This study is supported by an operating grant from the Canadian Institutes for Health Research to M.L.D. and P.L. The work presented here is supported by a John R. Evans Leader Fund award from the Canadian Foundation for Innovation and a matching fund from the Ontario Research Fund to P.L. Y.J. is supported by an MSc to PhD transfer scholarship from the Dean of the Schulich School of Medicine and Dentistry at The University of Western Ontario. S.D.G. is supported by a PhD Scholarship from ALS Canada.

1. Generation of New Fluorescently Tagged Httex1 Reporters for an Expression in Yeast
Note: This section has been modified from the protocol by Jiang et al.16 and Albakri et al.19.
<ol>
	<li>Design primers to amplify the sequence encoding the fluorescent protein or interest by PCR. The forward primer should include a leader sequence to assist the restriction enzyme during digestion (GATC), followed by a SpeI restric...

<ol>
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The authors have nothing to disclose.

In this article, various assays to measure the aggregation of Httex1 polyQ expansions and their effect on yeast growth were employed as a model to study how different fluorescent proteins alter their fusion partners in the context of fluorescent reporters. Using a GFP variant (ymsfGFP) as a positive control, we showed that this detects significant changes in polyQ toxicity and aggregation between different fluorescent tags and allows for a direct and rapid comparison of the polyQ-FP fusion performance against ...

Since the initial characterization of the green fluorescent protein (GFP) from Aequorea victoria1, a wide palette of genetically encoded FPs have been developed, allowing cell biologists to simultaneously localize and track multiple cellular events/proteins in living cells2,3. FPs are derived from multiple organisms, from jellyfish to coral, and therefore, display specific biophysical properties that divert extensively beyond their respective fluorescent spectrum. These properties include brightness, photostability, and a tendency to oligomerize among others2,4. Selecting monomeric FPs is an important aspect in the selection of a suitable tag when designing a fluorescent reporter, in order to minimize inappropriate interactions and alterations of the fusion partner&#39;s function and maximize the reporter efficiency for a given cellular compartment4,5,6. While GFP has, over time, been evolved to minimize the effect of adding the fluorescent tag to the fusion partner5,7,8, how new FP variants perform compared to GFP remains difficult to assess.
Few methods exist to characterize the behavior of FPs. Most of them involve testing biophysical properties of FPs using biochemical approaches, such as ultracentrifugation and gel filtration protocols9,10,11,12. Such methods have the caveat of using purified FPs in solution, offering little insight into their behavior in intact cells. The development of the organized smooth endoplasmic reticulum (OSER) assay offers a quantifiable assessment of FPs&#39; tendency to oligomerize in living cells13 by testing the ability of overexpressed FPs to reorganize endoplasmic reticulum tubules into OSER whorls14. This technique can successfully detect changes between monomeric and oligomeric variants of GFP and other FPs. However, it relies mostly on overexpression in transiently transfected cells, and the quantitation and image analysis can be time-consuming unless the technique is adopted as an automated data collection and analysis workflow.
In order to complement these approaches, we established an assay that takes advantage of the effect of fluorescent tags on the toxicity and aggregation of polyQ expansions in yeast15,16. The expansion of the polyQ stretch with more than 36 repeats within the first exon of the gene encoding the huntingtin protein (Htt) is associated with Huntington&#39;s disease17,18. The expression of expanded Httex1 in yeast results in a strong aggregation of the misfolded Htt protein coupled to a severe growth defect. Interestingly, these phenotypes are strongly influenced by the sequences flanking the polyQ stretch, including FPs15,16. It was rationalized that the different properties of FPs can differentially affect polyQ toxicity in yeast. Indeed, compared to GFP-like FPs, red fluorescent proteins and their evolved forms have shown a reduced toxicity and aggregation16. This manuscript provides a detailed protocol to assess the effect of the next generation of FPs on polyQ toxicity and aggregation in yeast. This assay allows for a rapid and potentially high-content analysis of FP variants that can be used in parallel with previously characterized techniques for the optimal characterization of new FPs and can assess how they perform compared to GFP.

