Function and regulation of kinesin motors in cells

基于微管的驱动蛋白和动力蛋白马达驱动大量的细胞过程，包括 细胞货物的细胞内运输、有丝分裂纺锤体的组装和功能以及纤毛功能。而 驱动蛋白的化学和物理性质在体外研究得很好，但对驱动蛋白的特异性 细胞中驱动蛋白马达的功能和调节。KIF 3A/KIF 3B/KAP电机，以下简称为 驱动蛋白-2，驱动各种货物的细胞内运输，也是鞭毛内运输（IFT）所必需的， 真核细胞纤毛内的一种特殊运输工具。纤毛是质膜的突起， 由一种称为轴丝的特殊微管结构支撑。初级纤毛是孤立的和不动的 以组织特异性方式感知各种刺激的纤毛。例如，它们可以感知到 发育过程中的形态发生素、鼻腔中的气味或肾小管中尿流的强度。 考虑到这些基本的感觉功能，许多疾病的基础是睫状体功能障碍， 被统称为纤毛病 在IFT期间，称为IFT列车的大蛋白质组件在纤毛内连续运输。的 IFT列车在纤毛基部装载有特定的货物，随后招募驱动蛋白-2马达， 沿着轴丝微管运输到纤毛顶端。在那里，驱动蛋白-2马达被释放， 卸载特定货物，并对列车进行改造，以便随后运输回睫状体基地， 动力蛋白-2。已经确定，驱动蛋白-2马达的任何亚基的缺失导致完全的细胞凋亡。 纤毛的缺失，IFT的干扰导致已经建立的纤毛的消失。从 用单细胞鞭毛虫衣原体进行的实验中，我们知道微管蛋白通过IFT流入纤毛， 作为纤毛长度的函数被调节。基于这一发现，最近的几个模型旨在解释 IFT对纤毛长度和纤毛维持的影响归因于纤毛微管蛋白的高度重要性 浓度.然而，在这些模型中微管蛋白浓度的变化不能解释所有的实验结果。 研究结果表明，除了微管蛋白输入外，IFT的其他方面对纤毛长度和 结构因此，IFT对于睫状结构的重要性和驱动蛋白-2马达对于IFT的调节是显而易见的。 只是不完全了解，特别是在哺乳动物系统中。在本提案中，我们将使用 生物化学和细胞分析，蛋白质和基因组工程，以及高分辨率显微镜，以研究如何 驱动蛋白-2在IFT中受到调控，并描述驱动蛋白-2驱动的IFT对哺乳动物组织结构的影响。 纤毛。我们方法的核心是工程化的驱动蛋白，其活性可以精确调节 在时间和空间上都是由调查员进行的本提案中列出的工作将阐明 和调节哺乳动物纤毛中的驱动蛋白马达，从而促进针对 减轻或治愈与运动蛋白相关的人类疾病。

Microtubule-based kinesin and dynein motors drive a plethora of cellular processes, including intracellular transport of cellular cargo, assembly and function of the mitotic spindle, and ciliary function. While the chemical and physical properties of kinesins are well studied in vitro, much less is known about the specific function and regulation of kinesin motors in cells. The KIF3A/KIF3B/KAP motor, subsequently referred to as kinesin-2, drives intracellular transport of various cargos and is also essential for intraflagellar transport (IFT), a specialized transport inside eukaryotic cilia. Cilia are protrusions of the plasma membrane that are supported by a specialized microtubule structure called the axoneme. Primary cilia are solitary and immotile cilia that sense various stimuli in a tissue-specific manner. They can, for instance, sense the presence of morphogens during development, odorants in the nasal cavity, or the strength of urine flow in kidney tubules. Given these essential sensory functions, it is not surprising that ciliary malfunction underlies many diseases that are collectively classified as ciliopathies. During IFT, large protein assemblies called IFT trains are continuously transported within cilia. The IFT trains are loaded with specific cargo at the ciliary base and subsequently recruit kinesin-2 motors for transport along the axonemal microtubules to the tip of the cilium. There, the kinesin-2 motors are released, specific cargo is unloaded, and the trains are remodeled for subsequent transport back to the ciliary base by dynein-2. It is well established that the loss of any subunit of the kinesin-2 motor leads to the complete absence of cilia, and interference with IFT leads to the disappearance of already established cilia. From experiments with the single-celled flagellate Chlamydomonas we know that tubulin influx into cilia via IFT is modulated as a function of cilium length. Based on this finding several recent models aimed at explaining the impact of IFT on cilium length and cilium maintenance attribute high importance to the ciliary tubulin concentration. However, the change in tubulin concentration in these models cannot explain all experimental findings and it is likely that other aspects of IFT in addition to tubulin import are important for ciliary length and structure. Thus, the importance of IFT for the ciliary structure and the regulation of kinesin-2 motor for IFT is only incompletely understood, especially in mammalian systems. In this proposal, we will use a combination of biochemical & cellular assays, protein & genome engineering, and high-resolution microscopy to study how kinesin-2 is regulated for IFT and to delineate the impact of kinesin-2 driven IFT on the structure of mammalian cilia. At the center of our approach are engineered kinesin proteins whose activity can be precisely regulated in time and space externally by the investigator. The work laid out in this proposal will shed light on the function and regulation of kinesin motors in mammalian cilia and thereby promote the development of therapies aimed at alleviating or curing motor protein-associated human diseases.