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This video demonstrates imaging and quantification of protein aggregates in genetically modified Drosophila brains. Brains are fixed, washed, and treated with an antifading agent before mounting. Images are captured using a confocal microscope and quantified to observe the transmission of mutant protein aggregates.
1. Coupling Gal4- and QF-mediated Htt Transgene Expression in Drosophila
2. Micro-dissection and Fixation of Adult Drosophila Brains
NOTE: This dissection procedure has been modified from a previous publication and can be used to prepare brains for imaging direct fluorescence signals from Htt-fluorescent protein fusions.
3. Whole Brain Mounting
4. Imaging and Quantifying Prion-like Transmission of Aggregates
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Figure 1. A genetic approach for coupled expression of mutant and wild-type Htt transgenes using the QF-QUAS and Gal4-UAS binary expression systems. In "cell A," mCherry-tagged mutant Htt protein containing a pathogenic-length polyQ stretch (Q91) is expressed using a QF driver located downstream of a tissue-specific promoter A ("PA"). In "cell B," a YFP-tagged wild-type Htt containing a normal polyQ stretch (Q25) is expressed via a Gal4 driver controlled by tissue-specific promoter B ("PB"). In Figures 2-4, Or67d-QF was used to drive QUAS-HttQ91-mCherry expression in DA1 ORNs, and repo-Gal4 was used to express UAS-HttQ25-YFP in all glia27. Importantly, HttQ91-mCherry is only expressed in QF-expressing cells by virtue of the QUAS sequence placed upstream of the transgene. Similarly, HttQ25-YFP is only expressed via Gal4, which specifically recognizes the UAS. If any overlap in the tissue distribution of the QF and Gal4 drivers is detected, transgenes encoding QS in Gal4-expressing cells and Gal80 in QF-expressing cells can be introduced. Appending fluorescent protein tags onto wild-type and mutant Htt allows for differentiation of the two proteins during imaging and the ability to measure FRET between appropriate donor/acceptor pairs (e.g., CFP/YFP or YFP/mCherry).
Figure 2. Confocal images of prion-like conversion of glial HttQ25-YFP by neuronal HttQ91-mCherry aggregates. (A) Maximum intensity projection of ~ 30 µm of confocal slices showing one antennal lobe from a male fly expressing HttQ91-mCherry (red) in DA1 ORN axons using Or67d-QF and HttQ25-YFP (green) in glia using repo-Gal4. The approximate boundaries of the antennal lobe and DA1 glomerulus, where DA1 ORN axons terminate, are indicated by the dotted and solid lines, respectively. (B) Maximum intensity projection from ~ 20 µm of confocal slices showing a magnified view of the DA1 glomerular region from A. (C) A single 0.35 µm confocal slice showing a single HttQ25-YFP puncta and its associated HttQ91-mCherry signal (indicated by the arrow in each channel). The signal in the red channel was enhanced to visualize co-localization between HttQ25-YFP and HttQ91-mCherry signals. All images were acquired using a 40X 1.4NA oil objective. Scale bars = 10 µm.
Figure 3. Three-dimensional analysis of HttQ91-mCherry aggregates in DA1 ORN axons. (A) A 3D depiction of HttQ91-mCherry aggregates expressed in the DA1 glomerulus via Or67d-QF using the same data shown in Figure 2B. (B) A screenshot showing individual objects or "spots" identified from the raw data in (A) using an image analysis software package. The software identified 56 objects of varying sizes in this channel/image. The spot indicated by the arrowhead in (B) was measured to have a diameter of ~ 1.2 µm. Arrows point to locations where two objects are inaccurately merged into one spot by the software, likely due to close proximity of the individual puncta. To overcome this, different thresholding settings should be tested in the software and/or merged spots should be separated manually if possible. Scale bars = 10 µm. (C) Histogram showing the distribution of diameters measured by the software for the HttQ91-mCherry "spots" shown in (B).
Figure 4. Co-localization and FRET analysis of induced HttQ25-YFP aggregates. (A) A montage of 4 individual 0.35 µm confocal z-slices from a male fly brain expressing HttQ91-mCherry in DA1 ORNs using Or67d-QF and HttQ25-YFP in glia using repo-Gal4. The signals were adjusted so that even small HttQ91-mCherry aggregates are visible and induced HttQ25-YFP aggregates stand out from the surrounding diffuse signal. The slices shown are each separated by ~ 1.0 µm (slice number indicated at the lower right corner of merged images) so that multiple aggregates can be observed. Arrows indicate HttQ25-YFP puncta that were determined to be in or near focus in that particular z-slice by manually moving through the z-stack. Of the seven HttQ25-YFP puncta indicated here, six have detectably associated HttQ91-mCherry signal (i.e., 86% of the HttQ25-YFP aggregates co-localize with HttQ91-mCherry). Note that the mCherry signal associated with HttQ25-YFP puncta is often weaker than the majority of mCherry-positive puncta in the DA1 glomerulus. Scale bar = 5 µm. (B) A HttQ25-YFP/HttQ91-mCherry-co-localized punctum before (left panels) and after (right panels) mCherry (acceptor) photobleaching. The resulting increase in YFP (donor) fluorescence was used to produce a pixel-by-pixel FRET efficiency (FRETeff) image using the AccPbFRET plug-in for ImageJ46. This particular aggregate has an overall FRETeff of 61%. Scale bar = 1 µm.
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Name | Company | Catalog Number | Comments |
Phosphate buffered saline (PBS), 10X, pH 7.4 | ThermoFisher Scientific | AM9625 | Dilute to 1X |
Triton X-100 | Sigma-Aldrich | T9284-1L | |
Kimwipes | Thomas Scientific | 2904F24 | |
20% paraformaldehyde (PFA) | Electron Microscopy Sciences | 15713-S | |
Normal Goat Serum (NGS), filtered | Lampire Biological Laboratories | 7332500 | Aliquot and freeze upon receipt |
Chicken anti-GFP | Aves Labs | GFP-1020 | Use at 1:500 dilution |
Rabbit anti-DsRed | Clontech | 632496 | Use at 1:2000 dilution; can recognize DsRed-based fluorescent proteins (e.g. mCherry, mStrawberry, tdTomato, etc.) |
Mouse anti-Bruchpilot | Developmental Studies Hybridoma Bank | nc82 | Use at 1:100 dilution; will label active pre-synaptic structures thoughout the fly brain |
FITC anti-chicken | ThermoFisher Scientific | SA1-7200 | Use at 1:250 dilution |
Alexa Fluor 568 anti-rabbit | Life Technologies | A11011 | Use at 1:250 dilution |
Alexa Fluor 647 anti-mouse antibody | Life Technologies | A21235 | Use at 1:250 dilution |
Slowfade Gold Antifade Reagent | Life Technologies | S36936 | |
Microscope Slides (25 x 75 x 1.0 mm) | Fisher Scientific | 12-550-143 | |
Cover Glass (22 x 22 mm) | Globe Scientific | 1404-15 | |
Dumont Biology Grade Forceps, Style 3 | Ted Pella | 503 | use in non-dominant hand |
Dumont Biology Grade Forceps, Style 5 | Ted Pella | 505 | use in dominant hand |
LAS X image analysis software | Leica | ||
Imaris image analysis software | Bitplane |
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Source: Donnelly, K. M., et al. Monitoring Cell-to-cell Transmission of Prion-like Protein Aggregates in Drosophila Melanogaster. J. Vis. Exp. (2018)
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