Institute of Biology, Department of Biochemistry, University of Hohenheim.
Institute of Biology, Department of Biochemistry, University of Hohenheim;
J Vis Exp. 2022 Jan 21(179). doi: 10.3791/63375.
Membrane protein trafficking regulates the incorporation and removal of receptors and ion channels into the plasma membrane. This process is fundamentally important for cell function and cell integrity of neurons. Drosophila photoreceptor cells have become a model for studying membrane protein trafficking. Besides rhodopsin, which upon illumination becomes internalized from the photoreceptor membrane and is degraded, the transient receptor potential-like (TRPL) ion channel in Drosophila exhibits a light-dependent translocation between the rhabdomeral photoreceptor membrane (where it is located in the dark) and the photoreceptor cell body (to which it is transported upon illumination). This intracellular transport of TRPL can be studied in a simple and non-invasive way by expressing eGFP-tagged TRPL in photoreceptor cells. The eGFP fluorescence can then be observed either in the deep pseudopupil or by water immersion microscopy. These methods allow detection of fluorescence in the intact eye and are therefore useful for high-throughput assays and genetic screens for Drosophila mutants defective in TRPL translocation. Here, the preparation of flies, the microscopic techniques, as well as quantification methods used to study this light-triggered translocation of TRPL are explained in detail. These methods can be applied also for trafficking studies on other Drosophila photoreceptor proteins, for example, rhodopsin. In addition, by using eGFP-tagged rhabdomeral proteins, these methods can be used to assess the degeneration of photoreceptor cells.
膜蛋白运输调节受体和离子通道进入质膜的整合和去除。这个过程对神经元的细胞功能和细胞完整性至关重要。果蝇光感受器细胞已成为研究膜蛋白运输的模型。除了光照时从光感受器膜内化并降解的视紫红质外，果蝇中的瞬时受体电位样（TRPL）离子通道在光感受器膜（在黑暗中位于其中）和光感受器细胞体（在光照时被转运到其中）之间表现出光依赖性易位。通过在光感受器细胞中表达 eGFP 标记的 TRPL，可以以简单且非侵入性的方式研究 TRPL 的这种细胞内运输。然后可以通过深伪瞳孔或水浸显微镜观察 eGFP 荧光。这些方法允许在完整的眼睛中检测荧光，因此对于 TRPL 易位缺陷的果蝇突变体的高通量测定和遗传筛选非常有用。本文详细介绍了用于研究这种光触发的 TRPL 易位的果蝇制备、显微镜技术以及定量方法。这些方法也可用于研究其他果蝇光感受器蛋白，例如视紫红质的运输研究。此外，通过使用 eGFP 标记的纤毛蛋白，这些方法可用于评估光感受器细胞的退化。
