کد مقاله | کد نشریه | سال انتشار | مقاله انگلیسی | نسخه تمام متن |
---|---|---|---|---|
10825724 | 1064667 | 2014 | 14 صفحه PDF | دانلود رایگان |
عنوان انگلیسی مقاله ISI
Automated selection of regions of interest for intensity-based FRET analysis of transferrin endocytic trafficking in normal vs. cancer cells
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کلمات کلیدی
STDTransferrin receptor (TfR)AF488ACCSNRSBTPBSHMETransferrinTFRROITFn - TFNAlexa Fluor 488 - الکسا فلور 488Förster resonance energy transfer - انتقال انرژی تابشی ForsterFörster resonance energy transfer (FRET) - انتقال انرژی حرارتی Förster (FRET)FRET - انتقال انرژی رزونانسی فورسترstandard deviation - انحراف معیارDonor - اهدا کنندهEnergy transfer efficiency - بازده انتقال انرژیEdge detection - تشخیص لبهHuman mammary epithelial cells - سلول های اپیتلیال پستان انسانی انسانیPhosphate buffered saline solution - محلول نمک فسفات بافرRegion of Interest (ROI) - منطقه سود (ROI)region of interest - منطقه مورد نظرsignal to noise ratio - نسبت سیگنال به نویزAcceptor - پذیرندهImage processing - پردازش تصویرArea fraction - کسر مساحتtransferrin receptor - گیرنده انتقالین
موضوعات مرتبط
علوم زیستی و بیوفناوری
بیوشیمی، ژنتیک و زیست شناسی مولکولی
زیست شیمی
پیش نمایش صفحه اول مقاله
چکیده انگلیسی
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.
ناشر
Database: Elsevier - ScienceDirect (ساینس دایرکت)
Journal: Methods - Volume 66, Issue 2, 15 March 2014, Pages 139-152
Journal: Methods - Volume 66, Issue 2, 15 March 2014, Pages 139-152
نویسندگان
Ronak Talati, Andrew Vanderpoel, Amina Eladdadi, Kate Anderson, Ken Abe, Margarida Barroso,