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

果蝇组织内源蛋白的动态和超分辨率成像

• 批准号: 7937884
• 负责人: Lynn COOLEY
• 金额: $ 45.35万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-30 至 2012-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7937884
• 关键词: Adult; Animals; Area; Behavior; Cell physiology; Cells; Cellular biology; Cloning; Communication; Communities; Confocal Microscopy; Cultured Cells; Cytoplasm; DNA; Data; Development; Disease; Drosophila genus; Drosophila inturned protein; Engineering; Epithelial Cells; Fluorescence; Fluorescence Microscopy; Generations; Genes; Genetic; Genome; Genomics; Giant Cells; Goals; Green Fluorescent Proteins; Image; Image Analysis; Integrase; Intercellular Junctions; Investigation; Libraries; Life; Location; Methodology; Methods; Microscopy; Molecular; Monitor; Movement; Mus; Mutation; National Institute of General Medical Sciences; Nature; Ovarian; Photons; Process; Property; Protein Analysis; Protein Dynamics; Proteins; Refractive Indices; Research; Resolution; Set protein; Somatic Cell; Structure; System; Technology; Testing; Thick; Time; Tissues; Transcriptional Regulation; Work; Zebrafish; animal tissue; egg; fly; imaginal disc; improved; insight; light microscopy; mutant; photoactivation; protein degradation; research study; tissue/cell culture; tool

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.

描述（由申请人提供）：在过去 15 年中，荧光蛋白标签的使用彻底改变了细胞生物学研究的各个方面，使得能够分析活细胞内的动态结构。绿色荧光蛋白 (GFP) 的克隆以及随后开发的具有广泛光谱特性的其他荧光蛋白使这场革命成为可能。最新版本的荧光蛋白可光激活 (PA)，从而使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以及活体组织中蛋白质随时间的运动观察来检查。 PA 蛋白还实现了超分辨率荧光显微镜的新方法，可产生分辨率低于 100 nm 的复合图像，突破了光学显微镜的衍射极限。该提案的目标是显着扩展 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，我们将在果蝇基因组背景下将 PA 标签工程化到果蝇基因中，以便分析在天然转录控制下在内源水平表达的蛋白质。我们将使用这些 PA 标记蛋白来开发完整卵巢卵室的超分辨率成像，为超分辨率显微镜提供一系列精美分级的挑战 - 在逐渐变厚的卵室内发现尺寸逐渐增大的种系环管，以及在卵室周围的上皮细胞内发现更小的环管。我们的具体目标是：1) 在果蝇基因组 DNA 的新 BAC 克隆中使用重组工程，产生内源表达 PA 标记蛋白的果蝇种群； 2) 使用全安装果蝇卵室的双平面荧光光激活定位显微镜（BP FPALM）开发用于分析环管的超分辨率成像； 3) 使用 PA 标记的环管蛋白的 2 光子激活和共聚焦显微镜来表征活体环管中的蛋白质动力学。该项目将通过极大扩展新兴超分辨率技术的应用来影响整个细胞生物学界。在果蝇、斑马鱼、小鼠和线虫等实验系统中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项目，将发现该项目生成的工具特别有用。 公共卫生相关性：观察细胞内部运作的能力对于理解正常条件和疾病过程中的功能至关重要。过去几年出现了超分辨率显微镜的新方法，极大地改善了细胞生物学的分析。该项目将显着扩展超分辨率显微镜的使用范围，以研究完整动物组织中的多种蛋白质，这将使对正常和患病组织中的细胞生物学进行更详细的分析成为可能。