Single-molecule analysis of kinesin motors in live cells

描述(申请人提供)：我们的长期目标是了解分子马达在拥挤的细胞环境中驱动沿细胞骨架细丝定向交通的机制。具体地说，我们的目标是了解运动蛋白马达，其生物物理特性已在体外得到很好的研究，在体内如何驱动细胞器和囊泡沿着微管轨迹运输。要做到这一点，需要新的技术来对细胞质中单个分子的分布、活动和相互作用进行纳米级跟踪。通过结合物理、化学、生物和数学科学的技术，我们开发了基于荧光蛋白遗传标记和全内反射荧光(TIRF)显微镜的新方法，首次提供了对活细胞细胞质中单个Kinesin-1马达的分析。使用这个系统，活细胞中的单分子成像(SMILE)，我们已经证明了单个截短的Kinesin-1马达的运动性不会受到拥挤的细胞环境的阻碍，也不会被未知的细胞因素上调(生物物理杂志，在出版中)。了解单个马达在体内是如何工作的，是回答细胞问题的关键的第一步，例如多个马达如何组装在一起，并整合它们的活动，以驱动沿着拥挤的微管的膜运输事件。在目标1中，我们将继续开发用于纳米级成像的SMILE方法，并使用这些方法测试其他系统(例如其他动蛋白、神经细胞)和各种条件(例如不同的温度和共表达的辅助蛋白)。在目标2中，我们将开发双色微笑方法来表征细胞拥挤对运动蛋白运动的影响。在目标3中，我们将使用两种颜色的方法来研究多个马达的相互作用和活动，以及它们如何协同驱动活细胞中单个货物的运动。这一应用的结果将为控制基于运动蛋白的运输的生物物理参数提供新的见解。在这一应用中提出的方法和途径将适用于广泛的生物系统(转录、翻译、突触传递、膜运输等)。它们是由数量惊人的极少分子的作用所驱动的。此外，这些结果将有助于设计工程和诊断设备，并开发神经退行性疾病、癌症和病毒感染的治疗方法。 项目简介：蛋白质、细胞器和其他细胞组件的运动是由分子马达驱动的。运动驱动的运输缺陷已被证明与神经退行性疾病、癌症和多囊肾病有关。了解分子马达在细胞中的功能将为针对这些疾病的治疗提供重要的新靶点。

DESCRIPTION (provided by applicant): Our long-term goal is to understand the mechanisms by which molecular motors drive directional traffic along cytoskeletal filaments in the crowded cellular milieu. Specifically, we aim to understand how kinesin motors, whose biophysical characteristics have been well-studied in vitro, drive the transport of organelles and vesicles along microtubule tracks in vivo. To do this requires new techniques for nanoscale tracking of the distribution, activity and interactions of individual molecules in the cytoplasm. By combining techniques from the physical, chemical, biological and mathematical sciences, we have developed new methodologies, based on genetic labeling with fluorescent proteins and total internal reflection fluorescence (TIRF) microscopy, that provide the first analysis of single Kinesin-1 motors in the cytoplasm of live cells. Using this system, single molecule imaging in live-cells (SMILe), we have shown that the motility of single truncated Kinesin-1 motors is not hindered by the crowded cellular environment nor upregulated by unknown cellular factors (Biophysical Journal, in press). Understanding how single motors work in vivo is an essential first step to answer cellular questions such as how multiple motors assemble together and integrate their activities to drive membrane trafficking events along crowded microtubules. In Aim 1, we will continue to develop SMILe methodologies for imaging at the nanoscale and use these methods to test other systems (e.g. other kinesins, neuronal cells) and various conditions (e.g. different temperatures and co-expressed accessory proteins). In Aim 2, we will develop two-color SMILe methodologies to characterize the influence of cellular crowding on kinesin motors. In Aim 3, we will use the two-color methodologies to examine the interactions and activities of multiple motors and how they cooperate to drive motility of individual cargoes in live cells. The results of this application will give new insights into the biophysical parameters that control motor protein-based transport. The methods and approaches advanced in this application will be applicable to a wide variety of biological systems (transcription, translation, synaptic transmission, membrane trafficking, etc.) that are driven by the action of a surprisingly low number of molecules. In addition, these results will aid in the design of engineering and diagnostic devices and the development of treatments for neurodegenerative diseases, cancer and viral infection. Project Narrative: The movement of proteins, organelles, and other cellular components is driven by molecular motors. Defects in motor-driven transport have been shown to be associated with neurodegenerative diseases, cancer, and polycystic kidney disease. Understanding how molecular motors function in cells will provide important new targets for therapies aimed at these diseases.