Development of green fluorescent protein technology

超分辨率技术，如光激活定位显微镜（PALM），使荧光蛋白嵌合体的成像，以揭示在纳米级的分子密度高到足以提供结构背景的遗传表达的蛋白质的组织。在PALM中，进行光活化荧光蛋白分子的许多稀疏子集的连续光活化和随后的漂白。然后，通过对单个分子的点扩散函数进行统计拟合来确定其荧光发射中心，从而以接近分子的分辨率定位单个分子。然后，来自所有子集的聚集位置信息被组装成超分辨率图像，其中单个荧光分子以高分子密度（高达10，000个分子/平方微米）被分离。我们以前已经证明了PALM成像的细胞内结构（包括溶酶体，高尔基体和线粒体）在冷冻制备的薄切片，以及在固定的细胞与TIRF激发，和相关的PALM/透射电子显微镜的线粒体标记蛋白质的黏着斑蛋白和肌动蛋白的成像。 我们已经开发了一种双标记PALM检测系统，使用两种不同的细胞内表达的光活化分子。此外，我们已经开发了一个系统，用于在活细胞中使用PALM进行单粒子跟踪，该系统允许在单分子水平上表征蛋白质扩散和固定。这项技术被称为单粒子跟踪PALM（sptPALM），涉及激活、定位和漂白活细胞中许多光活化荧光蛋白嵌合体的子集。单分子运动的空间分辨图可以通过用这种技术对膜蛋白进行成像来获得，比传统的单粒子跟踪提供每个细胞多几个数量级的轨迹。通过探测不同的分子子集，包括Gag和VSVG，我们证明了sptPALM可以为探索膜中时空异质性的起源提供有力的手段。 我们实验室开发的另一种荧光蛋白技术允许在活细胞中确定蛋白质拓扑结构。被称为荧光蛋白酶保护（FPP），该测定提供了一个荧光读数响应胰蛋白酶诱导的破坏GFP连接到一个蛋白质的目的质膜透化之前和之后。 在进行FPP测定时，将荧光蛋白连接至感兴趣的蛋白质的N-或C-末端。随后，表达融合蛋白的细胞在通过毛地黄皂苷透化质膜之前或之后暴露于胰蛋白酶。如果表达蛋白上的荧光蛋白部分面对暴露于胰蛋白酶的环境（即细胞质），则其荧光信号将丢失。相反，如果表达的蛋白质上的荧光蛋白部分面对免受胰蛋白酶保护的环境（即，隔室的内腔），则其荧光持续存在。考虑到这些结果和蛋白质内已知的工程化位置的荧光蛋白，可以推断蛋白质在脂质双层上的取向。我们证明了FPP的广泛适用性，通过使用它来定义定位于几个不同的细胞器，包括ER，高尔基体，线粒体，过氧化物酶体和自噬体的蛋白质的拓扑结构。

Superresolution techniques such as photoactivated localization microscopy (PALM) enable the imaging of fluorescent protein chimeras to reveal the organization of genetically-expressed proteins on the nanoscale with a density of molecules high enough to provide structural context. In PALM, serial photoactivation and subsequent bleaching of numerous sparse subsets of photoactivated fluorescent protein molecules is performed. Individual molecules are then localized at near molecular resolution by determining their centers of fluorescent emission via a statistical fit of their point-spread-function. The aggregate position information from all subsets is then assembled into a super-resolution image, in which individual fluorescent molecules are isolated at high molecular densities (up to 10,000 molecules/micron squared). We have previously demonstrated PALM imaging of intracellular structures (including lysosome, Golgi apparatus and mitochondria) in cryo-prepared thin sections, as well as imaging of vinculin and actin in fixed cells with TIRF excitation, and correlative PALM/transmission electron microscopy of a mitochondrial marker protein. We have developed a dual-label PALM assay system using two different photactivatable molecules expressed within cells. In addition, we have developed a system for doing single particle tracking using PALM in living cells that allows protein diffusion and immobilization to be characterized at the single molecule level. Called single particle tracking PALM (sptPALM), the technique involves activating, localizing and bleaching many subsets of photoactivatated fluorescent protein chimeras in live cells. Spatially-resolved maps of single molecule motions can be obtained by imaging membrane proteins with this technique, providing several orders of magnitude more trajectories per cell than by traditional single particle tracking. By probing distinct subsets of molecules, including Gag and VSVG, we demonstrated that sptPALM can provide a powerful means for exploring the origin of spatial and temporal heterogeneities in membranes. Another fluorescent protein technique developed in our lab allows protein topology to be determined in living cells. Termed fluorescence protease protection (FPP), the assay provides a fluorescent readout in response to trypsin-induced destruction of GFP attached to a protein-of-interest before and after plasma membrane permeabilization. In performing the FPP assay, a fluorescent protein is attached to the N- or C-terminus of a protein of interest. Subsequently, cells expressing the fusion protein are exposed to trypsin either before or after plasma membrane permeabilization by digitonin. If the fluourescent protein moiety on the expressed protein faces the environment exposed to trypsin (that is the cytoplasm), then its fluorescent signal will be lost. Conversely, if the fluorescent protein moiety on the expressed protein faces the environment protected from trypsin (that is, the lumen of a compartment) then its fluorescence persists. Given these outcomes and the fluorescent proteins known engineered position within the protein, it is possible to deduce the orientation of the protein across the lipid bilayer. We demonstrated the broad applicability of FPP by using it to define the topology of proteins localized to several different organelles, including the ER, Golgi apparatus, mitochondria, peroxisomes and autophagosomes.