How is membrane maintenance related to turnover of its primary protein, namely opsin? In retinoid deprived Drosophila, sensitivity, visual pigment, P-face particles, rhodopsin immunogold labeling and rhabdomere size are greatly reduced. Here is a Transmission electron micrograph showing that rhabdomeres of retinoid deprived Drosophila are smaller than those of replete flies (from Sapp et al., 1991). Recovery is rapid when deprived flies are given retinoid "replacement therapy." This was shown electrophysiologically in collaboration with De-Mao Chen in my laboratory, and using freeze-fracture electron microscopy in collaboration with Prof. Richard H. White, Department of Biology, University of Massachusetts - Boston (now retired). At one day of replacement, when opsin synthesis and deployment are high, high immunogold in the rough endoplasmic reticulum shows de novo opsin synthesis (from Sapp, Christianson & Stark, 1991) Using CAT as a reporter driven by the reporter for opsin's gene suggested that retinoid metabolites including retinoic acid activate transcription of the opsin gene directly. Here I present the demonstration that retinoic acid activates opsin's promoter just as well as carotenoid replacement with carrot juice (from Sun et al., 1993) This was a novel finding and one of particular interest since retinoic acid (RA), an important developmental regulator, was shown for the first time to directly regulate genes of the visual system. Our work goes beyond the manipulations that are possible in vertebrates since RA is neither toxic nor essential in Drosophila. I pursued my interests in retinoid regulation of the rhodopsin gene using northern and western blotting in collaboration with Dr. Wendy Picking. Here is a northern bolt showing the lack of opsin transcription in retinoid deprived Drosophila while the mRNA is present in replete flies raised on normal food and on media with various other activators of transcription (from Picking et al., 1996) We also obtained DNase I footprinting showing three protected regions around -583, -603 and -634 with flanking hypersensitive regions. Finally, we obtained these results from gel retardation: A consistent concentration of fly head extract retarded mobility of a labeled PCR fragment of the opsin promoter (-739 to -464); no retardarion for BSA control. Thesis projects for several students in my lab, D. Sun and R. D. Lee revolved around these retinoid experiments.
Most recently, using stocks made by Dr. Franck Pichaud in Prof.
laboratory, my students and I used GFP (green fluorescent protein)
to examine activation of transcription in rhodopsin genes (Stark
and Thomas, 2004). Because this paper is available on line, some
of the earler content of this web page has been deleted.
Papers on vitamin A deprivation and replacement in Drosophila:
Sapp, R, J., Christianson, J. S., Stark, W. S. Turnover of membrane and opsin in visual receptors of normal and mutant Drosophila. Journal of Neurocytology, 1991, 20, 597-608. PubMed
Sapp, R. J., Christianson, J. S., Maier, L., Studer, K. Stark, W. S. Carotenoid replacement therapy in Drosophila: Recovery of membrane, opsin and visual pigment. Experimental Eye Research, 1991, 53, 73-79. PubMed
Chen, D.-M., Stark, W. S. Electrophysiological sensitivity of carotenoid deficient and replaced Drosophila. Visual Neuroscience, 1992, 9,461-469. PubMed
Sun, D., Chen, D.-M., Harrelson, A., Stark, W. S. Enhancement of expression of chloramphenicol acetyltransferase (CAT) by carotenoid and retinoid replacement in opsin promoter - CAT reporter fusion Drosophila. Experimental Eye Research, 1993, 57, 177-187. PubMed
Stark, W. S., White, R. H. Carotenoid replacement in Drosophila. Freeze fracture electron microscopy. Journal of Neurocytology, 1996, 25,233-241. PubMed
Lee, R. D., Thomas, C. F., Marietta, R. G., Stark, W. S. Vitamin A, visual pigments and visual receptors in Drosophila. Microscopy Research and Technique (invited paper for issue devoted to visual receptors), 1996,35, 418-430. PubMed
Picking, W. L., Chen, D.-M., Lee, R. D., Vogt, M. E., Polizzi, J. L., Marietta, R. G., Stark, W. S. Control of Drosophila opsin gene expression by carotenoids and retinoic acid: Northern and Western analyses. Experimental Eye Research, 1996, 63, 493-500. PubMed
Stark, W. S. Comparative biology of receptor recovery by retinoid replacement in retinoid deprived flies and rodents. In Degenerative Retinal Diseases (eds. M. M. LaVail, J. G. Hollyfield, R. E. Anderson), New York, Plenum, 1997,135-143.
Stark, W. S., Thomas, C. F. Microscopy of multiple visual receptor
types in Drosophila, Molecular Vision, 2004, 2004; 10:943-955,
Full paper on line.
A demonstration of how computerized image analysis was used for morphometric measurements of photoreceptive rhabdomeres in Drosophila retinoid replacement experiments.
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This page was last updated June 27, 2005