Mechanisms of Granule Membrane Recapture following Exocytosis in Intact Mast Cells
Mechanisms of Granule Membrane Recapture following Exocytosis in Intact Mast Cells
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DOI：
10.1074/jbc.m113.459065
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发表时间：
2013-07-12
影响因子：
4.8
通讯作者：
Ales, Eva
中科院分区：
文献类型：
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作者：
Cabeza, Jose M.;Acosta, Jorge;Ales, Eva
在分泌细胞中，已经提出了几种与胞吐作用相关的内吞作用，包括网格蛋白介导的内吞作用、接吻运行内吞作用、腔捕获和大量内吞作用。这些形式的内吞作用可以在不同条件下诱导，但其详细的分子机制和功能很大程度上未知。我们使用膜片钳技术的穿孔贴片和全细胞配置，使用细胞电容测量与电流血清素检测相结合，研究了肥大细胞的胞吐作用和内吞作用。我们发现完整的肥大细胞表现出在化合物48/80诱导的胞吐作用之后的早期内吞作用。对个体胞吐和胞吞事件的直接观察显示，完整肥大细胞中的电容闪烁（27.3%）和失步（11.4%）百分比高于透析细胞（分别为5.4%和2.9%）。此外，我们观察到一种大片膜的内吞作用，这可能是由几种分泌颗粒与细胞膜累积融合形成的。我们还发现了大内吞事件后发生的“大电容闪烁”。孔导分析表明，这些瞬态事件可能代表“复合空捕获”，很可能是由于扩张的融合孔的闪烁所致。使用单个胞吐和内吞事件的荧光成像，我们观察到颗粒可以与已经与质膜融合的颗粒融合，然后融合颗粒的膜和致密核心被内化。总而言之，我们的结果表明，完整肥大细胞中受刺激的胞吐作用之后是几种形式的补偿性内吞作用，包括接吻运行内吞作用和通过单个膜裂变事件有效回收多个分泌颗粒的复合膜的机制。
In secretory cells, several exocytosis-coupled forms of endocytosis have been proposed including clathrin-mediated endocytosis, kiss-and-run endocytosis, cavicapture, and bulk endocytosis. These forms of endocytosis can be induced under different conditions, but their detailed molecular mechanisms and functions are largely unknown. We studied exocytosis and endocytosis in mast cells with both perforated-patch and whole-cell configurations of the patch clamp technique using cell capacitance measurements in combination with amperometric serotonin detection. We found that intact mast cells exhibit an early endocytosis that follows exocytosis induced by compound 48/80. Direct observation of individual exocytic and endocytic events showed a higher percentage of capacitance flickers (27.3%) and off-steps (11.4%) in intact mast cells than in dialyzed cells (5.4% and 2.9%, respectively). Moreover, we observed a type of endocytosis of large pieces of membrane that were likely formed by cumulative fusion of several secretory granules with the cell membrane. We also identified "large-capacitance flickers" that occur after large endocytosis events. Pore conductance analysis indicated that these transient events may represent "compound cavicapture," most likely due to the flickering of a dilated fusion pore. Using fluorescence imaging of individual exocytic and endocytic events we observed that granules can fuse to granules already fused with the plasma membrane, and then the membranes and dense cores of fused granules are internalized. Altogether, our results suggest that stimulated exocytosis in intact mast cells is followed by several forms of compensatory endocytosis, including kiss-and-run endocytosis and a mechanism for efficient retrieval of the compound membrane of several secretory granules through a single membrane fission event.