Single-molecule analysis of kinesin motors in live cells

描述（由申请人提供）：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。具体来说，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充分研究）如何在体内沿着微管轨道驱动细胞器和囊泡的运输。要做到这一点，需要新的纳米级技术来跟踪细胞质中单个分子的分布、活性和相互作用。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，首次对活细胞细胞质中的单个Kinesin-1马达进行了分析。使用该系统，活细胞中的单分子成像（SMILe），我们已经表明，单个截断的Kinesin-1马达的运动不会受到拥挤的细胞环境的阻碍，也不会受到未知细胞因素的上调（生物物理杂志，已出版）。了解单个马达如何在体内工作是回答细胞问题的重要的第一步，例如多个马达如何组装在一起并整合它们的活动来驱动拥挤的微管中的膜运输事件。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（例如其他动力蛋白，神经元细胞）和各种条件（例如不同温度和共表达的辅助蛋白）。在目标2中，我们将开发双色SMILe方法来表征细胞拥挤对运动蛋白马达的影响。在目标3中，我们将使用双色方法来检查多个马达的相互作用和活动，以及它们如何合作来驱动活细胞中单个货物的运动性。这项应用的结果将为控制运动蛋白转运的生物物理参数提供新的见解。在这个应用中先进的方法和方法将适用于各种各样的生物系统（转录、翻译、突触传递、膜运输等），这些系统是由数量少得惊人的分子驱动的。此外，这些结果将有助于工程和诊断设备的设计，以及神经退行性疾病、癌症和病毒感染的治疗方法的发展。

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.