Biomechanics of the axonemal nanomachine

描述（由申请人提供）：9+2轴体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，一个至关重要的装置是径向辐条（RS）复合体，它被假定为一个机械化学传感器，控制微管的电机驱动滑动。有趣的是，与camp依赖性蛋白激酶A （PKA）与A激酶锚定蛋白（AKAP）对接的结构域相似的分子基序存在于RS的四个组成成分中。然而，它们似乎与钙感知、机械转导和核苷酸代谢相关的关键机制有关，但与PKA无关。与这些RSPs类似的分子参与了不同生物体中不同的细胞反应，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多功能性远远超出了目前的认识。本研究旨在验证一种假设，即先前被证明是RS基础并被预测为辐条型AKAP的单一蛋白质实际上作为一种结构支架来锚定四种不同的非pka调节片段，这些片段分别赋予整个RS复合物刚性，以便在振荡搏动期间进行精确和重复的机械转导，或者通过钙和核苷酸衍生物的信号传导来调节RS以改变搏动。测试这一假设的策略是在鞭毛模型系统衣藻中突变这种支架蛋白的假定结合位点。体内实验是检验四种相似对接基序相互作用的必要条件。突变体中有缺陷的RS复合体可以通过运动、蛋白质生物化学和电子显微镜明确地定义。实验所需的试剂已从先前的RS系统蛋白质组学项目中生成。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。这些精心设计的、以假设为导向的项目将为研究生和本科生提供优秀的智力和实验训练。这些结果将1)揭示轴突纳米机器的控制机制，这是该领域长期存在的核心问题；2)为设计治疗不动纤毛相关疾病的新策略提供坚实的基础；3)揭示了化学信号与机械转导整合的新机制，这一机制正在迅速得到重视；4)证明了多种细胞反应中相似对接基序的差异识别。

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.