Probing Cellular Membrane Processes by Single Particle Orientation and Rotational Tracking

 描述（由申请人提供）：病毒、药物递送载体和其他外部颗粒在细胞膜上和细胞膜中表现出多种复杂行为，这些行为反映了它们在触发内化途径进入细胞之前的物理和化学性质，包括大小、形状、电荷和膜受体的可用性。了解这些细胞膜过程的动力学对于许多重要的人类健康相关问题至关重要，例如基于纳米颗粒的药物递送系统的合理设计以及传染性病原体的预防和控制。过去的努力提供了很好的细胞膜过程的可视化，但主要是翻译动力学。该建议的重点是利用最近开发的单粒子取向和旋转跟踪（SPORT）技术来阐明特征活细胞旋转动力学。SPORT同时提供高空间，角度和时间分辨率，用于在微分干涉对比（DIC）显微镜中观察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，拟议的研究将获得有关细胞膜过程的详细旋转动力学的新的基础知识，例如功能化纳米颗粒的粘附，运输和内吞作用，可能与药物输送和病毒进入有关。细胞的旋转模式 用于功能化金纳米棒的膜将被识别并与它们的横向运动和用荧光蛋白标记的相关功能生物分子的存在相关联。不同内化途径下货物的特征旋转运动 也将直接可视化，从而为理解不同途径中蛋白质模块的时序，信号传导以及化学和机械功能带来新的机会。将开发计算机模拟来了解纳米颗粒形状、尺寸和表面改性剂的影响。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，SPORT将用于研究癌细胞中适配体负载的金纳米星的摄取机制。拟议的研究可能会通过证明在单分子和纳米颗粒水平上旋转动力学的重要性，引发当前膜结构和功能研究范式的转变。对基本运动的透彻理解将有助于了解膜蛋白扩散和平行内化途径的分子机制的细节，这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.