Modeling capping protein FRAP and CALI experiments reveals in vivo regulation of actin dynamics.
Modeling capping protein FRAP and CALI experiments reveals in vivo regulation of actin dynamics.
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DOI：
10.1002/cm.20463
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发表时间：
2010-08
期刊：
影响因子：
2.9
通讯作者：
Jacobson, Ken
中科院分区：
文献类型：
--
作者：
Kapustina, Maryna;Vitriol, Eric;Elston, Timothy C.;Loew, Leslie M.;Jacobson, Ken
为了深入了解调控肌动蛋白聚合的细胞机制，我们使用虚拟细胞来模拟FRAP和发色团辅助激光失活(CALI)对EGFP封顶蛋白(EGFP-CP)的实验。FRAP动力学模型显示，肌动蛋白微丝中CP的体内解离速率(~0.1S−1)远快于体外的解离速率(0.01%~0.0004 S−1)。CALI模拟表明，为了诱导CP失活后细胞形态的持续变化，细胞应该表现出抗封顶能力。我们在模拟方案中加入了Vasp蛋白作为抗封顶剂。该模型预测Vasp对带刺末端的亲和力与Vasp-带刺末端复合体的浓度具有协同依赖关系。这种依赖关系产生了正反馈，稳定了络合物，并允许簇状细丝尖端的持续生长。我们分析了足以引起细胞形态变化的激光强度范围。这一分析表明，使用EGFP-CP的FRAP实验可以在不改变细胞形态的情况下安全地进行，因为光漂白光束的强度不够高，不足以在EGFP-CP发生扩散替换之前产生诱导丝状生长的自由带刺末端的临界浓度。
To gain insights on cellular mechanisms regulating actin polymerization, we used the Virtual Cell to model FRAP and chromophore assisted laser inactivation (CALI) experiments on EGFP-capping protein (EGFP-CP). Modeling the FRAP kinetics demonstrated that the in vivo rate for the dissociation of CP from actin filaments is much faster (~0.1 s−1) than that measured in vitro (0.01–0.0004 s−1). The CALI simulation revealed that in order to induce sustainable changes in cell morphology after CP inactivation, the cells should exhibit anti-capping ability. We included the VASP protein as the anti-capping agent in the modeling scheme. The model predicts that VASP affinity for barbed ends has a cooperative dependence on the concentration of VASP-barbed end complexes. This dependence produces a positive feedback that stabilizes the complexes and allows sustained growth at clustered filament tips. We analyzed the range of laser intensities that are sufficient to induce changes in cell morphology. This analysis demonstrates that FRAP experiments with EGFP-CP can be performed safely without changes in cell morphology, because, the intensity of the photobleaching beam is not high enough to produce the critical concentration of free barbed ends that will induce filament growth before diffusional replacement of EGFP-CP occurs.
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