Jun Sangmi, Zhao Gongpu, Ning Jiying, Gibson Gregory A, Watkins Simon C, Zhang Peijun
Department of Structural Biology, University of Pittsburgh School of Medicine, USA.
J Vis Exp. 2013 Jun 24(76):50386. doi: 10.3791/50386.
Cryo-electron tomography (cryoET) allows 3D visualization of cellular structures at molecular resolution in a close-to-physiological state(1). However, direct visualization of individual viral complexes in their host cellular environment with cryoET is challenging(2), due to the infrequent and dynamic nature of viral entry, particularly in the case of HIV-1. While time-lapse live-cell imaging has yielded a great deal of information about many aspects of the life cycle of HIV-1(3-7), the resolution afforded by live-cell microscopy is limited (~200 nm). Our work was aimed at developing a correlation method that permits direct visualization of early events of HIV-1 infection by combining live-cell fluorescent light microscopy, cryo-fluorescent microscopy, and cryoET. In this manner, live-cell and cryo-fluorescent signals can be used to accurately guide the sampling in cryoET. Furthermore, structural information obtained from cryoET can be complemented with the dynamic functional data gained through live-cell imaging of fluorescent labeled target. In this video article, we provide detailed methods and protocols for structural investigation of HIV-1 and host-cell interactions using 3D correlative high-speed live-cell imaging and high-resolution cryoET structural analysis. HeLa cells infected with HIV-1 particles were characterized first by confocal live-cell microscopy, and the region containing the same viral particle was then analyzed by cryo-electron tomography for 3D structural details. The correlation between two sets of imaging data, optical imaging and electron imaging, was achieved using a home-built cryo-fluorescence light microscopy stage. The approach detailed here will be valuable, not only for study of virus-host cell interactions, but also for broader applications in cell biology, such as cell signaling, membrane receptor trafficking, and many other dynamic cellular processes.
冷冻电子断层扫描（cryoET）能够在接近生理状态下以分子分辨率对细胞结构进行三维可视化（1）。然而，利用cryoET在宿主细胞环境中直接可视化单个病毒复合物具有挑战性（2），这是由于病毒进入的频率低且具有动态性，尤其是在HIV-1的情况下。虽然延时活细胞成像已经产生了大量关于HIV-1生命周期许多方面的信息（3-7），但活细胞显微镜提供的分辨率有限（约200纳米）。我们的工作旨在开发一种关联方法，通过结合活细胞荧光显微镜、冷冻荧光显微镜和cryoET来直接可视化HIV-1感染的早期事件。通过这种方式，活细胞和冷冻荧光信号可用于在cryoET中准确指导采样。此外，从cryoET获得的结构信息可以通过对荧光标记靶标的活细胞成像获得的动态功能数据进行补充。在这篇视频文章中，我们提供了使用三维相关高速活细胞成像和高分辨率cryoET结构分析对HIV-1与宿主细胞相互作用进行结构研究的详细方法和方案。首先通过共聚焦活细胞显微镜对感染HIV-1颗粒的HeLa细胞进行表征，然后通过冷冻电子断层扫描分析包含相同病毒颗粒的区域以获取三维结构细节。使用自制的冷冻荧光显微镜载物台实现了光学成像和电子成像这两组成像数据之间的关联。这里详细介绍的方法不仅对于研究病毒-宿主细胞相互作用有价值，而且对于细胞生物学中的更广泛应用，如细胞信号传导、膜受体运输以及许多其他动态细胞过程也有价值。
