Automatic and quantitative measurement of protein-protein colocalization in live cells
Automatic and quantitative measurement of protein-protein colocalization in live cells
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DOI：
10.1529/biophysj.103.038422
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发表时间：
2004-06-01
影响因子：
3.4
通讯作者：
Lockett, S
中科院分区：
文献类型：
--
作者：
Costes, SV;Daelemans, D;Lockett, S
我们介绍了一种新的统计方法，量化，第一次，在图像中的两个荧光标记的蛋白质的共定位量自动，消除视觉解释的偏见。这是通过同时估计每种颜色的最大强度阈值来完成的，低于该阈值的像素不显示任何统计相关性。该方法的灵敏度，说明了模拟数据的统计确认存在真正的共定位图像中，只有3%的共定位。然后在用CFP/YFP蛋白对共转染的固定细胞的大的三维集合上测试该方法，所述蛋白对是共区室化的、相互作用的或只是随机定位在核仁中。在该测试中，该算法成功地区分了随机颜色重叠与共定位，这是由于共区室化或相互作用，并且结果通过荧光共振能量转移进行了验证。我们的算法的准确性和一致性进一步说明了测量，首次在活细胞中，解离速率（k（d））的HIV-1 Rev/CRM 1输出复合物诱导的细胞毒素链霉素B。加入来普霉素B后，Rev/CRM 1在核仁中的共定位以1.25 x 10（-3）s（-1）的速率呈指数下降。更一般地说，该算法可以用来回答各种生物学问题，涉及蛋白质-蛋白质相互作用或共区室化，并可以推广到共定位的莫拉比两种颜色。
We introduce a novel statistical approach that quantifies, for the first time, the amount of colocalization of two fluorescent-labeled proteins in an image automatically, removing the bias of visual interpretation. This is done by estimating simultaneously the maximum threshold of intensity for each color below which pixels do not show any statistical correlation. The sensitivity of the method was illustrated on simulated data by statistically confirming the existence of true colocalization in images with as little as 3% colocalization. This method was then tested on a large three-dimensional set of fixed cells cotransfected with CFP/YFP pairs of proteins that either co-compartmentalized, interacted, or were just randomly localized in the nucleolus. In this test, the algorithm successfully distinguished random color overlap from colocalization due to either co-compartmentalization or interaction, and results were verified by fluorescence resonance energy transfer. The accuracy and consistency of our algorithm was further illustrated by measuring, for the first time in live cells, the dissociation rate (k(d)) of the HIV-1 Rev/CRM1 export complex induced by the cytotoxin leptomycin B. Rev/CRM1 colocalization in nucleoli dropped exponentially after addition of leptomycin B at a rate of 1.25 x 10(-3) s(-1). More generally, this algorithm can be used to answer a variety of biological questions involving protein-protein interactions or co-compartmentalization and can be generalized to colocalization of mora than two colors.