Investigating Mast Cell Secretory Granules; from Biosynthesis to Exocytosis
Investigating Mast Cell Secretory Granules; from Biosynthesis to Exocytosis
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DOI：
10.3791/52505
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发表时间：
2015-01-01
影响因子：
1.2
通讯作者：
Sagi-Eisenberg, Ronit
中科院分区：
文献类型：
--
作者：
Azouz, Nurit P.;Fukuda, Mitsunori;Sagi-Eisenberg, Ronit
肥大细胞（Mast Cells，MC）是免疫系统的分泌细胞，通过释放预先形成和新合成的过敏、炎症和免疫调节介质来完成其生理和病理功能。MC的介质影响多个组织和器官，最终导致过敏和免疫反应。MC介质的合成、储存和释放受到高度调节。预先形成的介质包装在细胞质分泌颗粒（SG）中，与质膜融合并通过调节胞吐作用释放其内容物。我们提出了一个协议，基于共同表达的一个感兴趣的基因与一个报告基因，有针对性的SGs和被释放在一个受管制的方式旁边的内源性SG介质。该方案能够实现MC SG的高分辨率四维共聚焦分析，并监测它们从生物发生到触发胞吐的时间轴。因此，使用该方案筛选感兴趣的基因的表型和功能的影响，允许破译的分子机制，管理MC SG的生物合成和胞吐作用，并确定涉及的监管机构。因此，应提供对MC在健康和疾病中的功能的细胞机制的进一步见解。
Mast Cells (MC) are secretory cells of the immune system that accomplish their physiological and pathological functions by releasing pre-formed and newly synthesized allergic, inflammatory and immunoregulatory mediators. MCs' mediators affect multiple tissues and organs culminating in allergic and immune responses. The synthesis, storage and release of the MC mediators are highly regulated. The pre-formed mediators are packed in cytoplasmic secretory granules (SG) that fuse with the plasma membrane and release their content by regulated exocytosis. We present a protocol, based on the co-expression of a gene of interest with a reporter gene that is targeted to the SGs and is released in a regulated fashion alongside the endogenous SG mediators. The protocol enables high resolution four dimensional confocal analyses of the MC SGs and monitoring their timeline from biogenesis to triggered exocytosis. Thus, using this protocol for screening genes of interest for their phenotypic and functional impact allows deciphering the molecular mechanisms that govern the biogenesis and exocytosis of the MC SGs and identifying the regulators involved. Thereby, further insights into the cellular mechanisms that account for MCs function in health and disease should be provided.