Structure and Function of Flagellar Central Pair Microtubule-Associated Complexes

纤毛和鞭毛是细长的圆柱形细胞表面延伸，它们有节奏地摆动或跳动，从而使细胞在液体环境中移动，例如在活动的原生动物或精子细胞中，或者使液体环境穿过细胞表面，例如在呼吸道的纤毛上皮中。纤毛或鞭毛内部的分子机制统称为轴突，主要由高度有序排列的专门结构元件（微管）和分子马达（动力蛋白）组成，微管以高度调节和有序的方式相互纵向滑动。这项工作将在该奖项的支持下进行，旨在了解真核生物纤毛和鞭毛运动过程中基于微管的运动动力蛋白的调节机制。该方法关注的是一种模式生物——光合原生生物莱茵衣藻（Chlamydomonas reinhardtii）的突变，这种突变通过破坏调节复合体（中央对装置）的组装来改变动力蛋白的活性。基于先前的许多研究，两个中心对微管的突起与径向辐条相互作用，径向辐条反过来将调节信号传递给双态微管相关的动力蛋白。蛋白激酶级联参与了这一信号传导过程，但中心对结构的作用尚未确定。虽然大多数影响中央对结构的突变完全阻断了运动，但最近描述的一种突变允许改变运动。这种突变，cpc1，特异性地阻止了一排中央对微管突起的组装。将在该奖项的支持下进行的实验将提供有关cpc1鞭毛中缺失的结构和功能的新信息。分离的，重新激活的cpc1鞭毛轴突的运动和蛋白酶处理轴突的微管滑动速率将被测量，以表征跳动频率，波形和滑动速度的变化。特异性蛋白激酶和蛋白磷酸酶抑制剂对这些参数的影响将通过cpc1轴突组、野生型轴突组和其他中心对缺陷菌株轴突组进行比较。cpc1和另一个中心对缺陷突变体pf6轴突体中保留的结构将通过电子显微镜进行表征。将固定、切片材料的图像与快速冻结、蚀刻材料的图像进行比较，建立中心对结构的三维模型。这一信息将阐明在鞭毛运动过程中，中心副突起和径向辐条之间可能的相互作用。分子方法将用于克隆CPC1基因，从可用的插入标记等位基因开始。相应的cDNA序列将用于预测基因产物的一级和二级结构，并与已知功能的蛋白质进行同源性鉴定。这项工作的结果将为鞭毛运动如何被调节提供新的见解。

Cilia and flagella are slender cylindrical surface extensions of cells that wave or beat rhythmically, thereby either moving the cell through its liquid environment , as for example in the case of motile protozoa or sperm cells, or moving the liquid environment across the surface of the cell, as for example in the case of ciliated epithelia in the respiratory tract. The molecular machinery inside the cilia or flagella is collectively called the axoneme, and consists mainly of a highly ordered arrangement of specialized structural elements (microtubules) and molecular motors (dyneins) that the microtubules to slide lengthwise relative to each other in a highly regulated and orderly fashion. The work that will be performed with support from this award is directed toward understanding the mechanisms that regulate the microtubule-based motor dynein during motility of eukaryotic cilia and flagella. The approach focuses on mutations in a model organism, the photosynthetic protist Chlamydomonas reinhardtii, that alter dynein activity by disrupting assembly of a regulatory complex, the central pair apparatus. Based on many previous studies, projections from the two central pair microtubules interact with radial spokes, which in turn transmit regulatory signals to doublet microtubule-associated dyneins. A cascade of protein kinases has been implicated in this signaling process, but the role of central pair structures has not been determined. Although most mutations affecting central pair structure completely block motility, a mutation was recently described that allows altered motility. This mutation, cpc1, specifically prevents assembly of one row of central pair microtubule projections. The experiments that will be performed with support from this award will provide new information about the structures and functions that are missing in cpc1 flagella.The motility of isolated, reactivated cpc1 flagellar axonemes and the microtubule sliding rate of protease treated axonemes will be measured to characterize changes in beat frequency, waveform, and sliding velocity. The effects of specific protein kinase and protein phosphatase inhibitors on these parameters will be compared using cpc1 axonemes, wild type axonemes and axonemes from other central pair defective strains. Structures retained in axonemes from cpc1 and another central pair defective mutant, pf6, will be characterized by electron microscopy. Images from fixed, sectioned material will be compared with images from rapidly frozen, etched material to build three-dimensional models of central pair structure. This information will clarify possible interactions between central pair projections and radial spokes during flagellar motility. Molecular approaches will be used to clone the CPC1 gene, starting from available insertionally tagged alleles. Sequence of the corresponding cDNA will be used to predict the primary and secondary structure of the gene product and identify homology with proteins of known function. The results from this work will provide new insight into how flagellar motility is regulated.