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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.
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.
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 trafficking2. This is not only due to the genetic versatility of Drosophila that allows effective genetic screens, but also because all essential components of the light-absorbing photoreceptor membrane are characterized in great detail and efficient microscopic techniques are available that can be applied to the fly eye. These techniques are the focus of this article.
In Drosophila photoreceptor cells, the apical plasma membrane forms a densely packed stack of microvilli along one side of the cell, termed rhabdomere. The rhabdomeres of photoreceptor cells R1-6 are arranged in a characteristic trapezoidal pattern while photoreceptor cells R7 and R8 form a single rhabdomere in the center of this trapezoid3. Membrane protein trafficking is needed for a regulated turnover of rhabdomeral membrane proteins such as rhodopsin and the light-activated TRP (transient receptor potential) and TRPL (TRP-like) ion channels to assure the proper amount of these phototransduction proteins in the rhabdomere. Photoreceptor membrane proteins are synthesized in the endoplasmic reticulum and transported via the Golgi apparatus to the rhabdomere. Following activation of rhodopsin by light, a rhodopsin molecule can either become inactivated by absorption of a second photon or can be removed from the rhabdomere by clathrin-mediated endocytosis. Endocytosed rhodopsin either becomes degraded in the lysosome or is recycled back to the rhabdomere4,5. The ion channel TRPL is also internalized following activation of the phototransduction cascade and undergoes a light-dependent translocation between the rhabdomere (where it is located when flies are kept in the dark) and an ER-enriched storage compartment in the cell body (to which it is transported within several hours upon illumination)6,7,8,9,10. In contrast to endocytosed rhodopsin, only small amounts of TRPL are degraded via the endolysosomal pathway, and the majority is stored intracellularly instead and recycled back to the rhabdomere upon dark adaptation6. TRPL can thus be used for analyzing light-triggered trafficking of plasma membrane proteins. Drosophila photoreceptor cells are also employed for studying neuronal degeneration. Photoreceptor cell degeneration is frequently determined by assessing the structure of rhabdomeres, which disintegrate as a result of degenerative processes5.
In order to study the subcellular localization of TRPL and rhodopsin in photoreceptor cells or photoreceptor cell degeneration, two fluorescence microscopy methods that differ with respect to analysis speed and resolution have been applied here. A very fast, non-invasive method that can be used for genetic screens but with a limited spatial resolution is the detection of fluorescence in the deep pseudopupil (DPP). The DPP is an optical phenomenon of arthropod compound eyes whose geometric origin has been explained in detail by Franceschini and Kirschfeld in 197111. In short, on several optical planes below the retina overlay-images of rhabdomeres from adjacent ommatidia can be observed. On a focal plane through the center of the eye's curvature, these superimposed projections form an image that resembles the trapezoidal layout of rhabdomeres in a single ommatidium only orders of magnitude larger. This phenomenon can also be observed independently of exogenous expression of fluorescence proteins (e.g. TRPL::eGFP8), which nonetheless make the DPP easier to detect (Figure 1A-A'')12. A second non-invasive method is water immersion microscopy that relies on imaging fluorescently tagged proteins after optically neutralizing the eyes' dioptric apparatus with water (Figure 1B-C'')12. Using the water immersion method, the relative amount of TRPL::eGFP in the rhabdomeres or cell body can be assessed quantitatively for individual photoreceptor cells. Furthermore, non-translocating fluorescence-tagged proteins can be utilized to evaluate rhabdomeral integrity and to determine the time course of a potential degeneration in a quantitative manner, as described here.
While recordings of the DPP are by far the easiest and fastest of these methods to perform, the spatial resolution of data they generate is limited. In addition, there are numerous reasons why a DPP may be absent, which are not necessarily discernible by DPP imaging itself. Since the DPP represents a summation of several ommatidia, information about individual cells is lost. Thus, low-resolution DPP imaging serves an important function in screening large numbers of flies but should generally be followed by higher resolution recordings by way of water immersion microscopy. Water immersion micrographs allow interpretations about individual cells, developmental defects, eye morphology, protein mislocalization or retinal degeneration as well as quantification of these effects. This Protocol describes these two techniques in detail.
Figure 1: Overview of microscopy variations for the Drosophila eye presented in this Protocol. Schematic representations and exemplary micrographs of (A-A'') fluorescent deep pseudopupil (DPP) imaging, (B-B'') lethal water immersion microscopy of fluorescent rhabdomeres, and (C-C'') non-lethal water drop microscopy of fluorescent rhabdomeres. Scale bar (A''): 100 µm. Scale bars (B''-C''): 10 µm. The figure has been modified from reference13. Please click here to view a larger version of this figure.
