We thank James Bardwell and Tiago F. Outeiro for kindly sharing the plasmids containing &#945;-synuclein. Changhan Lee received funding from the National Research Foundation of Korea (NRF) funded by the Korean government (MSIT) (grant 2021R1C1C1011690), the Basic Science Research Program through the NRF funded by the Ministry of Education (grant 2021R1A6A1A10044950), and the new faculty research fund of Ajou University.

1. Preparation of media and solutions
<ol>
	<li>Media preparation
	<ol>
		<li>To prepare YPD medium, dissolve 50 g of YPD powder in dH2O to make a final volume of 1 L. Autoclave for sterilization. Store at room temperature (RT).</li>
		<li>To make YPD agar medium, dissolve 50 g of YPD powder and 20 g of agar in 1 L of dH2O. Autoclave for sterilization. After cooling down, pour onto Petri dishes. Store at 4 &#730;C.</li>
		<li>To make SC with raffinose (SRd)-Ura medium, dissolve 6.7 g of yeast nitrogen base with ammonium sulfate and without amino acids in 795 mL of dH2O. Autoclave for sterilization. Add 100 mL of 20% raffinose, 5 mL of 20% glucose, and 100 mL of 10x -Ura drop-out before use.</li>
		<li>To make SD-Ura agar medium, dissolve 6.7 g of yeast nitrogen base with ammonium sulfate and without amino acids and 20 g of agar in 800 mL of dH2O. Autoclave in a flask with a volume over 2 L. Add 100 mL of 20% glucose and 100 mL of 10x -Ura drop-out. Mix well and pour onto Petri dishes. Store at 4 &#730;C.</li>
		<li>To make SC with galactose (SG)-Ura agar medium, dissolve 6.7 g of yeast nitrogen base with ammonium sulfate and without amino acids and 20 g of agar in 800 mL of dH2O. Autoclave in a flask with a volume over 2 L. Add 100 mL of 20% galactose and 100 mL of 10x -Ura drop-out. Mix well and pour on Petri dishes. Store at 4 &#730;C.</li>
	</ol>
	</li>
	<li>Stock solutions to use with SD media
	<ol>
		<li>To make a 20% carbon source (glucose, galactose, and raffinose), dissolve 100 g of glucose, galactose, or raffinose in dH2O to make a final volume of 500 mL. Autoclave and store at RT.</li>
		<li>To make 10x yeast synthetic drop-out medium supplements without uracil (10x-Ura drop-out), dissolve 19.2 g of &#34;yeast synthetic drop-out medium supplements without uracil&#34; in dH2O to make a final volume of 1 L. Autoclave and store at RT. 
		&#8203;NOTE: For the preparation of a medium containing agar, add a magnetic stir bar to the flask to mix all the solutions well before pouring onto Petri dishes.</li>
	</ol>
	</li>
</ol>
2. Yeast transformation
NOTE: The pRS426 plasmid was used to clone the &#945;-synuclein gene and contained the URA3 gene as a selectable marker. There are familial PD-associated variants of &#945;-synuclein that have severe disease phenotypes (e.g., cytotoxicity). These variants were also used here to monitor their cytotoxicity and aggregated foci formation. Use the same colony of yeast transformants for all the experiments to get consistent results.
<ol>
	<li>For the transformation of the yeast expression plasmids, use an empty vector (pRS426) as the control and &#945;-synuclein-containing plasmids. Referring to the standard lithium acetate (LiAc) method11, prepare six competent cells and 0.5 &#181;g of each plasmid, and perform the transformation.</li>
	<li>To select for yeast transformants containing&#160;plasmids, use the synthetic defined medium (SD medium) without uracil (SD-Ura), and incubate the plates at 30 &#730;C for 2-3 days.</li>
	<li>Patch three single colonies onto an SD-Ura agar plate for the selection of cells containing plasmids for further experiments. 
	&#8203;NOTE: To generate accurate and reliable results, we examined at least three biological replicates per strain.</li>
</ol>
3. Spotting assay
NOTE: The expression of GFP-tagged &#945;-synuclein in high-copy number plasmids is known to be associated with cytotoxicity in yeast10.
<ol>
	<li>Inoculate the three patched yeast transformants per strain into 3 mL of SRd-Ura for the selective growth of yeast containing the plasmids with 2% raffinose for inhibition of the induction of &#945;-synuclein under the GAL1 promoter and 0.1% glucose. Incubate the cell culture at 30 &#176;C under agitation at 200 rpm. 
	NOTE: Repeating all the experiments with different colonies is important. Obtaining the same results from over three independent colonies means the result is reliable.</li>
	<li>The next day, inoculate the overnight cultured cells into 3 mL of fresh SRd-Ura to an OD600 of 0.2, and incubate for 6 h at 30 &#176;C under agitation at 200 rpm.&#160;</li>
	<li>When the OD600 reaches 0.6-0.8, dilute the cells in 96-well plates for the spotting assay as follows.
	<ol>
		<li>Measure the OD600 of all the samples, and dilute the samples to an OD600 of 0.5 with sterile dH2O (the mixed volume is 100 &#181;L) in lane 1 of a 96-well plate to equalize the number of cells in each culture.</li>
