Studying Membrane Protein Trafficking in Drosophila Photoreceptor Cells Using eGFP-Tagged Proteins

Summary

Here, non-invasive methods are described for localization of photoreceptor membrane proteins and assessment of retinal degeneration in the Drosophila compound eye using eGFP fluorescence.

Abstract

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.

Introduction

By delivering and removing proteins to and from the plasma membrane, membrane protein trafficking in neurons controls the plasma membrane equipment with receptors as well as ion channels and, as a result, regulates neuronal function. Misregulation or defects in protein trafficking typically have detrimental effects on cells and result in neuronal degeneration. In humans, this may cause neurodegenerative diseases such as Alzheimer's and Parkinson's disease or Retinitis pigmentosa¹. Photoreceptors in the compound eye of Drosophila melanogaster have become an in vivo model system for studying membrane protein tra....

Protocol

1. General considerations

1. Use Drosophila stocks expressing a permanently rhabdomerally located fluorescence protein for morphological analysis (e.g., TRP::eGFP, eGFP::NINAC) and translocating proteins for analyses regarding protein trafficking (e.g., TRPL::eGFP, Arr2::eGFP).
2. Predetermine light exposure conditions of selected flies for the experimental approach.
   1. For dark-adaptation, keep the flies in dark boxes for the desired period at 25 °C. For illumination up to 16 h in translocation experiments (e.g., TRPL::eGFP expressing flies), keep flies under a fluorescent tube at room temperature.
   2. In....

Results

Transgenic Drosophila flies expressing a TRPL::eGFP fusion protein under the control of the rhodopsin 1 promoter have been generated. In these flies, TRPL::eGFP is expressed in photoreceptor cells R1-6 of the compound eye and displays an illumination-dependent localization. When flies are kept in the dark, TRPL::eGFP is incorporated into the outer rhabdomeres. After illumination for several hours, TRPL translocates into the cell body where it is stored in an ER-enriched compartment.^8.......

Discussion

The applicability of fluorescence proteins and simplicity of screening by DPP imaging and retinal water immersion microscopy has been proven to be successful by many groups¹². Strategies similar to the ones presented here have been used in several genetic screens to detect defects in rhodopsin expression levels, homeostasis, retinal organization, or cellular integrity with the help of Rh1::eGFP^17,18,19

Materials

Name	Company	Catalog Number	Comments
15 mL centrifuge tube	Greiner Bio-One	188271
CO2 anaesthesia fly pad	Flystuff	59-172
Cold light lamp (KL 1500 LCD)	Zeiss
Fiji/ImageJ	NIH
Fluorescence microscope with UV lamp, camera, filter set and software (AxioImager.Z1m, Axiocam 530 mono, 38 HE, ZEN2 blue edition)	Zeiss
Fluorescent tube (Lumilux T8, L 30W/840, 4000 K, G13) [1750 Lux, Ee470nm = 298 µW cm-2, Ee590nm = 215 µW cm-2] and [760 Lux, Ee470nm = 173 µW cm-2, Ee590nm = 147 µW cm-2]	Osram	4050300518039
Laboratory pipette (20-200 µL)	Eppendorf
Object slide	Roth	0656.1
Petri dish (94 mm)	Greiner Bio-One	633102
Pipette tips (200 µL)	Labsolute	7695844
Plasticine (Blu-Tack)	Bostik	30811745
Stereo microscope (SMZ445)	Nikon
Stereo microscope with UV lamp, camera, filer set and software (MZ16F, MC170 HD, GFP3, LAS 4.12)	Leica