Dynamic and super-resolution imaging of endogenous proteins in Drosophila tissues

描述(申请人提供)：在过去的15年里，荧光蛋白质标签的使用彻底改变了细胞生物学研究的方方面面，使分析活细胞内的动态结构成为可能。绿色荧光蛋白(GFP)的克隆和其他具有广泛光谱性质的荧光蛋白的开发使这场革命成为可能。最新版本的荧光蛋白是可光激活的(PA)，允许细胞中的蛋白质具有更高的动态和空间分辨率。标记蛋白质的动态行为可以通过分子的局部激活和观察蛋白质在活组织中随时间的移动来检验。PA-蛋白质还使超分辨率荧光显微镜的新方法成为可能，这种方法可以产生分辨率低于100 nm的复合图像，打破了光学显微镜的衍射极限。这项建议的目标是显着扩大PA蛋白在完整组织中动态细胞过程成像和超分辨率荧光显微镜的实验应用。为了实现这一目标，我们将在果蝇的基因组背景下将PA标签工程到它们的基因中，以便分析在天然转录控制下内源水平表达的蛋白质。我们将使用这些PA标记的蛋白质来开发完整的卵巢卵室的超分辨率成像，这为超分辨率显微镜提供了一组美丽的分级挑战-在逐渐厚的卵室中发现的逐渐增大的生殖系环管，以及在卵室周围的上皮细胞中发现的小得多的环管。我们的具体目标是：1)通过在新获得的果蝇基因组DNA的BAC克隆中进行重组工程，获得在内源水平表达PA标记蛋白的果蝇种群；2)利用全安装果蝇卵室的双平面荧光光活化定位显微镜(BP FPALM)开发用于环管分析的超分辨率成像；3)使用PA标记的环管蛋白的双光子激活和共聚焦显微镜来表征活的环管中的蛋白质动力学。该项目将极大地扩展新生的超分辨率技术的应用，从而影响整个细胞生物学领域。对果蝇、斑马鱼、小鼠和线虫等实验系统进行细胞生物学研究，这些系统拥有广泛的遗传工具和强大的基因组计划，将发现该项目产生的工具特别有用。 公共卫生相关性：观察细胞内部工作的能力对于理解正常条件下和疾病过程中的功能至关重要。最近几年出现了超分辨率显微镜的新方法，极大地改进了细胞生物学的分析。该项目将大大扩展超分辨率显微镜的使用，以研究完整动物组织中的各种蛋白质，这将使对正常和疾病组织中的细胞生物学进行更详细的分析成为可能。

DESCRIPTION (provided by applicant): The use of fluorescent protein tags has revolutionized all aspects of cell biology research during the last 15 years, enabling analysis of the dynamic structures within living cells. The cloning of Green Fluorescent Protein (GFP) and the subsequent development of additional fluorescent proteins with a wide range of spectral properties made this revolution possible. The newest versions of fluorescent proteins are photo-activatable (PA), allowing higher dynamic and spatial resolution of proteins in cells. The dynamic behavior of tagged proteins can be examined by local activation of molecules, and observation of protein movement over time in live tissue. PA-proteins have also enabled new methods of super-resolution fluorescence microscopy that yield composite images with sub-100 nm resolution, breaking the diffraction limit of light microscopy. The goal of this proposal is to significantly expand the experimental applications of PA proteins for imaging dynamic cellular processes and super-resolution fluorescence microscopy in intact tissues. To achieve this goal, we will engineer PA tags into Drosophila genes in their genomic context to allow analysis of proteins expressed at their endogenous levels under native transcriptional control. We will use these PA-tagged proteins to develop super-resolution imaging of intact ovarian egg chambers that provide a beautifully graded set of challenges for super-resolution microscopy - germline ring canals of gradually increasing size found within gradually thicker egg chambers, and much smaller ring canals within epithelial cells surrounding egg chambers. Our specific aims are: 1) produce Drosophila stocks expressing PA-tagged proteins at endogenous levels using recombineering in newly available BAC clones of Drosophila genomic DNA; 2) develop super-resolution imaging for analysis of ring canals using biplane fluorescence photoactivation localization microscopy (BP FPALM) of whole-mount Drosophila egg chambers; 3) characterize protein dynamics in living ring canals using 2-photon activation and confocal microscopy of PA-tagged ring canal proteins. This project will impact the entire cell biology community by dramatically expanding the applications of nascent super-resolution technology. Investigations of cell biology in experimental systems such as Drosophila, zebrafish, mouse and C. elegans that have extensive genetic tools and robust genome projects will find the tools generated by this project especially useful. PUBLIC HEALTH RELEVANCE: The ability to observe the inner workings of cells is crucial for understanding function in both normal conditions and disease processes. New methods for super-resolution microscopy have emerged in the last few years that dramatically improve analysis of cell biology. This project will significantly expand the use of super-resolution microscopy to the study of a wide range of proteins in intact animal tissues, which will enable much more detailed analysis of the cell biology in normal and diseased tissue.