Rhodopsin traffic investigation with the heat shock promoter.
Denny, George1 and William S. Stark2. 1Washington University School of Medicine, St. Louis, MO 63110, 2Department of Biology, Saint Louis University, St. Louis, MO 63103. e-mail email@example.com
This laboratory has a long-standing interest in rhodopsin turnover in Drosophila (Stark, et al., 1988); rhodopsin is cleared from the photoreceptive organelle (rhabdomere) via coated pits then multivesicular bodies (MVBs) and lysosomes and imported into rhabdomeres via membranous vesicles. This ultrastructural description has had many molecular elaborations since then (e.g. Chinchore et al., 2009) since our early work. Later, we showed that white-eyed flies maintained in the dark had considerably more rhodopsin than flies kept on a light-dark cycle (Zinkl, et al., 1990; Selimovic, et al., 2010). A white-eyed stock in which R1-6 rhodopsin (Rh1) attached to green fluorescent protein (GFP) was driven by a heat shock (hs) promoter (hs-Rh1-GFP, Belliveau, 2008) allowed us to visualize aspects of rhodopsin traffic using optical neutralization of the cornea in the confocal microscope (Stark and Thomas, 2004).
For heat shock, flies were lightly etherized and placed in a vial in a 37oC water bath for 1 hr. Then, based on what we already knew and how our pilot observations guided us, we put them in a food vial in the dark. R1-6 and R7 rhabdomeres show fluorescence (Figure, top left). Since it was the heat shock promoter, not Rh1Ős promoter (ninaE), R1-6Ős rhodopsin (Rh1) should be expected to be driven ectopically into R7 (and ocellar receptors) as well as into R1-6 (Belliveau, 2008). Some of the R1-6 rhabdomeres were dark, and this striking result was repeatable and unexplained. The fly (Figure, top left) had been maintained in the dark for 4 days, showing that, without light, the rhodopsin remains in the rhabdomere, and, in other work, we extended this obsevation out to 12 days. If, however, flies were kept in the dark for 3 days then in the light for 1 day (Figure, top right), the fluorescence was greatly diminished. This is in keeping with our earlier finding (Zinkl, et al., 1990) and Chinchore et al.Ős (2009) work and reinforces the notion that light is necessary to trigger the clearance of rhodopsin from the rhabdomere.
We sought to also investigate import of rhodopsin into the rhabdomere. In pilot work, we showed that there was no fluorescence 2 hr after heat shock while the fluorescence was nearly fully established at 5 hr. Dissecting this time span, we saw dim rhabdomere fluorescence at 3.5 hr (Figure, bottom right). For a control, we show a fly 26 hr after heat shock (Figure, bottom left); as stated above, both were kept in the dark after heat shock. The striking aspect of the 3.5 hr vista is the haze of fluorescent bodies seen in the cytoplasm of the retinula cells. We presume that we are visualizing membranous vehicles (and, perhaps Golgi apparatus) involved in the import of rhodopsin into the rhabdomere.
All 4 of our figures, and hundreds of other images we have obtained, show large fluorescent bodies that appear to be in pigment cells between ommatidia. We have always assumed, though we have not proven, that these are the giant unpigmented pigment granules of white eyes (Stark and Sapp, 1988). We thought we should not gloss over this point because, again, with techniques more sophisticated than our 1980Ős ultrastructural work, there has been a vastly renewed interest in eye color pigment granules.
We hope that our observations are of use to the many research groups using modern techniques and the accessibility of rhodopsin and the compound eye in Drosophila to study the broader issue of protein traffic.
Acknowledgements: Funding was from SLUŐs Beaumont and Presidential funds. We thank Prof Joseph OŐTousa of the University of Notre Dame for providing the white-eyed hs-Rh1-GFP stock.
References: Belliveau, B., Thesis, University of Notre Dame, 2008; Chinchore, Y., et al., 2009, PloS Genet. 5(2): e1000377; Selimovic, A., et al., 2010, Dros. Inf. Serv. 93: 1-2; Stark, W. S. et al., 1988, J. Neurocytol 17: 499-509; Stark, W. S. and R. Sapp, Can J. Zool. 66: 1301-1308; Stark, W. S. and C. F. Thomas 2004, Molec. Vision 10: 943-955 (on line at http://www.molvis.org/molvis/v10/a113); Zinkl, G., et al., 1990, Vis. Neurosci. 5: 429-439.