Department of Biochemistry, Institute of Physiology, University of Hohenheim, Stuttgart, Germany.
J Histochem Cytochem. 2019 Oct;67(10):745-757. doi: 10.1369/0022155419859870. Epub 2019 Jun 27.
photoreceptor cells are employed as a model system for studying membrane protein transport. Phototransduction proteins like rhodopsin and the light-activated TRPL ion channel are transported within the photoreceptor cell, and they change their subcellular distribution in a light-dependent way. Investigating the transport mechanisms for rhodopsin and ion channels requires accurate histochemical methods for protein localization. By using immunocytochemistry the light-triggered translocation of TRPL has been described as a two-stage process. In stage 1, TRPL accumulates at the rhabdomere base and the adjacent stalk membrane a few minutes after onset of illumination and is internalized in stage 2 by endocytosis after prolonged light exposure. Here, we show that a commonly observed crescent shaped antibody labeling pattern suggesting a fast translocation of rhodopsin, TRP, and TRPL to the rhabdomere base is a light-dependent antibody staining artifact. This artifact is most probably caused by the profound structural changes in the microvillar membranes of rhabdomeres that result from activation of the signaling cascade. By using alternative labeling methods, either eGFP-tags or the self-labeling SNAP-tag, we show that light activation of TRPL transport indeed results in fast changes of the TRPL distribution in the rhabdomere but not in the way described previously.
感光细胞被用作研究膜蛋白运输的模型系统。像视紫红质和光激活的 TRPL 离子通道这样的光转导蛋白在感光细胞内运输，并且它们以依赖于光的方式改变其亚细胞分布。研究视紫红质和离子通道的运输机制需要用于蛋白质定位的准确组织化学方法。通过免疫细胞化学，已经描述了 TRPL 的光触发易位是一个两阶段过程。在第一阶段，TRPL 在光照开始后几分钟在纤毛基部和相邻的柄膜中积累，并在长时间光照后通过内吞作用在第二阶段内化。在这里，我们表明，一种常见的观察到的新月形抗体标记模式表明视紫红质、TRP 和 TRPL 快速易位到纤毛基部，这是一种依赖于光的抗体染色假象。这种假象很可能是由于信号级联激活导致纤毛微绒毛膜的深刻结构变化引起的。通过使用替代标记方法，即 eGFP 标签或自标记 SNAP 标签，我们表明 TRPL 运输的光激活确实导致 TRPL 在纤毛中的分布快速变化，但不是以前描述的方式。
