mtFociCounter for automated single-cell mitochondrial nucleoid quantification and reproducible foci analysis-猫眼课题宝

mtFociCounter 用于自动单细胞线粒体核仁定量和可重复的焦点分析

DOI：

10.1101/2022.08.13.503663

发表时间：

2022

期刊：

影响因子：

通讯作者：

中科院分区：

文献类型：

作者：

Rey T

线粒体 DNA (mtDNA) 编码 OXPHOS 的核心亚基，这是几乎所有真核生物所必需的。线粒体 DNA 被包装在线粒体内的不同焦点（类核）中，不同细胞类型的线粒体 DNA 拷贝数不同，并且会受到多种人类疾病的影响。目前，常见的方案通过对大量样品进行测序或 qPCR 来估计每个细胞的 mtDNA 分子数量。然而，这不允许深入了解细胞间的异质性，并且可能掩盖表型亚群。在这里，我们介绍 mtFociCounter，这是一种单细胞图像分析工具，用于对类核和其他焦点进行可重复定量。mtFociCounter 是一款轻量级、开源免费软件，克服了当前线粒体焦点可重复单细胞分析的限制。我们通过分析 2165 个单个成纤维细胞来证明其用途，并观察到细胞间的核苷数量存在很大的异质性。此外，mtFoci 对线粒体含量进行了定量，我们的结果表明，类核数量和线粒体面积之间存在良好的相关性 (R=0.90)，并且我们发现类核密度的变化小于野生型细胞中的类核数量。最后，我们证明 mtFociCounter 可以轻松检测样品处理后焦点数量的差异，并适用于线粒体 RNA 颗粒和超分辨率显微镜。mtFociCounter 提供了一种多功能解决方案，可重复地量化单细胞中的细胞焦点，我们的结果强调了在线粒体焦点分析中考虑细胞间差异和线粒体背景的重要性。

Mitochondrial DNA (mtDNA) encodes the core subunits for OXPHOS, essential in near-all eukaryotes. Packed into distinct foci (nucleoids) inside mitochondria, the number of mtDNA copies differs between cell-types and is affected in several human diseases. Currently, common protocols estimate per-cell mtDNA-molecule numbers by sequencing or qPCR from bulk samples. However, this does not allow insight into cell-to-cell heterogeneity and can mask phenotypical sub-populations. Here, we presentmtFociCounter, a single-cell image analysis tool for reproducible quantification of nucleoids and other foci.mtFociCounteris a light-weight, open-source freeware and overcomes current limitations to reproducible single-cell analysis of mitochondrial foci. We demonstrate its use by analysing 2165 single fibroblasts, and observe a large cell-to-cell heterogeneity in nucleoid numbers. In addition,mtFociCounterquantifies mitochondrial content and our results show good correlation (R= 0.90) between nucleoid number and mitochondrial area, and we find nucleoid density is less variable than nucleoid numbers in wild-type cells. Finally, we demonstratemtFociCounterreadily detects differences in foci-numbers upon sample treatment, and applies to Mitochondrial RNA Granules and superresolution microscopy.mtFociCounterprovides a versatile solution to reproducibly quantify cellular foci in single cells and our results highlight the importance of accounting for cell-to-cell variance and mitochondrial context in mitochondrial foci analysis.