Collaboration with Joel Eissenberg

This collaboration started on June 20, 2008 when Joel Eissenberg asked me if I would be interested in his study, characterized at the time about being about "autophagy." I said I might have published the first paper (Stark, Sapp and Schilly, 1988) that used the words "autophagy" and "Drosophila" in the same paper, and he indicated that autophagy in Drosophila had become a big topic.

I was able to mobilize about a month of time Summer 2008

Present format of this report

More or less a diary, emphasizing findings, telegraphic on text.

Also somewhat rambling. Sometimes, I summarized the overall issue as trafficing in cells, "proteins and vesicles must be sent to the correct location, if everything is working properly, and so the cell must work like a post office." Autophagy would address the degrative pathways. However, the strategy of vitamin A deprivation followed by "replacement therapy" would assess export since replacement synchronizes a massive de novo rhodopsin synthesis and transport to the rhabdomeres.

Although there is work presented on this web page done primarily by Eissenberg (or Ryerse), I present only work for which I fell I had a significant input.

Blue cheese

Justified by recent literature (Khodosh et al 2006; Finley et al 2003; Simonsen, 2007). Since (BCO1) blue cheese overexpression eyes are abnormal when flies are reared at 18 degrees (left) and very abnormal when flies are reared at 27 degrees (right).

Other pictures demonstrate that (1) ocelli are fairly normal at cool and warm temperatures; (2) rhabdomeres are missing at both temperatures (or few and far between at low temperature); (3) there is a fine grained mosaicism of eye color pigment at low temperature; (4) there are large white patches in some flies at low temperatures; and (5) eye color is more even but not very dark at low temperature.

(BCO2) SEM of blue cheese overexpression clarifies the external anatomy (18 degrees (left), 27 degrees (right)) but also demonstrates that the ocelli (inserts) are not smooth.

References

Finley KD, Edeen PT, Cumming RC, Mardahl-Dumesnil MD, Taylor BJ, Rodriguez MH, Hwang CE, Benedetti M, McKeown M (2003) blue cheese mutations define a novel, conserved gene involved in progressive neural degeneration. J Neurosci 23(4): 1254-1264

Khodosh R, Augsburger A, Schwarz TL, Garrity PA (2006) Bchs, a BEACH domain protein, antagonizes Rab11 in synapse morphogenesis and other developmental events. Development 133(23): 4655-4665

Simonsen A, Cumming RC, Lindmo K, Galaviz V, Cheng S, Rusten TE, Finley KD (2007) Genetic modifiers of the Drosophila blue cheese gene link defects in lysosomal transport with decreased life span and altered ubiquitinated-protein profiles. Genetics 176(2): 1283-1297


GGA knockdown

My main focus was to show that, despite a fairly external appearance, the eye of GGA knockdown flies (driven by GMR and ey) was abnormal. Here is a plate that Eissenberg assembled from the micrographs I obtained comparing wild type (left) with GGAKD (right) (deep pseudopupil bottom, optical neutralization of the cornea top).

"LAMP"

Male yw;eyGAL4+LAMPGFP X Female ywGGA.Es
(Newly emerged) The deep pseudopupil (DPP) is terrible (left) but the rhabdomeres, as viewed by optical neutralization of the cornea (ON) seem to be OK (middle); conclusion, something is wrong with the optics of the eye, but the receptors are not degenerated. The fluorescence is excluded from rhabdomeres but surrounding them, probably in cell bodies (right).

(Day 4) Rhabdomeres, especially R1-6, are degenerating, ON, (lower mag left, higher mag middle) and fluorescence is not so neatly organized, DPP, (right)

(Day 8) Rhabdomeres, if any are visible, are hard to come by, ON, (left) and fluorescence is scattered around the eye, DPP, (right)

By the end of Summer 2008...

...as my teaching load became prohibitive (blocks of time required for laboratory research were not available), I had advised that histology be undertaken. With Eissenberg's seed money, he contracted to The Saint Louis University School of Medicine Department of Pathology Imaging Core. I also advised a fixation, embedding and sectioning that would allow untrastructure should the histology prove informative.

Fall semester 2008

My contributions were minimal, consulting on histological images and on drafts of a paper and grant that were largely prepared by Eissenberg

I was able to devote only a few hours of time to hands-on research and consulting on this project fall semester. However, I did prepare and submit an application for a sabbatical leave which was granted for Spring semester 2010 (letter from Provost Weixlmann dated Feb 22. 2009)

Major findings fall semester

Histology showed that rhabdomeres in GGA knockdowns were small in newly emerged flies, but the driver only control had similarly reduced rhabdomeres. Control rhabdomeres increased with age, but GGA knockdown rhqbdomeres did not. Knockdowns have rhabdomeres that appear to be (1) out of position, (2) split. and (3) missing. Some abnormal "shedding" of membranous material was also evident. A possible accumulation of autophagosomes in retinula cells prompted transmission electron microscopy, but these bodies were then judged to be intraretinular pigment granules. Curiously, autophagosomes were found in secondary pigment cells.

By the end of Fall semester,

we had submitted a paper:

JCEissenberg, AMIlvarsonn, WSStark, JHGrubb, RPohlmann, WSSly, AWaheed, & ACDennes, The sorting adaptor GGA is essential in Drosophila and is implicated in autophagy

and an NSF grant

Role of GGA in autophagy

Spring semester, 2009

Although my teaching load was high spring semester, I mobilized about 20% of my time for this project and enforced that research dedication by spending Mondays at the Doisy Research Center. Among my other accomplishments was to prepare and present a seminar of my research related to autophagy (Web site and PodCast are posted)

I contributed to work using fluorescent antibodies and reporters using the larval salivary gland and the imaginal disks to visualize Golgi vesicles and lococations of GGA protein in cells. This work was not primarily my work, and I have not yet organized a presentation of findings in this report.

Summer 2009

I estimate that I am able to mobilize 1 (out of 3) month of effort.

Because the pace of my work picked up and was less disjointed, I summarize progress of spring and summer together.

Reopening my lab

Because of the investment made in me, (1) productive and stimulating collaboration likely to eventually generate funds and publications; and (2) promise of release time for research in the sabbatical, I felt able to pay forward by resuming undergraduate research mentoring. George Denny volunteered in my lab this summer and has been very productive.

Confocal microscopy

I had hoped to regain a methodological competence my lab had used so productively in the past, namely confocal microscopy. To that end, I worked on several confocal microscopes, one under Ryerse's supervision in the Imaging Core, another adjacent to Eissenberg's lab, and the third under Prof Spencer in my department. Spencer's seemed to be the most user friendly, and, under Spencer's mentoring, George Denny picked up the essentials quickly. So, in conclusion, I (we) can just walk down the hall and look in the confocal.

Funding.

I mobilized about $650 (out of $1000) from the Chauncey E. Finch Award for excellence in mentoring in 2008. Because I had done so much work Summer 2008 for faculty mentoring and promotions summer 2008, the Biology Department gave me $500. Also, the Biology Department gave each faculty member $150.

Expenditures

Drosophila and lab supplies - about $400
External hard drive - about $110
Photoshop (a new version compatable with modern hardware)- about $135 (and, while I was installing programs, I obtained a new version of EndNote, a data reference manager)
The remainder was microscope fees

Expediting research

To assist in microscopy in Eissenberg's lab, I donated an electronic CCD camera and a Sony monitor.

I delivered a rhodopsin antibody and recipes for eventual western blotting.

DavitaWachsstock, a former STARS student, was doing behavioral assays, and I her set up to include phototaxis.

Using the nina E driver

UAS;;YFP.GGA;ninaE-Gal4

Here is a plate made by Eissemberg demonstrating eye abnormalities of interest. Compared with controls (without GGA), eyes were abnormal, worse at high temperature

(Fig) SEM showing that UAS;;YFP.GGA;ninaE-Gal4 is slightly abnormal at lower temperature (left) and extremely abnormal when raised at a higher temperature (right). Ocelli are normal in both cases.

Despite the ninaE driver, it was surprising to see fluorescence in pigment cells and a total wasteland in the receptors (data not shown).

GGA.E3xninaE

Here is a plate made by Eissenberg of optical neutralization we did on GGA.E3xninaE flies. Compared with wild-type, OregonR, rhabdomeres are "missing," more in the male than in the female.