		<li>Perform fivefold serial dilutions of the yeast cells by adding 160 &#181;L of sterile dH2O to the next five lanes. Ensure a total volume of 40 &#181;L when serially diluting the cells in each lane. 
		NOTE: Low dilution factors can produce larger differences between the samples. Sufficient pipetting is required to ensure uniform mixing when performing the serial dilutions. The pipette tip should be replaced at every dilution point. Otherwise, the cells attached to the surface of the tip could be transferred to the next well and affect the cell number.</li>
	</ol>
	</li>
	<li>Use a multichannel pipette to transfer 10 &#181;L from each well to spot the samples on SD-Ura agar plates for the controls and SG-Ura agar plates to monitor toxicity. 
	NOTE: The plates should be dried until the spotted samples are fully absorbed onto the plates.</li>
	<li>Incubate the spotted plates upside down at 30 &#176;C for 4 days.</li>
	<li>Take images of the plates every 24 h until day 4, and then analyze the data by comparing the growth differences between strains spotted by eye. Count the individual colonies in the most diluted sample, calculate the number of viable cells in the original culture for each strain, or compare the size of the single colonies.</li>
</ol>
4. Measurement of yeast growth using a microplate reader
<ol>
	<li>Inoculate the three patched yeast transformants per strain into 3 mL of SRd-Ura, and incubate at 30 &#176;C under agitation at 200 rpm overnight. 
	NOTE: To understand each colony phenotype of the humanized yeast strain expressing &#945;-synuclein, use the same transformants for all the experiments.</li>
	<li>The next day, inoculate the overnight cultured cells into a total of 200 &#181;L of fresh SD-Ura and SG-Ura in 96-well plates. Adjust the final volume, including the cells, to 200 &#181;L, and adjust the initial cell OD600 to 0.05.
	<ol>
		<li>Adjust the Program settings in the microtiter plate as follows: set the target temperature to 30 &#730; C, set the shaking (Linear) duration to 890 s, set the shaking (Linear) amplitude to 5 mm, set the interval time to 00:15:00, and set the measurement as absorbance at 600 nm.</li>
		<li>Incubate the plate for 2-3 days for all the strains to reach the stationary phase in a microplate reader.</li>
	</ol>
	</li>
	<li>Analyze the data by plotting the growth curve (e.g., using Workbook). Acquire the data points by averaging the values of absorbance from the three biological replicates, and indicate the error bars. Use the value of absorbance from only media as a control, and subtract this from the values from cultured cells (optional).</li>
</ol>
5. Fluorescence microscopy
<ol>
	<li>Inoculate the patched yeast transformants into 3 mL of SRd-Ura, and culture them overnight at 30 &#176;C under agitation at 200 rpm. 
	NOTE: To determine the correlation of the phenotype with the cytotoxicity and foci formation, use the same colonies for the experiment.</li>
	<li>The next day, reinoculate the overnight cultured cells into 3 mL of fresh SRd-Ura to an OD600 of 0.003. Incubate the culture until the OD600 reaches 0.2 at 30 &#176;C under agitation at 200 rpm (~18 h).</li>
	<li>When an OD600 of 0.2 is achieved, add 300 &#181;L of 20% galactose, and then incubate for another 6 h at 30 &#176;C under agitation at 200 rpm.</li>
	<li>Collect the cells by centrifugation at 800 x g for 5 min, and discard the supernatant.</li>
	<li>Resuspend the cell pellet gently in 30 &#181;L of distilled water.</li>
	<li>Load 5 &#956;L of the resuspended cells onto a slide glass. Prepare an agarose gel pad12 and put it onto the loaded cells to make it spread evenly.</li>
	<li>Perform microscopic imaging with a 100x objective using both brightfield and GFP-fluorescence filters.
	<ol>
		<li>First, use brightfield to focus the cell with a 100x objective using immersion oil. Use the screen capture option for generating the images using the program.</li>
		<li>Switch to a GFP-fluorescence filter. Adjust the image exposure time to between 50 ms and 300 ms to achieve a clear image by balancing the signal-to-noise ratio. Screen capture the image using the program.</li>
		<li>Image many cells from multiple points rather than just one.</li>
	</ol>
	</li>
	<li>Analyze the images by counting the percentage of cells containing foci of &#945;-synuclein aggregates using analytical software. Observe the differences between the soluble GFP protein signal and the foci-formed GFP signal, as well as the diffusion of GFP with the A30P variant form of &#945;-synuclein. 
	NOTE: Foci formation will be observed with the wild-type, E46K, and A53T variants of &#945;-synuclein.&#160;</li>
</ol>

