肌管自动定量分割揭示S6激酶激活的单细胞动力学。

IF 2 4区 生物学 Q4 CELL BIOLOGY
Cell structure and function Pub Date : 2018-08-31 Epub Date: 2018-07-26 DOI:10.1247/csf.18012
Haruki Inoue, Katsuyuki Kunida, Naoki Matsuda, Daisuke Hoshino, Takumi Wada, Hiromi Imamura, Hiroyuki Noji, Shinya Kuroda
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引用次数: 2

摘要

在活细胞荧光成像中,自动细胞分割是定量单细胞分辨率信号动力学的一种有效方法。单核细胞和圆形细胞的分割方法得到了广泛的发展。然而,一种细长多核细胞的分割方法,如分化的C2C12肌管,尚未开发。此外,肌管被未分化的储备细胞包围,使得难以识别背景区域和随后的定量。本文提出了一种基于二值化和图像分水岭分割的肌管自动定量分割方法。我们使用了稳定表达S6激酶(S6K)荧光共振能量转移(FRET)生物传感器evee-S6K的分化C2C12细胞的延时荧光图像。二值图像的总和增强了肌管和储备细胞之间的对比度,允许检测肌管和肌管中心。使用肌管中心代替细胞核,可以通过分水岭分割自动检测单个肌管。此外,使用双分量高斯混合模型的背景校正允许在单个肌管中自动量化信号强度。因此,我们提供了一种结合自动肌管检测和背景校正的自动定量分割方法。此外,该方法使我们能够量化单个肌管中的S6K活性,证明S6K活性的一些时间特性,如峰值时间和适应半衰期,在细胞群体和个体之间显示出不同的胰岛素剂量依赖性变化。关键词:延时图像,细胞分割,荧光共振能量传递,C2C12,肌管
本文章由计算机程序翻译,如有差异,请以英文原文为准。
Automatic Quantitative Segmentation of Myotubes Reveals Single-cell Dynamics of S6 Kinase Activation.

Automatic cell segmentation is a powerful method for quantifying signaling dynamics at single-cell resolution in live cell fluorescence imaging. Segmentation methods for mononuclear and round shape cells have been developed extensively. However, a segmentation method for elongated polynuclear cells, such as differentiated C2C12 myotubes, has yet to be developed. In addition, myotubes are surrounded by undifferentiated reserve cells, making it difficult to identify background regions and subsequent quantification. Here we developed an automatic quantitative segmentation method for myotubes using watershed segmentation of summed binary images and a two-component Gaussian mixture model. We used time-lapse fluorescence images of differentiated C2C12 cells stably expressing Eevee-S6K, a fluorescence resonance energy transfer (FRET) biosensor of S6 kinase (S6K). Summation of binary images enhanced the contrast between myotubes and reserve cells, permitting detection of a myotube and a myotube center. Using a myotube center instead of a nucleus, individual myotubes could be detected automatically by watershed segmentation. In addition, a background correction using the two-component Gaussian mixture model permitted automatic signal intensity quantification in individual myotubes. Thus, we provide an automatic quantitative segmentation method by combining automatic myotube detection and background correction. Furthermore, this method allowed us to quantify S6K activity in individual myotubes, demonstrating that some of the temporal properties of S6K activity such as peak time and half-life of adaptation show different dose-dependent changes of insulin between cell population and individuals.Key words: time lapse images, cell segmentation, fluorescence resonance energy transfer, C2C12, myotube.

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来源期刊
Cell structure and function
Cell structure and function 生物-细胞生物学
CiteScore
2.50
自引率
0.00%
发文量
6
审稿时长
>12 weeks
期刊介绍: Cell Structure and Function is a fully peer-reviewed, fully Open Access journal. As the official English-language journal of the Japan Society for Cell Biology, it is published continuously online and biannually in print. Cell Structure and Function publishes important, original contributions in all areas of molecular and cell biology. The journal welcomes the submission of manuscripts on research areas such as the cell nucleus, chromosomes, and gene expression; the cytoskeleton and cell motility; cell adhesion and the extracellular matrix; cell growth, differentiation and death; signal transduction; the protein life cycle; membrane traffic; and organelles.
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