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 restriction site (ACTAGT) and 20 bases downstream of the ATG (excluding ATG) of the fluorescent protein gene of interest. The reverse primer should include the leader sequence (GATC), followed by a SalI restriction site (GTCGAC) and the reverse complement of 20 bases upstream of the stop codon of the FP sequence (including stop).</li>
	<li>Using the primers designed in step 1.1, perform the PCR reaction using a thermocycler with the following settings: heat to 95 &#176;C for 1 min and cycle at 95 &#176;C for 30 s, 60 &#176;C for 30 s, and 72 &#176;C for 2 min per kB of PCR product. Performing 18 cycles is ample.</li>
	<li>Run the PCR reaction on an agarose gel (0.5% in Tris-acetate-EDTA). There should a single band corresponding to the expected product size. Isolate the fragment using a gel purification kit.</li>
	<li>The protocol employs an Httex1 vector carrying 25 (nontoxic) and 72 (HD-associated, displaying a strong aggregation) polyQ repeats. Digest both the PCR fragments and the vector with SpeI and SalI restriction enzymes for 3 h at 37 &#176;C.</li>
	<li>Purify the digested vector by running it on an agarose gel as in step 1.3.</li>
	<li>Purify the digested PCR fragment using a PCR purification kit.</li>
	<li>Ligate the resulting digested PCR fragment and p415-GAL1-FLAG-25/72QpolyQ plasmids1 using T4 ligase (1 h at room temperature). Use a 10 &#181;L reaction (1 &#181;L of T4 enzyme, 1 &#181;L of 10X buffer, 6 &#181;L of PCR fragment, and 2 &#181;L of vector).</li>
	<li>Transform 2 &#181;L of the ligation reaction into 50 &#181;L of Escherichia coli-competent cells and incubate them on ice for 30 min. Then, heat-shock the cells at 42 &#176;C for 30 s. Add 1 mL of SOC outgrowth media and incubate at 37 &#176;C for 1 h in a shaker. Plate 200 &#181;L of the reaction on an LB-agar plate containing 100 &#181;g/mL ampicillin. Incubate the plate at 37 &#176;C overnight.</li>
	<li>Select three individual bacterial colonies, grow them overnight in 3 mL of LB-broth containing 100 &#181;g/mL ampicillin at 37 &#176;C in a shaker and extract the plasmid DNA using a plasmid purification kit.</li>
	<li>Check the plasmid by digesting 500 ng of DNA using SpeI and SalI restriction enzymes for 1 h at 37 &#176;C and run the reaction on an agarose gel (0.5% in Tris-acetate-EDTA). There should be two bands at the right sizes of the vector (~7 kb) and the insert (size varies according to the gene of interest). Then, verify the plasmid by sequencing.</li>
	<li>Transform the p415-GAL1-FLAG-polyQ-FP plasmids into the yeast strain W303 following a standard yeast transformation protocol2.</li>
</ol>
2. Spotting Assay
<ol>
	<li>Streak the yeast clones carrying 25Q/72Q tagged with the FP of interest on an agar plate containing yeast selection media (synthetic complete-SC without leucine) with glucose as the carbon source. At the same time, also streak 25Q/72Q-ymsfGFP to serve as a positive control. 
	Note: 25Q/72Q constructs that do not contain a fluorescent tag are not toxic and can serve as negative control.</li>
	<li>Incubate the plates at 30 &#176;C for 2 - 3 d.</li>
	<li>Select up to three single colonies from the plate.</li>
	<li>Inoculate 5 mL of SC supplemented with 2% glucose as the carbon source.</li>
	<li>Pellet 200 &#181;L of each overnight culture and wash it 3x with sterile distilled water.</li>
	<li>Resuspend the cells in SC media containing 2% galactose as the carbon source to induce the expression of polyQ fusions. Incubate the galactose media overnight at 30 &#176;C in a tube rotator. As a control, repeat this step by using glucose-containing media.</li>
