Real-time imaging of the axonal transport of granules containing a tissue plasminogen activator green fluorescent protein hybrid
Real-time imaging of the axonal transport of granules containing a tissue plasminogen activator green fluorescent protein hybrid
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DOI：
10.1091/mbc.9.9.2463
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发表时间：
1998-09-01
影响因子：
3.3
通讯作者：
Scalettar, BA
中科院分区：
文献类型：
--
作者：
Lochner, JE;Kingma, M;Scalettar, BA
以大鼠组织纤溶酶原激活物(TPA)和绿色荧光蛋白(GFP)为载体，在PC12细胞中表达了tPA/GFP杂合蛋白，并用于研究含有tPA的分泌颗粒在神经元型活细胞中的分布、分泌行为和动力学。高分辨率图像显示tPA/GFP在分化的细胞中具有偏向生长锥的分布，并且tPA/GFP在与含有分泌型颗粒素II的颗粒共定位的调节分泌途径的颗粒中运输。分泌的时间推移图像显示分泌剂导致细胞内tPA/GFP荧光的大量丧失，最重要的是来自生长锥。含有tPA/GFP的颗粒轴突运输的时间推移图像揭示了颗粒动力学的惊人复杂性。一些颗粒经历典型的快速轴突运输；另一些颗粒移动较慢，特别是在强荧光神经突起中。最引人注目的是，颗粒沿着轴突双向运输，这在一定程度上取决于颗粒的堆积，单个颗粒可以逆转其运动方向。这种双向转运的逆行成分可能通过将多余的tPA/GFP转运回细胞体来帮助维持细胞的动态平衡。本文的结果为分泌颗粒的轴突运输提供了一个新的视角。此外，结果表明，tPA的靶向是调节分化细胞生长锥体的分泌，在轴突延长过程中战略性地定位tPA以降解细胞外屏障或激活其他屏障降解酶。
A hybrid protein, tPA/GFP, consisting of rat tissue plasminogen activator (tPA) and green fluorescent protein (GFP) was expressed in PC12 cells and used to study the distribution, secretory behavior, and dynamics of secretory granules containing tPA in living cells with a neuronal phenotype. High-resolution images demonstrate that tPA/ GFP has a growth cone-biased distribution in differentiated cells and that tPA/GFP is transported in granules of the regulated secretory pathway that colocalize with granules containing secretogranin II. Time-lapse images of secretion reveal that secretagogues induce substantial loss of cellular tPA/GFP fluorescence, most importantly from growth cones. Time-lapse images of the axonal transport of granules containing tPA/GFP reveal a surprising complexity to granule dynamics. Some granules undergo canonical fast axonal transport; others move somewhat more slowly, especially in highly fluorescent neurites. Most strikingly, granules traffic bidirectionally along neurites to an extent that depends on granule accumulation, and individual granules can reverse their direction of motion. The retrograde component of this bidirectional transport may help to maintain cellular homeostasis by transporting excess tPA/GFP back toward the cell body. The results presented here provide a novel view of the axonal transport of secretory granules. in addition, the results suggest that tPA is targeted for regulated secretion from growth cones of differentiated cells, strategically positioning tPA to degrade extracellular barriers or to activate other barrier-degrading proteases during axonal elongation.