Quantitative Imaging and Simulation Study for Analyzing Exocytosis in PC12 Cell

胞吐是一种重要的生物学现象，尤其是神经细胞和内分泌细胞。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，我们采用生物成像和随机模拟相结合的方法，开发了一种用于胞吐的系统生物学方法。对于定量模拟，需要同时成像细胞内钙升高作为胞吐的触发因素，也需要对胞吐事件进行定量计数。通过双视图技术的成像方法，我们可以通过荧光染料、Fura-red和gfp融合神经肽y的胞外分泌来观察细胞内Ca的反应，ATP的刺激诱导了Ca的快速和短暂的反应，随后的胞外分泌延迟了几秒钟。这表明我们可以从细胞内Ca的第一次触发反应到单细胞内神经递质释放的最后事件来追踪胞外分泌。接下来，我们开发了一种基于随机Gillespie方法的模拟技术。将胞吐的预融合过程分为5个阶段，并估计了过渡概率，以拟合实验数据。我们成功地揭示了胞内钙的短暂升高所引起的胞吐。这个随机模型包括调节胞吐作用的酶活性。最后，我们还成功地用单随机模型的双级联模拟了染色质细胞的顺序胞吐。我们认为同时成像和定量随机模拟为胞吐系统生物学提供了一种新的分析技术。

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.