Oheim M, Stühmer W
Max-Planck Institute for Experimental Medicine, Molecular Biology of Neuronal Signals, Göttingen, Germany.
Eur Biophys J. 2000;29(2):67-89. doi: 10.1007/s002490050253.
Quantitative time-lapse evanescent-wave imaging of individual fluorescently labelled chromaffin granules was used for kinetic analysis of granule trafficking through a approximately 300-nm(1/e2) optical section beneath the plasma membrane. The mean squared displacement (MSD) was used to estimate the three-dimensional diffusion coefficient (D(3)). We calculated the granules' speed, frame-to-frame displacement and direction and their autocorrelation to identify different stages of approach to the membrane. D(3) was about 10,000 times lower than expected for free diffusion. Granules located approximately 60 nm beneath the plasma membrane moved on random tracks (D(3) approximately 10(-10) cm(2)s(-1)) with several reversals in direction before they approached their docking site at angles larger than 45 degrees. Docking was observed as a loss of vesicle mobility by two orders of magnitude within <100 ms. For longer observation times the MSD saturated, as if the granules' movement was confined to a volume only slightly larger than the granule. Rarely, the local random motion was superimposed with a directed movement in a plane beneath the membrane. Stimulation of exocytosis selectively depleted the immobile, near-membrane granule population and caused a recruitment of distant granules to sites at the plasma membrane. Their absolute mobility levels were not significantly altered. Application of latrunculin or jasplakinolide to change F-actin polymerisation caused a change in D(3) of the mobile granule population as well as a reduction of the rate of release, suggesting that granule mobility is constrained by the filamentous actin meshwork and that stimulation-dependent changes in actin viscosity propel granules through the actin cortex.
对单个荧光标记的嗜铬颗粒进行定量延时倏逝波成像，用于动力学分析颗粒通过质膜下方约300纳米（1/e²）光学切片的运输。均方位移（MSD）用于估计三维扩散系数（D(3)）。我们计算了颗粒的速度、逐帧位移和方向及其自相关，以识别接近膜的不同阶段。D(3)比自由扩散预期值低约10000倍。位于质膜下方约60纳米处的颗粒在随机轨道上移动（D(3)约为10⁻¹⁰平方厘米·秒⁻¹），在以大于45度的角度接近其对接位点之前，方向有几次反转。对接表现为囊泡迁移率在<100毫秒内下降两个数量级。对于更长的观察时间，MSD饱和，就好像颗粒的运动局限于仅略大于颗粒的体积内。很少有局部随机运动与膜下方平面内的定向运动叠加。刺激胞吐作用选择性地耗尽了固定的、近膜颗粒群体，并导致远处颗粒募集到质膜位点。它们的绝对迁移率水平没有显著改变。应用拉春库林或茉莉素内酯改变F-肌动蛋白聚合，导致可移动颗粒群体的D(3)发生变化，以及释放速率降低，这表明颗粒迁移率受丝状肌动蛋白网络的限制，并且肌动蛋白粘度的刺激依赖性变化推动颗粒穿过肌动蛋白皮层。