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The authors have no conflicts of interest to disclose.

Given the complexity of various cellular systems in humans, it is advantageous to use yeast as a model for studying human neurodegenerative diseases. Although it is nearly impossible to investigate the complex cellular interactions of the human brain using yeast, from a single-cell perspective, yeast cells have a high level of similarity to human cells in terms of the genomic sequence homology and fundamental eukaryotic cellular processes8,13. Moreover, given the ease of genetic manipulation, yeast can facilitate the study of complex and demanding human systems. Therefore, yeast has been widely used as a model system to understand eukaryotic cellular processes and the pathogenesis of various diseases14,15,16. In particular, there are discrepancies between the in silico, in vitro, and in vivo aggregation properties of amyloidogenic proteins, including &#945;-synuclein17. Taking into account the complexity and unpredictability of protein behavior in the cell, it is important to study protein properties in vivo. Thus, to understand what is happening in a cell with &#945;-synuclein, it is necessary to investigate it in in vivo conditions, and yeast is thought to be a useful and powerful model system for this.
In this study, the properties of &#945;-synuclein in yeast in a highly overexpressed condition using a 2 micron plasmid were analyzed. The &#945;-synuclein level was controlled by an inducible GAL1 promoter. This promoter is activated when galactose is available as a nutrient, and thus, this system allows us to study phenotypes derived from specific levels of &#945;-synuclein protein expression. The overexpression of the wild-type and &#945;-synuclein variants, except for A30P, caused toxicity in the spotting assay, as well as in the growth curve analysis. These wild-type and &#945;-synuclein variants were examined using fluorescence microscopy to monitor the protein aggregates of &#945;-synuclein in the cells. From these experiments, the formation of foci derived from &#945;-synuclein aggregation was observed in the &#945;-synuclein wild-type and variants exhibiting cytotoxicity. These results confirm that the cellular toxicity and foci formation resulting from misfolded &#945;-synuclein protein aggregation are linked to one of the representative phenotypes of PD in neuronal cells18,19.
It is important to incubate the transformants for 3 days and to choose big and reddish colonies for further experiments. The W303a strain has an adenine mutation, which makes the cell turn red20. Therefore, choosing a white colony results in working with the wrong contaminant. Choosing the appropriate cell stage for the induction of &#945;-synuclein by adding galactose is important in order to observe a reproducible phenotype. For testing the effect of a specific protein on the cytotoxicity of &#945;-synuclein, other proteins can be co-expressed by co-transformation. However, plasmids with Ura markers must be avoided as the &#945;-synuclein plasmid is maintained with Ura.
This method is not limited to the study of the cellular characteristics of &#945;-synuclein itself and can be a useful tool to validate candidate genes that may influence the aggregation of &#945;-synuclein. Interestingly, various bacterial proteins (e.g., chaperones and curli) have recently been reported to affect the formation of &#945;-synuclein in vitro21,22. The effect of a candidate gene on &#945;-synuclein-derived toxicity or aggregate formation can be monitored by co-expression. For example, &#945;-synuclein maintained by the URA3 marker gene was co-expressed in cells with a candidate gene from the pRS425 plasmid, which contained a galactose-inducible promoter, and a LEU2 marker. Furthermore, this system could be used for the purpose of genetic screening using gene deletion or overexpression libraries, as well as chemical libraries, in yeast23. However, in genome-wide screening assays that aim to uncover genetic interactions, co-overexpression with other proteins in a plasmid will not be easy to achieve in terms of transformation efficiency. In such situations, it may be better to express &#945;-synuclein in the genome in multiple copies to manipulate the toxic form20. In conclusion, this yeast model system and protocol provide a straightforward and comprehensive platform to understand the physiology of &#945;-synuclein and evaluate genes with the potential ability to modulate &#945;-synuclein toxicity and aggregation (Figure 5).

