Structure and Function of Flagellar Central Pair Microtubule-Associated Complexes

纤毛和鞭毛是细胞的细长圆柱形表面延伸，其有节奏地摆动或跳动，从而使细胞移动通过其液体环境，例如在能动的原生动物或精子细胞的情况下，或者使液体环境移动穿过细胞的表面，例如在呼吸道中的纤毛上皮细胞的情况下。 纤毛或鞭毛内的分子机制统称为轴丝，主要由高度有序的特殊结构元件（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对于彼此纵向滑动。 该奖项的支持下，将进行的工作是针对了解的机制，调节微管为基础的运动动力蛋白在真核纤毛和鞭毛的运动。该方法的重点是突变的模式生物，光合原生生物莱茵衣藻，改变动力蛋白的活性，通过破坏组装的监管复合物，中央对装置。基于许多以前的研究，从两个中央对微管的预测与径向辐条，这反过来又传递调节信号的双峰微管相关的动力蛋白相互作用。一个级联的蛋白激酶已经牵连在这个信号传导过程中，但中心对结构的作用尚未确定。虽然大多数影响中心对结构的突变完全阻断运动性，但最近描述了允许改变运动性的突变。这种突变，cpc1，特别是防止组装一排中央对微管的预测。在该奖项的支持下进行的实验将为cpc1鞭毛缺失的结构和功能提供新的信息。将测量分离的、重新激活的cpc1鞭毛轴丝的运动性和蛋白酶处理的轴丝的微管滑动速率，以表征拍频、波形和滑动速度的变化。将使用cpc1轴丝、野生型轴丝和来自其他中心对缺陷株的轴丝比较特异性蛋白激酶和蛋白磷酸酶抑制剂对这些参数的影响。保留在cpc1和另一个中心对缺陷突变体pf6的轴丝中的结构将通过电子显微镜表征。将来自固定切片材料的图像与来自快速冷冻蚀刻材料的图像进行比较，以构建中心对结构的三维模型。这一信息将澄清可能的相互作用中心对预测和径向辐条在鞭毛运动。分子方法将用于克隆CPC 1基因，从可用的插入标记的等位基因开始。相应cDNA的序列将用于预测基因产物的一级和二级结构，并鉴定与已知功能蛋白的同源性。这项工作的结果将提供新的见解鞭毛运动是如何调节。

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.