Telomere analysis using 3D fluorescence microscopy suggests mammalian telomere clustering in hTERT-immortalized Hs68 fibroblasts
Telomere analysis using 3D fluorescence microscopy suggests mammalian telomere clustering in hTERT-immortalized Hs68 fibroblasts
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DOI：
10.1038/s42003-019-0692-z
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发表时间：
2019-12-04
影响因子：
5.9
通讯作者：
Beattie, Tara
中科院分区：
文献类型：
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作者：
Adam, Nancy;Degelman, Erin;Beattie, Tara
端粒长度和动力学是理解细胞衰老、基因组不稳定性和癌症的核心。目前，使用不同的细胞模型在3D中分析端粒特征的指导方针有限。用于端粒分析的图像处理在许多领域中越来越受关注，然而缺乏标准化可能会使比较和再现性成为问题。在这里，我们提供了一个用户的指导，定量免疫荧光显微镜端粒间期细胞，包括图像采集，处理和分析。使用正常人二倍体Hs68成纤维细胞鉴定用于确定端粒大小和数目的策略。我们演示了如何准确地确定端粒的数量，长度，体积和聚类程度使用定量免疫荧光。使用这种工作流程，我们意外地观察到，具有较长端粒的hTERT永生化的Hs68细胞在间期具有较少的可分辨端粒。严格的定量分析表明，这是由于端粒的聚集，导致系统低估端粒的数量和高估端粒的大小。
Telomere length and dynamics are central to understanding cell aging, genomic instability and cancer. Currently, there are limited guidelines for analyzing telomeric features in 3D using different cellular models. Image processing for telomere analysis is of increasing interest in many fields, however a lack of standardization can make comparisons and reproducibility an issue. Here we provide a user's guide for quantitative immunofluorescence microscopy of telomeres in interphase cells that covers image acquisition, processing and analysis. Strategies for determining telomere size and number are identified using normal human diploid Hs68 fibroblasts. We demonstrate how to accurately determine telomere number, length, volume, and degree of clustering using quantitative immunofluorescence. Using this workflow, we make the unexpected observation that hTERT-immortalized Hs68 cells with longer telomeres have fewer resolvable telomeres in interphase. Rigorous quantification indicates that this is due to telomeric clustering, leading to systematic underestimation of telomere number and overestimation of telomere size.