Quantitative Imaging and Simulation Study for Analyzing Exocytosis in PC12 Cell

胞吐作用是一种重要的生物学现象，特别是对于神经和内分泌细胞。尽管生物物理学、细胞生物学、生物化学和分子生物学等不同科学领域的实验技术已经被开发出来用于分析这一现象，但我们还没有达到对胞吐作用的定量理解。因此，我们发展了一个系统生物学的方法与生物成像和随机模拟的组合方法的胞吐。对于定量模拟，需要同时成像细胞内Ca增加作为胞吐的触发，并且还需要胞吐事件的定量计数。通过双视图技术的成像方法使我们能够通过荧光染料Fura红和GFP融合的神经肽Y的胞吐作用来可视化细胞内Ca响应。ATP的刺激引起了一个快速和短暂的反应的Ca和随后的胞吐与几秒钟的延迟。这表明，我们可以跟踪胞吐从细胞内钙的第一个触发反应和神经递质释放的最终事件在单个细胞。接下来，我们开发了一种新的模拟技术的基础上的随机吉莱斯皮方法。将胞吐融合前的过程分为5个阶段，并对各阶段的跃迁概率进行了估计，以拟合实验数据。该随机模型包括用于调节胞吐的酶活性。最后，我们还成功地模拟了单随机模型的双级联在嗜铬细胞中的顺序胞吐。我们的结论是，同时成像和定量随机模拟为我们提供了一个新的分析技术的系统生物学的胞吐。

Exocytosis is an important biological phenomenon, especially for neural and endocrine cells. Although several experimental techniques from various fields of sciences including biophysics, cell biology, biochemistry and also molecular biology have been developed for analyzing this phenomenon, we have not reached the quantitative understandings for exocytosis. We, therefore, developed a systems biological approach for exocytosis with the combinatorial method of biological imaging and stochastic simulation. For the quantitative simulation, simultaneous imaging for intracellular Ca increase as a trigger of exocytosis, and also for quantitative counting of events of exocytosis is required. An imaging method by a dual view technique enables us to visualize the intracellular Ca response by a fluorescent dye, Fura-red and exocytosis by GFP-fused neuropeptide Y. Stimulation by ATP induced a quick and transient response of Ca and following exocytosis with a several seconds delay. This indicates that we can trace the exocytosis from the first trigger response of intracellular Ca and the final event of neurotransmitter release in single cells. Next, we develop a new simulation technique based on the stochastic Gillespie method. Pre-fusion process of exocytosis was divided to 5 stages, and the transition probabilities were estimated to fit the experimental data We succeed to reveal the exocytosis induced by a transient intracellular Ca increase. This stochastic model includes the enzymatic activity for modulating exocytosis. Finally, we also succeed to simulate the sequential exocytosis reported in chromaffin cells with double cascade of the single stochastic model. We conclude that simultaneous imaging and quantitative stochastic simulation provide us a new analytical technique for systems biology of exocytosis.