pHusion: A robust and versatile toolset for automated detection and analysis of exocytosis.
pHusion: A robust and versatile toolset for automated detection and analysis of exocytosis.
复制标题
DOI：
10.1101/2023.07.25.550499
复制
发表时间：
2023-07
期刊：
影响因子：
--
通讯作者：
Ellen C. O’Shaughnessy;Mable Lam;Samantha E Ryken;Theresa Wiesner;Kimberly Lukasik;J. B. Zuchero;Christophe Leterrier;D. Adalsteinsson;S. Gupton
中科院分区：
文献类型：
--
作者：
Ellen C. O’Shaughnessy;Mable Lam;Samantha E Ryken;Theresa Wiesner;Kimberly Lukasik;J. B. Zuchero;Christophe Leterrier;D. Adalsteinsson;S. Gupton
胞吐作用是真核细胞用来调节质膜组成并促进细胞间通讯的基本过程。为了研究胞吐作用在神经元形态发生中的作用，我们之前开发了带有图形用户界面（GUI）的计算工具，以便能够从荧光延时图像中自动检测和分析胞吐事件（ADAE GUI）。尽管这些工具已被证明是有用的，但我们发现代码很脆弱并且不容易适应不同的实验条件。在这里，我们开发并验证了一个强大且多功能的工具包，名为 pHusion，用于分析用 ImageTank 编写的胞吐作用，ImageTank 是一种结合图像可视化和数值方法的图形编程语言。我们使用多种成像方式和 pH 敏感荧光团、多种细胞类型和各种胞吐标记物测试了该方法，以生成灵活直观的包。使用 pHusion，我们发现，与原代少突胶质细胞相比，VAMP3 介导的胞吐作用在黑色素瘤细胞中发生的频率高出 30 倍，成熟大鼠海马神经元中 VAMP2 介导的融合事件比未成熟小鼠皮质神经元中的融合事件持续时间更长，并且在发育中的皮质神经元中，胞吐事件在空间上聚集但在时间上随机。摘要陈述 胞吐作用是细胞改变形状、改变膜组成以及与其他细胞沟通的重要过程。尽管所有真核细胞都进行胞吐作用，但囊泡融合的调节、囊泡的货物以及胞吐作用在细胞命运中所起的作用在不同细胞类型中存在很大差异。在这里，我们开发了一套灵活而强大的工具，能够自动识别和分析各种细胞类型、囊泡类型和成像条件的胞吐事件。
Exocytosis is a fundamental process used by eukaryotic cells to regulate the composition of the plasma membrane and facilitate cell-cell communication. To investigate the role exocytosis plays in neuronal morphogenesis, previously we developed computational tools with a graphical user interface (GUI) to enable the automatic detection and analysis of exocytic events (ADAE GUI) from fluorescence timelapse images. Though these tools have proven useful, we found that the code was brittle and not easily adapted to different experimental conditions. Here, we have developed and validated a robust and versatile toolkit, named pHusion, for the analysis of exocytosis written in ImageTank, a graphical programming language that combines image visualization and numerical methods. We tested this method using a variety of imaging modalities and pH-sensitive fluorophores, diverse cell types, and various exocytic markers to generate a flexible and intuitive package. Using pHusion, we show that VAMP3-mediated exocytosis occurs 30-times more frequently in melanoma cells compared with primary oligodendrocytes, that VAMP2-mediated fusion events in mature rat hippocampal neurons are longer lasting than those in immature murine cortical neurons, and that exocytic events are clustered in space yet random in time in developing cortical neurons. Summary Statement Exocytosis is an essential process by which cells change shape, alter membrane composition, and communicate with other cells. Though all eukaryotic cells carry out exocytosis, the regulation of vesicle fusion, the cargo of vesicles, and the role exocytosis plays in cell fate differ greatly across cell types. Here, we developed a flexible and robust set of tools to enable automatic identification and analysis of exocytic events across a wide range of cell types, vesicle types, and imaging conditions.