations of isoforms.