Molecular counting by photobleaching in protein complexes with many subunits: best practices and application to the cellulose synthesis complex.
Molecular counting by photobleaching in protein complexes with many subunits: best practices and application to the cellulose synthesis complex.
复制标题
DOI：
10.1091/mbc.e14-06-1146
复制
发表时间：
2014-11-05
影响因子：
3.3
通讯作者：
Hancock WO
中科院分区：
文献类型：
--
作者：
Chen Y;Deffenbaugh NC;Anderson CT;Hancock WO
荧光光漂白用于估计拟南芥纤维素合成复合体中的纤维素合成酶亚基的数量。为了克服从光漂白痕迹中计数许多(>；10)荧光团的挑战，开发了一套通用的统计程序，用于客观检测步骤和准确估计高拷贝数。大的多亚单位蛋白质复合体的组成决定了它们在细胞中的功能，但在体内确定它们的精确分子组成是具有挑战性的。这种复合体的一个例子是纤维素合成复合体(CSC)，它在植物中合成纤维素，这是地球上最丰富的生物聚合物。在生长中的植物细胞中，CSC以六叶花环的形式存在于质膜中，其中至少含有三种不同的纤维素合成酶(CESA)亚型，但每个CSC中CESA的数量和化学计量尚不清楚。为了开始解决这个问题，我们使用可变角度荧光显微镜对活的拟南芥细胞中含有GFP标记的AtCESA3颗粒进行了定量光漂白，并开发了一套基于信息的步骤检测程序来估计每个颗粒中的GFP分子数量。阶跃检测算法考虑了由于荧光团数量的改变而引起的信号方差的变化，并且后续分析避免了与将多个高斯函数拟合到入库的直方图数据相关联的常见问题。分析表明，每个颗粒中至少存在10个GFP-AtCESA3分子。这些程序可以应用于任何含有大量荧光标记亚基的蛋白质复合体的光漂白数据，为探索复杂组成和化学计量提供了一种新的分析工具。
Fluorescence photobleaching is used to estimate the number of cellulose synthase subunits in Arabidopsis cellulose synthesis complexes. To overcome challenges with counting many (>10) fluorophores from photobleaching traces, a general set of statistical procedures is developed for objectively detecting steps and accurately estimating high copy numbers. The constituents of large, multisubunit protein complexes dictate their functions in cells, but determining their precise molecular makeup in vivo is challenging. One example of such a complex is the cellulose synthesis complex (CSC), which in plants synthesizes cellulose, the most abundant biopolymer on Earth. In growing plant cells, CSCs exist in the plasma membrane as six-lobed rosettes that contain at least three different cellulose synthase (CESA) isoforms, but the number and stoichiometry of CESAs in each CSC are unknown. To begin to address this question, we performed quantitative photobleaching of GFP-tagged AtCESA3-containing particles in living Arabidopsis thaliana cells using variable-angle epifluorescence microscopy and developed a set of information-based step detection procedures to estimate the number of GFP molecules in each particle. The step detection algorithms account for changes in signal variance due to changing numbers of fluorophores, and the subsequent analysis avoids common problems associated with fitting multiple Gaussian functions to binned histogram data. The analysis indicates that at least 10 GFP-AtCESA3 molecules can exist in each particle. These procedures can be applied to photobleaching data for any protein complex with large numbers of fluorescently tagged subunits, providing a new analytical tool with which to probe complex composition and stoichiometry.