It is surprising what a mess the deep pseudopupil is in these flies (Fig) [wild left, mutant male middle, mutant female right, transmitted light top, fluorescence bottom)

Histology

Here is a plate made by Eissenberg showing that (1) the driver control has small rhabdomeres when newly-emerged that recover after a week; and (2) the GGA knockdown has small rhabdomeres that (and other anomalies) when newly-emerged with a host of structural problems after a week

Ultrastructure (TEM)

Here is a comparison made by Ryerse of the ultrastructure of 8 day control (left) vs knockdown (right). The most striking finding is how "normal" both are based on the available literature. However, there are several differences: (1) There is more membranous debris in the intraommatidial matrix in knockdowns; (2) There are more dense bodies, possibly autophagic vesicles) in retinula cells (and pigment cells [not shown in this image]) of the knockdown; (3) Dense bodies about the size and location of intraretinular pigment granules are more numerous and more variable in size in the knockdown; and (4) Intraretinular membrane circles near the rhabdomeres, once hypothesized to be export vehicles, may be more numerous in the knockdown.

In August, 2009, I revisited these grids with Jan Ryerse, I wanted to obtain vistas missing from our earlier set and also to have sections where landmarks would identify the location, Here is GGA distal. Here is GGA spanning the basememt membrane. Here is GGA, an area with several abnormalities. Here is a GGAkd distal ommatidium with an abnormal rhabdomere count. Here is another. Here is an optic cartridge in GGA. Here is control at the plane of the R7 nucleus. Here is control with some interesting cellular details. Here is control in the area of the basememt membrane with optic cartridges.

Vitamin A deprivation and replacement

We had hypothesized that vitamin A deprivation might protect knockdown flies from the demise of photoreceptors. Here is a plate made by Eissenberg showing the results of our vitamin A experiment. Controls (driver-only - left) vs knockdown (right). Top - deprived from egg to emergence, then shifted to regular food supplemented with carrot juice for two weeks. Middle - reared and maintained as adults for 2 weeks on regular food. Bottom - Reared and maintained for 2 weeks as adults on vitamin A deprivation (ending with one day of replacement. We concluded that vitamin A deprivation did not protect GGA knockdowns. Long ago it had been shown that small rhabdomeres was one of deprivation's many effects (quickly recovered upon replacement); tentatively, we note that the knockdown does not recover size in one day of replacement.

work on Golgi apparatus (ARF72RFP)
(mostly done by George Denny, undergraduate research assistant)


Through Eissenberg, I obtained this stock from Joe O'Tousa at Notre Dame. The stock had flies that had fluorescence and flies that did not. Studies of this stock seemed the ideal starting project for a new undergraduate research assistant, George Denny. I hypothesized that Golgi apparatus might be in lower quantity in vitamin A deprived flies because of (1) the decreased rhodopsin and (2) we had always seen "ghosty cytoplasm" in the electron microscope in deprived flies.

To make the stock stronger, we etherized flies, placed them on a slide in the fluorescence microscope, and mated only ones that fluoresced. During this process, we noted that some flies had very white eyes while others had eyes with a slight pink tinge, and found that there was no strict correlation with whether they fluoresced.

Microscopy Here is a comparison of fluorescence in vitamin A replete vs deprived flies. We looked in the confocal (4/27/09), and here is the deep pseudopupil. In the area of the deep pseudopupil, fluorescence is excluded from where the rhabdomeres are imaged, but present in an area around that (presumably the retinula cell bodies. I have no explanation for the highlights, the 7 radiating spokes at the bottom of the figure.Optical neutralization is shown here. We obtained a z-series (stack) that presumably traces the idivieual Golgi complexes in retinula cell bodies surrounding the dark exclusion of fluorescence for the rhabdomeres

microphotometry We set up to quantify fluorescence with the microscope photometer system. Fortunately George noticed right away that that fluorescence increased with age (Figs 1 & 2). Because of this, he studied only newly eclosed flies.

The vitamin A deprivation study There was a slightly higher fluorescence in flies reared on regular food than in flies reared on vitamin A deprivational medium (Fig). There was a higher autofluorescence (presumably the background, the fluorescence of the flies that did not have fluorescence of Golgi) in the deprived flies (Fig). About 1/3 of the flies reared on the regular food showed fluorescence of Golgi (Fig). A lower fraction (about 1/6) of the vitamin A deprived flies showed fluorescence of Golgi (Fig).

Genetics

With Eissenberg's help, we attempted a "genetic" attempt to "purify" the stock, making:
Arf72-RFP/CyO;Rh1-Gal4/TM3-y+
Most but not all the flies have curly wings. Unlike the original Golgi stock, most of the flies have eye color pigment while some are pure white. Most have red fluorescence, but nor all, and it is not correlated with eye color

ERGs of GGA knockdowns and controls

GGA knockdown - Female GGAE3 X Male eyGal4+GMRGal4. The appropriate control was driver only. The control for the control was wild-type.

Methods. The tutorial I gave when I taught the ERG at the "Neurobiology of Drosophila" course at Cold Spring Harbor gives many details. Here, I will document some specifics for this work. To photograph the deep pseudopupil (DPP) before running the ERG, the cover slip the flies were fixed to was optically fused to a microscope slide using a drop of water, and the DPP was viewed with a 10x air objective. For the ERG, a long wavelength, 585 nm, was chosen for two reasons: (1) potential differences between the effects of eye color pigments of experimental and control flies would be minimized; and (2) visualization of on- and off transients would be optimized. Data acquisition was expedited by quickly obtaining responses to a defined sequence of seven stimuli of 1.0 s 0.3 to 0.6 log units apart (an intensity response sequence). After obtaining ERGs, the cover slip was again placed on a slide, and rhabdomeres were photographed using oil immersion and a 40x objective.

Results. ERGs of GGA knock-downs were surprisingly consistent with the expectation for "normal" flies. Near threshold, i.e. when the ERG component attributed to receptor depolarization was 0-3 mV, there were on- and off-transients that were fairly large. For higher intensities, the receptor wave was larger, and the transients were diminished. Ath the highest intensities, the receptor component sometimes settled back rather than just swing up to a steady state. Newly emerged GGA knockdown flies were run as well as flies of various ages out to 22 days, and they all had similar ERG responsivities and sensitivities. ERG on- and off-transients not only imply a high level of R1-6 function but also that the synaptic connections of R1-6 in the first order optic neuropile, the lamina ganglionaris, are functional.

The optics were slightly variable when several trustworthy flies of the same age post-eclosion were run, but were surprisingly constant across ages. The DPP was fuzzy, especially where R1-6 should have been imaged (with R7/8 being less degraded). Under optical neutralization, some rhabdomeres, especially R7, were quite clearly visualized while several per ommatidium were not seen.

"eyGal4 driver stock" (control). Optics were wild-type, clearly better than GGAkd.

A typical result is shown. The knockdown at 6 days is compared with the driver (bottom). Stimuli are 585 nm starting at 12.58, and increasing in the same 0.3 or 0.6 log unit steps to 14.7 (top) or 15.4 (bottom) (log quanta per square cm per second). Larger responses to the same stimuli indicate higher sensitivity in the driver-only stock.

Representative experiments for GGA knock downs, (top) and driver-only controls (second row) are shown. Newly emerged (left), 6-8 days (second column), 15 days (3rd column) and 22 days (right). For comparison, wild-type newly emerged is at the bottom left. The vertical scales have been adjusted to be equivalent

Newly eclosed, knockdown and driver are about the same sensitivity and about 0.6 to 0.9 log units than wild type, consistent with the small rhabdomeres. With age, the knockdown stays about the same while the driver only stock increases to wild type, consistent with the increase in rhabdomere size in only the driver-only stock.

Initial plans for Fall 2009

Here is a confocal image of a fly obtained from O'Tousa that has Rh1 marked with GFP. We (George Denny, Jan Ryerse and I) have several anecdotal observations worth pursuing. Light reared have dimmer fluorescence than dark reared (preliminary observation); also they have more cellular fluorescence. We want to quantify the former observation. Also, we are rearing vitamin A deproved flies to see if we can use GFP fluorescence to observe rhodopsin synthesis and export.

Progress, Fall, 2009

A proposal for a seed grant
was prepared and submitted, and of particular note, in my opinion, were these two figures:

Confocal microscopy of flies in which rhodopsin is labeled with green fluorescent protein (GFP). Optical neutralization of the cornea allowed visualization of rhabdomeres in each ommatidium (while color inserts, upper left, show deep pseudopupil views of entire eye). A comparison of dark reared flies (left) and light reared flies (right) suggests that light triggers autophagy of rhodopsin (hence the decrease in fluorescence). (BOTTOM LEFT INSERT) Vitamin A deprived flies undergoing vitamin A replacement therapy demonstrate visualization of export of rhodopsin to the rhabdomeres.

