Deciphering the molecular rules of evolutionary diversification of Intraflagellar Transport

纤毛(又名鞭毛)是从大多数真核细胞表面伸出的普遍存在的细胞器，包括人体内的细胞类型。几乎所有纤毛的结构和功能都需要非膜结合的鞭毛内转运(IFT)。IFT是由动蛋白-2和动力蛋白-2马达提供动力的所谓IFT列车对睫状结构积木的连续运输。IFT的一个特点是睫状体底部和顶端两个相反方向的马达的位置特异性和互斥的激活和失活。越来越清楚的是，IFT列车的分级组装执行了IFT过程中严格有序的事件。在纤毛底部，从IFT-B综合体开始组装大型多兆道尔顿IFT列车。这个IFT-B复合体构成了IFT-A复合体和动力蛋白-2马达的骨架。在组装的最后一步，激动素-2马达被招募来将列车移动到睫状尖。虽然IFT-B和IFT-A复合体在体内与动蛋白-2和动力蛋白-2的活性有关，但它们在调节IFT马达方面的具体作用在分子水平上仍不清楚。还很明显的是，各IFT-B和IFT-A复合体的组成因物种而异，具有高度的多样性。后者引发了这样的问题：各种生物如何部署各自的IFT亚单位，以协调体内IFT过程的有序步骤。在这里，我们调查和对比嗜热嗜热嗜铬杆菌和拟态嗜铬杆菌的IFT。尽管这些生物是同一个进化超级群体的成员，但它们部署了一套令人惊讶的不同的IFT亚基和Kinesin-2发动机，但最终建立了基本上相同的细胞超微结构，即可移动的纤毛。假单胞菌模型特别令人感兴趣，因为它消除了大多数IFT亚基，但保留了仅使用IFT-B复合体中的亚基子集来协调IFT过程的能力。这个“最小”的IFT模型可能会告诉我们功能灵活性的局限性，这些功能灵活性可以编码到一组特定的蛋白质中，以创造复杂的生物功能。从我们的初步数据中已经可以看出，来自‘最小’假丝虫和‘经典’嗜热链霉菌模型的kinesin-2同源基因表现出非常不同的自我调节和动力学特性。然而，所有的Kinesin-2马达都应该遵守IFT高度有序的步骤规则。描述这些关键步骤的分子要求将揭示将马达与特定的IFT亚基联系起来的规则，这些规则最终分别在“最小的”假丝虫和“典型的”嗜热链霉菌模型中导致IFT过程中事件的严格年代表。这一提议将使我们更接近于根据IFT亚基的集合和它所部署的Kinesin-2的类型(S)在给定的生物体中建立有序的IFT过程的规则。

Cilia (aka flagella) are ubiquitous organelles that project from the surface of most eukaryotic cells, including the cell types in the human body. Virtually all cilia require the non-membrane bound IntraFlagellar Transport (IFT) for their construction and function. IFT is a continuous transport of ciliary building blocks by the so-called IFT trains that are powered by the kinesin-2 and dynein-2 motors. A hallmark of IFT is the site-specific and mutually exclusive activation and deactivation of the two oppositely directed motors at the ciliary base and tip. It is becoming increasingly clear that a hierarchical assembly of the IFT trains enforces the strictly ordered events of the IFT process. At the ciliary base, large multi mega-Dalton IFT trains are assembled starting with the IFT-B complex. This IFT-B complex constitutes the backbone that scaffolds the IFT-A complex and the dynein-2 motor. In the last step of the assembly, the kinesin-2 motor is recruited to move the trains towards the ciliary tip. Whereas IFT-B and IFT-A complexes have been linked to the kinesin-2 and dynein-2 activities in vivo, their specific contributions to the regulation of IFT motors remain largely unknown at the molecular level. It also became clear that the composition of the respective IFT-B and IFT-A complexes are species-specific and highly diverse. The latter provokes the question of how the various organisms deploy the respective IFT subunits to orchestrate the orderly steps of the IFT process in vivo. Here, we investigate and contrast IFT of the Chromalveolates T. thermophila and T. pseudonana. Despite being members of the same evolutionary super group, these organisms deploy an astonishingly different set of IFT subunits and kinesin-2 motors, yet end up building essentially the same cellular ultrastructure, a motile cilium. T. pseudonana model is of particular interest as it has eliminated most IFT subunits but retained the ability to orchestrate the IFT process using only a subset of subunits from the IFT-B complex. This ‘minimal’ IFT model will likely teach us the limits of functional flexibility that can be coded into a specific set of proteins to create complex biological function. As already evident from our preliminary data, the kinesin-2 orthologs from the ‘minimal’ T. pseudonana and ‘canonical’ T. thermophila models display vastly different self-regulatory and kinetic properties. Yet all kinesin-2 motors are expected to obey the rules of the highly orderly steps of IFT. Delineating the molecular requirements of these key steps will expose the rules of linking the motors to specific IFT subunits that ultimately give rise to the strict chronology of events during the IFT process in the ‘minimal’ T. pseudonana and ‘canonical’ T. thermophila models, respectively. This proposal will bring us closer to derive the rules of establishing an orderly IFT process in a given organism based on the set of IFT subunits and the type(s) of kinesin-2 that it deploys.