	<li>The next morning, equalize the cell densities to optical density at 600 nm (OD600) of 0.2 in 100 &#181;L of SC media in a sterile 96-well plate.</li>
	<li>Prepare four fivefold dilutions of each sample with sterile water by pipetting 20 &#181;L of the sample from the previous well into 80 &#181;L of media in the next well.</li>
	<li>Use a yeast pinning tool to spot the cells onto selective plates (containing glucose or galactose) and incubate at 30 &#176;C for 2 d.</li>
	<li>Image the plates with an image documentation device.</li>
</ol>
3. Quantification of Cell Growth in Liquid Culture
<ol>
	<li>Prepare the cell cultures, following steps 2.1 - 2.5 of this protocol.</li>
	<li>Measure the OD600 using a spectrophotometer.</li>
	<li>Dilute the cells to an OD600 of 0.1 in 300 &#181;L of media in a 96-well plate.</li>
	<li>Run each sample in triplicate.</li>
	<li>Incubate the plate in a plate reader/incubator with shaking capabilities. Set the number of samples, the temperature at 30 &#176;C, the absorbance at 600 nm, the length of the experiments to 24 h, and the measurement intervals to 15 min, and select the continuous shaking mode.</li>
	<li>Create the growth curve and quantify the area under the curve using scientific graphing software. The GraphPad Prism 7 is recommended. 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.</li>
</ol>
4. Fluorescent Microscopy
<ol>
	<li>Prepare the cell cultures, following steps 2.1 - 2.5 of this protocol.</li>
	<li>Dilute the cells 10x in growth media and transfer 200 &#181;L of each sample to 8-well imaging chambers.</li>
	<li>Image the cells using a confocal microscope equipped with a 63X Plan Aprochromoat objective (1.4 NA) at room temperature. 
	Note: The usage of a confocal microscope is optional. A standard wide-field fluorescent microscope can also be employed.</li>
	<li>Adjust the pinhole and laser power for optimal image acquisition. Since the 72Q aggregates are much brighter than the diffuse 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.</li>
	<li>Process the images using ImageJ20 or another image-processing software. At this step, the percentage of cells that display aggregate can be calculated manually is desired.</li>
</ol>
5. Dot Blot
Note: In this protocol, dot blot is used to examine the protein expression levels. Prepare the cell cultures, following steps 2.1 - 2.5 of this protocol.
<ol>
	<li>Generate protein lysates using glass beads in lysis buffer (100 mM Tris, pH 7.5; 200 mM NaCl; 1 mM EDTA; 5% glycerol, 1 mM dithiothreitol [DTT]). Add protease inhibitors, 4 mM phenylmethylsulfonyl fluoride (PSMF) and protease inhibitor cocktail, directly before use. Pellet 5 mL of the overnight culture and resuspend it in 200 &#181;L of glass beads and 200 &#181;L of lysis buffer. Vortex 30 s for 12 rounds. Centrifuge at 12,000 x g at 4 &#176;C for 10 min and collect the supernatant.</li>
	<li>Spot an equal amount of total proteins on a nitrocellulose membrane using a microfiltration apparatus. Prewet the membrane with PBS and assemble the apparatus. Connect to a vacuum source and make sure the screws are tightened. Turn on the vacuum and let the sample filter through the membrane by gravity.</li>
	<li>Block the membrane in PBS- 0.05% Tween/5% fat-free milk.</li>
	<li>Incubate the membrane with primary anti-FLAG antibody overnight at 4 &#176;C. The monoclonal anti-FLAG M1 is recommended.</li>
	<li>Wash the membrane 3x for 10 min each with PSB- 0.05% Tween.</li>
	<li>Incubate the membrane with a fluorescently labeled secondary antibody (anti-mouse IgG) for 1 h at room temperature in PBS- 0.05% Tween/5% fat-free milk.</li>