Transmission electron microscopy (TEM). Control (left) and GGA knockdown (right). Flies were aged eight days in normal room lighting after emergence as adults from the pupa case. Roughly equivalent fields of distal retina (top) and receptor axons projecting to the first optic neuropil ("brain") (bottom) were selected for comparison. There is only one striking abnormality, and that concerns the rhabdomeres, the rhodopsin-containing photoreceptive organelles (dark circles in the retina at the top): The number, shape, size and organization for the GGA knockdown (right) does not match the tidy organization of 7 rhabdomeres per ommatidium (facet in the compound eye) for the control (left).

A research note was submitted and accepted:
Reference: Drosophila Information Service 2009, 92:117-119
text.
figure.
link.

Endnote. Working with IT (Eric Kaufman), some complicated bugs were worked out, permitting utilization of Word with Endnote

SPRING SEMESTER is my sabbatical, progress will be identified by month

Personnel

myself
collaborator Joel Eissenberg
collaborator Abdul Waheed
collaborator Jan Ryerse
research microscopy rechnician Barbara Nagel
undergraduate research assistant George Denny (presently in charge of confocal microscopy)
MS student (and salaried technician in Dr Eissenberg's lab) Anne Ilvarsonn
On Feb 10, Shan Luan, a PhD student, joined the lab.
undergraduate research assistant Asmir Selimovic (presently in charge of photometry)
undergraduate work study research assistant Imran Shaikh

Funding

WSStark sabbatical from SLU Spring 2010
Presidential seed grant ($25,000) Feb 2010 - Feb 2011
Shan Luan is supported by a Biology Department Teaching Assistantship
Imran Shaikh is funded by the Biology department's work-study support

General methods,

Photometer
My microscope was set up as a photometer. Asmir Selimovovic, an undergraduate senior, contributed to the photometry project. The fundamentals of technique are one of this laboratory's cottage industries (Stark et al, 1985). For readout these days is with a PowerLab 410 (ADInstruments) using TChart v4.0.1 into a Mac computer using OS 8.6.

Reference

Stark WS, Walker KD, Eidel JM (1985) Ultraviolet and blue light induced damage to the Drosophila retina: Microspectrophotometry and electrophysiology. Curr Eye Res 4(10): 1059-1075

Fluorescence microscopy was done largely with a Zeiss Axioskop2 with an AxioCam camera and AxioVision 4.6 software
Confocal microscopy is the research project of undergraduate student George Denny using the ZeisssPascal confocal with LSM software
Funding $220,000 NSF grant 0421383 (S. Spencer PI, W. S. Stark one of several contributers[users]) was used to obtain the Fluorescence and confocal microscopes.

Progress, January 2010

A plate was made from the optical images obtained during last summer's ERG work. Driver control (left), 1 day GGA knockdown (middle) and 22 day knockdown (right), deep pseudopupil (top), optical neutralization of the cornea (bottom). Of note is that the abnormal optics for 1 and 22 days are about the same

The importance of dark rearing

Pursuing this observation presented above, I realized that it was already known that the concentration of visual pigment was higher in dark-reared flies than in flies that were in the light (Zinkl et al, 1990). As photometry and microscopy of vitamin A induced recovery commenced, we set up dark reared flies. Here is a confocal micrograph obtained by George Denny showing the difference for dark reared flies and flies that had been moved from dark to light for 6 days. These are matched (pinhole, gain, etc). In addition to the difference in intensity of GFP emission, note also the cytoplasmic localization of rhodopsin in the flies that had been kept in the light.

In this experiment, ninaERh1GFP llies that had been dark reared were either kept in the dark or subjected to bright blue light for 1 hr after being fixed to a microscope slide. Then both fly types were maintained for another 1 hr before being examined in the confocal microscope. No change in the bright rhabdomeres and dark retinula cells was observed indicating that this combination of light treatment and time period was not sufficient to induce clearance from the rhabdomere.

Reference

Zinkl G, Maier L, Studer K, Sapp R, Chen DM, Stark WS (1990) Microphotometric, ultrastructural and electrophysiological analyses of light dependent processes on visual receptors in white-eyed wild-type and norpA (no receptor potential) mutant Drosophila. Vis Neurosc 5: 429-439

GGA Overexpression

A major breakthrough was the availability of a GGA overexpression stock. The eyes were so abnormal that this picture, with no rhabdomeres, came as no surprise. In the process, I found that ocelli were externally normal,

Heat Shock

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.

Progress, February 2010

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.

Mosaics of GGA mutant

Since the GGA mutant is lethal, its affect on visual cells must be studied in mosaics. Here is a picture shot by Prof Eissenberg of such an eye. White (w) marked wild-type, and rhabdomeres were completely normal. Red identified either heterozygous tissue or homozygous GGA mutant tissue. Rhabdomeres appeared mostly murky or absent in red tissue. Here is an ommatidium at a border where the clear ability to focus some rhabdomeres validates the conclusion that others are missing. These flies were maintained in the dark, so, clearly, light was not required for importing Rh1-GFP into the rhabdomere.

Some text with references that I have been preparing:

How membrane is internalized into Drosophila photoreceptors

Autophagy

Vitamin A deprivation and replacement

Heterozygotes of the GGA mutant

y w GGAP1/FM7,yw+Bar (white-eyed)
GGAP1/FM7 GFP (red eyed)

Preliminary results revealed that rhabdomeres were missing in white-eyed flies kept in the light for 14 days after eclosion but present in newly emerged flies kept in the dark. Red-eyed flies did have rhabdomeres even out to 14 days in the light.

Asmir Selimovic did photometry, quantifying visual pigment, comparing newly emerged vs aged white-eyed heterozygotes. This preliminary result is very encouraging in the suggestion that photoreceptors are lost as a function of light exposure in white-eyed GGA heterozygotes.

White-eyed Rh1-GFP with (experimental) vs without (control) knockdown

Control: virgin female w; cn bw ninaE-Gal4; UAS-Rh1-GFP x male w, cn bw;Sb/TM3,ubx
Knockdown: virgin female w; cn bw ninaE-Gal4; UAS-Rh1-GFP x male w; cn bw; GGA.E3

We wanted to have GFP reporter in GGA knockdowns. Also, we wanted to move our work to white eyes. Our earlier work involved the ey-Gal4+GMR-Gal4 driver combination, which drives in most or all cells in the eye disc beginning in first instar. This experiment involved the NinaE-Gal4 driver, which only starts in the last day of pupation. For this latter reason, flies were vitamin A deprived then maintained for two days on vitamin A deficient medium to insure the knockdown could take effect. Then flies were transferred to replacement for various time periods

Here is a picture comparing knockdown vs control at 4 days of replacement (optical neutralizatio of the cornea).
Here is a picture comparing knockdown with control at 3 days of replacement (deep pseudopupil)

Organization of rhabdomeres is fairly regular, fluorescence is lower than control.

"sectored"

females varigated for ubiGFP
males solid for ubiGFP

Here is the first picture

female - variagate for ubiGFP
We did see rhabdomeres in pigmented and white areas, though good areas
of the eye were harder to find than they should have been if the eye were
completely normal. Oddly, when we looked for fluorescence, there was less
from pigmented areas than white areas. There may have even been rhabdomere
fluorescence, but not in the clear R1-6 plus R7 array. The distribution of eye
color pigment is very weird! Some pigment seems to be localized more distal
than where the primary pigment cells would be, and in multiple small patches
(in contrast to the prinary pigment cell that would be semi-lunar in shape

male - solid for ubiGFP
Rhabddomeres seemed pretty normal in the R1-6 plus R7 trapezoid. Oddly, a
deep pseudopupil did not focus well implying that the optics of the eye were
disrupted. Again, the odd distribution of eye color pigments was noted. We not
only did not see fluorescence in rhabdomeres and retinula cells, but we noted, in
a fluorescent deep pseudopupil, exclusion of fluorescence from the rhabdomeres
against a background of autofluorescence

Purifying stocks

Photoreceptor fluorescence can be considered to be a "visible trait" in our lab. Etherized flies can be tested for fluorescence and set back in a vial to make stock. We had been having trouble with several stocks which we have purified and started expanding in February

(1) white-eyed ARF72RFP
and
(2) white-eyed ninaERh1-GFP

Degeneration or "stationary" defect?

Upon re-examination of whether the GGA knockdown driven by the Eyless GMR promoter had degeneration, I am obtaining images of every age in staged isolates provided by Anne Ilvarson. So far (in February) I have done days 0 through 10. Here is day 0. Here is day 10.. The entire series, pictures of deep pseudopupil and optical neutralization of the cornea, support a stationary rather than a degenerative defect.

