FRET imaging of protein-protein interactions inside living cells

活细胞内蛋白质-蛋白质相互作用的 FRET 成像

• 批准号: 7732133
• 负责人: Steven S Vogel
• 金额: $ 127.15万
• 依托单位: NATIONAL INSTITUTE ON ALCOHOL ABUSE AND ALCOHOLISM
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7732133
• 关键词: Address; Anisotropy; Austria; Behavior; Biological; Biological Assay; Biology; Biophotonics; Book Chapters; Calcium; Calcium Channel; Calcium Signaling; Canada; Cell division; Cells; Complex; Consult; DYSF gene; Denmark; Development; Down-Regulation; Dynamin; Embryo; Endocytosis; Energy Transfer; Environment; Enzymes; Equilibrium; Exocytosis; Fluorescence Anisotropy; Fluorescence Microscopy; Foundations; Funding; Germany; Gifts; Green Fluorescent Proteins; Homo; Image; Imaging Techniques; Injection of therapeutic agent; Intramural Research Training Award; Investigation; Label; Laboratories; Lasers; Learning; Life; Manuscripts; Measures; Mechanics; Mediating; Membrane; Memory; Methodological Studies; Methods; Microscope; Microscopy; Monitor; Muscular Dystrophies; Netherlands; Neurons; P2X-receptor; Peptides; Phase; Phosphoric Monoester Hydrolases; Phosphotransferases; Play; Protein Tyrosine Kinase; Protein Tyrosine Phosphatase; Proteins; Publishing; Reference Standards; Reporting; Research; Research Design; Role; Scanning; Sea Urchins; Singapore; Spain; Standards of Weights and Measures; Structure; Switzerland; Synapses; Technology; Temperature; Testing; Thinking; Time; Variant; Visit; Work; Wound Healing; base; beta barrel; calmodulin-dependent protein kinase II; fluorescence imaging; fluorophore; follow-up; inhibitor/antagonist; interest; protein protein interaction; protein structure; quantum; quantum computer; research study; src-Family Kinases; stoichiometry; synaptic function; theories; two-photon; voltage; Address; Anisotropy; Austria; Behavior; Biological; Biological Assay; Biology; Biophotonics; Book Chapters; Calcium; Calcium Channel; Calcium Signaling; Canada; Cell division; Cells; Complex; Consult; DYSF gene; Denmark; Development; Down-Regulation; Dynamin; Embryo; Endocytosis; Energy Transfer; Environment; Enzymes; Equilibrium; Exocytosis; Fluorescence Anisotropy; Fluorescence Microscopy; Foundations; Funding; Germany; Gifts; Green Fluorescent Proteins; Homo; Image; Imaging Techniques; Injection of therapeutic agent; Intramural Research Training Award; Investigation; Label; Laboratories; Lasers; Learning; Life; Manuscripts; Measures; Mechanics; Mediating; Membrane; Memory; Methodological Studies; Methods; Microscope; Microscopy; Monitor; Muscular Dystrophies; Netherlands; Neurons; P2X-receptor; Peptides; Phase; Phosphoric Monoester Hydrolases; Phosphotransferases; Play; Protein Tyrosine Kinase; Protein Tyrosine Phosphatase; Proteins; Publishing; Reference Standards; Reporting; Research; Research Design; Role; Scanning; Sea Urchins; Singapore; Spain; Standards of Weights and Measures; Structure; Switzerland; Synapses; Technology; Temperature; Testing; Thinking; Time; Variant; Visit; Work; Wound Healing; base; beta barrel; calmodulin-dependent protein kinase II; fluorescence imaging; fluorophore; follow-up; inhibitor/antagonist; interest; protein protein interaction; protein structure; quantum; quantum computer; research study; src-Family Kinases; stoichiometry; synaptic function; theories; two-photon; voltage

The principal aim of the Section on Cellular Biophotonics is to use imaging techniques, such as two-photon microscopy, spectral imaging, fluorescence lifetime microscopy, and fluorescence anisotropy analysis to study how protein complexes regulate synaptic function in living cells. Recently, we have concentrated our efforts on utilizing Forsters Resonance Energy Transfer (FRET) to monitor protein-protein interactions. This method has great potential for studying protein interactions because it is sensitive to changes in the distance separating two fluorophores on the 1-10 nm scale. FRET imaging in conjunction with the development of spectral variants of Green Fluorescent Protein (GFP) provides the opportunity to genetically tag synaptic proteins of interest and monitor their interactions with other labeled proteins in real time. Our Section is comprised of Drs. Steven Vogel (Chief), Srinagesh Koushik (Research Fellow), Christopher Thaler (Postdoctoral IRTA), and Jose Fernando Covian-Nares (Visiting Fellow). Our Sections initial efforts concentrated on 1. Building and testing a laser scanning microscope specifically designed for studying protein-protein interactions in living cells, 2. Develop new methods for measuring FRET, and 3. Overcoming some of the practical limitations of FRET imaging. The microscope we have constructed is a fully functional laser scanning two-photon microscope, with the additional capabilities