Talati Ronak, Vanderpoel Andrew, Eladdadi Amina, Anderson Kate, Abe Ken, Barroso Margarida
Center for Cardiovascular Sciences, Albany Medical College, 47 New Scotland Avenue, Albany, NY 12208, USA.
Department of Mathematics, The College of Saint Rose, 432 Western Avenue, Albany, NY 12203, USA.
Methods. 2014 Mar 15;66(2):139-52. doi: 10.1016/j.ymeth.2013.08.017. Epub 2013 Aug 28.
The overexpression of certain membrane-bound receptors is a hallmark of cancer progression and it has been suggested to affect the organization, activation, recycling and down-regulation of receptor-ligand complexes in human cancer cells. Thus, comparing receptor trafficking pathways in normal vs. cancer cells requires the ability to image cells expressing dramatically different receptor expression levels. Here, we have presented a significant technical advance to the analysis and processing of images collected using intensity based Förster resonance energy transfer (FRET) confocal microscopy. An automated Image J macro was developed to select region of interests (ROI) based on intensity and statistical-based thresholds within cellular images with reduced FRET signal. Furthermore, SSMD (strictly standardized mean differences), a statistical signal-to-noise ratio (SNR) evaluation parameter, was used to validate the quality of FRET analysis, in particular of ROI database selection. The Image J ROI selection macro together with SSMD as an evaluation parameter of SNR levels, were used to investigate the endocytic recycling of Tfn-TFR complexes at nanometer range resolution in human normal vs. breast cancer cells expressing significantly different levels of endogenous TFR. Here, the FRET-based assay demonstrates that Tfn-TFR complexes in normal epithelial vs. breast cancer cells show a significantly different E% behavior during their endocytic recycling pathway. Since E% is a relative measure of distance, we propose that these changes in E% levels represent conformational changes in Tfn-TFR complexes during endocytic pathway. Thus, our results indicate that Tfn-TFR complexes undergo different conformational changes in normal vs. cancer cells, indicating that the organization of Tfn-TFR complexes at the nanometer range is significantly altered during the endocytic recycling pathway in cancer cells. In summary, improvements in the automated selection of FRET ROI datasets allowed us to detect significant changes in E% with potential biological significance in human normal vs. cancer cells.
某些膜结合受体的过表达是癌症进展的一个标志，并且有人提出它会影响人类癌细胞中受体 - 配体复合物的组织、激活、再循环和下调。因此，比较正常细胞与癌细胞中的受体运输途径需要能够对表达水平差异极大的受体进行成像。在这里，我们在基于强度的福斯特共振能量转移（FRET）共聚焦显微镜收集的图像分析和处理方面取得了重大技术进展。开发了一个自动化的Image J宏，用于在具有降低的FRET信号的细胞图像中基于强度和基于统计的阈值选择感兴趣区域（ROI）。此外，严格标准化平均差异（SSMD），一种统计信噪比（SNR）评估参数，用于验证FRET分析的质量，特别是ROI数据库选择的质量。Image J ROI选择宏与作为SNR水平评估参数的SSMD一起，用于在纳米级分辨率下研究人正常细胞与表达内源性TFR水平差异显著的乳腺癌细胞中Tfn - TFR复合物的内吞再循环。在这里，基于FRET的分析表明，正常上皮细胞与乳腺癌细胞中的Tfn - TFR复合物在其内吞再循环途径中表现出显著不同的E%行为。由于E%是距离的相对度量，我们提出E%水平的这些变化代表内吞途径中Tfn - TFR复合物的构象变化。因此，我们的结果表明，Tfn - TFR复合物在正常细胞与癌细胞中经历不同的构象变化，表明在癌细胞的内吞再循环途径中，Tfn - TFR复合物在纳米级的组织发生了显著改变。总之，FRET ROI数据集自动选择的改进使我们能够检测到人类正常细胞与癌细胞中具有潜在生物学意义的E%的显著变化。