Due to a problem with the stock, the study of controls was discontinued until the problem is resolved

Progress in March

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]

Heterozygote of GGA mutant

Here is a graph Asmir produced of rhodopsin measurements in newly-emerged dark-reared white-eyed GGA-mutant heterozygotes. Asmir noted that about 1/5 of the flies did not have rhodopsin, so he also did the analysis splitting the flies into ones with rhodopsin vs without.

Research Assistant (RA) Application

March saw the deadline for graduate students to compete for the limited allotment of RA. Graduate students are expected to write a proposal. Working with Shan Luan gave me the opportunity to mentor her in (1) writing; and (2) the nature of her project. (Also I needed to submit a letter of support.) The RA position, if she obtains it, would give her better blocks of time for her research by eliminating her TA obligations, typically 20 hr/wk.

On the basis of this competition, Shan was awarded an RA position (letter of March 31)

Undergraduate Keath research award

I worked with George Denny to help prepare his poster ("Timeline of rhodopsin traffickingwithin Drosophila retinula cells and GGA knockdown's effect upon it") for the undergraduate research award.

On the basis of a competition among 9 students on March 31, George was awarded second place and further he received an invitation to participate in the Senior Legacy Symposium.

The most striking feature of the ninaE driven GGA knockdown is how normal the arrangement of rhabdomeres and ommatidia is; this is in contrast with the structural abnormalities seen in the eyGMR driven GGA knockdowns. We presented this in February (Figs 1 and 2), but George's poster really drives home the point.

One of the unique contributions George made in the final data collections before printing his poster was to test the hypothesis that pertains GGA knockdown contributes to clearance (from the rhabdomere) (as opposed to import to the rhabdomere, the other limb of the protein/membrane turnover process). My own work and that of (Satoh et al, 2005) agree that light is needed for clearance. All of our work to date on the ninaE-driven GGA knockdown (where Rh1-GFP allowed measurement) was on vitamin A deprived flies given vitamin A replacement in the dark; there would be import (but no clearance), and the time series shows that GGA interferes with rhodopsin import (confirming Asmir's findings from quantitative thodopsin photometry). However, a series from dark-reared flies moved to the light disproves the hypothesis that GGA knockdown interferes with clearance.

reference

Satoh AK, O'Tousa JE, Ozaki K, Ready DF (2005) Rab11 mediates post-Golgi trafficking of rhodopsin to the photosensitive apical membrane of Drosophila photoreceptors. Development 132(7): 1487-1497

GGA knockdown interferes with rhodopsin import to the rhabdomere

Here are data of rhodopsin photometry obtained by Asmir on 5 to 6 day vitamin A replaced flies without (control) vs with (experimental) GGA k.d. The ninaE driver drove Rh1-GFP into R1-6 (as well as driving the knockdown). Previously, in the confocal microscope, we had wondered why so many experimental flies were negative for fluorescence, but now we may speculate that they are low in Rh1-GFP because of the GGA knockdown. Note that these data quantify what was observed with confocal microscopy (and presented above).

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.

Degeneration or "stationary" defect? (re-re-visited)

(revisiting images obtained summer 2009, driver left, knockdown 1 day middle, knockdown 22day right)
(extending data processed by the end of February, day 0 and day 10)
I confirm and extend the finding from the end of February concerning the F1 from male eyGMR driver crossed with female GGA.E3. Here I show that zero day dark reared retina looks the same as 29 day retina. (In summary, the organization of the rhabdomeres is shows similar disorganization regardless of light or days post-eclosion but that the rhabdomeres survive [for over 4 weeks]).

I added the zero-day dark-reared manipulation to test the hypothesis that clearance (from the rhabdomere) mediated the structural abnormalities of eyGMR GGA knockdowns. My own work agrees with that of (Satoh et al, 2005) that light is required for clearance. To date, we had only investigated light-reared flies for which clearance (from the rhabdomere) and import (to the rhabdomere) would be confounded. The finding of abnormal rhabdomere structure in zero-day dark-reared flies disproves the hypothesis that the clearance (endocytosis) limb of protein/membrane turnover is the cause of the knockdown's abnormalities.

reference:

Satoh AK, O'Tousa JE, Ozaki K, Ready DF (2005) Rab11 mediates post-Golgi trafficking of rhodopsin to the photosensitive apical membrane of Drosophila photoreceptors. Development 132(7): 1487-1497

Importantly,

By the end of March, we have three converging lines of evidence that GGA interferes with import (to the rhabdomere), not clearance (from the rhabdomere).

Controls on the GGA knockdown experiment

Three controls have been run to datee. controls are being run:

(1) At first, the controls showed a phenotype where R7 was missing (as seen in this picture from 3 days); we learned that the flies were bride of sevenless (boss) homozygotes, so we discontinued this time series after one week

(2) GGA.E3/+
These flies have apricot eyes darkening (but never to a deep red) with age. They have excellent structure as witnessed by this picture from 7 days

(3) eyGMRdriver;boss/+

most of the flies showed normal structure as witnessed by this picture from 19 days

either some of these flies are not driver only or the range of phenotypes of the driver only fly's phenotypes overlaps more than we had thought as witnessed by this picture from 9 days

Molecular biology

Shan Luan launched her project.

The overall plan is to rescue the GGA mutant with the wild type gene, and, assuming that works, "rescue" with constructs engineered to affect specific GGA domains.

So far (March), she has PCRed the gene (4460 bp) after consultation with fly base and Prof Eissenberg to get optimal primers. With TOPO cloning, she ligated the DNA into plasmids and inserted the plasmids into competent cells. There were enough (8) colonies so that there is a high probability that at least one colony will have DNA that does not have Taq errors. Minipreps produced 6 plasmids that were confirmed by restriction digestion by EcoR1 to have GGA and 6 were sent off to sequencing. and they are being sent off for sequencing now (beginning of April)

Consulting on histology

(done by Prof Eissenberg with the help of Jan and Barb at the microscopy resource)

The precipitation event was the need to revise the manuscript for Traffic. I was gratified that my finding suggesting structural abnormalities in eyes of mosaic GGA mutants was confirmed. Also, the abnormalities in the optics of newly emerged GGA knockdown flies was explained in sections showing supernumerary rhabdomeres.

Results for April

Asmir is wrapping up a study on GGA mutant heterozygotes.Here white-eyed heterozygous female (Bar/+ GGAP1/+) of two strains (A&B) were combined. Absorbance difference is plotted against days post eclosion to see whether rearing in the light diminished the level of visual pigment. According to Asmir, the 7 vs 9+10 day comparison is the most important: (1) these days are close together; and (2) the n is high (n=34 for light and n=29 for dark). Although there is a definite light/dark difference, this is on the order of that found for flies that do not carry a GGA mutation. In the meantime. Prof Eissenberg did histology (including flies aged in the light) and saw no obvious degeneration.

On the basis of these two converging lines of evidence disproving the hypothesis that white-eyed GGA heterozygotes have light induced retinal degeneration, we see no point in further study of the structuree / function of the heterozygotes.

Photometry catch-up

Asmir had completed several studies (in February and March) that have not been posted yet:

(1) norpA vitamin A replacement

Work my lab published long ago showed that norpA flies lost visual pigment as a function of time in the light (Zinkl et al., 1990). There has been recent interest in norpA's defect in rhodopsin trafficking leading to its demise in the light (Chimchore et al., 2009). We hypothesized that norpA's defect extended to rhodopsin import to the rhabdomere. On the basis of these data, we reject this hypothesis. (The n's for norpA were 11 for 3 day 16 for 7 days, high enough to be fairly confident.)

Reference

Zinkl G, Maier L, Studer K, Sapp R, Chen DM, Stark WS (1990) Microphotometric, ultrastructural and electrophysiological analyses of light dependent processes on visual receptors in white-eyed wild-type and norpA (no receptor potential) mutant Drosophila. Vis Neurosc 5: 429-439

(2) (ninaE driven) Rh1-GFP

(Because we had obtained ninaE Rh1-GFP from Joe O'Touse at Notre Dame, we referred to these flies as "O'Tousa.) We hypothesized that the attachment of GFP to Rh1 might interfere with rhodopsin's incorporation into the rhabdomere. On the basis of these data, we reject this hypothesis.

GGA overexpression

In January, I obtained pictures of the abnormal eye and normal ocelli of GGA overexpression. Here is an SEM plate. Ocelli are normal. Although completely abnormal, the anterior of the eye has facet lenses while the posterior does not. At the borderline, facet lenses look like volcanoes. At a high magnification, even the totally abnormal posterior of the eye shows the surface roughening (corneal nipples) that serves as an antireflection coating; this is a diagnostic feature of the eye surface.

