Transduced viral IL-10 is exocytosed from lacrimal acinar secretory vesicles in a myosin-dependent manner in response to carbachol.
Transduced viral IL-10 is exocytosed from lacrimal acinar secretory vesicles in a myosin-dependent manner in response to carbachol.
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DOI：
10.1016/j.exer.2008.10.023
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发表时间：
2009-03
影响因子：
3.4
通讯作者：
Trousdale, Melvin D.
中科院分区：
文献类型：
--
作者：
Xie, Jiansong;Marchelletta, Ronald R.;Thomas, Padmaja B.;Jacobs, Damon T.;Yarber, Francie A.;Cheney, Richard E.;Hamm-Alvarez, Sarah F.;Trousdale, Melvin D.
本研究的目的是确定细胞内运输和释放途径的治疗蛋白，病毒IL-10（vIL-10），从转导腺泡上皮细胞从兔泪腺。原代培养的兔泪腺腺泡细胞（LGAC）用含病毒白细胞介素-10（AdvIL-10）的腺病毒血清型5转导。共聚焦荧光显微镜观察vIL-10的分布。通过ELISA定量卡巴胆碱（CCH）刺激的vIL-10释放。响应于用调节基于肌动蛋白和肌球蛋白的转运的试剂处理，探测vIL-10定位和胞吐作用。在转导的LGAC中的大胞内囊泡中检测到vIL-10免疫反应性。vIL-10与生物合成标记物部分共定位，但不与内体区室标记物共定位。vIL-10释放对CCH敏感，释放动力学显示与分泌蛋白β-氨基己糖苷酶相似但不相同的初始爆发相。用latrunculin B包裹肌动蛋白丝显著增加了CCH刺激的vIL-10分泌，表明vIL-10是从位于亚顶端肌动蛋白屏障下的储存物中释放的。这种释放需要肌动蛋白依赖性肌球蛋白马达的活性，以前参与分泌囊泡胞吐的研究结果证实，CCH刺激的vIL-10释放减少抑制非肌肉肌球蛋白2和肌球蛋白5c的功能，分别使用ML-7和显性负性肌球蛋白5c的过度表达。这些结果表明，大多数vIL-10转基因产物被包装到分泌囊泡的亚群中，所述分泌囊泡利用肌动蛋白依赖性肌球蛋白马达进行肌动蛋白涂层组装、化合物融合和顶端质膜处的胞吐作用以响应CCH刺激。
The purpose of this study was to determine the intracellular trafficking and release pathways for the therapeutic protein, viral IL-10 (vIL-10), from transduced acinar epithelial cells from rabbit lacrimal gland. Primary cultured rabbit lacrimal gland acinar cells (LGACs) were transduced with adenovirus serotype 5 containing viral interleukin-10 (AdvIL-10). The distribution of vIL-10 was assessed by confocal fluorescence microscopy. Carbachol (CCH)-stimulated release of vIL-10 was quantified by ELISA. vIL-10 localization and exocytosis was probed in response to treatments with agents modulating actin- and myosin-based transport. vIL-10 immunoreactivity was detected in large intracellular vesicles in transduced LGAC. vIL-10 was partially co-localized with biosynthetic but not endosomal compartment markers. vIL-10 release was sensitive to CCH, and the kinetics of release showed an initial burst phase that was similar but not identical to that of the secretory protein, β-hexosaminidase. Disassembly of actin filaments with latrunculin B significantly increased CCH-stimulated vIL-10 secretion, suggesting that vIL-10 was released from stores sequestered beneath the subapical actin barrier. That release required the activity of actin-dependent myosin motors previously implicated in secretory vesicle exocytosis was confirmed by findings that CCH-stimulated vIL-10 release was reduced by inhibition of non-muscle myosin 2 and myosin 5c function, using ML-7 and overexpression of dominant negative myosin 5c, respectively. These results suggest that the majority of vIL-10 transgene product is packaged into a subpopulation of secretory vesicles that utilize actin-dependent myosin motors for aspects of actin coat assembly, compound fusion and exocytosis at the apical plasma membrane in response to CCH stimulation.
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影响因子：
27.4
作者：
Evans, CH;Whalen, JD;Robbins, PD
通讯作者：
Robbins, PD
影响因子：
5.5
作者：
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作者：
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3.3
作者：
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影响因子：
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作者：
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