We thank Sue Lindquist, Aaron Gitler, and Martin Duennwald for kindly sharing reagents. Our studies were supported by: an American Heart Association Post-Doctoral Fellowship (M.E.J); NIH Director's New Innovator Award DP2OD002177, NIH grants R21NS067354, R21HD074510, and R01GM099836, a Muscular Dystrophy Association Research Award (MDA277268), Packard Center for ALS Research at Johns Hopkins University, Target ALS, and an Ellison Medical Foundation New Scholar in Aging Award (J.S.).

1. Library Generation
<ol>
	<li>To construct libraries of Hsp104 using domain-specific error-prone PCR, first amplify the domain of interest with an error prone DNA polymerase20.</li>
	<li>Purify the PCR product by gel extraction.</li>
	<li>Perform a megaprimer extension step using a standard site-directed mutagenesis protocol: combine 50 ng template plasmid, 250 ng megaprimer, 200 &#956;M dNTPs, and high-fidelity DNA polymerase in PCR buffer, and dilute to 50 &#956;l total volume with PCR grade water20. Run a standard PCR program. 
	NOTE: Specific primers used will vary based on the particular region of the gene that is to be amplified.
	<ol>
		<li>Following PCR, digest parental template DNA with 1 &#956;l DpnI restriction enzyme for 2 hr at 37 &#176;C.</li>
	</ol>
	</li>
	<li>Transform the library by electroporation or other means in ultracompetent E. coli and purify it by miniprep. 
	NOTE: Library generation varies substantially based upon the aims of a given experiment. Hsp104 is a very large protein, so it is impractical to randomize the entire gene, which is why we utilize domain-specific error prone PCR. Additionally, the structure of Hsp104 remains poorly understood, making the design of directed libraries challenging21. Libraries can be constructed using directed or random approaches to mutagenesis, with the only restriction being that the template plasmid backbone should contain the URA3 gene to allow for 5-FOA counter selection on dextrose media.</li>
</ol>
2. Transformation of the Hsp104 Library
<ol>
	<li>Integrate the disease-associated substrate into W303a&#916;hsp104 yeast using a standard lithium acetate/PEG transformation protocol22. Select single colonies and screen them for toxicity to isolate a strain with high toxicity of the disease associated substrate23. 
	NOTE: Use an integrated strain and isolate a single colony to ensure equal expression in all cells. Clone the disease substrate into a plasmid allowing integration of the gene under any marker other than uracil (we use histidine) and the Hsp104 library into the pAG416GAL plasmid (uracil marker)24. To allow for 5-FOA counterselection, ensure that the library is expressed from a plasmid with the uracil marker. Other yeast strains can also be employed. We have noted a similar toxicity suppression phenotype using W303a and BY4741 yeast strains in both WT and &#916;hsp104 backgrounds (M.E.J. and J.S., unpublished observations).</li>
	<li>Transform the Hsp104 library into this strain using the same lithium acetate/PEG transformation protocol22. Scale up the transformation appropriately to maintain the sequence space of the library and to preserve the predicted library size.</li>
	<li>Plate the transformation mixture onto noninducing, selective plates (e.g. SD-His-Ura) using enough plates to ensure growth of a large number of colonies. Use large petri dishes (150 mm) to minimize the number of plates needed. Recovering transformants using plates allows transformation efficiency to be assessed.</li>
	<li>Transform Hsp104WT and vector negative controls in parallel.</li>
</ol>
3. Screening for Suppression of Proteotoxicity
<ol>
	<li>Wash the colonies off the plates using raffinose supplemented dropout media (e.g. SRaff-His-Ura). Use a serological pipette and sterile wooden applicators to loosen the colonies from the plates. Transfer the liquid washes to a 50 ml conical tube and vortex thoroughly to separate any clumps of cells. Dilute to a slightly cloudy culture, OD600 ~0.025. 
	NOTE: Here raffinose serves to relieve the cells of glucose-mediated repression of induction, thus priming the cells for galactose-mediated induction.</li>
	<li>Grow the diluted culture overnight in raffinose dropout media with shaking at 30 &#186;C. Grow the Hsp104WT and vector controls in parallel.</li>
	<li>The following morning, plate a range of concentrations onto individual galactose (e.g. SGal-His-Ura) plates (1 &#956;l to 2 ml concentrated culture in 400 &#956;l total volume). Plating a broad range of concentrations will ensure that a plate will have single colonies. The actual concentration required depends upon the toxicity of the disease substrate of interest.</li>
	<li>Normalize the vector and Hsp104WT cultures to the OD600 of the library and plate equal volumes to compare growth of the library relative to the controls.</li>
	<li>To assess selection stringency, plate the library on glucose (repressing) media. Incubate plates for 2-3 days at 30 &#186;C, until colonies appear (Figure 2). Assess the resulting colonies and compare to the controls. 
