Protein mobility within secretory granules.
Protein mobility within secretory granules.
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分泌颗粒内的蛋白质流动性。
DOI：
10.1016/j.bpj.2014.04.063
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发表时间：
2014
影响因子：
3.4
通讯作者：
Axelrod,Daniel
中科院分区：
文献类型：
--
作者：
Weiss,AnnitaNgatchou;Bittner,MaryA;Holz,RonaldW;Axelrod,Daniel
我们研究了以前的观察结果，荧光标记的神经肽Y（NPY）通常在融合后200毫秒内释放，而标记的组织纤溶酶原激活剂（tPA）往往在几秒钟内放电的基础。我们发现tPA和NPY在培养的牛嗜铬细胞的小的和不同的亚群中内源性表达。我们测量了这些蛋白质的流动性（荧光标记）在活嗜铬细胞中的单个分泌颗粒的管腔内，并将其流动性融合后释放动力学。开发了一种不受标准光学分辨率限制的方法，其中由全内反射（TIR）产生的强烈衰减消逝场（~ 64 nm指数衰减常数）的明亮闪光选择性地漂白靠近玻璃盖片的天蓝色标记蛋白质单个颗粒内。当未漂白的蛋白质从300 nm颗粒内的远端区域扩散到漂白的近端区域时，发生荧光恢复。当随后用TIR激发探测时，tPA-天蓝色（tPA-cer）的部分漂白比用落射荧光探测时更大，表明tPA-cer迁移率低。当用TIR和落射荧光探测时，几乎相等的NPY-cer漂白表明NPY-cer在300 ms漂白脉冲内平衡，因此具有比tPA-cer更大的迁移率。光漂白后的TIR荧光恢复显示漂白后几百毫秒内tPA-cer（但不是NPY-cer）荧光的显着恢复。考虑到漂白持续时间、颗粒直径和颗粒中有限数量的荧光团，数值模拟结果与tPA-cer是100%移动的，扩散系数为2 × 10− 10 cm 2/s（水溶液中类似大小蛋白质的1/3000）的结论一致。然而，tPA的低扩散迁移率不能单独解释其缓慢的融合后释放。在随后的研究中，我们认为，此外，tPA本身稳定的融合孔的尺寸，限制自己的出口。
We investigated the basis for previous observations that fluorescent-labeled neuropeptide Y (NPY) is usually released within 200 ms after fusion, whereas labeled tissue plasminogen activator (tPA) is often discharged over many seconds. We found that tPA and NPY are endogenously expressed in small and different subpopulations of bovine chromaffin cells in culture. We measured the mobility of these proteins (tagged with fluorophore) within the lumen of individual secretory granules in living chromaffin cells, and related their mobilities to postfusion release kinetics. A method was developed that is not limited by standard optical resolution, in which a bright flash of strongly decaying evanescent field (∼64 nm exponential decay constant) produced by total internal reflection (TIR) selectively bleaches cerulean-labeled protein proximal to the glass coverslip within individual granules. Fluorescence recovery occurred as unbleached protein from distal regions within the 300 nm granule diffused into the bleached proximal regions. The fractional bleaching of tPA-cerulean (tPA-cer) was greater when subsequently probed with TIR excitation than with epifluorescence, indicating that tPA-cer mobility was low. The almost equal NPY-cer bleaching when probed with TIR and epifluorescence indicated that NPY-cer equilibrated within the 300 ms bleach pulse, and therefore had a greater mobility than tPA-cer. TIR-fluorescence recovery after photobleaching revealed a significant recovery of tPA-cer (but not NPY-cer) fluorescence within several hundred milliseconds after bleaching. Numerical simulations, which take into account bleach duration, granule diameter, and the limited number of fluorophores in a granule, are consistent with tPA-cer being 100% mobile, with a diffusion coefficient of 2 × 10−10cm2/s (∼1/3000 of that for a protein of similar size in aqueous solution). However, the low diffusive mobility of tPA cannot alone explain its slow postfusion release. In the accompanying study, we suggest that, additionally, tPA itself stabilizes the fusion pore with dimensions that restrict its own exit.