Function and regulation of kinesin motors in cells

细胞中驱动蛋白马达的功能和调节

• 批准号: 10501529
• 负责人: Martin F. Engelke
• 金额: $ 21.39万
• 依托单位: ILLINOIS STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-09-01 至 2027-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10501529
• 关键词: Back; Biochemical; Cell membrane; Cell physiology; Cells; Cellular Assay; Chlamydomonas; Cilia; Development; Disease; Dynein ATPase; Engineering; Genome engineering; In Vitro; Intracellular Transport; Kinesin; Length; Light; Maintenance; Mastigophora; Microscopy; Microtubules; Mitotic spindle; Modeling; Motor; Nasal cavity; Organelles; Physiology; Process; Protein Engineering; Proteins; Regulation; Renal tubule structure; Research Personnel; Resolution; Sensory; Stimulus; Structure; System; Time; Tissues; Training; Tubulin; Urine; Work; base; chemical property; ciliopathy; experimental study; human disease; morphogens; novel; physical property; recruit; therapy development

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.

基于微管的驱动蛋白和动力蛋白电动机驱动了许多细胞过程，包括 细胞内货物的细胞内转运，有丝分裂纺锤体的组装和功能以及睫状功能。尽管 在体外对驱动蛋白的化学和物理特性进行了充分的研究，对特定的知之甚少 细胞中驱动蛋白电动机的功能和调节。 KIF3A/KIF3B/KAP电机，随后称为 驱动蛋白2，驱动各种嘉戈斯的细胞内转运，对于flagellar运输（IFT），也是必不可少的 真核纤毛内的专门运输。纤毛是质膜的突出 由称为Axoneme的专门微管结构支持。原发性纤毛是孤独的 纤毛在组织特异性的方式中感觉到各种刺激。例如，他们可以感觉到 发育过程中的形态剂，鼻腔中的气味剂或肾小管中的尿液流量。 鉴于这些基本的感觉功能，睫毛故障构成了许多疾病，这并不奇怪 统称为纤毛病。 在IFT期间，称为IFT火车的大蛋白质组件在纤毛内连续运输。这 IFT火车在睫状底座装载了特定的货物，随后招募了驱动蛋白-2电动机 沿着轴突微管沿纤毛尖端运输。在那里，释放了运动蛋白-2电动机， 特定的货物被卸载，火车被重塑，以便随后通过 Dynein-2。众所周知，驱动蛋白-2电动机的任何亚基的损失导致完整 缺乏纤毛，干扰IFT会导致已经建立的纤毛的消失。从 对单细胞鞭毛衣原体的实验，我们知道小管蛋白通过IFT涌入Cilia是 根据纤毛长度调制。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非常重要地属于睫状微管蛋白 专注。但是，这些模型中微管蛋白浓度的变化无法解释所有实验 调查结果，除小管蛋白进口外，IFT的其他方面可能对睫状长度和 结构。因此，IFT对睫状结构的重要性和驱动蛋白-2电动机对IFT的调节是 只有不完全理解，尤其是在哺乳动物系统中。在此提案中，我们将使用一个组合 生化和细胞测定，蛋白质和基因组工程以及高分辨率显微镜的研究 驱动蛋白-2用于IFT，并描述驱动IFT对哺乳动物结构的影响 纤毛。我们方法的中心是工程设计的动力蛋白蛋白，其活性可以受到精确调节 在调查员的外部时间和空间上。该提案中规定的工作将阐明该功能 和调节哺乳动物纤毛的动力素电动机，从而促进针对疗法的发展 减轻或治愈与运动蛋白相关的人类疾病时。