Rapamycin-Based Inducible Translocation Systems for Studying Phagocytosis
Rapamycin-Based Inducible Translocation Systems for Studying Phagocytosis
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DOI：
10.1007/978-1-61779-139-0_13
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发表时间：
2011-01-01
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影响因子：
--
通讯作者：
Fairn, Gregory D.
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文献类型：
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作者：
Bohdanowicz, Michel;Fairn, Gregory D.
吞噬作用是一种免疫受体介导的细胞吞噬大颗粒的过程。这个过程是动态的，需要几个局部因子与免疫受体协同作用，并在免疫受体参与后依次作用，以包裹颗粒。一旦颗粒被内化，新生的吞噬体经历一系列事件，导致其成熟为杀微生物的吞噬溶酶体。在活细胞中研究这些动态和时间控制的事件系列需要非侵入性方法。快速募集目标蛋白质到吞噬作用位点或新生吞噬体的能力将有助于剖析吞噬作用期间涉及的调节机制。在这里，我们描述了一个通用的方法来表达RAW264.7小鼠巨噬细胞，基因编码的雷帕霉素诱导异源二聚化系统。在雷帕霉素存在下，观察到FK506结合蛋白（FKBP）和FKBP雷帕霉素结合蛋白（FRB）之间的紧密结合。基于该原理，由连接至FKBP的靶向结构域组成的合成系统可以在添加雷帕霉素后募集融合至FRB的感兴趣蛋白。以前，这种技术已被用于靶向脂质修饰酶和小GTP酶的吞噬体或质膜。如果荧光蛋白与FRB序列融合，则可以通过荧光显微镜监测FRB模块的募集。虽然本章的重点是吞噬事件，这种方法可以用来研究任何感兴趣的细胞器时，使用适当的靶向序列。
Phagocytosis is an immune receptor-mediated process whereby cells engulf large particles. The process is dynamic and requires several localized factors acting in concert with and sequentially after the engagement of immune receptors to envelope the particle. Once the particle is internalized, the nascent phagosome undergoes a series of events leading to its maturation to the microbicidal phagolysosome. Investigating these dynamic and temporally controlled series of events in live cells requires noninvasive methods. The ability to rapidly recruit the proteins of interest to the sites of phagocytosis or to nascent phagosomes would help dissect the regulatory mechanisms involved during phagocytosis. Here, we describe a general approach to express in RAW264.7 murine macrophages, a genetically encoded rapamycin-induced heterodimerization system. In the presence of rapamycin, tight association between FK506-binding protein (FKBP) and FKBP rapamycin-binding protein (FRB) is observed. Based on this principle, a synthetic system consisting of a targeting domain attached to FKBP can recruit a protein of interest fused to FRB upon the addition of rapamycin. Previously, this technique has been used to target lipid-modifying enzymes and small GTPases to the phagosome or plasma membrane. The recruitment of the FRB module can be monitored by fluorescent microscopy if a fluorescent protein is fused to the FRB sequence. While the focus of this chapter is on phagocytic events, this method can be employed to study any organelle of interest when the appropriate targeting sequence is used.