I began work on a white-eyed fly stock with Rh1-GFP driven by a heat shock promoter. This work was inspirted by Brian Belliveau 's thesis from Joe O'Toiusa's lab at the University of Notre Dame "AN ANALYSIS OF RHODOPSIN TRAFFICKING IN THE SECRETORY PATHWAY." Keeping flies at 37 o C for one hour caused rhodopsin to be imported into the rhabdomeres. Since the heat shock promoter would not be specific to R1-6 (as the ninaE promoter would be) fluorescence increased in R7 (and ocelli) as well as in R1-6. This picture shows 1 day replaced, optical neutralization. It is curious that some R1-6 rhabdomeres are dark.

Import and maintenance of Rh1-GFP into rhabdomeres

Since rhabdomeres lit up clearly at one day, we went to shorter times. This picture shows some R1-6 rhabdomeres fluorescing at 5.5 hours (in the dark), but the most interesting observation was how clearly R7 rhabdomeres showed fluorescence. If maintained in the dark, Rh1-GFP was tidily maintained in the rhabdomeres.

Clearance of Rh1-GFP from the rhabdomere

I hypothesized that light might be required for the clearance of rhodopsin from rhabdomeres. Flies moved from dark to light showed diminution of fluorescence by 3 days of light treatment (Figure).

Is vitamin A needed for rhodopsin import to rhabdomeres?

Yes (pic). Heat shocked vitamin A deprived flies do not show GFP.

Heat Shock

Here is heat shock, maintained in dark 4 days vs 3 days in the dark and moved to the light for 1.

[In summary, rhodopsin import to rhabdomeres has not started at 2 hrs but is well under way at 5.5 hrs. Clearance from rhabdomeres is well under way at 1 day in the light. By the way, Shan helped to determine that rhabdomeres showed no GFP after 7 days in the light. Every picture we shoot has that odd appearance of dark rhabdomeres (even though transmitted light shows that those rhabdomeres are present). Rh1 is imported into R7 and ocelli]

Timing when rhodopsin vesicles are poised for rhodopsin import to rhabdomeres

Here is a figure comparing heat shock flies 3.5 hr after heat shock vs 26 hr after heat shock. Note the vesicles and dimly fluorescent rhabdomeres in the 3.5 hr and the bright rhabdomeres and lack of vesicles in the 26 hr animal.

Undergraduate Keath research award

George was awarded second place and further he received an invitation to participate in the Senior Legacy Symposium.

George Denny's poster ("Timeline of rhodopsin traffickingwithin Drosophila retinula cells and GGA knockdown's effect upon it") [bottom, middle to right] presents his confocal work on the timing of import to the rhabdomere and clearance from the rhabdomere usng heat shock.