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。尽管 驱动蛋白的化学和物理特性在体外已得到充分研究，但对其具体特性知之甚少。 细胞中驱动蛋白马达的功能和调节。 KIF3A/KIF3B/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，对于鞭毛内运输 (IFT) 也至关重要， 真核纤毛内的特殊运输。纤毛是质膜的突起， 由称为轴丝的特殊微管结构支撑。初级纤毛是孤立的且不动的 纤毛以组织特异性方式感知各种刺激。例如，他们可以感觉到 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。 鉴于这些基本的感觉功能，纤毛功能障碍是许多疾病的根源也就不足为奇了 统称为纤毛病。 在 IFT 过程中，称为 IFT 序列的大型蛋白质组件在纤毛内持续运输。这 IFT 列车在睫状体基部装载特定货物，随后招募驱动蛋白 2 电机 沿着轴丝微管运输至纤毛尖端。在那里，kinesin-2 马达被释放， 卸载特定货物，并对火车进行改造，以便随后运回睫状基部 动力蛋白-2。众所周知，kinesin-2 马达的任何亚基的丢失都会导致完全的 纤毛缺失，干扰 IFT 会导致已形成的纤毛消失。从 通过单细胞鞭毛衣藻实验，我们知道微管蛋白通过 IFT 流入纤毛是 作为纤毛长度的函数进行调节。基于这一发现，最近有几个模型旨在解释 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 专注。然而，这些模型中微管蛋白浓度的变化并不能解释所有的实验结果。 研究结果表明，除微管蛋白输入外，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对于纤毛结构的重要性以及驱动蛋白2马达对IFT的调节 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来研究如何 kinesin-2 受 IFT 调节，并描述了 kinesin-2 驱动的 IFT 对哺乳动物结构的影响 纤毛。我们方法的核心是工程驱动蛋白，其活性可以精确调节 研究者在外部的时间和空间上。本提案中阐述的工作将阐明该功能 和哺乳动物纤毛驱动蛋白马达的调节，从而促进旨在治疗的开发 减轻或治愈与运动蛋白相关的人类疾病。