<table>
	<tbody>
		<tr>
			<td>5-alpha Competent E. coli (High efficiency)</td>
			<td>New Englanfd Biolab</td>
			<td>C2987</td>
			<td></td>
		</tr>
		<tr>
			<td>SpeI-HF</td>
			<td>New Englanfd Biolab</td>
			<td>R3133</td>
			<td>High fidelity enzymes are preferred</td>
		</tr>
		<tr>
			<td>SalI-HF</td>
			<td>New Englanfd Biolab</td>
			<td>R0138</td>
			<td>High fidelity enzymes are preferred</td>
		</tr>
		<tr>
			<td>Agarose</td>
			<td>Fisher Scientific</td>
			<td>BP160</td>
			<td></td>
		</tr>
		<tr>
			<td>LB-Agar</td>
			<td>Fisher Scientific</td>
			<td>BP1425</td>
			<td></td>
		</tr>
		<tr>
			<td>LB-Broth</td>
			<td>Fisher Scientific</td>
			<td>BP1426</td>
			<td></td>
		</tr>
		<tr>
			<td>Ampicilin</td>
			<td>Fisher Scientific</td>
			<td>BP1760</td>
			<td></td>
		</tr>
		<tr>
			<td>PfuUltra High-fidelity DNA Polymerase</td>
			<td>Agilent Technologies</td>
			<td>600382</td>
			<td></td>
		</tr>
		<tr>
			<td>EPOCH2 microplate spectrophotometer</td>
			<td>BioTek Instruments inc</td>
			<td>EPOCH2TC</td>
			<td></td>
		</tr>
		<tr>
			<td>Yeast Pin Replicator</td>
			<td>V&#38;P Scientific inc.</td>
			<td>VP407AH</td>
			<td></td>
		</tr>
		<tr>
			<td>SPI imager</td>
			<td>S&#38;P Robotics inc.</td>
			<td>spImager-M</td>
			<td></td>
		</tr>
		<tr>
			<td>Zeiss LSM 800 confocal with AryScan</td>
			<td>Carl Zeiss Microscopy</td>
			<td>LSM 800</td>
			<td></td>
		</tr>
		<tr>
			<td>8 well Lab-Tek imaging chambers</td>
			<td>Fisher Scientific</td>
			<td>12565470</td>
			<td></td>
		</tr>
		<tr>
			<td>Bio-Dot apparatus</td>
			<td>Bio-Rad</td>
			<td>1706545</td>
			<td></td>
		</tr>
		<tr>
			<td>Chemi Doc XRS+</td>
			<td>Bio-Rad</td>
			<td>1708265</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-FLAG M1 antibody</td>
			<td>Sigma-Aldrich</td>
			<td>F3040</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti-mouse IgG alexa 555 secondary antibody</td>
			<td>Thermo&#160;</td>
			<td>A32727</td>
			<td></td>
		</tr>
		<tr>
			<td>Plasmid MiniPrep Kit</td>
			<td>Fisher Scientific</td>
			<td>K0503</td>
			<td></td>
		</tr>
		<tr>
			<td>Plasmid Gel extraction Kit</td>
			<td>Fisher Scientific</td>
			<td>K0831</td>
			<td></td>
		</tr>
		<tr>
			<td>PCR Purification Kit</td>
			<td>Fisher Scientific</td>
			<td>K0702</td>
			<td></td>
		</tr>
		<tr>
			<td>Prizm</td>
			<td>GraphPad</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>TAE (Tris-Acetate-EDTA)</td>
			<td>Fisher Scientific</td>
			<td>BP13354</td>
			<td></td>
		</tr>
	</tbody>
</table>

effect of fluorescent proteins on fusion partners using polyglutamine toxicity assays in yeast

For the investigation of protein localization and trafficking using live cell imaging, researchers often rely on fusing their protein of interest to a fluorescent reporter. The constantly evolving list of genetically encoded fluorescent proteins (FPs) presents users with several alternatives when it comes to fluorescent fusion design. Each FP has specific optical and biophysical properties that can affect the biochemical, cellular, and functional properties of the resulting fluorescent fusions. For instance, several FPs tend to form nonspecific oligomers that are susceptible to impede on the function of the fusion partner. Unfortunately, only a few methods exist to test the impact of FPs on the behavior of the fluorescent reporter. Here, we describe a simple method that enables the rapid assessment of the impact of FPs using polyglutamine (polyQ) toxicity assays in the budding yeast Saccharomyces cerevisiae. PolyQ-expanded huntingtin proteins are associated with the onset of Huntington&#39;s disease (HD), where the expanded huntingtin aggregates into toxic oligomers and inclusion bodies. The aggregation and toxicity of polyQ expansions in yeast are highly dependent on the sequences flanking the polyQ region, including the presence of fluorescent tags, thus providing an ideal experimental platform to study the impact of FPs on the behavior of their fusion partner.

