Co-localization analysis of complex formation among membrane proteins by computerized fluorescence microscopy: application to immunofluorescence co-patching studies
Co-localization analysis of complex formation among membrane proteins by computerized fluorescence microscopy: application to immunofluorescence co-patching studies
复制标题
DOI：
10.1046/j.1365-2818.2003.01239.x
复制
发表时间：
2003-11-01
影响因子：
2
通讯作者：
Weiss, AM
中科院分区：
文献类型：
--
作者：
Lachmanovich, E;Shvartsman, DE;Weiss, AM
两个独立标记的蛋白质的荧光成像通常被用来确定它们在细胞中的共同定位。抗体介导的交联会介导这些蛋白质在细胞表面的修补，它们的共同定位可以确定它们之间的复合体的形成。然而，对这类研究的人工分析既繁琐又主观。在这里，我们提出了一种数字共定位分析，它独立于荧光强度，在观察者之间高度一致和可重复性，并极大地减少了分析时间。提出的方法基于分割过程，该分割过程创建二进制对象，然后确定属于两个不同组(例如，绿色标记和红色标记)的对象是否被共同局部化。使用两种方法来确定共本地化。如果一个物体的质心落在另一个物体的区域内，‘重叠’分析将两个物体定义为共同定位的。如果两个物体的中心在成像模式确定的阈值距离内，则最近邻距离分析认为两个物体是共同定位的。为了测试结果的重要性，将实际图像的分析与通过创建具有来自两组的对象的不相关分布的图像的方法生成的随机化图像进行测试。通过对流感血凝素突变体的联合补丁研究，证明了所提出的算法在活细胞中研究蛋白质相互作用的适用性，这些突变体通过与AP-2适配器复合体结合而在细胞表面形成或不结合成相互的低聚物。该方法有可能适用于其他方法(如电子显微镜)的共定位研究，最近邻距离法也可用于研究相关放置现象。
Fluorescence imaging of two independently labelled proteins is commonly used to determine their co-localization in cells. Antibody-mediated crosslinking can mediate the patching of such proteins at the cell surface, and their co-localization can serve to determine complex formation among them. However, manual analysis of such studies is both tedious and subjective. Here we present a digital co-localization analysis that is independent of the fluorescence intensity, is highly consistent and reproducible between observers, and dramatically reduces the analysis time. The approach presented is based on a segmentation procedure that creates binary objects, and then determines whether objects belonging to two different groups (e.g. green- and red-labelled) are co-localized. Two methods are used to determine co-localization. The 'overlap' analysis defines two objects as co-localized if the centre of mass of one falls within the area of the other. The 'nearest-neighbour distance' analysis considers two objects as co-localized if their centres are within a threshold distance determined by the imaging modality. To test the significance of the results, the analysis of the actual images is tested against randomized images generated by a method that creates images with uncorrelated distributions of objects from the two groups. The applicability of the algorithms presented to study protein interactions in live cells is demonstrated by co-patching studies on influenza haemagglutinin mutants that do or do not associate into mutual oligomers at the cell surface via binding to AP-2 adaptor complexes. The approach presented is potentially applicable to studies of co-localization by other methods (e.g. electron microscopy), and the nearest-neighbour distance method can also be adapted to study phenomena of correlated placement.