	<li>Wash membrane 3x 10 min with PSB- 0.05% Tween.</li>
	<li>Image-blot using an immunoblot documentation system.</li>
</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 GFP-tagged constructs16,19.
While the present protocol focuses on fluorescent proteins, various parts of the protocol could be readily adapted to test the effects of other protein tags. In addition, the present protocol employs low-copy yeast centromeric vectors that can vary in terms of copy numbers (generally one to two copies) present in cells25. Using integrative vectors to ensure a uniform expression across experimental conditions could circumvent this problem. While this protocol has been optimized for use in the W303 background, other S. cerevisiae strains can be employed. However, susceptibility to polyQ toxicity should be determined using the ymsfGFP-tagged vectors prior to designing new constructs. In certain cases, it may be appropriate to employ high-copy (2&#181;) vectors to generate a significant growth defect. It is also suggested to test multiple isolates following the yeast transformation with polyQ vectors to avoid selecting spontaneous suppressors showing a reduced polyQ toxicity. Of note, the W303 yeast strain26 is usually used as it is more sensitive to polyQ toxicity than other S288C derivatives, such as BY4741/BY474227, thus allowing for a wider range of growth phenotypes. Importantly, strains employed for this assay need to carry the Rnq1 prion protein since rnq1&#916; cells do not display polyQ toxicity and aggregation28. It is also important to use Httex1 constructs carrying the amino-terminal FLAG tag and lacking the proline-rich domain. Other variations of the fusion design may alter toxic phenotypes15. Finally, the induction of the polyQ fusion expression in galactose-containing media is a critical step of the protocol21. When transferring cells from glucose- to galactose-containing media, it is important to wash the cells at least three times with sterile water to eliminate all traces of glucose that could contribute to repressing the induction of the Gal1 promoter29. When performing spotting assays, culturing the cells overnight in galactose media to induce the expression of the fusion can exacerbate the toxic phenotype of the 72Q fusion and help discriminate changes in growth across different fusions16.
As a limitation, previous studies did not observe differential effects between a nonmonomeric version of CFP (a GFP derivative) and ymsfGFP16. Thus, at least for GFP variants, this assay may not be sensitive enough to discriminate between monomeric and oligomeric variants, highlighting the need to complement the polyQ toxicity assays with other standard methods, such as the OSER assay13 and biochemical analysis9,10,11,12 that can directly assess oligomerization. Also, it should be noted that FPs can behave differently in yeast compared to in vitro assays or their expression in other organisms23.
Collectively, these methods allow researchers to rapidly characterize new FPs and measure their effect on their fusion partner. In the future, this protocol will enable the quick screening of new derivatives of previously characterized FPs to identify mutants that behave similarly to GFP variants, which are still the gold standard measure for FP reporters. While this protocol focuses on fluorescent proteins, it can easily be adapted to screen for the effects of other genetically encoded tags, such as SNAP-tag30 and SunTag31.
In conclusion, this protocol provides a rapid and easily scalable assay to enable further characterization of the new generation of FPs and other genetically encoded tags to guide research in fusion protein design.