of measuring florescent emission spectra (spectral imaging), fluorescent lifetime decays (FLIM), and fluorescent anisotropy lifetime decays (rFLIM). These added capabilities make it specifically useful for monitoring FRET between either dissimilar (Hetero-FRET) or similar (Homo-FRET) fluorophores. Since our last report we have published two new manuscripts. Additionally we currently have a book chapter in press, and a manuscript under review. We have also produced a set of FRET standards in our section and have provided these FRET standards to over 60 research groups including laboratories in Germany, Spain, Canada, Netherlands, Austria, Singapore, Switzerland, and Denmark. Currently we have 5 working projects in the lab. The first project is involved in concluding our feasibility and methodological studies on FRET imaging. The last four projects initiate the next phase in our Sections activities where our microscopes unique capabilities are utilized to address biological questions: 1. We have generated Homo-FRET reference standards that will be used in interpreting anisotropy decay experiments. This relatively new method has the potential for monitoring how proteins form multimeric structures, and their stoichiometry in living cells. 2. The second project has been following up on an observation we have made where we measure more and faster FRET between GFP type fluorophores than are predicted by FRET theory. After consulting with FRET experts from around the world, our current working hypothesis is that because GFP fluorophores are protected from the external environment by its beta-barrel protein structure, vibrational dequenching of excited states are slowed and thus allow for coherent energy transfer between FPs. If our hypothesis is correct, this would be one of the first examples of a biology adaptation to exploit quantum mechanical behavior at room temperature. This phenomenon might also be useful for generating components required for building quantum computers. 3. Our third project uses anisotropy lifetime decay analysis to monitor changes in the multimeric structure of Cam kinase-II. This abundant synaptic enzyme has been shown to play a pivotal role in learning and memory. It is believed that long lived structural changes in this protein complex might be the embodiment of some forms of memory. Our results indicate that structural changes associated with Cam kinase-II activation can be detected with anisotropy imaging. 4. In our fourth project we are using two-photon microscopy to investigating the roles of Dynamin (a protein that assembles into a coiled structure and is directly involved in membrane scission ) in regulating endocytosis and cell division. We have developed a new imaging based assay for testing the effects of either over expression of exogenous proteins, or down regulation of endogenous proteins, on compensatory endocytosis and cell division in developing sea urchin embryos. Using this assay we have found a surprising connection between endocytosis and cell division. We have previously shown that over expression of Src kinase, inhibits compensatory endocytosis and blocks cell division. Inhibitors of tyrosine phosphatase had similar effects, suggesting that the balance of tyrosine kinase and phosphatase activity plays a key role in how cells regulate endocytosis and cell division. Injection of an anti-sense morpholino against Dynamin also blocked endocytosis and cell division, and treatment with a peptide that blocks Dynamins interactions with other proteins through its SH3 (Src homology 3) domain also blocked endocytosis and cell division. Injection of an anti-sense morpholino against Src inhibited cell division, but did not inhibit endocytosis. These experiments suggest that dynamin-dependent endocytosis is required for cell division. 5. Finally, the Jain foundation has generously established a gift fund in support of our laboratories preliminary investigations into the functions of Dysferlin, a protein that is known to be responsible for LGMD2B/Miyoshi muscular dystrophy. Our preliminary experiments indicated that anti-sense morpholinos against Dysferlin injected into developing sea urchin embryos inhibit cell division and wound healing. Accordingly, we expanded this study to investigate the role of Dysferlin in calcium signaling. We have found that upon wounding, cells depolarize and secrete ATP by a mechanism involving agatoxin sensitive voltage-gated calcium channels. Neighboring cells respond to the secreted ATP by a mechanism thought to involve P2X receptors as well as voltage-gated calcium channels. Anti-sense morpholinos against Dysferlin block the secretion of ATP supporting the hypothesis that Dysferlin might act by mediating calcium triggered exocytosis.

