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物的化学计量。蛋白质是生命的基本组成部分之一。在活细胞中，蛋白质通常组装成大分子复合物以执行目标任务。了解蛋白质组装的位置和确切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，人们对活细胞中这些复合物的组成知之甚少，很明显，这些复合物的组成可以根据细胞环境而变化。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装中的亚基，并能够收集有关天然非侵入性环境中生物过程的非常详细的信息。该项目结合了生物、数学和物理科学的专业知识，为学生和博士后提供了卓越的跨学科培训机会。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。该项目将直接从称为PALM（光激活定位显微镜）的超分辨率显微镜技术中提取蛋白质复合物化学计量。PALM的工作原理是用可光切换荧光蛋白（FP）标签对感兴趣的蛋白质进行基因编码。在足够低的光照下，每个FP光激活并随后光漂白。因此，在空间的一个小区域上的荧光峰的数量应该与局限于该区域的蛋白质亚基的数量一致（通常局限于特定的蛋白质复合体）。在实践中，FPs荧光的开启和关闭（即它们“闪烁”）与闪烁属性取决于局部细胞环境。这种闪烁是使用PALM作为定量工具的主要挑战。该项目将采用离子通道膜片钳实验分析中使用的数学和统计推断工具，直接从PALM数据确定蛋白质复合物的化学计量（目标1）。为了更准确地确定复合化学计量，将在确定复合化学计量的同时提取FP的闪烁属性（目标2）。该方法将在合成和真实数据集上进行测试，其中蛋白质复合物化学计量学已经已知。然后将分析扩展到化学计量学仍在研究中的蛋白质复合物（SpoIIIE和着丝点）。如果成功，这种计数方法将比现有的计数方法具有几个决定性的优势，现有的计数方法假设FP的特定光物理性质或不随机处理FP的性质。该基金由分子和细胞生物科学部的细胞动力学项目和物理部的生命系统物理学项目共同资助。

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.