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 (Figure 1). A non-HD-associated polyQ length (25Q) is used as a negative control and does not display significant toxicity or aggregation15,16,21,22. 72Q is employed to obtain the HD-like phenotypes, including strong growth inhibition and polyQ aggregation. Importantly, the Httex1 coding sequence employed lack the proline-rich domain that follows the polyQ stretch. In the presence of the proline-rich domain, Httex1 is not toxic in yeast15. In this assay, an Httex1 fused to a yeast-optimized monomeric variant of superfolder GFP12 (ymsfGFP)16 is used as a positive control as previously described16. The constructs also contain a FLAG epitope tag at the N-terminus of Httex1. This allows detection of the different fusions with the same antibody (anti-FLAG) for biochemical analysis. As a proof-of-principle, 72Q Httex1 fused to yeast-optimized TagBFP2 (yomTagBFP2)23 does not result in slow growth measured by either spot assays on agar plates or growth in liquid media (Figure 2), indicating that the nature of the fluorescent tag can indeed impede polyQ expansion behavior in cells.
Aggregation of the fluorescent polyQ fusions can be assessed using fluorescent microscopy. 72Q-ymsfGFP displays significant aggregation compared to 25Q. However, the 72-yomTagBFP fluorescent signal remains diffused throughout the cytoplasm (Figure 3). In most of the cases, it is not recommended to use the same image acquisition settings (laser power, exposure time) to acquire both 25Q and 72Q images. The aggregates in the 72Q-expressing cells are much brighter than the diffused 25Q signal. Therefore, under imaging conditions used to acquire 72Q images, the diffused 25Q signal may appear very weak or not be visible at all. Appropriate acquisition settings should also be applied to minimize the saturation during the imaging of the 72Q-expressing cells.
Expression levels of the various polyQ fusions could affect toxicity. Detergent-insoluble amyloids, such as polyQ aggregates, are notoriously difficult to study biochemically and are not suitable for an analysis by standerd SDS-PAGE. Therefore, dot blots can be performed to assess protein levels. The inclusion of the FLAG tag at the amino terminus end of Httex1 allows detection of all the fluorescent fusions simultaneously, despite the presence of FPs (Figure 4). Alternatively, semi-denaturing detergent agarose gel electrophoresis (SDD-AGE) can be performed to assess the formation of polyQ oligomers16. A detailed protocol and video are available in Halfmann and Lindquist24.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/58748/58748fig1.jpg" /> 
Figure 1: Workflow diagram for the analysis of the effect of fluorescent protein tag on the aggregation and toxicity of polyQ expansion proteins in yeast. First, FPs are cloned into yeast expression vectors encoding a galactose-inducible version of FLAG-tagged Httex1 harboring either 25Q (nontoxic) or 72Q (HD-associated, aggregating and toxic) repeats. Clones are selected and verified by sequencing and, subsequently, transformed in yeast. Following the induction of polyQ fusion expression by incubation in galactose-containing media, either spotting assays on agar plates or growth liquid media can assess the polyQ toxicity. PolyQ aggregation is analyzed by fluorescent microscopy. A relative expression of the different constructs is assessed using dot blot. <a href="https://www.jove.com/files/ftp_upload/58748/58748fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/58748/58748fig2.jpg" /> 
Figure 2: Representative growth assay results following the expression of Httex1 fluorescent fusions in yeast. Yeast expressing either 25Q or 72Q Httex1 fused to ymsfGFP or yomTagBFP was cultured in glucose (control) or galactose media (polyQ-induced) overnight and either (A) spotted on agar plates or (B) incubated further in liquid media to assess growth under the different conditions. While 72Q-ymsfGFP induces a significant growth defect, 72Q-yomTagBFP displays a growth phenotype similar to the nontoxic 25Q counterparts. <a href="https://www.jove.com/files/ftp_upload/58748/58748fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/58748/58748fig3.jpg" /> 
Figure 3: Representative fluorescent images of Httex1 fluorescent fusions in yeast. Yeast expressing either 25Q or 72Q Httex1 fused to ymsfGFP or yomTagBFP was cultured in glucose (control) or galactose media (polyQ-induced) overnight and imaged with a confocal microscope. While the 72Q-ymsfGFP expression results in a strong polyQ protein aggregation, 72Q-yomTagBFP displays a diffused cytoplasmic signal similar to the nontoxic 25Q counterparts. <a href="https://www.jove.com/files/ftp_upload/58748/58748fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/58748/58748fig4.jpg" /> 
Figure 4: Representative dot blot analysis of Httex1 fluorescent fusion expression in yeast. Yeast expressing 25Q, 46Q, 72Q, or 103Q Httex1 fused to CFP was cultured in galactose media (polyQ-induced) overnight and processed for dot blot analysis. Fivefold dilutions of the cell lysates are shown. <a href="https://www.jove.com/files/ftp_upload/58748/58748fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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 ...

