Using formins to build distinct structures in cardiomyocytes

项目总结/摘要 每个细胞都有一个独特的肌动蛋白为基础的结构，发挥特定的作用。这些结构有助于 细胞保持完整性和极性，它们驱动胞吞和胞质分裂，在运动和肌肉的情况下， 细胞，它们是收缩机制的主要组成部分。肌动蛋白成核剂，刺激形成的蛋白质 是肌动蛋白结构的基本决定因素。了解设计原则， 区分成核剂，如formin，提供了对它们如何构建不同结构的深入了解。Formins in the 形成蛋白同源结构域（Fhod）-家族在发育、免疫、肌肉维持、 和其他过程。哺乳动物有两种Fhod家族的形式，Fhod 1和Fhod 3。FHOD 3在肌肉中富集 细胞，包括心肌细胞，其中它对肌节发育和维持至关重要。FHOD 1是 主要在非肌肉细胞中表达，但也在肌肉细胞中表达。在心肌细胞中，Fhod 1是 与非肌节结构相关，包括肋肌和闰盘，这些结构对于 细胞完整性和机械传导。因此，心肌细胞是研究心肌细胞的理想细胞类型 两种密切相关的形式蛋白如何构建不同的结构。此外，Fhod 1和Fhod 3两者都涉及人类免疫缺陷。 心肌病Fhod 3的多态性被认为占肥厚型心肌病的1-2% 例在扩张型心肌病中，检测到Fhod 1表达增加和Fhod 3水平降低， 这与两种形式素发挥不同作用的观点一致。早期的研究得出结论，Fhod家族形成蛋白 不是肌动蛋白成核剂，不像迄今为止研究的所有其他形式。提出的模型中， 封盖和/或细丝成束是重要的FHOD活动。然而，我们最近证明， 事实上，果蝇Fhod和人类Fhod 1是有效的成核剂。有趣的是，它们允许有倒刺的末端 延长，但不加速生长速度，与大多数formin相反。进行性伸长是一个标志 被认为是一种机制，通过这种机制，formin构建的肌动蛋白丝比由 其他核。Fhod 1和Fhod 3都与小的肌动蛋白密集结构有关，其中一些结构 含有特定长度的肌动蛋白丝，如应力纤维和肌节。我们认为成核剂 用来制造更短的肌动蛋白丝的设计是构建这些结构所必需的。我们还发现Fhod 1 区别肌动蛋白亚型，成核非肌肉肌动蛋白，但不是肌肉肌动蛋白。因此，我们有一个新的 从这个角度来研究这一类重要的formins。我们将检验那些假设 成核、弱延伸和同种型特异性都是Fhod 1和Fhod 3构建所必需的活性 肌肉细胞中独特的肌动蛋白结构。为此，我们将联合收割机结合生化分析和功能分析， 在干细胞衍生的人类心肌细胞中进行测试。这些细胞是一个强大的模型， 肌肉发育、功能和疾病。因此，这项工作可以帮助我们了解如何Fhod的功能和 与心肌病有关

Project Summary/Abstract Every cell has a complement of distinct actin-based structures that play specific roles. These structures help cells maintain integrity and polarity, they drive endocytosis and cytokinesis, and in the cases of motile and muscle cells, they are a major component of the contractile machinery. Actin nucleators, proteins that stimulate formation of new filaments, are essential determinants of actin architecture. Understanding the design principles that distinguish nucleators, such as formins, provides insight into how they build distinct structures. Formins in the Formin HOmology Domain (Fhod)-family play important roles in development, immunity, muscle maintenance, and other processes. Mammals have two Fhod-family formins, Fhod1 and Fhod3. Fhod3 is enriched in muscle cells, including cardiomyocytes, where it is critical for sarcomere development and maintenance. Fhod1 is predominantly expressed in non-muscle cells but is also expressed in muscle cells. In cardiomyocytes, Fhod1 is associated with non-sarcomeric structures, including costameres and intercalated discs, structures important for cellular integrity and mechanotransduction. Therefore, the cardiomyocyte is an ideal cell type in which to study how two closely related formins build distinct structures. Further, both Fhod1 and Fhod3 are implicated in human cardiomyopathies. Polymorphisms in Fhod3 are thought to account for 1-2% of hypertrophic cardiomyopathy cases. In dilated cardiomyopathies, increased expression of Fhod1 and decreased levels of Fhod3 are detected, consistent with the idea that the two formins play distinct roles. Early studies concluded that Fhod-family formins are not actin nucleators, unlike all other formins studied to date. Models were proposed in which barbed end capping and/or filament bundling are important Fhod activities. However, we recently demonstrated that Drosophila Fhod and human Fhod1 are, in fact, potent nucleators. Interestingly, they permit barbed end elongation but do not accelerate the growth rate, in contrast to most formins. Processive elongation is a hallmark of formins and is considered a mechanism by which formins build longer actin filaments than can be made by other nucleators. Both Fhod1 and Fhod3 are associated with small, actin-dense structures, some of which contain actin filaments of specific length, such as stress fibers and sarcomeres. We propose that nucleators designed to create shorter actin filaments are necessary to build these structures. We also discovered that Fhod1 discriminates between actin isoforms, nucleating non-muscle actin but not muscle actin. Thus we have a new perspective from which to study this important class of formins. We will test the hypotheses that strong nucleation, weak elongation, and isoform specificity are all activities necessary for Fhod1 and Fhod3 to build distinct actin-based structures in muscle cells. To do so, we will combine biochemical analysis with functional tests in human cardiomyocytes derived from stem cells. These cells are a powerful model in which to study muscle development, function, and disease. Thus this work can help us understand how Fhods function and are associated with cardiomyopathies.