Spatial Intensity Distribution Analysis Reveals Abnormal Oligomerization of Proteins in Single Cells
Spatial Intensity Distribution Analysis Reveals Abnormal Oligomerization of Proteins in Single Cells
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DOI：
10.1016/j.bpj.2015.06.068
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发表时间：
2015-08-18
影响因子：
3.4
通讯作者：
Wisemant, Paul W.
中科院分区：
文献类型：
--
作者：
Godin, Antoine G.;Rappaz, Benjamin;Wisemant, Paul W.
膜受体组织的知识对于理解细胞信号传导和运输机制的初始步骤是必不可少的，但是在单细胞水平和不同细胞区室中受体相互作用的定量分析仍然具有高度挑战性。为了实现这一目标，我们应用定量图像分析技术-空间强度分布分析（SpIDA）-可以测量活细胞中不同亚细胞区室内的荧光颗粒浓度和寡聚化状态。基于荧光显微镜的寡聚化测量所面临的一个重要技术挑战是受体标记的保真度。在实践中，不完美的标签偏见的分布在聚集系统内测量的低聚状态。我们扩展SpIDA，使高阶低聚物的荧光显微镜图像的分析，包括一个概率加权校正算法nonemitting标签。我们证明，这部分的nonemitting探针可以估计在单细胞中使用SpIDA测量模型系统与已知的oligonnerization状态。以前，这种伪影是使用单步光漂白测量的。使用计算机模拟数据验证了这种方法，并在具有已知寡聚体亚基计数的离子通道的细胞中定量了不完美的标记。然后将其应用于量化COS-7细胞中表达的蛋白脂质蛋白（PLP）在不同细胞隔室中的寡聚化状态。与受损的运输相关的突变体PLP的表达导致在内质网中持续存在的PLP四聚体的检测，而在野生型和突变的PLP的分布之间的膜处没有测量到差异。我们的研究结果表明，SpIDA允许测量完整细胞的不同隔室中的蛋白质寡聚化，即使在成像过程中发生分数错误标记以及光漂白，并揭示了对PLP受损贩运机制的见解。
Knowledge of membrane receptor organization is essential for understanding the initial steps in cell signaling and trafficking mechanisms, but quantitative analysis of receptor interactions at the single-cell level and in different cellular compartments has remained highly challenging. To achieve this, we apply a quantitative image analysis technique-spatial intensity distribution analysis (SpIDA)-that can measure fluorescent particle concentrations and oligomerization states within different subcellular compartments in live cells. An important technical challenge faced by fluorescence microscopy-based measurement of oligonnerization is the fidelity of receptor labeling. In practice, imperfect labeling biases the distribution of oligomeric states measured within an aggregated system. We extend SpIDA to enable analysis of high-order oligomers from fluorescence microscopy images, by including a probability weighted correction algorithm for nonemitting labels. We demonstrated that this fraction of nonemitting probes could be estimated in single cells using SpIDA measurements on model systems with known oligonnerization state. Previously, this artifact was measured using single-step photobleaching. This approach was validated using computer-simulated data and the imperfect labeling was quantified in cells with ion channels of known oligomer subunit count. It was then applied to quantify the oligomerization states in different cell compartments of the proteolipid protein (PLP) expressed in COS-7 cells. Expression of a mutant PLP linked to impaired trafficking resulted in the detection of PLP tetramers that persist in the endoplasmic reticulum, while no difference was measured at the membrane between the distributions of wild-type and mutated PLPs. Our results demonstrate that SpIDA allows measurement of protein oligomerization in different compartments of intact cells, even when fractional mislabeling occurs as well as photobleaching during the imaging process, and reveals insights into the mechanism underlying impaired trafficking of PLP.