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

Nanyang Technological University

Research

JoVE Journal

Biology

6.7K Views.  University of Western Ontario. 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.

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

Immunohistochemistry on Paraffin Sections of Mouse Epidermis Using Fluorescent Antibodies

In the epidermis, immunohistochemistry is an efficient means of localizing specific proteins to their relative expression compartment; namely the basal, suprabasal, and stratum corneum layers. The precise localization within the epidermis of a particular protein lends clues toward its functional role within the epidermis. In this chapter, we describe a reliable method for immunolocalization within the epidermis modified for both frozen and paraffin sections that we use very routinely in our laboratory. Paraffin sections generally provide much better morphology, hence, superior results and photographs; however, not all antibodies will work with the harsh fixation and treatment involved in their processing. Therefore, the protocol for frozen sectioning is also included. Within paraffin sectioning, two fixation protocols are described (Bouin's and paraformaldehyde); the choice of fixative will be directly related to the antibody specifications and may require another fixing method.

We describe a reliable method for immunolocalization within the epidermis modified for both frozen and paraffin sections that we use very routinely in our laboratory.

In the epidermis, immunohistochemistry is an efficient means of localizing specific proteins to their relative expression compartment; namely the basal, ...

Genetic Studies of Human DNA Repair Proteins Using Yeast as a Model System 

Understanding the roles of human DNA repair proteins in genetic pathways is a formidable challenge to many researchers. Genetic studies in mammalian systems have been limited due to the lack of readily available tools including defined mutant genetic cell lines, regulatory expression systems, and appropriate selectable markers. To circumvent these difficulties, model genetic systems in lower eukaryotes have become an attractive choice for the study of functionally conserved DNA repair proteins and pathways. We have developed a model yeast system to study the poorly defined genetic functions of the Werner syndrome helicase-nuclease (WRN) in nucleic acid metabolism. Cellular phenotypes associated with defined genetic mutant backgrounds can be investigated to clarify the cellular and molecular functions of WRN through its catalytic activities and protein interactions. The human WRN gene and associated variants, cloned into DNA plasmids for expression in yeast, can be placed under the control of a regulatory plasmid element. The expression construct can then be transformed into the appropriate yeast mutant background, and genetic function assayed by a variety of methodologies. Using this approach, we determined that WRN, like its related RecQ family members BLM and Sgs1, operates in a Top3-dependent pathway that is likely to be important for genomic stability. This is described in our recent publication [1] at <a href="http://www.impactaging.com">www.impactaging.com</a>. Detailed methods of specific assays for genetic complementation studies in yeast are provided in this paper.

Genetic Studies of Human DNA Repair Proteins Using Yeast as a Model System

Genetic studies in yeast can be employed to investigate the molecular and cellular functions of human genes in cellular DNA metabolism. Methods are described for the genetic characterization of the human WRN gene product defective in the premature aging disorder Werner syndrome in functionally conserved pathways using yeast as a tractable model system.

Understanding the roles of human DNA repair proteins in genetic pathways is a formidable challenge to many researchers.  Genetic studies in mammalian ...

Expression of Recombinant Proteins in the Methylotrophic Yeast Pichia pastoris

Protein expression in the microbial eukaryotic host Pichia pastoris offers the possibility to generate high amounts of recombinant protein in a fast and easy to use expression system.
As a single-celled microorganism P. pastoris is easy to manipulate and grows rapidly on inexpensive media at high cell densities. Being a eukaryote, P. pastoris is able to perform many of the post-translational modifications performed by higher eukaryotic cells and the obtained recombinant proteins undergo protein folding, proteolytic processing, disulfide bond formation and glycosylation [1].
As a methylotrophic yeast P. pastoris is capable of metabolizing methanol as its sole carbon source. The strong promoter for alcohol oxidase, AOX1, is tightly regulated and induced by methanol and it is used for the expression of the gene of interest. Accordingly, the expression of the foreign protein can be induced by adding methanol to the growth medium [2; 3].
Another important advantage is the secretion of the recombinant protein into the growth medium, using a signal sequence to target the foreign protein to the secretory pathway of P. pastoris. With only low levels of endogenous protein secreted to the media by the yeast itself and no added proteins to the media, a heterologous protein builds the majority of the total protein in the medium and facilitates following protein purification steps [3; 4].
The vector used here (pPICZ&alpha;A) contains the AOX1 promoter for tightly regulated, methanol-induced expression of the gene of interest; the &alpha;-factor secretion signal for secretion of the recombinant protein, a Zeocin resistance gene for selection in both E. coli and Pichia and a C-terminal peptide containing the c-myc epitope and a polyhistidine (6xHis) tag for detection and purification of a recombinant protein. We also show western blot analysis of the recombinant protein using the specific Anti-myc-HRP antibody recognizing the c-myc epitope on the parent vector.

Expression of Recombinant Proteins in the Methylotrophic Yeast Pichia pastoris

The protocol describes protein expression using the methylotrophic yeast Pichia pastoris. The preparation of electrocompetent yeast cells, transformation of the vector with the gene of interest into P. pastoris and yeast DNA purification are also performed. Western blot analysis and protein purification build the last steps in this protein expression protocol.

Protein expression in the microbial eukaryotic host Pichia pastoris offers the possibility to generate high amounts of recombinant protein in a fast and ...