Using regular food instead of vitamin A replacement for ninaE Rh1-GFP

Here we compared control and Experimental flies, maintained in the dark vs moved to the light 26 hr earlier. We saw no striking differences. On this day, about half the flies were negative for GFP; However, this was not nearly the problem it might have been for, say, 1 day replaced where it would be uncertain whether the fly was negative or simply showing little Rh1-GFP import into the rhabdomere.

Even though the 4 selected images in the above confocal plate indicate no major differences, Asmir's photometry does indicate a much lower rhodopsin content in the experimental vs control flies maintained in the dark.

Interestingly, George's confocal work on regular food knockdowns flies that had been in the light 17 days earlier shows substantial structural abnormality

Control flies raised on regular food kept in the light 17 days look abnormal also (fig)

knockdown flies kept in the dark 17-26 days were fairly normal (fig)

Interaction of GGA mutant with EGFR Ellipse

SEM. The left (I think, I'm checking) is Ellipse withourt mutant, then the right would show the interaction with GGA.P1/+

Importance of dark rearing (revisited)

A paper by Chinchore et al. (2009) compares the rich density of Rab7-positive rhodopsin containing endosomes in flies maintained in the light vs paucity when flies had been moved to the dark for 13 hr. We hypothesized rhodopsin should be higher in flies returned to the dark because clearance from the rhabdomere would be stopped while import of rhodopsin to the rhabdomere would be maintained. Here is a graph Asmir drew from his photometry data confirming our expectation.

Reference

Chinchore Y, Mitra A, Dolph PJ (2009) Accumulation of rhodopsin in late endosomes triggers photoreceptor cell degeneration. PLoS Genet 5(2): e1000377

Toward a GGA kd paper

4/26 draft of optics section
4/27 draft of electrophysiology section
4/27 draft rationalizing vitamin A deprivation and replacement
5/4 draft of ultrastructure

In preparation of a heat shock GGA kd stock...

...to test whether the knockdown alters the timing of rhodopsin import to the rhabdomere or clearance from the rhabdomere, here I gather together the semester's work using Rh1-GFP driven by a heat shock promoter

Molecular biology

(refer back to molecular biology at the end of March, 6 sequences were being obtained)

By the end of April, Shan Luan found this interesting result, that all 6 sequences found in March had the same six "mutations" (polymorphisms). One caused an amino acid change: His562(CAT) changed to Asp(GAT). Efforts to clone into CaSpeR (digest, phosphatase treatment, GGA insertion, and ligation) ran into a number of complications and failed. Possibly the phosphatase failed or the vector religated. TOPO cloning was started again and 3 plasmids are being sequenced. It would be interesting if these do not have the mutation found earlier because that would mean that one altered sequence is already available.

Falling out

For all intents and purposes, a failure to communication led to irreconcilable differences between Joel and me by April, and interactions, though cordial, were strained ever since.

Results for May

Confocal


The disorganization of rhabdomeres (see above George's confocal) is shown to depend on light, not on time (here [not ready yet]) is a picture from a fly 26-17 days aged but kept in the dark showing perfect organization)

Writing

discussion of ey GMR vs ninaE driven GGA knockdown

Photometry

The graph Asmir made (above "importance of dark rearing (revisited)) was remade after a more sufficient number of flies was run

Histology

The availability of ey GMR GGA kd flies, staged isolates brought to a ripe old age, invited a fixation and sectioning. Here is 24 day aged distal showing cornea, pseudocone, Semper cells, rhabdomeres, rhabdomere caps, and primary and secondary pigment cells. Here is GGA kd 24 days showing a large expanse of retina from cornea to near the basement membrane; the most notable feature is the abnormal rhabdomere count in many ommatidia. Here is GGA kd 24 day proximal retina and lamina ganglionaris; note that some (but not all) ommatidia have abnormal rhabdomere counts while the optic cartridges look fairly normal. Here is a large expanse from GGA kd 37 days with incorrect rhabdomere counts but normal cartridges in the optic neuropil.

New Q Imaging camera in my microscope lab

here is a movie I made of rhodopsin to metarhodopsin conversion with blue light
Here is a movie I made of the autofluorescence increase induced by bright blue light
Here is a movie I made of a focus series for GFP in ninaE Rh1-GFP flies

References refer to http://starklab.slu.edu/CV/publications.htm)

(on autofluorescence), 12, 15, 29, 33

(on rhodopsin - metarhodopsin conversions) 18


TEM of 37 day ey GMR GGA knockdown

Many of the descriptions for 8 day apply here

Bodies somewhat like MVBs but with double membrane bounded vesicles and pits
Here is shown a tremendous quantity of membrane circles and intraretinular pigment granules in a plane near the basement membrane; see discussion of circles here.
Here we saw one (and only one) retinula cell that appeared to be dead or dying.
Here is shown a peculiar basement membrane, very swolen
Here is a distal section, oblique (nearly longitudinal) showing the rhabdomere split and looking like beads on a string
The optic cartridge is shown here with fairly normal structure and conspicuous T-synapses

TEM of 24 day ey GMR GGA knockdown

Here is the abnormal accumulation of circles and pigment granules
Bodies somewhat like MVBs but with double membrane bounded vesicles and pits (as in 37 day)
Here is an indication of normal synaptic structure

Reminder

The importance of the "circles" is discussed in a paragraph in this text, first written with the 8 day aged fly in mind. These circles are very abundant in 24 and 37 day GGA kd flies! They are presumably rhodopsin-carying vesicles blocked from their rhabdomeric destination.

Molecular biology

By the end of May, Shan Luan had successfully inserted GGA into pCaSpeR, and this had been sent to BestGene to make transgenic flies

Sabbatical is over

Here is my summary to the dean

June 2010

As the summer began...


George was salaried on the Seed Grant
Imran was salaried on work-study and the Seed Grant
Microscopy was mostly moved to the new Q-capture camera afrom the Seed Grant and the Stark lab microscopes

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.

We were testing the hypothisis, based on Asmir's earlier photometry and on George's earlier confocal microscopy, that GGA kd delayed rhodopsin deployment to the rhabdomere. We thought we were confirming this hypothesis. However, a time course for GFP increase in rhabdomeres was confounded by GFP loss from rhabdomeres.

There are so many bewildering contradictions in the data obtained from these new stocks that we feel obliged to put these studies on the back burner for the time being.

Histological follow-up on unusual optics found in confocal of Rh1-GFP for GGA kd driven by ninaE

To pursue the finding of disorganized fluorescent rhabdomeres in Rh1-GFP flies with GGA kd that had been left in the light for 17 days, we obtained micrographs of histological sections. Some cells appeared to have degeneration. Our excitement was short-lived when we found that controls (23 days in the light) also had some cells with degeneration. There was one other control (knockdowns kept in the dark 20-29 days) where receptors were pretty much normal.

We had Barbara Nagel section the available blocks to the righe depth to decide blocks voted most likely to succeed for follow-up TEM. In the process, we asked her to make half-micron unstained sections to test the hypothesis that phase contrast microscopy would produce better micrographs; we decided that finding the sections was so difficult that this approach was counterproductive.

TEM follow-up

ninaE-driven Rh1-GFP flies with GGA kd that had been left in the light for 17 days:
Cells that still appeared to be alive were filled with rough endoplasmic reticulum
Some cells looked like they were apoptotic
We did not see the abundance of circles that accumulated in the ey GMR driven GGA kd.

Vistas of abnormal and degenerate cells were reminiscent of micrographs presented for rdgB that has light induced retinal degeneration

ninaE-driven Rh1-GFP flies without GGA kd (control) flies that had been left in the light 23 days:
Degenerating cells were noted as anticipated from the light microscopy

ninaE-driven Rh1-GFP flies with GGA kd that had been kept in the dark 20-29 days:
Ommatidia were largely normal

Reference

Stark, W.S. and Carlson, S.D. Ultrastructural pathology of the compound eye and optic neuropiles of the retinal degeneration mutant (w rdgBKS222) Drosophila melanogaster. Cell and Tissue Research, 1982, 225, 11-22.

Degeneration in white-eyed ninaE driven GGA kd but not in red-eyed ey GMR driven GGA kd

The work outlined in the previous section, combined with a substantial body of ERG, EM and optical work over the previous year, led us to hypothesize that GFP or white eyes, not GGA knockdown, led to degeneration. Interestingly, we had presented in an abstract and poster the finding that photoreceptors still worked even if they were full of GFP. But our recent work took flies out to ages never studied previously. (Also, that GFP was not attached to rhodopsin, while it is in this case.) To test our hypothesis, we collected and staged the aging in room light of white-eyed ninaE driven Rh1-GFP flies. Our first efforts, using standard and confocal fluorescence microscopies, verified the suggestion that GFP or white in prolonged room light led to abnormalities.

