Supersensitive Ras activation in dendrites and spines revealed by two-photon fluorescence lifetime imaging
Supersensitive Ras activation in dendrites and spines revealed by two-photon fluorescence lifetime imaging
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DOI：
10.1038/nn1635
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发表时间：
2006-02-01
影响因子：
25
通讯作者：
Svoboda, K
中科院分区：
文献类型：
--
作者：
Yasuda, R;Harvey, CD;Svoboda, K
为了了解神经活动调控的生化信号，有必要测量神经元微室（如轴突、树突及其棘）中的蛋白质-蛋白质相互作用和酶活性。我们将双光子激发激光扫描与荧光寿命成像相结合，以高分辨率测量脑切片的荧光共振能量转移。我们还开发了基于荧光蛋白的敏感传感器，用于激活小GTPase蛋白Ras，具有慢（FRas）和快（FRas- f）动力学。使用FRas-F，我们发现在CA1海马神经元中，反向传播动作电位的训练快速且可逆地激活了树突和棘中的Ras。放电速率与Ras激活呈高度非线性关系（Hill系数接近5）。这种高度依赖是由钙离子（Ca2+）和Ras激活剂之间的高度合作相互作用引起的。因此，Ras通路作为神经活动和Ca2+浓度的超敏感阈值检测器。
To understand the biochemical signals regulated by neural activity, it is necessary to measure protein-protein interactions and enzymatic activity in neuronal microcompartments such as axons, dendrites and their spines. We combined two-photon excitation laser scanning with fluorescence lifetime imaging to measure fluorescence resonance energy transfer at high resolutions in brain slices. We also developed sensitive fluorescent protein-based sensors for the activation of the small GTPase protein Ras with slow ( FRas) and fast ( FRas-F) kinetics. Using FRas-F, we found in CA1 hippocampal neurons that trains of back-propagating action potentials rapidly and reversibly activated Ras in dendrites and spines. The relationship between firing rate and Ras activation was highly nonlinear ( Hill coefficient similar to 5). This steep dependence was caused by a highly cooperative interaction between calcium ions ( Ca2+) and Ras activators. The Ras pathway therefore functions as a supersensitive threshold detector for neural activity and Ca2+ concentration.