Biomechanics of the axonemal nanomachine

描述（由申请人提供）：9+2轴丝是一种基于微管的非凡纳米机器，为运动纤毛和鞭毛的振荡跳动提供动力。严格控制的运动对于重要器官的正常功能至关重要。这种纳米机器的缺陷导致了常见的先天性疾病和严重的慢性呼吸道感染。在这个复杂的生物机器中，一个关键的装置是径向辐条（RS）复合体，它被假定为控制微管马达驱动滑动的机械化学换能器。有趣的是，类似于将cAMP依赖性蛋白激酶A（PKA）对接到A-激酶锚定蛋白（AKAP）的结构域的分子基序存在于RS的四个组成部分中。然而，它们似乎与钙敏感、机械转导和核苷酸代谢相关但独立于PKA的关键机制有关。类似于这些RSP的分子参与不同生物体中的不同细胞反应，但它们的作用在很大程度上仍然未知。因此，这些对接基序的普遍性和通用性远远超出了目前的认识。该提议试图检验以下假设，即先前显示为RS的基础并预测为辐条AKAP的单个蛋白质实际上充当结构支架以锚四种不同的非蛋白质。PKA调节部分，其分别赋予整个RS复合物刚性以在振荡搏动期间进行精确和重复的机械转导，或在钙和核苷酸衍生物的信号传导后调节RS来改变跳动测试这一假设的策略是在鞭毛模型系统衣原体中突变该支架蛋白中的假定结合位点。体内实验对于测试涉及四个相似对接基序的相互作用是必不可少的。突变体中的缺陷RS复合物可以通过运动性、蛋白质生物化学和电子显微镜明确地定义。实验所需的试剂已经从RS以前的系统蛋白质组学项目中产生。第一代突变菌株的初步数据有力地支持了这一假设，并证实了实验方法。精心设计的，假设驱动的项目将提供优秀的培训，智力和实验，为研究生和本科生。这些结果将1）揭示轴丝纳米机器的控制机制，这是该领域长期存在的中心问题; 2）为设计治疗与不动纤毛相关疾病的新策略提供坚实的基础; 3）揭示了一种新的机制，在整合化学信号与机械转导，这是获得迅速赞赏和4）证明了各种各样的细胞反应的类似对接基序的差异化表达。 公共卫生相关性：拟议的实验将阐明一种机制，曾经只锚定一个关键的蛋白激酶，是如何被采用来整合机械调节，化学信号和核苷酸代谢，以局部控制鞭毛跳动和许多其他对人类健康至关重要的细胞反应。

DESCRIPTION (provided by applicant): The 9+2 axoneme is a microtubule-based extraordinary nanomachine that powers the oscillatory beating of motile cilia and flagella. The tightly controlled locomotion is crucial for the normal function of vital organs. Defects in this nanomachine caused common congenital disorders and severe chronic respiratory tract infections. A crucial device in this intricate biological machine is the radial spoke (RS) complex that is postulated as a mechanochemical transducer controlling the motor-driven sliding of the microtubules. Intriguingly, the molecular motifs resembling the domain for docking cAMP-dependent protein kinase A (PKA) to A-kinase anchoring protein (AKAP) are present in four constitutive components in the RS. However, they appear to tether crucial mechanisms related to calcium sensing, mechanic transduction and nucleotide metabolism but independent to PKA. Molecules analogous to these RSPs are involved in distinct cellular reactions in diverse organisms and yet their roles remain largely unknown. Hence, the ubiquity and versatility of these docking motifs are far beyond recognized currently. This proposal seeks to test the hypothesis that a single protein previously shown to be the base of the RS and predicted to be a spoke AKAP actually serve as a structural scaffold to anchor the four different non-PKA regulatory moieties that respectively confer the rigidity to the entire RS complex for the precise and repetitive mechanic transduction during oscillatory beating or modulate the RS upon the signaling of calcium and nucleotide derivatives to alter the beating. The strategy for testing this hypothesis is to mutate the putative binding sites in this scaffold protein in the flagellar model systems, Chlamydomonas. The in vivo experiments are essential for testing the interactions involving the four similar docking motifs. The defective RS complex in the mutants can be unequivocally defined by motility, protein biochemistry and electron microscopy. The reagents necessary for the experiments have been generated from a previous systematic proteomic project of the RS. The preliminary data of first-generation mutant strains strongly support the hypothesis and confirmed the experimental approach. The carefully designed, hypothesis-driven projects will offer excellent training, intellectually and experimentally, for both graduate and undergraduate students. These results will 1) shed crucial insight on the control mechanism of the axonemal nanomachine, the long standing central question in the field; 2) provide a solid foundation for designing new strategies for treating diseases related to immotile cilia; 3) reveal a novel mechanism in integration of chemical signaling with mechanic transduction that is gaining appreciation rapidly and 4) demonstrate the differential recognitions of the similar docking motifs for a wide variety of cellular reactions. PUBLIC HEALTH RELEVANCE: The proposed experiment will elucidate how a mechanism, once known for only anchoring a crucial protein kinase, is adopted to integrate mechanic regulation, chemical signaling and nucleotide metabolism for local control of flagellar beating and like many other cellular reactions that are vital for human health.