A high mag from the bottom right shows the constellation of abnormalities in the 23 day aged ninaE Rh1 GFP flies

Reference

Stark, W. S. Green fluorescent protein (GFP) expression does not interfere with photoreceptor function in Drosophila. ARVO 2005, (IOVS 2005, 46)

Specific light treatments

Imran was set up to follow up on Asmir's photometry work using ninaE-driven Rh1-GFP flies. This initiative was undertaken to pursue our long-standing resolve to take advantage of the Stark lab's knowledge about the effects of specific wavelengths and intensities of light on Drosophila visual receptors. Therefore, in addition to room light and dark, we initially put flies in UV (ultraviolet) and blue light. Then, based on little-referenced findings by Schewemer & Henning (see also Smakman & Stavenga and Schwemer and Spengler, 1992), follolwed up only one time in our lab (Stark, 1994), we added green light; green was thought to activate rhodopsin to the more labile metarhodopsin but deprive eyes of the blue light needed by the photoisomerase to maintain active chromophore. Here is Imran's first graph on the effects of these light treatments on rhodopsin level. Part of Imran's new initiative was to examine GFP fluorescence. Here is that graph. Although we might have expected the fluorescence to decrease in the light treatments, it did not.

References

Schwemer & Henning 1984, Cell Tiss Res, 236, 293-303

Smakman & Stavenga, 1986 Vision Res, 26, 1019-1025

Schwemer & Spengler 1992, in Str. Fcn. Ret. Proteins, ed J. L. Rigaud, Colloq INSERM

Stark, W. S. R1-6 visual pigment is reduced by green light in Drosophila, 11 Internsat Cong Eye Res 1994 New Delhi, Exp Eye Res 59 S.33)

Lyso-tracker red

George undertook to set up eye injections. Earlier, we had used fluorescent dye to demonstrate that light-induced uptake was blocked by norpA(Zinkl et al, 1990). Also, we had used tunicamycin to interfere with rhodopsin in rhabdomeres (Stark et al., 1991). Encouraged by these findings and the ERG associated methodology and apparatus (micropipettes and micromanipulators), he thought he could make important and unique contributions.

Here is one of his first pictures, Rh1GFP on the left, LR on the right

With this technique, taking advantage of the fact that it marks rhabdomeres, George verified the expectation that there were no other rhabdomeres in ninaE Rh1-GFP flies which had apparently lost GFP fluorescing rhabdomeres (fig)

References

Stark WS, Christianson JS, Maier L, Chen D-M (1991) Inherited and environmentally induced retinal degenerations in Drosophila. In Retinal Degenerations, Anderson RE, Hollyfield JG, LaVail MM (eds), pp 61-75. New York: CRC Press, Inc.

Zinkl G, Maier L, Studer K, Sapp R, Chen DM, Stark WS (1990) Microphotometric, ultrastructural and electrophysiological analyses of light dependent processes on visual receptors in white-eyed wild-type and norpA (no receptor potential) mutant Drosophila. Vis Neurosc 5: 429-439

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

Imran's next knockdown data - coming attractions

Flies that George prepared became available:
Control: virgin female w; cn bw ninaE-Gal4; UAS-Rh1-GFP x male w, cn bw;Sb/TM3,ubx
Knockdown: virgin female w; cn bw ninaE-Gal4; UAS-Rh1-GFP x male w; cn bw; GGA.E3
Imran compared these flies for dark vs 2 day green light treatment

female w;Arf72RFP;ninaEGal4 x male GFPGGAE3

Here is a plate George made from phaerate adults that Eissenberg prepared (description will be ready soon). Bottom is Oregon-R (wild type), top three were different samples of w;Arf72RFP;ninaEGal4 x male GFPGGAE3. The reason to use phaerate adults was because it was believed the line would be lethal and not produce adults. However, we did get adults (see next plate)
Here is a plate George made from newly emerged adults (top is GGAGFP ARF72RFP cross, middle white-eyed otherwise wild type, bottom Oregon-R, all taken at the same settings) The channels are virtually identical on the merge. Question: Is the red channel really picking up RFP or just the tail of the GFP emission? Another question: Did nina E have a chance in phaerate adults and newly emerged flies to drive expression?
Here is a related image, one of our earlier pictures of white-eyed ARF72-RFP. The point of including this micrograph here is to emphasize the point that the RFP localization on the above plate (top, middle) looks very different.

Although the female w;Arf72RFP;ninaEGal4 x male GFPGGAE3 flies in the second figure look different from the white eyed and red eyed controls, there seem to be too many problems to pursue this for now

GGA kd interferes with Rhodopsin levels

Here are Imran's and Asmir's data based on 31 experimental vs 18 control flies reared on regular food and maintained in the dark. Non-overlapping error bars (95% confidence levels) indicate that the knockdown has significantly less rhodopsin.

Clathrin knockdown

I looked at clathrin knockdowns and found them to be normal (example):
VKK106632 x w-;cnbw ninaE Fal4; UAS Rh1 GFP
VKK103383 x cnbw ninaE Fal4; UAS Rh1 GFP
(The controls were:
VKK106632 x Oregon R
VKK103383 x Oregon R)

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

Coming attractions:

GGA kd and control flies are already being run

We plan to do an additional control: Heat shock of white-eyed otherwise wild-type flies

New careful intensity control:

It does not matter much for rhodopsin levels, since a ratio is calculated, but George designed an easy way to calibrate intensity of the fluorescence excitation beam. Davita had noticed a change in fluorescence readings which was attributed to a change in alignment of the beam.

GGA[P1] is not lethal

In separate experiments, Prof Eissenberg found that lethality was not caused by the P1 GGA mutant (and thus that GGA is not essential). As a result of this important finding, an extension was sought and granted on the resubmission of the manuscript to Traffic.

GGA P1 is not a protein null

With ehe help and expertise of Abdul Waheed and Joel Eissenberg, Shan Luan was recruited to do western blots.

Here is the second blot she obtained. Top is GGA, bottom is actin loading control, left is wild type

Middle is the mutant line that Eissenberg obtained by transposon insertion at GGA translation region. Western blot is supposed to show noGGA if the line is null mutant.

Right lane is the GGAP1 rescued larvae. Eissenberg was able to rescue GGAP1 by crossing it with Dp(1;3)DC203 which carries x-Chromosome duplication. Therefore, we are expecting not to see GGA as well since p(1;3)DC203 does not contain the GGA gene.

Eye optics of a more specific GGA P1 mutant

Male GGAP1;30327. Eyes of flies at least 12 days post eclosion look normal (figures to be hyperlinked soon) (Examples: (deep pseudopupil and optical neutralization of the cornea)

More comprehensive GGA kd photometry

Imran's new data, as with Davita's, now features a higher n and 95% confidence interval error bars

Reminder:
Control: virgin female w; cn bw ninaE-Gal4; UAS-Rh1-GFP x male w, cn bw;Sb/TM3,ubx
Knockdown: virgin female w; cn bw ninaE-Gal4; UAS-Rh1-GFP x male w; cn bw; GGA.E3
"O'Tousa" is our lab jargon for ninaE-Rh1-GFP since we got the stock from him. O'Tousa is expected to be the same as GGA control.

Rhodopsin Levels here.
Conclusions:
(1) O'Tousa, knockdown and control do not differ significantly
(2) there is significantly more rhodopsin for dark reared flies
(3) Rhodopsin level does not continue its decrease between 24 and 48 hours of green light

Fluorescence levels here.
Conclusions: Oddly, all levels are similar except for higher fluorescence for the knockdown in the dark

End of the summer

ninaE-Rh1-GFP (Imran's work)


Specific light treatments all decreased visualpigment (fig). By contrast, light treatments do not alter fluorescence (fig). Could it be that the visual pigment that is decreased by light treatments, the visualpigment that is measureable on the basis of rhodopsin - metarhodopsin conversions, is only the native visual pigment and not the Rh1 attached to GFP?

Fall 2010

Seminar


I gave the biology department seminar on September 10 (.pdf here).

End of summer 2010 report

Fall semester found the starklab with me teaching again, George and Imran back but with very limited time since they are students, Davita gone to college in Israel, and Katelyn Anderson signed up for a few credits of research.

At the end of the summer, despite many things that were done in better quality and quantity than ever, there were worries about the photometry data:
(1) Absorbance difference values seemed low
(2) variability was high despite high n's
(3) numbers that ought to have been similar were sometimes different
(4) fluorescence data seemed unreliable
and
(5) in the GGA kd vs control experiments, control vials were not productive and so many planned experiments went without the appropriate comparisons.