1. General considerations
2. DPP imaging
Figure 2: DPP imaging workspace. Materials needed are (A) CO2 anesthesia apparatus, (B) stereo microscope with a UV lamp and fluorescence filter set, (C) light source, (D) microscope-mounted camera with (E) software, (F) paint brush, (G) black cardboard, and (H) fly vial. Please click here to view a larger version of this figure.
Figure 3: Positioning of the fly under the stereomicroscope for DPP imaging. (A) Illustration of the fly on its side with one eye facing the microscope objective radially. (B) The fly head needs to be turned slightly up or down such that the objective focuses on a point slightly above or below the eye's equator as indicated by the red arrows. Please click here to view a larger version of this figure.
Figure 4: Illustration of DPP and fluorescent DPP imaging. Exemplary images of Drosophila eyes under conventional and UV illumination with GFP filter set, taken with varying focal planes illustrated in schematic cross-sections through the eye. (A) Micrograph recorded with bright settings of a conventional light source, 30 ms exposure time, 1x gain, deep depth of field, and focal plane near the surface of the cornea as illustrated in (A'). (B) Micrograph recorded with bright settings of a conventional light source, 30 ms exposure time, 1x gain, shallow depth of field, and focal plane approximately 180 µm below the surface of the cornea as illustrated in (B'). DPP indicated. (C-E) Micrograph recorded with high-intensity settings of UV light source and GFP filter set, 80 ms exposure time, 12x gain, shallow depth of field, and the focal plane (C') near, (D') slightly below, or (E') approximately 180 µm below the surface of the cornea. Fluorescent DPP is indicated with a curved arrow. Scale bar 100 µm. Please click here to view a larger version of this figure.
3. Water immersion microscopy
Figure 5: Water immersion microscopy workspace. Materials needed are: (A) 15 mL centrifuge tube, (B) ice flakes, (C) chilled distilled water, (D) stereomicroscope, (E) Petri dish, (F) plasticine, (G) object slide, (H) insect pins or pipette tips and scalpel, (I) fluorescence microscope with (J) software. Please click here to view a larger version of this figure.
Figure 6: Preparation for lethal water immersion microscopy. Illustration of (A) plasticine-coated object slide and Petri dish, (B) pinning a fly through thorax on plasticine ground, (C) fly orientation on the plasticine-coated object slide, and (D) final experimental setup. Please click here to view a larger version of this figure.
Figure 7: Preparation for non-lethal water drop microscopy. Illustration of (A) a cold-anesthetized fly fixed inside a 200 µL pipette tip mounted on plasticine-coated object slide and (B) the application of chilled water drop on the underside of the water immersion objective. Please click here to view a larger version of this figure.
Figure 8: Positioning of the fly under the fluorescence microscope for water immersion imaging. Setup and final orientation for image acquisition using the (A) lethal or (B) non-lethal fly preparation protocols. (C) Illustration of fly orientation for obtaining best results of water immersion microscopy images. The ideal point to focus on the eye is not the exact center with respect to the anterior/posterior and dorsal/ventral axes but is slightly above the eye's equator, as indicated by the red arrow. (D) Example of water immersion image for a perfectly positioned eye. All three symmetry axes of the hexagonal ommatidial tiling appear as straight lines and the maximum amount of ommatidia can be in focus at the same time. (E) Example of water immersion image of an improperly positioned eye. The image contains curved axes and a shallow depth of field. Scale bar: 20 µm. Please click here to view a larger version of this figure.
Figure 9: Quantification of relative rhabdomeral fluorescence for translocation studies. An illustration regarding quantification of relative eGFP fluorescence in the rhabdomeres by measuring the fluorescence intensity of rhabdomere (r), cell body (c), and background (b) of three different representative ommatidia (white circles) in one water immersion microscopy image; scale bar: 10 µm. A magnified ommatidium is shown on the right; scale bar: 2 µm. Please click here to view a larger version of this figure.
Figure 10: Quantification via rhabdomere evaluation for degeneration studies. An illustration regarding quantification of eye morphology by scoring the rhabdomeres of three different representative ommatidia (white circles) in one water immersion microscopy image with values of 2 (clearly visible; blue circle), 1 (weakly visible; orange circle), or 0 (absent; red circle). Scale bar: 10 µm. A magnified ommatidium is shown on the right; scale bar: 2 µm. Please click here to view a larger version of this figure.
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...
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::eGFP17,18,19
The authors have nothing to disclose.
We would like to thank our student researchers over the years. In particular, Nina Meyer, Sibylle Mayer, Juliane Kaim, and Laura Jaggy, whose data have been utilized in this protocol as representative results. Research of our group presented here was funded by grants from the Deutsche Forschungsgemeinschaft (Hu 839/2-4, Hu 839/7-1) to Armin Huber.
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 |
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