Laboratory of Experimental Biophysics, Ecole Polytechnique Fédérale de Lausanne, Lausanne, Switzerland.
Nano Lett. 2012 Sep 12;12(9):4705-10. doi: 10.1021/nl3021076. Epub 2012 Aug 27.
The HIV structural protein Gag assembles to form spherical particles of radius ∼70 nm. During the assembly process, the number of Gag proteins increases over several orders of magnitude from a few at nucleation to thousands at completion. The challenge in studying protein assembly lies in the fact that current methods such as standard fluorescence or electron microscopy techniques cannot access all stages of the assembly process in a cellular context. Here, we demonstrate an approach using super-resolution fluorescence imaging that permits quantitative morphological and molecular counting analysis over a wide range of protein cluster sizes. We applied this technique to the analysis of hundreds of HIV-Gag clusters at the cellular plasma membrane, thus elucidating how different fluorescent labels can change the assembly of virions.
HIV 结构蛋白 Gag 组装形成半径约为 70nm 的球形颗粒。在组装过程中，Gag 蛋白的数量从最初的几个增加到数千个，增加了几个数量级。研究蛋白质组装的挑战在于，目前的方法，如标准荧光或电子显微镜技术，无法在细胞环境中获得组装过程的所有阶段。在这里，我们展示了一种使用超分辨率荧光成像的方法，该方法允许对广泛的蛋白质簇大小进行定量形态和分子计数分析。我们将该技术应用于数百个 HIV-Gag 簇在细胞膜上的分析，从而阐明了不同荧光标记如何改变病毒体的组装。