Fluorescent Labeling of COS-7 Expressing SNAP-tag Fusion Proteins for Live Cell Imaging

SNAP-tag and CLIP-tag protein labeling systems enable the specific, covalent attachment of molecules, including fluorescent dyes, to a protein of interest in live cells. These systems offer a broad selection of fluorescent substrates optimized for a range of imaging instrumentation. Once cloned and expressed, the tagged protein can be used with a variety of substrates for numerous downstream applications without having to clone again. There are two steps to using this system: cloning and expression of the protein of interest as a SNAP-tag fusion, and labeling of the fusion with the SNAP-tag substrate of choice. The SNAP-tag is a small protein based on human O6-alkylguanine-DNA-alkyltransferase (hAGT), a DNA repair protein. SNAP-tag labels are dyes conjugated to guanine or chloropyrimidine leaving groups via a benzyl linker. In the labeling reaction, the substituted benzyl group of the substrate is covalently attached to the SNAP-tag. CLIP-tag is a modified version of SNAP-tag, engineered to react with benzylcytosine rather than benzylguanine derivatives. When used in conjunction with SNAP-tag, CLIP-tag enables the orthogonal and complementary labeling of two proteins simultaneously in the same cells.

SNAP-tag and CLIP-tag protein labeling systems enable the specific, covalent attachment of molecules, including fluorescent dyes, to a protein of interest in live cells. Once cloned and expressed, the tagged protein can be used with a variety of substrates for numerous downstream applications without having to clone again.

SNAP-tag and CLIP-tag protein labeling systems enable the specific, covalent attachment of molecules, including fluorescent dyes, to a protein of interest ...

Rapid Homogeneous Detection of Biological Assays Using Magnetic Modulation Biosensing System

A magnetic modulation biosensing system (MMB) [1,2] rapidly and homogeneously detected biological targets at low concentrations without any washing or separation step. When the IL-8 target was present, a 'sandwich'-based assay attached magnetic beads with IL-8 capture antibody to streptavidin coupled fluorescent protein via the IL-8 target and a biotinylated IL-8 antibody. The magnetic beads are maneuvered into oscillatory motion by applying an alternating magnetic field gradient through two electromagnetic poles. The fluorescent proteins, which are attached to the magnetic beads are condensed into the detection area and their movement in and out of an orthogonal laser beam produces a periodic fluorescent signal that is demodulated using synchronous detection. The magnetic modulation biosensing system was previously used to detect the coding sequences of the non-structural Ibaraki virus protein 3 (NS3) complementary DNA (cDNA) [2]. The techniques that are demonstrated in this work for external manipulation and condensation of particles may be used for other applications, e.g. delivery of magnetically-coupled drugs in-vivo or enhancing the contrast for in-vivo imaging applications.

Magnetic modulation biosensing system is utilized to rapidly, sensitively and simply detect biological assays, such as DNA molecules and proteins.

A magnetic modulation biosensing system (MMB) [1,2] rapidly and homogeneously detected biological targets at low concentrations without any washing or ...

Growth Assays to Assess Polyglutamine Toxicity in Yeast

Protein misfolding is associated with many human diseases, particularly neurodegenerative diseases, such as Alzheimer&#x2019;s disease, Parkinson's disease, and Huntington's disease 1. Huntington's disease (HD) is caused by the abnormal expansion of a polyglutamine (polyQ) region within the protein huntingtin. The polyQ-expanded huntingtin protein attains an aberrant conformation (i.e. it misfolds) and causes cellular toxicity 2. At least eight further neurodegenerative diseases are caused by polyQ-expansions, including the Spinocerebellar Ataxias and Kennedy&#x2019;s disease 3.
The model organism yeast has facilitated significant insights into the cellular and molecular basis of polyQ-toxicity, including the impact of intra- and inter-molecular factors of polyQ-toxicity, and the identification of cellular pathways that are impaired in cells expressing polyQ-expansion proteins 3-8. Importantly, many aspects of polyQ-toxicity that were found in yeast were reproduced in other experimental systems and to some extent in samples from HD patients, thus demonstrating the significance of the yeast model for the discovery of basic mechanisms underpinning polyQ-toxicity. 
A direct and relatively simple way to determine polyQ-toxicity in yeast is to measure growth defects of yeast cells expressing polyQ-expansion proteins. This manuscript describes three complementary experimental approaches to determine polyQ-toxicity in yeast by measuring the growth of yeast cells expressing polyQ-expansion proteins. The first two experimental approaches monitor yeast growth on plates, the third approach monitors the growth of liquid yeast cultures using the BioscreenC instrument. 
Furthermore, this manuscript describes experimental difficulties that can occur when handling yeast polyQ models and outlines strategies that will help to avoid or minimize these difficulties. The protocols described here can be used to identify and to characterize genetic pathways and small molecules that modulate polyQ-toxicity. Moreover, the described assays may serve as templates for accurate analyses of the toxicity caused by other disease-associated misfolded proteins in yeast models.