细胞生物光谱学部分的主要目的是使用成像技术，例如两光子显微镜，光谱成像，荧光寿命显微镜和荧光各向异性分析来研究蛋白质复合物如何调节活细胞中的突触功能。最近，我们将精力集中在利用Forsters共振能量转移（FRET）上监测蛋白质蛋白质相互作用。该方法具有研究蛋白质相互作用的巨大潜力，因为它对距离的变化敏感，将两个荧光团以1-10 nm的比例分开。 FRET成像结合了绿色荧光蛋白（GFP）的光谱变体的发展提供了机会，可以实时对具有遗传标记感兴趣的突触蛋白进行遗传标记突触蛋白，并实时监测其与其他标记蛋白的相互作用。我们的部分由Drs组成。史蒂文·沃格尔（酋长），斯里纳奇·库希克（研究研究员），克里斯托弗·塔勒（Christopher Thaler）（博士后IRTA）和Jose Fernando Covian-Nares（访问者）。 我们的初步努力集中在1。建立和测试激光扫描显微镜专为研究活细胞中蛋白质 - 蛋白质相互作用而设计的激光扫描显微镜，2。开发用于测量FRET的新方法，3。克服FRET成像的一些实际限制。我们构造的显微镜是一种功能齐全的激光扫描两光子显微镜，具有测量荧光发射光谱（光谱成像），荧光寿命衰减（FLIM）和荧光各向异性各向异性生命周期（Rflim）的其他功能。这些增加的功能使其可用于监测不同（异源）或类似（Homo-fret）荧光团之间的fret。自上次报告以来，我们发表了两个新手稿。此外，我们目前在新闻中有一章，并正在审查手稿。我们还在我们的部分中制定了一套肉质标准，并为60多个研究小组提供了这些FRET标准，包括德国，西班牙，加拿大，荷兰，奥地利，新加坡，瑞士和丹麦的实验室。 目前，我们在实验室中有5个工作项目。第一个项目参与了我们对FRET成像的可行性和方法论研究的结论。最后四个项目在我们的各节活动中启动了下一阶段，我们的显微镜独特功能可用于解决生物学问题： 1。我们已经生成了HOMO-FRET参考标准，这些标准将用于解释各向异性衰减实验。这种相对新的方法具有监测蛋白质在活细胞中如何形成多聚体结构及其化学计量法的潜力。 2。第二个项目一直在跟进我们所进行的观察结果，在GFP型荧光团之间，我们比FRET理论预测的是GFP型荧光团之间更快，更快的。在与来自世界各地的FRET专家进行了咨询之后，我们目前的工作假设是，由于GFP荧光团通过其β-桶蛋白结构保护了外部环境，因此激发态的振动去quinceration振动会减慢，因此可以在FPS之间进行连贯的能量传递。如果我们的假设是正确的，那么这将是生物学适应以在室温下利用量子机械行为的第一个例子之一。这种现象对于生成构建量子计算机所需的组件也可能很有用。 3。我们的第三个项目使用各向异性寿命衰减分析来监测CAM激酶-II多聚体结构的变化。这种丰富的突触酶已被证明在学习和记忆中起着关键作用。人们认为，这种蛋白质复合物中长期存在的结构变化可能是某些形式的记忆的实施例。我们的结果表明，可以通过各向异性成像检测与CAM激酶-II激活相关的结构变化。 4。在我们的第四个项目中，我们使用两光子显微镜研究Dynamin（一种聚集成盘绕结构并直接参与膜分裂的蛋白质的作用）在调节内吞作用和细胞分裂中的作用。我们开发了一种新的基于成像的测定方法，用于测试外源蛋白质过度表达的影响，或下调内源性蛋白对发展中海胆胚胎的代偿性内吞作用和细胞分裂的影响。使用此测定，我们发现内吞作用与细胞分裂之间存在令人惊讶的联系。我们先前已经表明，SRC激酶的表达过多，抑制补偿性内吞作用并阻止细胞分裂。酪氨酸磷酸酶的抑制剂具有相似的作用，表明酪氨酸激酶和磷酸酶活性的平衡在细胞调节内吞作用和细胞分裂的方式中起关键作用。注射抗敏感性的形态蛋白还阻断了内吞作用和细胞分裂，并用肽治疗通过其SH3（SRC同源3）结构域阻止了动力蛋白与其他蛋白质的相互作用，也阻塞了内吞和细胞分裂。注射抗敏感性的形态侵害SRC抑制细胞分裂，但没有抑制内吞作用。这些实验表明细胞分裂需要依赖性蛋白依赖性内吞作用。 5。最后，Ja那教基金会慷慨地建立了一项礼物基金，以支持我们实验室对Dysferlin的功能进行初步研究，Dysferlin是一种蛋白质，该蛋白质造成LGMD2B/Miyoshi肌肉营养不良。我们的初步实验表明，针对Dysferlin的反义形态学注射到发展中的海胆胚胎中，会抑制细胞分裂和伤口愈合。因此，我们扩展了这项研究，以研究dysferlin在钙信号传导中的作用。我们发现，在受伤后，细胞通过涉及阿加毒素敏感电压门控钙通道的机制去极化和分泌ATP。相邻的细胞通过一种被认为涉及P2X受体以及电压门控钙通道的机制对分泌的ATP做出反应。反义的形态学反对dysferlin阻断了ATP的分泌，支持dysferlin可能通过介导钙触发的胞吐作用的假设。