Building a molecular machine: analysis of co-chaperones for assembly of ciliary dynein motor complexes

你身体的几乎每个细胞都有一个细长的毛发状突起，叫做纤毛。某些类型的纤毛能够弯曲或击打，并参与液体运动。例如，在我们呼吸道内用于粘液运动的细胞，以及用于将新卵子飘向子宫的输卵管上，都可以发现这种“活动纤毛”。此外，精子细胞通过跳动的鞭毛游泳，鞭毛本质上是一种长长的可移动的纤毛。所有这些纤毛都通过它们内部的“马达蛋白质”银行的作用而弯曲。这些马达蛋白形成了巨大的分子机器--其中一些是自然界已知的最大的分子机器。也许并不令人惊讶的是，组装这些马达蛋白质机器是极其复杂的，需要其他专门的蛋白质充当分子伴侣，以确保它们在细胞构建纤毛的过程中被正确构建。这项建议涉及到伴侣蛋白的鉴定和分析它们在运动组装中的作用。运动组装的重要性通过出现问题时发生的情况来说明：原发性睫状肌运动障碍(PCD)是一种人类遗传性疾病，由于这些运动蛋白的失效，纤毛不能运动。其结果是，患者出现了与不孕不育和清除粘液困难有关的症状，例如导致频繁和破坏性的胸部感染。严重者还会出现内翻--即内部器官定位紊乱(例如，心脏不再位于胸部左侧)。许多不同基因的突变会导致PCD。一些突变存在于编码分子伴侣蛋白的基因中。这些伴侣蛋白是如何发挥作用的尚不清楚。此外，伴侣蛋白并不是人类独有的--运动组装的整个途径非常古老，从原生动物(例如游泳的草履虫)到更高的生物体中都有这种蛋白的存在。为了进一步了解马达蛋白是如何组装的，我们的策略是研究果蝇--黑腹果蝇。果蝇很容易饲养和研究。复杂的遗传和细胞方法可以用在果蝇身上，以发现运动组装所需的基因。我们将研究扰乱这些基因功能的影响。这很容易实现，因为在果蝇中，只有感觉和精子才需要运动纤毛，所以运动纤毛有缺陷的果蝇很容易通过明显的感觉缺陷和雄性不育而被发现。此外，遗传和显微工具的最新进展意味着，我们可以在整个有机体的背景下，开发出比迄今在动物中可能的更复杂的方法来探测运动组装途径。仅靠基因发现和分析是不够的。同样重要的是，纤毛的分子结构在昆虫和高等动物之间是完全保守的。因此，在果蝇中发现的伴侣机制可能对动物和人类也很重要。对于纤毛生物学的研究，在可能的情况下，使用果蝇比使用更复杂的生物更具成本效益和伦理上的可接受性。

Almost every cell of your body has a thin, hair-like outgrowth called a cilium. Some types of cilia are capable of bending or beating and are involved in fluid movement. Such 'motile cilia' are found for example on cells lining our airways for mucus movement as well as the fallopian tubes for wafting a new egg towards the uterus. Moreover, sperm cells swim by means of a beating flagellum, which is essentially a long motile cilium. All these cilia bend through the action of banks of 'motor proteins' within them. These motor proteins form huge molecular machines - some of the largest known in nature. Perhaps not surprisingly, assembling these motor protein machines is extremely complex and requires other dedicated proteins that act as 'molecular chaperones' to ensure they are built correctly during the construction of the cilium by the cell. This proposal concerns the identification of the chaperone proteins and analysing how they function in motor assembly.The importance of motor assembly is illustrated by what happens when it goes wrong: primary ciliary dyskinesia (PCD) is a human inherited disease in which cilia are immotile due to failure of these motor proteins. The result of this is the patient has symptoms related to infertility and difficulties in clearing mucus, leading for instance to frequent and damaging chest infections. Severe cases also have situs inversus - in which internal organ positioning is disrupted (e.g. the heart is no longer on the left side of the chest). Mutations in many different genes cause PCD. Some mutations are in genes that code for the molecular chaperone proteins. How these chaperone proteins work is not clear. Moreover, the chaperone proteins are not unique to humans - the entire pathway of motor assembly is very ancient, and is found in organisms from protozoa (e.g. the swimming Paramecium) upwards. To further our knowledge of how motor proteins assemble, our strategy is to look in the fruit fly, Drosophila melanogaster. The fruit fly is easy to rear and to study. Sophisticated genetic and cellular approaches can be used in Drosophila to discover genes that are required for motor assembly. We shall examine the effect of disrupting the function of these genes. This is quite straightforward to achieve because in Drosophila, motile cilia are required only for senses and sperm, and so flies with defective motile cilia are easy to spot through obvious sensory deficits and male infertility. Moreover, recent advances in genetic and microscopy tools mean that we can develop much more sophisticated ways of probing the motor assembly pathway, within the context of a whole organism, than has been hitherto possible in animals.Ease of gene discovery and analysis is not sufficient. Just as important is the fact that the molecular machinery of the cilium is completely conserved between insects and 'higher' animals. Therefore, the chaperone mechanisms discovered in Drosophila are likely to be important in animals and humans too. For studies into cilium biology it is cost-effective and ethically more acceptable to use Drosophila than more complex organisms where possible.