Determining in Vivo Protein Complex Stoichiometry from Superresolution Microscopy

用超分辨显微镜测定体内蛋白质复合体的化学计量。蛋白质是构成生命的基本构件之一。在活细胞中，蛋白质通常组装成大分子复合体，以执行有针对性的任务。了解蛋白质组件的位置和准确组成使研究人员能够了解其在关键细胞事件(如分裂或运动)中活动的分子机制。不幸的是，人们对活细胞中这种复合体的组成知之甚少，而且很明显，这种组成可以根据细胞环境的不同而变化。该项目将开发一种光学/计算方法来计算活细胞中蛋白质组件中的亚基，并能够收集关于自然、非侵入性环境中生物过程的异常详细的信息。该项目结合了生物、数学和物理科学的专业知识，因此为学生和博士后提供了特殊的跨学科培训机会。此外，该项目将产生在光学生物科学中具有广泛适用性的方法。该项目将直接从名为Palm(光活化定位显微镜)的超分辨率显微镜技术中提取蛋白质复杂化学计量比。Palm的工作原理是利用光开关荧光蛋白(FP)标签对感兴趣的蛋白质进行基因编码。在足够弱的光线下，每个FP都会被光激活，然后发生光漂白。因此，一个小空间区域上的荧光尖峰的数量应该与限制在该区域的蛋白质亚单位的数量一致(通常限制在特定的蛋白质复合体中)。在实践中，FP会根据局部细胞环境的不同而发出荧光(即，它们会“闪烁”)，其闪烁特性也会有所不同。这种眨眼一直是将Palm用作量化工具的主要挑战。该项目将采用用于分析离子通道膜片钳实验的数学和统计推断工具，以直接从Palm数据确定蛋白质复合体的化学计量比(目标1)。为了更准确地确定复杂的化学计量比，将同时提取FP的闪烁特性，同时确定复杂的化学计量比(目标2)。该方法将在已知蛋白质络合物化学计量学的合成和真实数据集上进行测试。然后，分析将扩展到化学计量比仍在进行当前研究的蛋白质复合体(SpoIIIE和动粒)。如果成功，这种计数方法将比现有的计数方法有几个决定性的优势，现有的计数方法假设特定的FP光物理性质或不随机处理FP性质。这项拨款由分子和细胞生物科学部的细胞动力学计划和物理部的生命系统物理计划联合资助。

Determining in vivo protein complex stoichiometry from superresolution microscopy.Proteins form one of the basic building blocks of life. In living cells, proteins typically assemble into macromolecular complexes in order to perform targeted tasks. Knowing the location and exact composition of protein assemblies enables investigators to understand the molecular mechanisms underlying their activity during key cellular events such as division or movement. Unfortunately, relatively little is known about the composition of such complexes in living cells, and it is clear that composition can vary depending on the cellular environment. This project will develop an optical/computational method to count subunits in protein assemblies in living cells, and enable exceptionally detailed information to be gathered about biological processes in native, non-invasive environments. This project combines expertise from the biological, mathematical, and physical sciences and as such provides exceptional interdisciplinary training opportunities for students and postdocs. Moreover, the project will yield methods that will have broad applicability in the optical biological sciences.This project will extract protein complex stoichiometry directly from the superresolution microscopy technique called PALM (PhotoActivated Localization Microscopy). PALM works by genetically encoding proteins of interest with photoswitchable fluorescing protein (FP) tags. Under sufficiently low light, each FP photoactivates and subsequently photobleaches. Thus, the number of fluorescence spikes over one small region of space should coincide with the number of protein subunits confined to that region (and normally confined to a specific protein complex). In practice, FPs fluoresce on and off (i.e. they 'blink') with blinking properties dependent on the local cellular environment. This blinking has been a major challenge in using PALM as a quantitative tool. The project will adapt the mathematical and statistical inference tools used in the analysis of ion channel patch clamp experiments to determine protein complex stoichiometry directly from PALM data (Goal 1). To determine the complex stoichiometry more accurately, the FP's blinking properties will be simultaneously extracted while determining the complex stoichiometry (Goal 2). The method will be tested on synthetic as well as real data sets where the protein complex stoichiometry is already known. The analysis will then be extended to protein complexes where the stoichiometry is still under current investigation (SpoIIIE and the kinetochore). If successful, this counting method will have several decisive advantages over existing counting methods, which assume specific FP photophysical properties or do not treat FP properties stochastically.This grant is funded jointly by the Cellular Dynamics Program in the Division of Molecular and Cellular Biosciences and the Physics of Living Systems Program in the Division of Physics.