Parkinson&#39;s disease (PD) is a serious problem for aging societies worldwide. The aggregation of &#945;-synuclein is closely associated with PD, and protein aggregates of &#945;-synuclein are widely used as a molecular biomarker for diagnosing the disease1. &#945;-synuclein is a small acidic protein (140 amino acids in length) with three domains, namely the N-terminal lipid-binding &#945;-helix, the amyloid-binding central domain (NAC), and the C-terminal acidic tail2. The misfolding of &#945;-synuclein can occur spontaneously and eventually leads to the formation of amyloid aggregates called Lewy bodies3. &#945;-synuclein may contribute to the pathogenesis of PD in several ways. In general, it is thought that its abnormal, soluble oligomeric forms called protofibrils are toxic species that cause neuronal cell death by affecting various cellular targets, including synaptic function3.
The biological models used to study neurodegenerative diseases must be relevant to humans with respect to their genome and cellular biology. The best models would be human neuronal cell lines. However, these cell lines are associated with several technical issues, such as difficulties in the maintenance of cultures, low efficiency of transfection, and high expense4. For these reasons, an easy and reliable tool is required to accelerate the progress in this research area. Importantly, the tool should be easy to use for analyzing the collected data. From these perspectives, various model organisms have been widely used, including Drosophila, Caenorhabditis elegans, Danio rerio, yeast, and rodents5. Among them, yeast is the best model organism because genetic manipulation is easy, and it is cheaper than the other model organisms. Most importantly, yeast has high similarities to human cells, such as 60% sequence homology to human orthologs and 25% close homology with human disease-related genes6, and they also share fundamental eukaryotic cell biology. Yeast contains many proteins with similar sequences and analogous functions to those in human cells7. Indeed, yeast expressing human genes has been widely used as a model system to elucidate cellular processes8. This yeast strain is called humanized yeast and is a useful tool for exploring the function of human genes9. Humanized yeast has merit for studying genetic interactions because genetic manipulation is well established in yeast.
In this study, we used the yeast Saccharomyces cerevisiae as a model organism to study the pathogenesis of PD, particularly for investigating &#945;-synuclein aggregate formation and cytotoxicity10. For the expression of &#945;-synuclein in the budding yeast, the W303a strain was used for transformation with plasmids encoding for the wild-type and familial PD-associated variants of &#945;-synuclein. As the W303a strain has an auxotrophic mutation on URA3, it is applicable for the selection of cells containing plasmids with URA3. The expression of &#945;-synuclein encoded in a plasmid is regulated under the GAL1 promoter. Thus, the expression level of &#945;-synuclein can be controlled. In addition, the fusion of green fluorescent protein (GFP) at the C-terminal region of &#945;-synuclein allows the monitoring of &#945;-synuclein foci formation. To understand the characteristics of the familial PD-associated variants of &#945;-synuclein, we also expressed these variants in yeast and examined their cellular effects. This system is a straightforward tool for screening compounds or genes exhibiting protective roles against the cytotoxicity of &#945;-synuclein.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>96 well plate</td>
			<td>SPL</td>
			<td>30096</td>
			<td></td>
		</tr>
		<tr>
			<td>Agarose</td>
			<td>TAESHIN</td>
			<td>0158</td>
			<td></td>
		</tr>
		<tr>
			<td>Bacto Agar</td>
			<td>BD Difco</td>
			<td>214010</td>
			<td></td>
		</tr>
		<tr>
			<td>Breathe-easy</td>
			<td>diversified biotech</td>
			<td>BEM-1</td>
			<td>Gas permeable sealing membrane for microtiter plates</td>
		</tr>
		<tr>
			<td>cover glasses</td>
			<td>Marienfeld</td>
			<td></td>
			<td>24 x 60 mm</td>
		</tr>
		<tr>
			<td>Culture tube</td>
			<td>SPL</td>
			<td>40014</td>
			<td></td>
		</tr>
		<tr>
			<td>Cuvette</td>
			<td>ratiolab</td>
			<td>2712120</td>
			<td></td>
		</tr>
		<tr>
			<td>D-(+)-Galactose</td>
			<td>sigma</td>
			<td>G0625</td>
			<td></td>
		</tr>
		<tr>
			<td>D-(+)-Glucose</td>
			<td>sigma</td>
			<td>G8270</td>
			<td></td>
		</tr>
		<tr>
			<td>D-(+)-Raffinose pentahydrate</td>
			<td>Daejung</td>
			<td>6638-4105</td>
			<td></td>
		</tr>
		<tr>
			<td>Incubator (shaking)</td>
			<td>Labtron</td>
			<td></td>
			<td>model: SHI1</td>
		</tr>
		<tr>
			<td>Incubator (static)</td>
			<td>Vision scientific</td>
			<td></td>
			<td>model: VS-1203PV-O</td>
		</tr>
		<tr>
			<td>LiAc</td>
			<td>sigma</td>
			<td>L6883</td>
			<td></td>
		</tr>
		<tr>
			<td>Microplate reader</td>
			<td>Tecan</td>
			<td>30050303 01</td>
			<td>Model: Infinite 200 pro</td>
		</tr>
		<tr>
			<td>multichannel pipette 20-200 &#181;L</td>
			<td>gilson</td>
			<td>FA10011</td>
			<td></td>
		</tr>
		<tr>
			<td>multichannel pipette 2-20 &#181;L</td>
			<td>gilson</td>
			<td>FA10009</td>
			<td></td>
		</tr>
		<tr>
			<td>Olympus microscope</td>
			<td>Olympus</td>
			<td>IX-53</td>
			<td></td>
		</tr>
		<tr>
			<td>PEG</td>
			<td>sigma</td>
			<td>P4338</td>
			<td>average mol wt 3,350</td>
		</tr>
		<tr>
			<td>Petridish</td>
			<td>SPL</td>
			<td>10090</td>
			<td></td>
		</tr>
		<tr>
			<td>pRS426</td>
			<td></td>
			<td></td>
			<td>Christianson, T. W., Sikorski, R. S., Dante, M., Shero, J. H. &#38; Hieter, P. Multifunctional yeast high-copy-number shuttle vectors. Gene. 110 (1), 119-122 (1992).</td>
		</tr>
		<tr>
			<td>pRS426 GAL1 promoter &#945;-synuclein A30P</td>
			<td></td>
			<td></td>
			<td>Outeiro, T. F. &#38; Lindquist, S. Yeast cells provide insight into alpha-synuclein biology and pathobiology. Science. 302 (5651), 1772-1775 (2003)</td>
		</tr>
		<tr>
			<td>pRS426 GAL1 promoter &#945;-synuclein A53T</td>
			<td></td>
			<td></td>
			<td>Outeiro, T. F. &#38; Lindquist, S. Yeast cells provide insight into alpha-synuclein biology and pathobiology. Science. 302 (5651), 1772-1775 (2003)</td>
		</tr>
		<tr>
			<td>pRS426 GAL1 promoter &#945;-synuclein E46K</td>
			<td></td>
			<td></td>
			<td>Outeiro, T. F. &#38; Lindquist, S. Yeast cells provide insight into alpha-synuclein biology and pathobiology. Science. 302 (5651), 1772-1775 (2003)</td>
		</tr>
		<tr>
			<td>pRS426 GAL1 promoter &#945;-synuclein WT</td>
			<td></td>
			<td></td>
			<td>Outeiro, T. F. &#38; Lindquist, S. Yeast cells provide insight into alpha-synuclein biology and pathobiology. Science. 302 (5651), 1772-1775 (2003)</td>
		</tr>
		<tr>
			<td>Reservoir</td>
			<td>SPL</td>
			<td>23050</td>
			<td></td>
		</tr>
		<tr>
			<td>Spectrophotometer</td>
			<td>eppendorf</td>
			<td>6131 05560</td>
			<td></td>
		</tr>
		<tr>
			<td>W303a</td>
			<td></td>
			<td></td>
			<td>Present from James Bardwell</td>
		</tr>
		<tr>
			<td>Yeast nitrogen base w/o amino acids</td>
			<td>Difco</td>
			<td>291940</td>
			<td></td>
		</tr>
		<tr>
			<td>Yeast synthetic drop-out medium supplements without uracil</td>
			<td>sigma</td>
			<td>Y1501</td>
			<td></td>
		</tr>
		<tr>
			<td>YPD</td>
			<td>Condalab</td>
			<td>1547.00</td>
			<td></td>
		</tr>
	</tbody>
</table>