FPs have different biophysical properties, including their tendency to oligomerize, that can affect the behavior of their fusion partners in the context of fluorescent reporters. This protocol describes a simple method where multiple FPs can be fused to toxic polyQ expansions. Since polyQ toxicity is highly dependent on the sequences flanking the polyQ stretch15, this assay allows a rapid and direct comparison of fluorescent polyQ fusion reporters (<strong class="x...

Watch this Scientific Journal Video about Effect of Fluorescent Proteins on Fusion Partners Using Polyglutamine Toxicity Assays in Yeast at JoVE.com

Effect of Fluorescent Proteins on Fusion Partners Using Polyglutamine Toxicity Assays in Yeast

This article describes protocols to assess the effect of fluorescent proteins on the aggregation and toxicity of misfolded polyglutamine expansion for the rapid evaluation of a newly uncharacterized fluorescent protein in the context of fluorescent reporters.

For the investigation of protein localization and trafficking using live cell imaging, researchers often rely on fusing their protein of interest to a ...

This method can help answer key questions in the field of fluorescent proteins such as how fluorescent proteins can affect their fusion partners. The main advantage of this technique is that it provides a rapid and easily scalable assessment of the effects of the fluorescent proteins and their fusion partners. Though this method can provide insight into fluorescent protein behavior, it can also be applied to other genetically encoded tags.Also demonstrating this procedure will be Sonja Di Gregorio, who is a graduate student at Western University. Each fluorescent protein that is tested by this method is first cloned into a yeast expression vector that encodes a galactose inducible version of FLAG-tagged HTT exon one harboring either the non-toxic 25Q repeat or the Huntington Disease associated toxic 72Q repeat. Clones are selected and verified by sequencing and subsequently transformed into yeast.To prepare cell cultures for the various assays, streak the yeast clones carrying 25Q or 72Q tag with a fluorescence protein of interest on an agar plate containing yeast selection medium with glucose as the carbon source. At the same time, streak yeast carrying 25Q or 72Q yeast optimized monomeric superfolder GFP to serve as a positive control. Incubate the plates at 30 degrees Celsius for two to three days.Select up to the three single colonies from each plate and inoculate five milliliters of synthetic complete medium supplemented with 2%glucose as the carbon source. Incubate the cultures at 30 degrees Celsius overnight. On the following day, transfer 200 microliters of each overnight culture to a microcentrifuge tube and centrifuge to pellet the cells.Wash at least three times with sterile distilled water. It's important to wash the cells well to eliminate all traces of glucose that could contribute to repressing the induction of the GAL1 promoter. Prepare the cells as demonstrated previously and re-suspend them in synthetic complete medium containing 2%galactose as the carbon source to induce the expression of polyQ fusions.As a control, re-suspend the cells in glucose-containing media. Incubate the cells at 30 degrees Celsius in a tube rotator overnight. On the following morning, measure the optical density at 600 nanometers of each culture using a spectrophotometer.Equalize the cell densities to an optical density 600 of 2 in 100 microliters of synthetic complete medium in a sterile 96-well plate. Prepare four five-fold dilutions of each sample by pipetting 20 microliters of the sample from the previous well into 80 microliters of media in the next well. Use a yeast pinning tool to spot the cells onto selective plates and incubate at 30 degrees Celsius for two days.Image the plates with an image documentation device. Prepare the cell cultures for this assay and then measure the OD600 of each culture using a spectrophotometer. Dilute the cells to an OD600 of 1 in 300 microliters of media in the 96-well plate.Prepare each sample in triplicate. Incubate the plate in a plate reader incubator with shaking capabilities. Set the number of samples, the temperature at 30 degrees Celsius, the absorbance at 600 nanometers, the length of the experiments to 24 hours, and the measurement intervals to 15 minutes.Select the