"Data mining" (Davita's work)

Some of Davita's work was in my seminar

Davita certainly was driven when it came to collecting data, so summer ended without her work completly entered into the spread sheet from the "polygraph records." Katelyn (Katie) was willing to work on this. The first data that is ready (here) relates to this text presented above concerning vitamin A replacement in deprived flies. It replicates the photometry in the Sapp et al (1991) paper.

"Data mining" (Imran's work)

Some of Imran's work was in my seminar

As with Davita, Imran's spread sheet was massive. A few graphs for Imran's work are shown above (end of summer). A re-evaluation led us to present the GGA k.d. vs. control data differently (here) [the zero data point is dark reared, while the X-axis presents time with green light treatment]. These data validate the impression we had (see George's poster, bottom left): (1) Fluorescence is not very different for KD vs Control, (2) light decreases but does not eliminate fluorescence; and (3) fluorescence stays the same with prolonged light treatment. Here, the appropriate controls were run but for George's poster, all we had ready as control flies were Rh1-Gfp (driven by ninaE).

Fluorescence work, presented above, was more debatable, but, interpreted with other data from this spread sheet, we guess that the fluorescence measurements mirror the absorbance difference measurements

Shan's work

She has been moved to a project where she is attempting to knock out GGA.

In August, she had a committee meeting for her progress report, presented here.

In December, she passed her PhD oral exam in front of her committee with this slide show, a Ph.D. Dissertation proposal, indicating her intent to generate a GGA knockout fly and to rescue this knockout mutant.

Publication

This research note is in press in Drosophila Information Service (2010 vol 93) [here is the figure]
link.

Spring, 2011

My teaching load was, for all intents and purposes, full time, and my research productivity was essentially zero.

Beaumont

In February, I told Joel that I had the possibility of funding from a source that he was not eligible for but that I had minimum time to work on the proposal. Thus, at a "cost" (to me) of only about one day of work, he prepared a proposal for our joint efforts, "Molecular genetic dissection of GGA, a protein essential for targeting vesicular transport." It was funded (for $4954 July 1, 2011 - June 30, 2012). Approximately 80% of the budget is for supplies for Shan Luan's dissertation research, as stated in the Beaumont specific aim to "engineer site-specific mutations in the GGA transgene construct to replace key amino acids with alanine, generate transgenic animals expressing the mutant GGA, and test the ability of the mutant GGA to complement the GGA mutation in lysosomal hydrolase and rhodopsin trafficking."

Collins award

I nominated George Denny for this "outstanding senior in Biology" award, and he won it. (He will be attending Wash U med school.)

From the archive

Here is a plate from DIS (Drosophila Information Service) 61, 1985, 162-164, Stark and Carlson, "Retinal degeneration in rdgBKS222 is blocked by oraJK84 which lacks photoreceptor organelles." I wanted to put this picture up after I hooked my scanner back up.

The thing I like about this picture is that is shows that the retinula cells of the ommatidium (top), the axons (middle right) and the R1-6 terminals (bottom) are pristine despite the lack of R1-6 opsin (ora was later shown by the O'Tousa lab to be a nonsense mutant at about 220 in the coding sequence).

Arrows (in the top figure) are belt desmosomes. Semper (cone) cell processes are clearly seen just in from the belt desmosomes between R8&R2, R3&R4, and R5&R6; A 4th is seen (but less clearly) between R7 and R1.

Arrows in the bottom are sites of synapses with T-shaped membrane specializations.

May 2011 presentation to St. Louis chapter of UW alumni

Here is "Using the fruit fly Drosophila to understand normal vision and what goes wrong in blindness"

Summer, 2011

Yeast


On the basis of findings from my Picking et al. (1995) paper, Joel had decided that yeast rearing offered a useful vitamin A deprivation manipulation, contrasting with rearing on Sang's medium in that the opsin gene would be activated. In pursuit, he mobilized Shan Luan to obtain numerous images (optical neutralization of the cornea). In this work, they made the surprising discovery that yeast-reared Rh1-GFP constructs (flies to test several hairpins for RNAi) showed GFP fluorescence in R1-6. I offered to replicate (extend?) their work. At my suggestion (because Rh1-GFP flies have light-induced receptor degeneration) Joel wrapped the vials in foil. I confirmed that there is Rh1-GFP fluorescence in yeast reared:
Rh1GFP (typical figure)
Rh1GFP x yw
Arf51F x Rh1GFP (typical bright figure out of bright, medium and low)
Arf102F x Rh1GFP
Arf79F 103572 x Rh1GFP
GGAE3 x Rh1GFP
ck100367 x Rh1GFP
(The control (Rh1GFP) had about 10x as much fluorescence)
All these observations were based on the deep pseudopupil, In my hands, only Rh1GFP on yeast had sufficient GFP fluorescence (and sufficiently low interference from eye color pigments) for me to see rhabdomere fluorescence in optical neutralization. None had noticible R<->M conversion, as expected on the basis of lack of chromophore precursor in the diet; furthermore, eye color pigments would have interfered with such an observation.

Oddities:

(1) The yeast food was not as dark as it used to be

(2) The Yeast ninaERh1GFP flies had short wings

Conclusions

It seems that Rh1GFP presence in yeast flies either (1) contradicts
a firm statement that vitamin A (or any related nutrient that may be
in yeast) controls opsin transcription through the ninaE promoter; or
(2) indicates that Rh1-GFP is controlled differently from Rh1-chromophore

Idea:

To answer the questions about whether degeneration of retinula cells in ninaE-Rh1-GFP AND hs-Rh1-GFP is due to stimulation of Rh1-chromophore or Rh1-GFP, the yeast rearing seems an interesting manipulation since they have only Rh1-GFP.

Does Yeast or retinoic acid rearing increase the rate of R<->M accumulation?

the answer is no

Here is R<->M assay (pseudopupil darkening) [left and middle] and fluorescence [right] for vitamin A deprived [top] and retinoic acid reared [bottom] replaced on carrot juice for 44 hr.

Here is a similar comparison [with yeast glucose rearing on the bottom] vitamin A replaced with carrot juice for about 25 hours

Katelyn Anderson expanded on the above observations by running out to longer time for:
Vitamin A replaced vitamin A deprived here
Vitamin A replaced retinoic acid here
The impression is that the R<->M (pseudopupil darkening) recovers better without retinoic acid and that the fluorescence recovers in both, perhaps more in retinoic acid.

Inverse relationship of absorbance difference and autofluorescence

George Denny, now a SLU alumuus heading for Wash U Med School, returned for part time work. He worked to help me post this serendipidous finding he made nearly one year ago

Does ninaE-Rh1GFP have less R<->M than control?

Does hs-Rh1GFP have less R<->M than control?

These two questions are justified since (1) possibly Rh1GFP does not photointerconvert leading to the increase in absorbance of 579 nm light (near metarhodopsin's peak) observed when 436 nm light converts much of the 480 nm-absorbing rhodopsin to 580 nm absorbing metarhodopsin; and (2) possibly Rh1GFP occupies space in the rhabdomeres lowering the level of native Rh1.

As Katelyn Anderson started her summer work in the lab, these were the first questions she addressed. Initial results suggest that the difference, if any, is subtle.

Figure. Flies were maintained in the dark and prepared under dim red light. Yellow transmission through the deep pseudopupil without (left) vs with (right) blue actinic stimulation. Top - white (control). Middle - heat shock. Bottom - ninaERhiGFP.

What are the time courses for degeneration in Rh1GFP?

We are REVISITING this topic! Here, from George Denny and Katie Anderson, is the disruption in ninE-Rh1-GFP flies moved from dark to light then maintained in the light for 9 days.

Does vitamin A deprivation protect Rh1GFP from degeneration?


The answer is yes. At first, Deep pseudopupil and Optical neutralization of the cornea observations of Sang's reared flies kept in the light sufficed for this demonstration at first.

Here, from George Denny, are confocal images showing intact receptor organization in Sang's-reared flies kept in the light then moved to carrot juice in the dark for 72 hours.

Here, from Katie Anderson, is the positive result for R<->M (pseudopupil darkening), and the very bright fluorescence (exposure dimmed to 40%) for flies ninaE-Rh1-Gfp flies raised on Sangs, aged 5 days in the light, then replaced in the dark on carrot juice for 3 days

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 retinular cells for 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 from the dark to the light is damaging.

