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，其有节奏地波动或跳动，从而使细胞在其液体环境中移动，例如在可移动的原生动物或精子细胞的情况下，或者在液体环境在细胞表面上移动，例如在呼吸道中的纤毛上皮的情况下。纤毛或鞭毛内部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，这些分子马达使微管以高度有序的方式相对于彼此纵向滑动。在该奖项的支持下，将开展的工作旨在了解真核生物纤毛和鞭毛运动过程中调节基于微管的运动动力蛋白的机制。该方法关注的是模式生物中的突变，即光合作用原生生物莱茵衣藻，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。基于许多以前的研究，来自两对中央微管的投射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重微管相关的动力蛋白。一系列的蛋白激酶参与了这一信号传递过程，但中央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性，但最近发现了一种允许改变运动性的突变。这种名为cpc1的突变特别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关于cpc1鞭毛缺失的结构和功能的新信息。将测量分离的、重新激活的cpc1鞭毛轴丝的运动性和经蛋白酶处理的轴丝的微管滑动率，以表征节拍频率、波形和滑动速度的变化。我们将使用cpc1轴丝、野生型轴丝和来自其他中心配对缺陷菌株的轴丝来比较特定的蛋白激酶和蛋白磷酸酶抑制剂对这些参数的影响。Cpc1和另一个中心对缺陷突变体pf6的轴丝中保留的结构将通过电子显微镜进行表征。来自固定的切片材料的图像将与快速冻结的蚀刻材料的图像进行比较，以建立中心对结构的三维模型。这一信息将阐明鞭毛运动过程中中央对投影和径向辐条之间可能的相互作用。将使用分子方法从可用的插入标记等位基因开始克隆CPC1基因。相应的基因序列将被用来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是如何调节的提供新的见解。

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.