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.

GGA affect in heat shock strain

We started of these flies:
knockdown: cnbw ninaE Gal4/cnbw;hs Rh1GFP/UASGGA.E3
control: ninaE Gal4/cnbw/hs Rh1 GFP / Sb p TM2 Ubx
Here is a plate of some of the first flies run, 23 hr after heat shock

Eventually, a plate with a higher n showed a better match between experimental and control

The abnormal dark areas ("islands") in the knockdown prompted us to check the eye for abnormal eye surface (rough eye phenotype), but we found the eye to be normal. These flies have normal rhodopsin-metarhodopsin conversion, and the rhabdomeres look fairly normal in optical neutralization

Heat shock Rh1-GFP with vs without GGA kd

George continued to work independently to obtain several time points after heat shock. At first we used flies collected by Prof Eissenberg from the appropriate crosses. Then, we tooled up to have George set up crosses and and collect flies.

We continued to be struk by the presence of dark "islands" between fluorescent ommatidia, but we also noted that these were present in controls as well as knockdowns. Therefore, we decided that these were probably a result of the particular genotype we were studying and had little relevance to our interest in GGA.

In the non-shocked control, the expected findings were obtained, autofluorescence in cell bodies, no fluorescence in rhabdomeres because there was no heat shock, and dark "islands"

Here is a plate for flies shocked and put in the dark for 5 days. Both experimental (with GGA kd) and control (without GGA kd) lose rhabdomere fluorescence, in contrast with the previously studied line of hs-Rh1-GFP flies. This is very troublesome. There is some unknown mechanism causing rhodopsin turnover in the absence of light. The "islands" are noted in the experimental and control but not in the previously studied heat shock flies.

Davita

On 6/21, Davita Wachsstock joined the lab (again - she worked with Eissenberg the previous two summers)

Davita was given the heat shock project. She was practicing her microscopy and photometry skills on h.s. Rh1-GFP. She measured visual pigment levels in 1 Day shocked flies vs non shocked flies with the same background (her first figure)

Soon , we will return to:
knockdown: cnbw ninaE Gal4/cnbw;hs Rh1GFP/UASGGA.E3
control: ninaE Gal4/cnbw/hs Rh1 GFP / Sb p TM2 Ubx
(these flies being provided by George after he took over the crosses from Eissenberg)


Heat shock with and without GGA

Here is the assay of rhodopsin - metarhodopsin conversion for controls
Here is the assay of rhodopsin - metarhodopsin conversion for experimentals
Here is a movie I made to show that the surface of the heat shock control eye is fairly normal
Here is a movie I made to show that the surface of the heat shock control eye is fairly normal

Davita's further work on heat shock

The effort was made to run a sufficient number of flies for a test of statistical significance. A comparison of non heat shocked vs heat shocked hs-Rh1-GFP flies had been initiated in the hope of testing whether GFP-labeled Rh1 was deficient in the rhodopsin-metarhodopsin photoconversions that enabled photometric determinations of visual pigment levels. Davita's inquiry about why the hs-Rh1-GFP flies were not dark reared before the heat shock treatment led to experiments to test the hypothesis that import of GFP-rhodopsin to rhabdomeres would be impeded if the rhabdomeres were more replete with native rhodopsin by virtue of the dark rearing. Thus, a comparison of heat shocked flies that had been previously maintained in light vs dark was added to her comparison of rhodopsin levels in heat shocked vs non heat shocked hs-Rh1-GFP flies.

This comparison (that heat shocked flies moved from the light to the dark have less measurable visual pigment than non shocked flies, also moved from the light to the dark) implies that the Rh1-GFP does not have normal rhodopsin-metarhodopsin photoconversions (or that the de novo Rh1-GFP interferes with conversions in the native photopigment). The same graph (comparison of dark- vs light-reared heat shocked flies) disproves the hypothesis that the higher rhodopsin in dark reared flies interferes with import into the rhabdomere of the new Rh1-GFP.

Error bars are 95% confidence intervals

The minimal differences in fluorescence measurements suggest that Rh1-GFP adds only slightly to the autofluorescence (fig).

During the summer heat wave, the air conditioning in Macelwane Hall became occasionally unreliable, so we moved heat shock flies to Prof Spencer's 18 degree incubator

Can we see rhodopsin transport vesicles?

I fixed heat shock flies, Barbara Nagel sectioned them, and Katelyn Anderson photographed sections. Here is a tantilizing plate suggesting more vesicles at an optimum time point (3.25 hr) than in control (non-shocked). Here we also see a cytoplasmic (maybe even intraommatidail) richness at another optimum time point (4.75 hr) while there are missing rhabdomeres, holes, and degeneration retinulafor 26 hr.

Transmission electron microscopy

We were disappointed in our quest for ultrastructural correlates of rhodopsin transport vesicles

hs-Rh1-GFP stock not shocked (light reared, unknown age)
There are many ommatidia with normal R1-7 geometry and ghosty cytoplasm; are these numerous MVBs?; not rough endoplasmic reticulum (bottom middle) (figure)
Here is an ommatidium where R3 has degenerated
A small fraction of ommatidia had large central rhabdomeres; ghosty cytoplasm plagued our fixation (figure)

3.25 hours after heat shock (light reared, unknown age)
Even in rare areas where the cytoplasm was not ghosty, there wee no extraordinary structures in the cytoplasm (figure)
Here are a few of the occasional large "central" rhabdomeres (R8 top right, R7 bottom left)

4.75 hours after heat shock (light reared unknown age)
Here is a typical vista of ommatidia with normal geometry at the plane of R1-6 nuclei
Ultrastructure was unremarkable, microvilli, submicrovillar cisterns, belt desmosomes, rough endoplasmic reticulum; note degrading MVB (figure)
Here is an unusual vista, rhabdomere caps one of which has an MVB
There were occasionally missing rhabdomeres and areas of ghosty cytoplasm (figure)

26 hr post hs (light reared, unknown age)
Here is normal R1-6 and R7, but rhabdomeres have vacuoles
Occasionally, there were swollen "central" rhabdomeres (R8 in this case) (figure); ghosty cytoplasm was the hallmark
Here is a degenerating R6 retinular cell


Further follow-up (on retinal disorganization)

Heat shocked flies, maintained in the dark are moved back in the light to lose their fluorescence. We heat shocked them again. Though we expected to see total devastation, structure was not as bad as we thought it would be (Figure). However, there are missing rhabdomeres amd holes where ommatidia ought to be.

Starting this work, here, from George Denny, is a plate showing that rhabdomere fluorescence is gone after 3 days in the light, confirming and further delimiting the time course observations from George Denny's poster. Notice the big hole.

Does a breif (1.25) hr light treatment eliminate Rh1GFP in hsRh1GFP? - -
the answer is no

As a control, we heat shocked white (non heat shock) flies; previously, Katie Anderson and George Denny had verified that the autofluorescence is sufficient to view rhabdomeres in the confocal microscope. Here, they showed that (1) heat shock does not disrupt white flies kept in the dark, (2) [oh, no not another new finding!] ommatidia are disrupted if these flies are moved to the light when heat shocked (3) flies kept in the light and not heat shocked are largely normal. CONCLUSION moving flies fro the dark to the light is damaging.