This manuscript describes three complementary protocols for assessing the toxicity of polyglutamine (polyQ)-expansion proteins in the yeast Saccharomyces cerevisiae. These protocols can easily be modified to monitor the toxicity of other misfolded proteins in yeast.

Protein misfolding is associated with many human diseases, particularly neurodegenerative diseases, such as Alzheimer&#x2019;s disease, Parkinson's ...

Identification of Protein Interacting Partners Using Tandem Affinity Purification

A critical and often limiting step in understanding the function of host and viral proteins is the identification of interacting cellular or viral protein partners. There are many approaches that allow the identification of interacting partners, including the yeast two hybrid system, as well as pull down assays using recombinant proteins and immunoprecipitation of endogenous proteins followed by mass spectrometry identification1. Recent studies have highlighted the utility of double-affinity tag mediated purification, coupled with two specific elution steps in the identification of interacting proteins. This approach, termed Tandem Affinity Purification (TAP), was initially used in yeast2,3 but more recently has been adapted to use in mammalian cells4-8.
As proof-of-concept we have established a tandem affinity purification (TAP) method using the well-characterized eukaryotic translation initiation factor eIF4E9,10.The cellular translation factor eIF4E is a critical component of the cellular eIF4F complex involved in cap-dependent translation initiation10. The TAP tag used in the current study is composed of two Protein G units and a streptavidin binding peptide separated by a Tobacco Etch Virus (TEV) protease cleavage sequence. The TAP tag used in the current study is composed of two Protein G units and a streptavidin binding peptide separated by a Tobacco Etch Virus (TEV) protease cleavage sequence8. To forgo the need for the generation of clonal cell lines, we developed a rapid system that relies on the expression of the TAP-tagged bait protein from an episomally maintained plasmid based on pMEP4 (Invitrogen). Expression of tagged murine eIF4E from this plasmid was controlled using the cadmium chloride inducible metallothionein promoter.
Lysis of the expressing cells and subsequent affinity purification via binding to rabbit IgG agarose, TEV protease cleavage, binding to streptavidin linked agarose and subsequent biotin elution identified numerous proteins apparently specific to the eIF4E pull-down (when compared to control cell lines expressing the TAP tag alone). The identities of the proteins were obtained by excision of the bands from 1D SDS-PAGE and subsequent tandem mass spectrometry. The identified components included the known eIF4E binding proteins eIF4G and 4EBP-1. In addition, other components of the eIF4F complex, of which eIF4E is a component were identified, namely eIF4A and Poly-A binding protein. The ability to identify not only known direct binding partners as well as secondary interacting proteins, further highlights the utility of this approach in the characterization of proteins of unknown function.

Tandem affinity purification is a robust approach for the identification of protein binding partners. As proof of concept, this methodology was applied to the well-characterized translation initiation factor eIF4E to co-precipitate the host cell factors involved in translation initiation. This method is easily adapted to any cellular or viral protein.

A critical and often limiting step in understanding the function of host and viral proteins is the identification of interacting cellular or viral protein ...