a method to study &#945;-synuclein toxicity and aggregation using a humanized yeast model

Parkinson's disease is the second most common neurodegenerative disorder and is characterized by progressive cell death caused by the formation of Lewy bodies containing misfolded and aggregated &#945;-synuclein. &#945;-synuclein is an abundant presynaptic protein that regulates synaptic vesicle trafficking, but the accumulation of its proteinaceous inclusions results in neurotoxicity. Recent studies have revealed that various genetic factors, including bacterial chaperones, could reduce the formation of &#945;-synuclein aggregates in vitro. However, it is also important to monitor the anti-aggregation effect in the cell to apply this as a potential treatment for the patients. It would be ideal to use neuronal cells, but these cells are difficult to handle and take a long time to exhibit the anti-aggregation phenotype. Therefore, a quick and effective in vivo tool is required for the further evaluation of in vivo anti-aggregation activity. The method described here was used to monitor and analyze the anti-aggregation phenotype in the humanized yeast Saccharomyces cerevisiae, which expressed human &#945;-synuclein. This protocol demonstrates in vivo tools that could be used for monitoring &#945;-synuclein-induced cellular toxicity, as well as the formation of &#945;-synuclein aggregates in cells.

The high expression of &#945;-synuclein is known to be linked to neuronal cell death and PD in model systems of PD. This study describes three methods to monitor the cytotoxicity of &#945;-synuclein and the foci formation of aggregated &#945;-synuclein in yeast. Here, the &#945;-synuclein was overexpressed in yeast, and the phenotypes of wild-type &#945;-synuclein and three variants of &#945;-synuclein known as familial mutants of PD were examined (Figure 1 and the Table of Materials).
The &#945;-synuclein expression under the GAL1 promoter in the pRS426 vector showed significant growth retardation on the agar plate containing galactose as an inducer (Figure 2). The difference in growth by &#945;-synuclein expression was also observed in liquid cultures (Figure 3). In both culture conditions, wild-type &#945;-synuclein and variants with E46K or A53T mutations showed growth defects due to &#945;-synuclein toxicity. However, the &#945;-synuclein A30P variant, which does not form aggregates, showed a non-toxic phenotype.
To understand the relationship between cytotoxicity and the aggregation of &#945;-synuclein, a method to monitor the status of &#945;-synuclein in yeast is necessary. The &#945;-synuclein was tagged by a GFP protein at the C-terminus to monitor the status of &#945;-synuclein in live yeast cells by detecting the GFP signal using fluorescence microscopy. The strains exhibiting severe cytotoxicity showed foci of &#945;-synuclein aggregates, but in the A30P variant, a less toxic form of &#945;-synuclein was diffused throughout the cells (Figure 4).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/64418/64418fig01.jpg" /> 
Figure 1: &#945;-synuclein domains and constructs used in this study. (A) The structure of human &#945;-synuclein with the three distinct domains-the N-terminal domain, the NAC domain, and the C-terminal domain. The amino acid residues are indicated at the bottom. The orange lines indicate the mutated sites. (B) The recombinant plasmid constructs used in this study. The pRS426 plasmid was used as a backbone. &#945;-synuclein was fused with a GFP tag at the C-terminus, and its expression was controlled by the GAL1 promoter. Abbreviations: NAC = non-amyloid-&#946; component; GFP = green fluorescent protein. <a href="https://www.jove.com/files/ftp_upload/64418/64418fig01large.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/64418/64418fig02.jpg" /> 
Figure 2: Cytotoxicity of &#945;-synuclein on agar due to the expression of high-copy number&#160;plasmids in yeast. Yeast cells expressing GAL1-driven &#945;-synuclein-GFP variants in pRS426 plasmids were spotted in fivefold dilutions on a selective yeast medium containing 2% glucose or 2% galactose. The cells were incubated for 3 days at 30 &#176;C, and the plates were photographed. Abbreviations: GFP = green fluorescent protein; EV = empty vector; WT = wild-type; &#945;-Syn = &#945;-synuclein. <a href="https://www.jove.com/files/ftp_upload/64418/64418fig02large.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/64418/64418fig03.jpg" /> 
Figure 3: Cytotoxicity of &#945;-synuclein in liquid media due to the expression of high-copy number plasmids in yeast. Yeast cells expressing GAL1-driven &#945;-synuclein-GFP variants were cultured in liquid media in a 96-well plate at 30 &#176;C for 2 days, and the growth was monitored using a microplate reader. Abbreviations: GFP = green fluorescent protein; EV = empty vector; WT = wild-type; &#945;-Syn = &#945;-synuclein. <a href="https://www.jove.com/files/ftp_upload/64418/64418fig03large.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/64418/64418fig04.jpg" /> 
Figure 4: Analysis of yeast cells expressing &#945;-synuclein-GFP by fluorescence microscopy. &#945;-synuclein-GFP variants were induced by adding galactose and incubating for 6 h. The confirmation of &#945;-synuclein-GFP was done by fluorescence microscopy. Scale bar = 10 &#181;m. Abbreviations: GFP = green fluorescent protein; EV = empty vector; WT = wild-type; &#945;-Syn = &#945;-synuclein. <a href="https://www.jove.com/files/ftp_upload/64418/64418fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/64418/64418fig05.jpg" /> 
Figure 5: Summary of this method for measuring cytotoxicity and aggregate formation of &#945;-synuclein using humanized yeast. <a href="https://www.jove.com/files/ftp_upload/64418/64418fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about A Method to Study ÃŽÂ±-Synuclein Toxicity and Aggregation Using a Humanized Yeast Model at JoVE.com

