Cytoskeletal Architecture of T-Tubules in Heart

描述（由申请人提供）：心脏细胞骨架结构缺陷与扩张型心肌病相关。在这里，我们专注于细胞骨架元素，稳定的横（t）管的心肌，并将它们连接到附近的肌质/内质网（SR/ER）。最近的研究结果表明，t-小管和SR/ER之间的界面是由至少两种不同的蛋白质阵列。一种是由兴奋-收缩偶联的经典介质，t-小管中的电压门控Ca通道和SR/ER中的Ca释放通道（ryanodine受体）组成。 第二种是由t-小管和SR/ER的其他整合膜蛋白组成，通过血影蛋白和锚蛋白的膜结合细胞骨架网络连接。Spectrin及其相关蛋白也可能在不与SR/ER膜紧密贴壁的位点结合并稳定t-管膜。我们建议测试的假设，t-小管的细胞骨架是由两个不同的血影蛋白网络，一个是帮助组织和稳定的t-管膜，另一个连接到SR/ER在一个复杂的磷酸化调节。我们将使用分子，细胞生物学，超微结构，蛋白质组学和生理学方法的组合来解决这个假设。我们有五个具体目标：（1）表征α II-cardi+，一种选择性表达于心脏的血影蛋白的选择性剪接形式及其形成的复合物;（2）表征其他血影蛋白及其在心脏t-小管膜上形成的复合物;（3）鉴定将t-小管连接至SR/ER膜的血影蛋白和锚蛋白，以及与它们相互作用的蛋白质;（4）研究磷酸化对t-小管处血影蛋白复合物的影响;（5）评估改变t-小管处血影蛋白网络对心肌细胞的形态学和生理学的影响。这些目标中的每一个都得到了我们的初步结果的支持，这表明t-小管及其与SR/ER的相互作用是由独特的血影蛋白复合物协调的，由局部信号级联调节。定义这些相互作用和控制它们的机制应该提供独特的见解，了解心脏如何正常工作，以及t-小管细胞骨架结构的变化如何导致心肌病。

DESCRIPTION (provided by applicant): Defects in the cytoskeletal architecture of the heart are associated with dilated cardiomyopathies. Here, we focus on cytoskeletal elements that stabilize the transverse (t) tubules of cardiac muscle and that link them to the nearby sarcoplasmic/endoplasmic reticulum (SR/ER). Recent results suggest that the interface between the t-tubules and the SR/ER is composed of at least two different protein arrays. One is composed of the classic mediators of excitation-contraction coupling, voltage-gated Ca channels in the t-tubule and Ca release channels (ryanodine receptors) in the SR/ER. The second is composed of other integral membrane proteins of the t-tubule and the SR/ER, linked by a membrane-bound cytoskeletal network of spectrin and ankyrin. Spectrin and its associated proteins are also likely to bind to and stabilize the t-tubular membrane at sites that are not closely apposed to the SR/ER membrane. We propose to test the hypothesis that the cytoskeleton of t-tubules is composed of two distinct spectrin networks, one that helps organize and stabilize the t-tubular membranes, and another that links them to the SR/ER in a complex that is regulated by phosphorylation. We will use a combination of molecular, cell biological, ultrastructural, proteomics and physiological approaches to address this hypothesis. We have five specific aims: (1) to characterize alphaII-cardi+, an alternatively spliced form of spectrin that is selectively expressed in the heart, and the complexes it forms; (2) to characterize other spectrins and the complexes they form at the cardiac t-tubule membrane; (3) to identify the spectrins and ankyrins that link the t-tubule to the SR/ER membrane, and the proteins with which they interact; (4) to study the effects of phosphorylation on the spectrin complexes at t-tubules; (5) to assess the effects of altering the spectrin network at t-tubules on the morphology and physiology of cardiocytes. Each of these aims is supported by our preliminary results, which suggest that t-tubules and their interactions with the SR/ER are coordinated by unique spectrin complexes, regulated by local signaling cascades. Defining these interactions and the mechanisms that control them should provide unique insights into how the heart functions normally, and how changes in cytoskeletal architecture at the t-tubules can result in cardiomyopathy.