Automated selection of regions of interest for intensity-based FRET analysis of transferrin endocytic trafficking in normal vs. cancer cells.
Automated selection of regions of interest for intensity-based FRET analysis of transferrin endocytic trafficking in normal vs. cancer cells.
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DOI：
10.1016/j.ymeth.2013.08.017
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发表时间：
2014-03-15
期刊：
影响因子：
4.8
通讯作者：
Barroso, Margarida
中科院分区：
文献类型：
--
作者：
Talati, Ronak;Vanderpoel, Andrew;Eladdadi, Amina;Anderson, Kate;Abe, Ken;Barroso, Margarida
关键词：
某些膜结合受体的过表达是癌症进展的标志，并且已经表明其影响人癌细胞中受体-配体复合物的组织、活化、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对表达显著不同的受体表达水平的细胞进行成像的能力。在这里，我们提出了一个显着的技术进步，使用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的分析和处理。开发了自动化Image J宏，以基于FRET信号降低的细胞图像内的强度和基于阈值的阈值来选择感兴趣区域（ROI）。此外，SSMD（严格标准化的平均差异），统计信噪比（SNR）的评价参数，用于验证FRET分析的质量，特别是ROI数据库的选择。Image J ROI选择宏与作为SNR水平的评价参数的SSMD一起用于在表达显著不同水平的内源性TFR的人正常细胞与乳腺癌细胞中以纳米范围分辨率研究Tfn-TFR复合物的内吞再循环。在此，基于FRET的测定表明，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期间显示出显著不同的E%行为。由于E%是距离的相对量度，因此我们提出E%水平的这些变化代表内吞途径期间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期间，Tfn-TFR复合物在纳米范围内的组织发生了显著改变。总之，FRET ROI数据集的自动选择的改进允许我们检测E%的显著变化，其在人类正常细胞与癌细胞中具有潜在的生物学意义。
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.
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影响因子：
3
作者：
Daniels, Tracy R.;Bernabeu, Ezequiel;Rodriguez, Jose A.;Patel, Shabnum;Kozman, Maggie;Chiappetta, Diego A.;Holler, Eggehard;Ljubimova, Julia Y.;Helguera, Gustavo;Penichet, Manuel L.
通讯作者：
Penichet, Manuel L.
影响因子：
5.3
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通讯作者：
NICOLSON, GL
影响因子：
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GATTER, KC;BROWN, G;MASON, DY
通讯作者：
MASON, DY