A Method to Study &#945;-Synuclein Toxicity and Aggregation Using a Humanized Yeast Model

An in vivo physiological model of &#945;-synuclein is required to study and understand the pathogenesis of Parkinson's disease. We describe a method to monitor the cytotoxicity and aggregate formation of &#945;-synuclein using a humanized yeast model.

Parkinson's disease is the second most common neurodegenerative disorder and is characterized by progressive cell death caused by the formation of Lewy ...

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Nanyang Technological University

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2.1K Views.  Ajou University. An in vivo physiological model of α-synuclein is required to study and understand the pathogenesis of Parkinson's disease. We describe a method to monitor the cytotoxicity and aggregate formation of α-synuclein using a humanized yeast model.

Video: A Method to Study α-Synuclein Toxicity and Aggregation Using a Humanized Yeast Model

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 ...

Platelet Adhesion and Aggregation Under Flow using Microfluidic Flow Cells

Platelet aggregation occurs in response to vascular injury where the extracellular matrix below the endothelium has been exposed. The platelet adhesion cascade takes place in the presence of shear flow, a factor not accounted for in conventional (static) well-plate assays. This article reports on a platelet-aggregation assay utilizing a microfluidic well-plate format to emulate physiological shear flow conditions. Extracellular proteins, collagen I or von Willebrand factor are deposited within the microfluidic channel using active perfusion with a pneumatic pump. The matrix proteins are then washed with buffer and blocked to prepare the microfluidic channel for platelet interactions. Whole blood labeled with fluorescent dye is perfused through the channel at various flow rates in order to achieve platelet activation and aggregation. Inhibitors of platelet aggregation can be added prior to the flow cell experiment to generate IC50 dose response data.

The platelet adhesion cascade takes place in the presence of shear flow, a factor not accounted for in conventional (static) well-plate assays. This article reports on a platelet-aggregation assay utilizing a microfluidic well-plate format to emulate physiological shear flow conditions.

Platelet aggregation occurs in response to vascular injury where the extracellular matrix below the endothelium has been exposed. The platelet adhesion ...

Hi-C: A Method to Study the Three-dimensional Architecture of Genomes.

The three-dimensional folding of chromosomes compartmentalizes the genome and and can bring distant functional elements, such as promoters and enhancers, into close spatial proximity 2-6. Deciphering the relationship between chromosome organization and genome activity will aid in understanding genomic processes, like transcription and replication. However, little is known about how chromosomes fold. Microscopy is unable to distinguish large numbers of loci simultaneously or at high resolution. To date, the detection of chromosomal interactions using chromosome conformation capture (3C) and its subsequent adaptations required the choice of a set of target loci, making genome-wide studies impossible 7-10.
We developed Hi-C, an extension of 3C that is capable of identifying long range interactions in an unbiased, genome-wide fashion. In Hi-C, cells are fixed with formaldehyde, causing interacting loci to be bound to one another by means of covalent DNA-protein cross-links. When the DNA is subsequently fragmented with a restriction enzyme, these loci remain linked. A biotinylated residue is incorporated as the 5' overhangs are filled in. Next, blunt-end ligation is performed under dilute conditions that favor ligation events between cross-linked DNA fragments. This results in a genome-wide library of ligation products, corresponding to pairs of fragments that were originally in close proximity to each other in the nucleus. Each ligation product is marked with biotin at the site of the junction. The library is sheared, and the junctions are pulled-down with streptavidin beads. The purified junctions can subsequently be analyzed using a high-throughput sequencer, resulting in a catalog of interacting fragments.
Direct analysis of the resulting contact matrix reveals numerous features of genomic organization, such as the presence of chromosome territories and the preferential association of small gene-rich chromosomes. Correlation analysis can be applied to the contact matrix, demonstrating that the human genome is segregated into two compartments: a less densely packed compartment containing open, accessible, and active chromatin and a more dense compartment containing closed, inaccessible, and inactive chromatin regions. Finally, ensemble analysis of the contact matrix, coupled with theoretical derivations and computational simulations, revealed that at the megabase scale Hi-C reveals features consistent with a fractal globule conformation.

