Probing Cellular Membrane Processes by Single Particle Orientation and Rotational Tracking

 描述(申请人提供)：病毒、药物输送载体和其他外部颗粒在细胞膜上和细胞膜内表现出各种复杂的行为，这些行为反映了它们的物理和化学性质，包括大小、形状、电荷和膜受体的可用性，然后它们触发内化途径进入细胞。了解这些细胞膜过程的动力学对于许多与人类健康相关的重要问题是至关重要的，例如基于纳米颗粒的药物传递系统的合理设计和传染病病原体的预防和控制。过去的努力为细胞膜过程提供了很好的可视化，但主要是为了翻译动力学。这项建议的重点是利用最近发展的单粒子定向和旋转跟踪(SPORT)技术来阐明活细胞旋转动力学的特征。SPORT同时提供了高的空间、角度和时间分辨率，用于在微分干涉衬度(DIC)显微镜下可视化活细胞中各向异性等离子体金纳米棒的旋转动力学。通过使用SPORT，拟议的研究将获得关于细胞膜过程的详细旋转动力学的新的基础知识，如功能纳米颗粒的黏附、运输和内吞作用，这些可能与药物输送和病毒进入有关。细胞上的旋转图案 功能化金纳米棒的膜将被识别，并与它们的侧向运动和相关功能生物分子的存在相关联，这些生物分子标记有荧光蛋白。货物在不同内化路径中的旋转特征 也将直接可视化，为理解不同途径中涉及的蛋白质模块的计时、信号以及化学和机械功能提供了新的机会。将开发计算机模拟来了解纳米颗粒形状、大小和表面修饰剂的影响。这些模拟将通过为进一步的信息实验提供建议来帮助该项目。最后，将利用SPORT来研究适体负载金纳米星在癌细胞中的摄取机制。这项拟议的研究可能通过证明旋转动力学在单分子和纳米颗粒水平上的重要性，启动当前膜结构和功能研究范式的转变。彻底了解证据中的基本运动将有助于了解膜蛋白扩散所涉及的分子机制和平行的内化途径的细节，这些途径对于更好地设计抗病毒药物以及开发靶向递送载体和抗癌药物至关重要。

 DESCRIPTION (provided by applicant): Viruses, drug delivery vectors, and other external particles exhibit a variety of complex behaviors on and in the cell membrane that are reflective of their physical and chemical properties, including size, shape, charge and the availability of membrane receptors, before they trigger internalization pathways to enter the cell. Understanding the dynamics of these cellular membrane processes is essential for many important human health related problems, such as the rational design of nanoparticle-based drug delivery systems and the prevention and control of infectious pathogens. The past efforts provided excellent visualization of cellular membrane processes, but primarily for translational dynamics. This proposal focuses on utilizing the recently-developed single particle orientation and rotational tracking (SPORT) technique to elucidate the characteristic live-cell rotational dynamics. SPORT affords high spatial, angular, and temporal resolutions simultaneously, for visualizing the rotational dynamics of anisotropic plasmonic gold nanorods in live cells in differential interference contrast (DIC) microscopy. By using SPORT, the proposed research will acquire new fundamental knowledge about the detailed rotational dynamics of cellular membrane processes, such as adhesion, transport, and endocytosis of functionalized nanoparticles, as may be relevant to drug delivery and viral entry. The rotational patterns on cell membranes for functionalized gold nanorods will be identified and correlated with their lateral movements and the presence of relevant functional biomolecules tagged with fluorescent proteins. The characteristic rotational motions of cargos during different internalization pathways will also be visualized directly, leading to new opportunities for understanding the timing, signaling and chemical and mechanical functions of protein modules involved in different pathways. Computer simulations will be developed to understand the effects of nanoparticle shapes, sizes and surface modifiers. The simulations will aid the project by providing suggestions for further informative experiments. Finally, SPORT will be utilized to study the uptake mechanism of aptamer-loaded gold nanostars in cancer cells. The proposed research may initiate a shift in the current research paradigm on membrane structure and function by demonstrating the importance of rotational dynamics at the single molecule and nanoparticle level. A thorough understanding of the fundamental motions in evidence will inform about the details of the molecular mechanisms involved in the diffusion of membrane proteins and parallel internalization pathways that will be critical for the better design of antiviral drugs, as well asthe development of targeted delivery vehicles and anti-cancer medicines.