Function and regulation of kinesin motors in cells

基于微管的动蛋白和动力蛋白马达驱动过多的细胞过程，包括 细胞内货物的运输，有丝分裂纺锤体的组装和功能，以及纤毛功能。而当 在体外对动蛋白的化学和物理性质进行了很好的研究，但对其特异性的了解更少。 细胞内动蛋白马达的功能和调节。KIF3A/KIF3B/KAP电机，以下简称 Kinesin-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， 真核生物纤毛内的一种特殊的运输。纤毛是质膜的突起， 由一种称为轴丝的特殊微管结构支撑。初生纤毛为单生和静止的。 纤毛以组织特有的方式感知各种刺激。例如，它们可以感觉到 发育过程中的形态原、鼻腔内的气味或肾小管内尿流的强度。 考虑到这些基本的感觉功能，睫状体功能障碍是许多疾病的基础也就不足为奇了。 被统一归类为纤毛病。 在IFT过程中，被称为IFT序列的大型蛋白质组件在纤毛内持续运输。这个 IFT列车在睫状基底部装载特定的货物，随后招募Kinesin-2发动机 沿轴丝微管向纤毛尖端运输。在那里，Kinesin-2马达被释放， 特定的货物被卸下来，列车被改装，以便随后通过以下方式运输回纤毛基地 动力蛋白-2。众所周知，Kinesin-2马达的任何亚单位的丢失都会导致完整的 纤毛的缺失和对IFT的干扰导致已经建立的纤毛的消失。从… 用单细胞鞭毛衣藻的实验我们知道微管蛋白通过IFT进入纤毛 作为纤毛长度的函数进行调制。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纤毛微管的重要性 集中精神。然而，这些模型中微管蛋白浓度的变化不能解释所有的实验。 除了微管蛋白输入，IFT的其他方面可能对纤毛长度和纤毛长度也很重要 结构。因此，IFT对纤毛结构和驱动蛋白-2马达的调节在IFT中的重要性是 只是不完全了解，特别是在哺乳动物系统中。在这个提案中，我们将使用组合 生化和细胞分析，蛋白质和基因组工程，以及高分辨率显微镜，以研究如何 Kinesin-2对IFT的调节以及Kinesin-2对哺乳动物IFT结构的影响 纤毛。我们方法的中心是可以精确调控其活性的工程动蛋白。 在时间和空间上由调查人员进行外部分析。这项提案中列出的工作将阐明该功能 和调节哺乳动物纤毛中的运动蛋白马达，从而促进旨在治疗的发展 在缓解或治愈与运动蛋白相关的人类疾病方面。

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.