The Hi-C method allows unbiased, genome-wide identification of chromatin interactions (1). Hi-C couples proximity ligation and massively parallel sequencing. The resulting data can be used to study genomic architecture at multiple scales: initial results identified features such as chromosome territories, segregation of open and closed chromatin, and chromatin structure at the megabase scale.

The three-dimensional folding of chromosomes compartmentalizes the genome and and can bring distant functional elements, such as promoters and enhancers, ...

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 ...

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the function and regulation of protein folding was studied in vitro, in isolated tissue culture cells and in unicellular organisms. Recent studies have uncovered links between protein homeostasis (proteostasis), metabolism, development, aging, and temperature-sensing. These findings have led to the development of new tools for monitoring protein folding in the model metazoan organism Caenorhabditis elegans. In our laboratory, we combine behavioral assays, imaging and biochemical approaches using temperature-sensitive or naturally occurring metastable proteins as sensors of the folding environment to monitor protein misfolding. Behavioral assays that are associated with the misfolding of a specific protein provide a simple and powerful readout for protein folding, allowing for the fast screening of genes and conditions that modulate folding. Likewise, such misfolding can be associated with protein mislocalization in the cell. Monitoring protein localization can, therefore, highlight changes in cellular folding capacity occurring in different tissues, at various stages of development and in the face of changing conditions. Finally, using biochemical tools ex vivo, we can directly monitor protein stability and conformation. Thus, by combining behavioral assays, imaging and biochemical techniques, we are able to monitor protein misfolding at the resolution of the organism, the cell, and the protein, respectively.

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

To study the relationship between protein homeostasis, stress and aging, we monitored changes in protein folding by following protein dysfunction, protein localization in the cell and protein stability at the organismal, cellular and protein levels, using the genetically tractable metazoan Caenorhabditis elegans as a model system.

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the ...

Generating a &#34;Humanized&#34; Drosophila S2 Cell Line Sensitive to Pharmacological Inhibition of Kinesin-5

Kinetochores are large protein-based structures that assemble on centromeres during cell division and link chromosomes to spindle microtubules. Proper distribution of the genetic material requires that sister kinetochores on every chromosome become bioriented by attaching to microtubules from opposite spindle poles before progressing into anaphase. However, erroneous, non-bioriented attachment states are common and cellular pathways exist to both detect and correct such attachments during cell division. The process by which improper kinetochore-microtubule interactions are destabilized is referred to as error correction. To study error correction in living cells, incorrect attachments are purposely generated via chemical inhibition of kinesin-5 motor, which leads to monopolar spindle assembly, and the transition from mal-orientation to biorientation is observed following drug washout. The large number of chromosomes in many model tissue culture cell types poses a challenge in observing individual error correction events. Drosophila S2 cells are better subjects for such studies as they possess as few as 4 pairs of chromosomes. However, small molecule kinesin-5 inhibitors are ineffective against Drosophila kinesin-5 (Klp61F). Here we describe how to build a Drosophila cell line that effectively replaces Klp61F with human kinesin-5, which renders the cells sensitive to pharmacological inhibition of the motor and suitable for use in the cell-based error correction assay.

Generating a &quot;Humanized&quot; Drosophila S2 Cell Line Sensitive to Pharmacological Inhibition of Kinesin-5

This protocol describes how to generate a Drosophila S2 cell line that is sensitive to small molecule inhibitors of kinesin-5. The use of these cells in a cell-based error correction assay is also outlined.

Kinetochores are large protein-based structures that assemble on centromeres during cell division and link chromosomes to spindle microtubules. Proper ...

Using a Whole-mount Immunohistochemical Method to Study the Innervation of the Biliary Tract in Suncus murinus

This work describes the whole-mount immunohistochemistry staining method in detail, using neurofilament protein antibody to label the innervation of the biliary tract in Suncus murinus (S. murinus&#160;). First, the specimen was dissected from S. murinus and fixed in 4% paraformaldehyde (PFA). Second, an enzymatic treatment and potential endogenous peroxidase inactivation were performed. The specimen was then exposed to the primary antibody, anti-neurofilament protein antibody, for 3-6 days. It was then incubated with the secondary antibody conjugated with horseradish peroxidase. The color reaction was revealed by reacting the specimen with a 3,3&#39;-diaminobenzidine (DAB) substrate. This method can be applied to analyze the innervation of all visceral organs. Furthermore, this protocol can also be adapted to test other neuronal antibodies, but optimization of the antibodies should be done first. This method was originally introduced by Kuratani and Tanaka1,2,3.

