Visualization of regulated exocytosis with a granule membrane probe using total internal reflection microscopy
Visualization of regulated exocytosis with a granule membrane probe using total internal reflection microscopy
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DOI：
10.1091/mbc.e04-02-0149
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发表时间：
2004-10-01
影响因子：
3.3
通讯作者：
Holz, RW
中科院分区：
文献类型：
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作者：
Allersma, MW;Wang, L;Holz, RW
当通过全内反射荧光显微镜观察时，用Vamp-绿色荧光蛋白（GFP）标记的分泌颗粒在胞吐后显示出不同的特征。在类似于90%的融合事件中，我们观察到荧光强度的大幅增加，伴随着从小的点状外观到更大的扩散云的过渡，Vamp-GFP自由扩散到质膜中。定量表明，这些事件反映了随着Vamp-GFP同时扩散通过融合结，最初融合的球形颗粒变平进入质膜平面的进展。大约10%的事件显示出从点状结构到环状结构的转变，几乎没有或没有扩散。环状图像定量地对应于颗粒融合和保留颗粒（类似于200 nm的凹陷）。大多数融合事件涉及存在于渐逝场中至少12 s的颗粒。然而，类似于20%的事件涉及颗粒存在于渐逝场中不超过0.3秒，表明颗粒与质膜的相互作用导致胞吐作用可以在该时间内发生。此外，类似于10%的胞吐部位更可能发生在先前事件的颗粒直径内，而不是偶然发生，这表明在其他细胞中观察到的顺序（背驮式）胞吐。在融合之前和融合期间的整体颗粒行为与先前在组成性分泌途径中描述的胞吐作用惊人地相似。
Secretory granules labeled with Vamp-green fluorescent protein (GFP) showed distinct signatures upon exocytosis when viewed by total internal reflection fluorescence microscopy. In similar to90% of fusion events, we observed a large increase in fluorescence intensity coupled with a transition from a small punctate appearance to a larger, spreading cloud with free diffusion of the Vamp-GFP into the plasma membrane. Quantitation suggests that these events reflect the progression of an initially fused and spherical granule flattening into the plane of the plasma membrane as the Vamp-GFP simultaneously diffuses through the fusion junction. Approximately 10% of the events showed a transition from puncta to ring-like structures coupled with little or no spreading. The ring-like images correspond quantitatively to granules fusing and retaining concavity (recess of similar to200 nm). A majority of fusion events involved granules that were present in the evanescent field for at least 12 s. However, similar to20% of the events involved granules that were present in the evanescent field for no more than 0.3 s, indicating that the interaction of the granule with the plasma membrane that leads to exocytosis can occur within that time. In addition, similar to10% of the exocytotic sites were much more likely to occur within a granule diameter of a previous event than can be accounted for by chance, suggestive of sequential (piggy-back) exocytosis that has been observed in other cells. Overall granule behavior before and during fusion is strikingly similar to exocytosis previously described in the constitutive secretory pathway.