continuous shaking mode. When the experiment is done, create the growth curve and quantify the area under the curve using scientific graphing software. Paste the data into an XY table with three replicate values.The growth curve will be shown under the graphs folder at the left side. To quantify the area under the curve, select analyze at the top left and click area under curve in XY analyses. Start this procedure by diluting the prepared cells 10-fold in growth medium.Transfer 200 microliters of each sample to an eight-well imaging chamber. Image the cells using a standard wide-field fluorescent microscope. Adjust the imaging settings for image acquisition.Since the 72Q aggregates are much brighter than the diffused 25Q signal, it is often required to use a different acquisition setting between the different plasmids in order to avoid saturation of the fluorescent signal. Process the images using a suitable image-processing software. In this protocol, dot blot is used to examine protein expression levels.Prepare the buffer for generating protein lysates by adding four micromolar of phenylmethylsulfonyl fluoride and a protease inhibitor cocktail to the lysis buffer. Pellet five milliliters of each overnight culture by centrifugation. Re-suspend the cells in 200 microliters of glass beads and 200 microliters of lysis buffer.Vortex for 30 seconds for 12 rounds, icing in between rounds. Centrifuge at 12, 000 times G at four degrees Celsius for 10 minutes and collect the supernatant. Use a microfiltration apparatus to spot equal amounts of total protein on a nitrocellulose membrane.Pre-wet the membrane with PBS and assemble the apparatus. Connect it to a vacuum source and make sure the screws are tightened. Load the samples, turn on the vacuum, and let the sample filter through the membrane by gravity.Blot the membrane in PBS 05%Tween 5%fat-free milk for 30 minutes. Incubate the membrane with primary anti-flag antibody at four degrees Celsius overnight. On the following day, wash the membrane three times for 10 minutes each with PBS 05%Tween.Incubate the membrane with a fluorescently labeled secondary antibody in PBS 05%Tween 5%fat free milk at room temperature for one hour. Wash the membrane three times for 10 minutes each with PBS 05%Tween. Subsequently, image the membrane using an amino blot documentation system.Yeast expressing either 25Q or 72Q HTT exon one fused to yeast optimized monomeric superfolder GFP or yeast optimized monomeric tag BFP2 was cultured in glucose or galactose medium overnight and either spotted on agar plates or incubated further in liquid media. While 72Q yeast optimized monomeric superfolder GFP induces a significant growth defect. 72Q yeast optimized monomeric tag BFP2 displays a growth phenotype similar to the non-toxic 25Q counterparts indicating that the nature of the fluorescent tag can impede polyQ expansion behavior in cells.Assessment of aggregation of the fluorescent polyQ fusions by fluorescent microscopy shows that 72Q yeast optimized monomeric superfolder GFP displays significant aggregation, while 72Q yeast optimized monomeric tag BFP2 displays a diffused cytoplasmic signal similar to the non-toxic 25Q counterparts. Since expression levels of the various polyQ fusions could affect toxicity, dot blots were performed to assess protein levels. Results from five-fold dilutions of the cell lysates are shown.Don't forget that this technique allows for rapid comparison of uncharacterized and fluorescent proteins with the GFP variants, but it cannot directly assess oligomerization. Following this procedure, other measures, for example, the ulcer assay in mammalian cells can be performed to answer additional questions such as the monomeric status of the fluorescent proteins.

effect-fluorescent-proteins-on-fusion-partners-using-polyglutamine

Research

JoVE Journal

Biology

6.7K vues.  University of Western Ontario. Cette méthode peut aider à répondre à des questions clés dans le domaine des protéines fluorescentes telles que la façon dont les protéines fluorescentes peuvent affecter leurs partenaires de fusion. Le principal avantage de cette technique est qu’elle fournit une évaluation rapide et facilement évolutive des effets des protéines fluorescentes et de leurs partenaires de fusion. Bien que cette méthode puisse fournir un aperçu du comportement des protéines fluorescentes, elle pe...