Analysis of SNARE-mediated Membrane Fusion Using an Enzymatic Cell Fusion Assay

The interactions of SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins on vesicles (v-SNAREs) and on target membranes (t-SNAREs) catalyze intracellular vesicle fusion1-4. Reconstitution assays are essential for dissecting the mechanism and regulation of SNARE-mediated membrane fusion5. In a cell fusion assay6,7, SNARE proteins are expressed ectopically at the cell surface. These "flipped" SNARE proteins drive cell-cell fusion, demonstrating that SNAREs are sufficient to fuse cellular membranes. Because the cell fusion assay is based on microscopic analysis, it is less efficient when used to analyze multiple v- and t-SNARE interactions quantitatively.
Here we describe a new assay8 that quantifies SNARE-mediated cell fusion events by activated expression of &beta;-galactosidase. Two components of the Tet-Off gene expression system9 are used as a readout system: the tetracycline-controlled transactivator (tTA) and a reporter plasmid that encodes the LacZ gene under control of the tetracycline-response element (TRE-LacZ). We transfect tTA into COS-7 cells that express flipped v-SNARE proteins at the cell surface (v-cells) and transfect TRE-LacZ into COS-7 cells that express flipped t-SNARE proteins at the cell surface (t-cells). SNARE-dependent fusion of the v- and t-cells results in the binding of tTA to TRE, the transcriptional activation of LacZ and expression of &beta;-galactosidase. The activity of &beta;-galactosidase is quantified using a colorimetric method by absorbance at 420 nm.
The vesicle-associated membrane proteins (VAMPs) are v-SNAREs that reside in various post-Golgi vesicular compartments10-15. By expressing VAMPs 1, 3, 4, 5, 7 and 8 at the same level, we compare their membrane fusion activities using the enzymatic cell fusion assay. Based on spectrometric measurement, this assay offers a quantitative approach for analyzing SNARE-mediated membrane fusion and for high-throughput studies.

We have developed a cell fusion assay that quantifies SNARE-mediated membrane fusion events by activated expression of &beta;-galactosidase.

The interactions of SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor)  proteins on vesicles (v-SNAREs) and on target membranes ...

Investigating the Spreading and Toxicity of Prion-like Proteins Using the Metazoan Model Organism C. elegans

Prions are unconventional self-propagating proteinaceous particles, devoid of any coding nucleic acid. These proteinaceous seeds serve as templates for the conversion and replication of their benign cellular isoform. Accumulating evidence suggests that many protein aggregates can act as self-propagating templates and corrupt the folding of cognate proteins. Although aggregates can be functional under certain circumstances, this process often leads to the disruption of the cellular protein homeostasis (proteostasis), eventually leading to devastating diseases such as Alzheimer&#8217;s disease (AD), Parkinson&#8217;s disease (PD), Amyotrophic lateral sclerosis (ALS), or transmissible spongiform encephalopathies (TSEs). The exact mechanisms of prion propagation and cell-to-cell spreading of protein aggregates are still subjects of intense investigation. To further this knowledge, recently a new metazoan model in Caenorhabditis elegans, for expression of the prion domain of the cytosolic yeast prion protein Sup35 has been established. This prion model offers several advantages, as it allows direct monitoring of the fluorescently tagged prion domain in living animals and ease of genetic approaches. Described here are methods to study prion-like behavior of protein aggregates and to identify modifiers of prion-induced toxicity using C. elegans.

Investigating the Spreading and Toxicity of Prion-like Proteins Using the Metazoan Model Organism C. elegans

Prion-like propagation of protein aggregates has recently emerged as being implicated in many neurodegenerative diseases. The goal of this protocol is to describe, how to use the nematode C. elegans as a model system to monitor protein spreading and to investigate prion-like phenomena.

Prions are unconventional self-propagating proteinaceous particles, devoid of any coding nucleic acid. These proteinaceous seeds serve as templates for ...

Comparing the Affinity of GTPase-binding Proteins using Competition Assays

In this protocol we demonstrate a method for comparing the competition between GTPase-binding proteins. Such an approach is important for determining the binding capabilities of GTPases for two reasons: The fact that all interactions involve the same face of the GTPases means that binding events must be considered in the context of competitors, and the fact that the bound nucleotide must also be controlled means that conventional approaches such as immunoprecipitation are unsuitable for GTPase biochemistry. The assay relies on the use of purified proteins. Purified Rac1 immobilized on beads is used as the bait protein, and can be loaded with GDP, a non-hydrolyzable version of GTP or left nucleotide free, so that the signaling stage to be investigated can be controlled. The binding proteins to be investigated are purified from mammalian cells, to allow correct folding, by means of a GFP tag. Use of the same tag on both proteins is important because not only does it allow rapid purification and elution, but also allows detection of both competitors with the same antibody during elution. This means that the relative amounts of the two bound proteins can be determined accurately.

This protocol compares the relative affinities of binding partners for Rho-family GTPases, including Rac1. In vivo, Rac1-binding proteins compete for a single binding interface, the conformation of which is dictated by a bound nucleotide. The nucleotide is both important and difficult to control experimentally, due to the high hydrolysis rate.

In this protocol we demonstrate a method for comparing the competition between GTPase-binding proteins. Such an approach is important for determining the ...