Using a Whole-mount Immunohistochemical Method to Study the Innervation of the Biliary Tract in Suncus murinus

A whole-mount immunohistochemical approach, to visualize neurofilament protein expression in the extrahepatic biliary tract in Suncus murinus. is presented here. This protocol can be used to analyze the innervation of all visceral organs in S. murinus or other species.

This work describes the whole-mount immunohistochemistry staining method in detail, using neurofilament protein antibody to label the innervation of the ...

Methods to Study Changes in Inherent Protein Aggregation with Age in Caenorhabditis elegans

In the last decades, the prevalence of neurodegenerative disorders, such as Alzheimer's disease (AD) and Parkinson's disease (PD), has grown. These age-associated disorders are characterized by the appearance of protein aggregates with fibrillary structure in the brains of these patients. Exactly why normally soluble proteins undergo an aggregation process remains poorly understood. The discovery that protein aggregation is not limited to disease processes and instead part of the normal aging process enables the study of the molecular and cellular mechanisms that regulate protein aggregation, without using ectopically expressed human disease-associated proteins. Here we describe methodologies to examine inherent protein aggregation in Caenorhabditis elegans through complementary approaches. First, we examine how to grow large numbers of age-synchronized C. elegans to obtain aged animals and we present the biochemical procedures to isolate highly-insoluble-large aggregates. In combination with a targeted genetic knockdown, it is possible to dissect the role of a gene of interest in promoting or preventing age-dependent protein aggregation by using either a comprehensive analysis with quantitative mass spectrometry or a candidate-based analysis with antibodies. These findings are then confirmed by in vivo analysis with transgenic animals expressing fluorescent-tagged aggregation-prone proteins. These methods should help clarify why certain proteins are prone to aggregate with age and ultimately how to keep these proteins fully functional.

Methods to Study Changes in Inherent Protein Aggregation with Age in Caenorhabditis elegans

The goal of the method presented here is to explore protein aggregation during normal aging in the model organism C. elegans. The protocol represents a powerful tool to study the highly insoluble large aggregates that form with age and to determine how changes in proteostasis impact protein aggregation.

In the last decades, the prevalence of neurodegenerative disorders, such as Alzheimer's disease (AD) and Parkinson's disease (PD), has grown. These ...

Protocols for Testing the Toxicity of Novel Insecticidal Chemistries to Mosquitoes

New classes of insecticides with novel modes of action are needed to control insecticide resistant populations of mosquitoes that transmit diseases such as Zika, dengue and malaria. Assays for rapid, high-throughput analyses of unformulated novel chemistries against mosquito larvae and adults are presented. We describe protocols for single point-dose and dose response assays to evaluate the toxicity of small molecule chemistries to the Aedes aegypti vector of Zika, dengue and yellow fever,&#160;the malaria vector, Anopheles gambiae and the northern house mosquito, Culex quinquefasciatus, on contact and via ingestion. As an example, we evaluated the toxicity of amitriptyline, a small molecule antagonist of G protein-coupled receptors, via larval, adult topical and adult blood-feeding assay. The protocols provide a starting point to investigate insecticide potential. Results are discussed in the context of additional experiments to explore product applications and mechanisms for delivery.

Protocols are described for assessing the toxicity of chemistries to immature and adult mosquitoes for development as new classes of larvicides, adulticides and endectocides. The protocols enable high-throughput testing of multiple chemistries at single-point dose and subsequent evaluation via dose response assay to determine toxicity on contact or via ingestion.

New classes of insecticides with novel modes of action are needed to control insecticide resistant populations of mosquitoes that transmit diseases such ...

A Proximal Culture Method to Study Paracrine Signaling Between Cells

Intercellular interactions play an important role in many biological processes, including tumor progression, immune responses, angiogenesis, and development. Paracrine or juxtacrine signaling mediates such interactions. The use of a conditioned medium and coculture studies are the most common methods to discriminate between these two types of interactions. However, the effect of localized high concentrations of secreted factors in the microenvironment during the paracrine interactions is not accurately recapitulated by conditioned medium and, thus, may lead to imprecise conclusions. To overcome this problem, we have devised a proximal culture method to study paracrine signaling. The two cell types are grown on either surface of a 10 &#181;m-thick polycarbonate membrane with 0.4 &#181;m pores. The pores allow the exchange of secreted factors and, at the same time, inhibit juxtacrine signaling. The cells can be collected and lysed at the endpoint to determine the effects of the paracrine signaling. In addition to allowing for localized concentration gradients of secreted factors, this method is amenable to experiments involving prolonged periods of culture, as well as the use of inhibitors. While we use this method to study the interactions between ovarian cancer cells and the mesothelial cells they encounter at the site of metastasis, it can be adapted to any two adherent cell types for researchers to study paracrine signaling in various fields, including tumor microenvironment, immunology, and development.

Paracrine and juxtacrine cellular interactions play an important role in many biological processes, including tumor progression, immune responses, angiogenesis, and development. Here, a proximal culture method is used to study paracrine signaling where the localized concentrations of the secreted factors are maintained while preventing direct cellular contact.