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有关。在这里，我们重点关注稳定心肌横(t)小管并将其连接到附近的肌浆/内质网（SR/ER）的细胞骨架元件。最近的研究结果表明，t小管与SR/ER之间的界面至少由两种不同的蛋白质阵列组成。一种是由经典的兴奋-收缩耦合介质，t小管中的电压门控钙通道和SR/ER中的钙释放通道（ryanodine受体）组成。第二种是由t小管和SR/ER的其他完整膜蛋白组成，通过谱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与SR/ER膜不密切相关的部位结合并稳定t管膜。我们建议测试t小管的细胞骨架由两个不同的谱蛋白网络组成的假设，一个帮助组织和稳定t小管膜，另一个将它们连接到由磷酸化调节的复合物中的SR/ER。我们将结合分子、细胞生物学、超微结构、蛋白质组学和生理学的方法来解决这一假设。我们有五个具体目标：(1)表征alphaII-cardi+，这是一种在心脏中选择性表达的选择性剪接形式的谱蛋白，以及它形成的复合物；(2)表征其他谱蛋白及其在心脏t小管膜上形成的复合物；(3)鉴定连接t小管与SR/ER膜的谱蛋白和锚蛋白，以及与它们相互作用的蛋白质；(4)研究磷酸化对t小管处谱蛋白复合物的影响；(5)评估改变t小管谱蛋白网络对心肌细胞形态和生理的影响。这些目标都得到了我们的初步结果的支持，这些结果表明t小管及其与SR/ER的相互作用是由独特的谱蛋白复合物协调的，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为心脏如何正常运作以及t小管细胞骨架结构的变化如何导致心肌病提供独特的见解。

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.