A follow-up to that last statement

Katelyn Anderson obtained better R<->M conversions (pseudopupil darkenings on white-eyed flies maintained in the light than in white eyed flies that had been moved from the dark to the light 4 days ago (example)

She did this again, obtaining better images, for flies moved to the light 22.5 hours earlier vs always in the light (example). The "moved to the light" had greater pseudopupil darkening than the always in the light." They were both negative for R1-6 fluorescence

White eyed (non heat shock) flies that she heat shocked

By now, the quality of the photos was publishable.

White flies heat shocked and put in the dark for one day then examined (dark reared top, light reared bottom) (figure). Note that the darkening and the fluorescence are more for the dark reared flies. Note also that the expected autofluorescence of R1-6 is quite noticeable (see above)

Light-reared white flies heat shocked and put in the dark for one day then moved into light for 0 days (top row), 1 day (second row), 2days (3rd row) and 3 days (bottom row). [Note that the top row is the same as the bottom row on the above.] (figure) Conclusions: pseudopupil darkening and fluorescence are lost as flies were in the lighr (as already suggested by Asmir's work)

Dark-reared white flies heat shocked and put in the dark for one day then moved into light for 0 days (top row), 1 day (second row), 2days (3rd row) and 3 days (bottom row).. [Note that the top row is the same as the top row on the one before the above.] (figure) Conclusions: pseudopupil darkening and fluorescence are also lost, a similar trend as the previous entry (as already suggested by Asmir's work)

Heat shock flies

Here Katelyn compared dark-reared heat shocked flies shocked 8 days earlier in the dark and maintained in the dark with the same flies moved to the light 20 hours earlier. For R<->M (pseudopupil darkening), the dark maintained flies seemed subtly better, though both had profound darkenings. At first, it was surprising that there was no R1-6 rhabdomere fluorescence. The same result was replicated several times. We now feel fairly certain that the stock is bad. We screened several different lines and we are expanding the best one

Here are light reared heat shock flies, heat shocked, put in the dark for 24 hours (top) and compared with the same flies that had then been moved to the light for 23 hours (bottom). Note decrease in R<->M (pseudopupil darkening) and decrease in fluorescence (as expected though perhaps earlier than previously found)

More TEM

not shocked

here is a picture including, oddly, an ommatidium lacking a rhabdomere. bad fixation and ghosty cytoplasm plagued this set
Terrible fixation notwithstanding, there are a surprising number of features that can be declaired normal in this picture

3.25 hr
Area with large R7 and R8 cells, some good fixation, some ghosty cytoplasm, no missing rhabdomeres (Figure)
Am area of good fixation with all the usual (but no unusual) cytoplasmic features (figure)
Another such area. If anything there is a lot of rough ER (figure)
Another such area. (figure)

4.75 hr
Area with large R7 and R8 cells, some good fixation, some ghosty cytoplasm, no missing rhabdomeres (Figure)
Area of good fixation showing mormal structure, MVBs, Golgi, RER (Figure)
This figure shows the circles I have always syspected as rhodopsin/membrane vehicles for transport into the rhabdomere, but, since they did not present regularly, I conclude there are no special characteristics

26 hr

With the giant central rhabdomeres as diagnostic of the heat shock, we can seewith fair certainty a degenerated rhabdomere despite bad fixation (figure)
Here is an ommatidium one shy of the proper number of retinula cells (and rhabdomeres)
Here is one of a number of ommatidia we habe documented with degeneration

Overall conclusions (TEM of heat shock) (taking into account both sets of grids)

(1) For several sets, especially 4.75, we have really nice pictures
(2) Expected transport vesicles were definitely not obvious
(3) rough ER was abundant during the time hsRh1GFP was expected to be sent to rhabdomeres
(4) An odd scattering of giant R7 and R8 rhabdomeres was discovered
(5) Despite bad fixation, there was definitely degeneration in flies at 26 hr post heat shock


Heat shock (cumulative)

Here, I have assembled a file with ALL our studies on heat shock

Fall, 2011

I am back to teaching, and so, a precipitous drop in my productivity from the summer, especially with Katelyn Anderson, very productive over 2010-2011, especially the summer, taking a full course load plus 3 jobs, and George Denny, so productive for >2 years, gone to Wash U med school.

President's research fund is over

Here is my report to the VP for research: (1) cover letter, (2) technical report , (3) budget (summary sans the 3 annual printouts), and (4) funding, publications and presentations report.

Shan Luan's molecular work

This from her research mentor, Prof Eissenberg, "She just showed me results that strongly support the conclusion that she has several knockouts of GGA" (Oct 12)

New students

Two sophomores, Chintan Shah and Nihar Shah started practicing the optical manipulations. first picture here: The top is R<->M (pseudopupil darkening) for a typical light reared white eyed fly, while a representative dark reared fly is on the bottom. Clearly, since these two students replicated the expected finding (that light reared flies have less rhodopsin), they are nearing mastery of the technique

Here is their first attempt to compare dark reared (bottom) with light reared (top) ninaE-Rh1-GFP flies for R<->M (pseudopupil darkening) (left and middle) and fluorescence (right)

Degeneration in ninaE-Rh1-GFP flies in the light

Here is a montage comparing dark reared controls (bottom) vs flies moved to the light for 5 days (top). We strategized that Chintan's and Nihar's now well-practiced talents shoule be applied to a follow-up of the work starting at the end of April 2010 (confocal, histology, EM) showing the demise. Rhodopsin <-> metarhodopsin conversion is less, the fluorescent deep pseudopupil is not as clear, and the optical neutralized view is not as crisp,

Shan's presentation

Shan went to the 2011 Midwest Drosophila research conference Nov 4-5, 2011 in Allerton Park Conference Center Illinois and presented this poster. Of particular note was her own work obtaining a knockout of GGA as well as the demonstration that wild-type transgenes rescue the mutant.

Dark reared ninaE-Rh1-GFP rhodopsin comparison

Chintan and Nihar did not find a difference between ninaE-Rh1-GFP (top) vs white controls (bottom) (fig) (confirming what Katie Anderson had found earlier).

Shan's Progress

Her progress report for Dec 2011 is here

A research note

was submitted, accepted at Drosophila Information Service (vol 44, 2011, pp80-82)
text here
figure here

Paper

JCEissenberg, AMIlvarsonn,WSSly,AWaheed,RPohlmann, DWaschkau, DKretzschmar and ACDennes, Drosophila GGA-model: an ultimate gateway to GGA analysis, Traffic, 2011 December, 12(12) 1821-1838. (Although it was originally planned that I would be co-author, I thought that moving myself to the acknowledgements was the only ethical action since the paper had none of my data; also, since the paper contradicted my findings, I did not feel it was appropriate to endorse the paper with my authorship.)

2012

Follow-up on "holes" ("islands")


Katie Anderson obtained these data that confirmed our hypothesis that holes were more prevalent in white-eyed flies that had been moved to the light (this series is 1, 3, 4, 6, 8 and 11 days in the light)

Katie Anderson and Imran Shaikh went to SLU Medical School

Shan's Progress

Her progress report for Oct 2012 is here

She published a paper

Work with undergraduates

Two research notes were submitted and are published in 2013

(1) pdf, text, figure
(2) pdf, text, figure

Beaumont

after a budget extension, the grant is over and here is the final report

2013

Shan Luan's
successful dissertation defense April 30 2013 (pdf)

Summer 2013

I am only now getting around to looking at work I did May (5/17) 2012 relating to the yeast work presented Summer 2011 above. Here is a comparison of ninaE-Rh1-GFP vs HS-Rh1-GFP (14 days) [both raised on yeast food].

Recipe: 100 ml water, 10 g brewers yeast, 10 g glucose, 1.7 g agar, .69 g mold inhibitor, .8 ml propionic acid

President's seed grant

...
ended August, 2011. Here is the final report

Acknowledgements.
The hairpin construct allowing RNAi for GGA was provided to Prof. Eissenberg by Andre C. Dennes, DanielaWaschkau and Regina Pohlmann at the UKM Munster. A stock with Rh1 attached to GFP driven by the ninaE promoter (w;cn bw ninaE GAL4; UASRh1GFP) was obtained from Prof Joe O'Tousa at the University of Notre Dame. He also provided a white-eyed stock in which Rh1 is driven by the heat shock promoter (w; cn bw; hsRh1GFP). Also, he provided a white-eyed stock in which the ARF72 promoter drives RFP (ARF72RFP). SEMs were produced in collaboration with Dr. Jan Ryerse of SLU's Imaging Core. Histology and electron microscopy done by Joel Eissenberg was done in collaboration with Dr. Jan Ryerse and Ms Barbara Nagel of SLU's Imaging Core. Golgi microphotometry was done by George Denny, an undergraduate Biology major working in my laboratory

This page was last updated 9/11/13