Monitoring Chemokine Receptor Trafficking by Confocal Immunofluorescence Microscopy.
Monitoring Chemokine Receptor Trafficking by Confocal Immunofluorescence Microscopy.
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DOI：
10.1016/bs.mie.2015.10.004
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发表时间：
2016
影响因子：
--
通讯作者：
Marchese A
中科院分区：
文献类型：
--
作者：
Marchese A
在这里，我们描述了一个协议，检测趋化因子受体CXCR 4共聚焦免疫荧光显微镜在HeLa细胞处理其趋化因子配体CXCL 12。通常，配体激活的趋化因子受体经历脱敏和/或从质膜内化的多步骤过程以终止信号传导。一旦内化到内体，趋化因子受体容易进入再循环途径并返回到细胞表面，引起信号传导的再敏感化。趋化因子受体CXCR 4在被CXCL 12激活时也被内化到内体，但与许多趋化因子受体相反，它主要被分选到降解途径，导致CXCR 4的细胞补体损失和信号传导的长期下调。CXCR 4从早期内体到溶酶体的运输可以通过共聚焦免疫荧光显微镜通过对固定的细胞进行受体的免疫染色和这些囊泡隔室的标记而容易地检测到。这种方法是有利的，因为它可用于鉴定调节CXCR 4从早期内体到溶酶体的运输的因子。这里描述的方案侧重于CXCR 4，但它可以很容易地适应其他趋化因子受体。
Here, we describe a protocol to detect chemokine receptor CXCR4 by confocal immunofluorescence microscopy in HeLa cells treated with its chemokine ligand CXCL12. Typically, ligand-activated chemokine receptors undergo a multistep process of desensitization and/or internalization from the plasma membrane in order to terminate signaling. Once internalized to endosomes, chemokine receptors readily enter the recycling pathway and return to the cell surface, giving rise to resensitization of signaling. The chemokine receptor CXCR4, when activated by CXCL12 is also internalized to endosomes, but in contrast to many chemokine receptors it is mainly sorted to the degradative pathway, contributing to a loss in the cellular complement of CXCR4 and long-term downregulation of signaling. The trafficking of CXCR4 from early endosomes to lysosomes can be easily detected by confocal immunofluorescence microscopy by immunostaining fixed cells for the receptor and with markers of these vesicular compartments. This approach is advantageous because it can be used to identify factors that regulate the trafficking of CXCR4 from early endosomes to lysosomes. The protocol described here focuses on CXCR4, but it can be easily adapted to other chemokine receptors.
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影响因子：
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