Johns L M, Levitan E S, Shelden E A, Holz R W, Axelrod D
Department of Pharmacology, The University of Michigan, Ann Arbor, Michigan 48109, USA.
J Cell Biol. 2001 Apr 2;153(1):177-90. doi: 10.1083/jcb.153.1.177.
We used total internal reflection fluorescence microscopy to study quantitatively the motion and distribution of secretory granules near the plasma membrane (PM) of living bovine chromaffin cells. Within the approximately 300-nm region measurably illuminated by the evanescent field resulting from total internal reflection, granules are preferentially concentrated close to the PM. Granule motion normal to the substrate (the z direction) is much slower than would be expected from free Brownian motion, is strongly restricted over tens of nanometer distances, and tends to reverse directions within 0.5 s. The z-direction diffusion coefficients of granules decrease continuously by two orders of magnitude within less than a granule diameter of the PM as granules approach the PM. These analyses suggest that a system of tethers or a heterogeneous matrix severely limits granule motion in the immediate vicinity of the PM. Transient expression of the light chains of tetanus toxin and botulinum toxin A did not disrupt the restricted motion of granules near the PM, indicating that SNARE proteins SNAP-25 and VAMP are not necessary for the decreased mobility. However, the lack of functional SNAREs on the plasma or granule membranes in such cells reduces the time that some granules spend immediately adjacent to the PM.
我们使用全内反射荧光显微镜对活牛嗜铬细胞质膜（PM）附近分泌颗粒的运动和分布进行了定量研究。在由全内反射产生的倏逝场可测量照亮的约300纳米区域内，颗粒优先集中在靠近质膜的位置。颗粒垂直于底物（z方向）的运动比自由布朗运动预期的要慢得多，在几十纳米的距离上受到强烈限制，并且倾向于在0.5秒内反向。随着颗粒靠近质膜，颗粒在z方向的扩散系数在小于颗粒直径的距离内连续下降两个数量级。这些分析表明，一种拴系系统或异质基质严重限制了颗粒在质膜紧邻区域的运动。破伤风毒素和肉毒杆菌毒素A轻链的瞬时表达并未破坏颗粒在质膜附近的受限运动，表明SNARE蛋白SNAP-25和VAMP对于降低的流动性不是必需的。然而，此类细胞中质膜或颗粒膜上缺乏功能性SNARE会减少一些颗粒紧邻质膜的停留时间。
