Molecular Analysis of Flagellar Dynein Function

动力蛋白分子马达是许多基本的运动细胞过程所必需的。在 纤毛/鞭毛，动力蛋白形成为这些细胞器提供动力的内外臂;缺陷 在这些酶和它们的调节系统中， 人类的多种表型，如不孕症、支气管扩张症和内脏逆位。 动力蛋白马达是高度复杂的大分子组装体， 它们在轴丝中的组装以及它们如何产生 响应于各种信号的特定波形非常不清楚。在本申请中， 我们将解决两个关键问题，动力蛋白力学，调节和 组装件.外臂重链（HC）似乎是一个关键的调控节点 其是Ca 2+、氧化还原和机械感觉信号的最终目标。第一个目标 将集中在Lis 1，这是已知的调节细胞质动力蛋白运动活性。Lis 1是 也存在于纤毛/鞭毛和鞭毛内水平的Lis 1被调节， 鞭毛搏动参数的改变。例如，Lis 1级别非常高， 当鞭毛被置于高粘性负荷下时， 鞭毛固有的跳动频率因此，细胞具有以前未识别的 一种监测运动性的机制，可以控制Lis 1进入或保留在细胞内。 鞭毛和修改功率输出。在衣原体中，大多数Lis 1位于 基底体区域，存在于几个不同的细胞质复合体中。这些将 进行纯化，以确定其组成和遗传和生物化学方法， 测试它们在控制负载敏感响应中的作用。第二个目标，将解决 外臂动力蛋白是如何在轴丝内的特定位点组装的。虽然 对接复合体和ODA 5是外臂动力蛋白组装所必需的。 鞭毛，现在很清楚，它们不足以作为两个新的因子（CCDC 103和 FBB 18）也是必要的。CCDC 103与轴丝紧密相关， 微管，并具有非凡的生物物理特性，这表明它可能自我- 组装可能充当“分子统治者”，以帮助基本的96纳米图案 轴丝重复含有这些蛋白质的细胞质和鞭毛复合体将被 分析以确定它们如何与动力蛋白和其他轴丝相互作用 件.这将使重要的，以前未被怀疑的作用，这些 蛋白质在动力蛋白组装过程中的作用有待确定。

Dynein molecular motors are required for many essential motile cellular processes. In cilia/flagella, dyneins form the inner and outer arms that power these organelles; defects in these enzymes and their regulatory systems lead to a broad array of severe and diverse phenotypes in humans such as infertility, bronchiectasis and situs inversus. Dynein motors are highly complex macromolecular assemblies and the mechanisms by which they are assembled within the axoneme and how they function to generate specific waveforms in response to various signals are very unclear. In this application, we will address two key questions concerning dynein mechanics, regulation and assembly. The outer arm ¿ heavy chain (HC) appears to act as a key regulatory node which is the ultimate target of Ca2+, redox and mechanosensory signals. The first aim will focus on Lis1 which is known to regulate cytoplasmic dynein motor activity. Lis1 is also present in cilia/flagella and intra-flagella levels of Lis1 are modulated in response to imposed alterations in flagella beat parameters. For example, Lis1 levels are highly elevated when flagella are placed under high viscous load which leads to an increase in the intrinsic flagella beat frequency. Thus, the cell has a previously unrecognized mechanism for monitoring motility that can control Lis1 entry into or retention within the flagellum and modify power output. In Chlamydomonas, most Lis1 is located in the basal body region and is present in several distinct cytoplasmic complexes. These will be purified to define their composition and genetic and biochemical approaches used to test their role in controlling the load-sensitive response. The second aim, will address how outer arm dynein is assembled at specific sites within the axoneme. Although the docking complex and ODA5 are required for outer arm dynein assembly within the flagellum, it is now clear that they are not sufficient as two novel factors (CCDC103 and FBB18) are also necessary. CCDC103 is very tightly associated with the axonemal microtubules and has extraordinary biophysical properties that suggest it may self- assemble potentially acting as a "molecular ruler" to help pattern the fundamental 96-nm axonemal repeat. Cytoplasmic and flagella complexes containing these proteins will be analyzed to determine how they interact with both dynein and other axonemal components. This will allow the essential, and previously unsuspected, role these proteins play in the dynein assembly process to be defined.