	NOTE: Often both large and small colonies are obtained, but no correlation between colony size and activity is observed. Screen these potential hits using a 5-FOA counter selection technique (Step 4) to minimize false positives carried over to sequencing.</li>
</ol>
4. 5-FOA Counterselection and Spotting to Eliminate False Positives
<ol>
	<li>Streak out single colonies in duplicate onto double and single dropout media (e.g. SD-His-Ura and SD-His plates) and grow overnight at 30 &#186;C. Repeat with vector and Hsp104WT controls. Plating on SD-His prior to 5-FOA increases the efficiency of the 5-FOA step by increasing the likelihood that cells will have lost the Hsp104 plasmid when they are plated on 5-FOA.</li>
	<li>To prevent plates from drying out, wrap the SD-His-Ura plates in parafilm and store at 4 &#186;C while performing the 5-FOA screen. These plates will eventually be used for sequencing.</li>
	<li>Streak the colonies from the SD-His plates to single colonies on 5-FOA plates (YPD media + 1g/L 5-FOA) and incubate for 1-2 days at 30 &#186;C until single colonies appear. Here, 5-FOA is converted into a toxic product (5-fluorodeoxyuridine) in cells that contain the URA3 gene. Thus, any cells still harboring the Hsp104 plasmid will die.</li>
	<li>Streak the single colonies from the 5-FOA plates in duplicate onto SD-Ura and SD-His plates. Test 3 colonies for each hit to increase the likelihood that at least one colony for each hit will have lost the Hsp104 plasmid. Incubate for 1-2 days at 30 &#176;C. Colonies that have lost the Hsp104 plasmid will grow on SD-His plates but not SD-Ura plates.</li>
	<li>Grow the strains that have lost the Hsp104 plasmids for spotting assays. Grow the strains to saturation in raffinose dropout media (e.g. SRaff-His) in 96 DeepWell 2 ml plates overnight at 30 &#186;C with shaking. Be sure to include the 5-FOA treated control strains.</li>
	<li>Prepare plates for spotting assay. Using a multichannel pipette, aliquot 200 &#181;l raffinose dropout media (e.g. SRaff-His) per well of a 96 well plate, reserving columns 1 and 7 for the neat cultures. Aliquot 250 &#181;l of each of the 16 saturated cultures into columns 1 and 7 of the plate.</li>
	<li>Serially dilute the cultures 5 fold into each column of the plate, mixing thoroughly with a multichannel pipette. Remove 50 &#181;l of the diluted culture from columns 6 and 12 to ensure the final volume of each well is 200 &#181;l. This is essential to ensure even spotting.</li>
	<li>Using a 96-bolt replicator tool (frogger), spot the cultures in duplicate onto SD-His and SGal-His plates. Incubate the plates at 30 &#186;C for 2-3 days.</li>
	<li>Select for sequencing strains that exhibit toxicity on the SGal-His plates similar to that of the controls. Discard as false positives strains that are not as toxic as the controls. 5-FOA is also often toxic on its own, so discard strains that exhibit a growth defect on SD-His plates or that exhibit substantially greater toxicity than the disease substrate alone (Figure 3).</li>
</ol>
5. Sequencing the Hsp104 Variants by Colony PCR
<ol>
	<li>Scrape ~10 &#956;l yeast from the SD-His-Ura plates in 3 &#956;l 20 mM NaOH in PCR strip tubes and lyse the cells by freeze-thaw. Place the strip tubes in a -80 &#186;C freezer for 10 min or in liquid nitrogen, then incubate at 99 &#186;C in a thermocycler for 10 min. Dilute the strains to 100 &#181;l with PCR grade water.</li>
	<li>Prepare PCR reactions: 5 &#956;l PCR template, 0.5 &#956;l 100 &#956;M each primer, 0.5 &#956;l 10mM dNTPs, 0.25 &#956;l DNA polymerase in PCR buffer, and make up to 25 &#956;l total volume with PCR grade water. Design primers to amplify the randomized region plus approximately 100 bp on either end. Minimize the size of the amplified region to increase the likelihood of successful amplification. Run a standard PCR program.</li>
	<li>Analyze samples by agarose gel electrophoresis to confirm amplification of a PCR product of the appropriate size. Repeat PCR if no product is present. 
	NOTE: PCR failure is typically due to too much yeast being used in step 5.1.</li>
	<li>Prepare samples for DNA sequencing. Remove PCR primers either by PCR purification or using a PCR product cleanup reagent such as Exonuclease I and Shrimp Alkaline Phosphatase enzyme (ExoSAP-IT) to degrade single stranded DNA. This reagent enzymatically degrades unincorporated primers and dNTPs, allowing for higher throughput.</li>
	<li>Sequence the PCR products. Be sure to thoroughly analyze sequencing chromatograms for mutations. Mutations may be observed as a mixture of two nucleotides at a given site (Figure 4), as yeast often harbor multiple plasmids.</li>
</ol>

<ol>
	<li>Johnson, B. S., McCaffery, J. M., Lindquist, S., Gitler, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+yeast+TDP-43+proteinopathy+model:+Exploring+the+molecular+determinants+of+TDP-43+aggregation+and+cellular+toxicity.">A yeast TDP-43 proteinopathy model: Exploring the molecular determinants of TDP-43 aggregation and cellular toxicity.</a> Proc Natl Acad Sci USA. 105 (17), 6439-6444 (2008).</li><li>Sun, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+Determinants+and+Genetic+Modifiers+of+Aggregation+and+Toxicity+for+the+ALS+Disease+Protein+FUS/TLS.">Molecular Determinants and Genetic Modifiers of Aggregation and Toxicity for the ALS Disease Protein FUS/TLS.</a> PLoS Biol. 9 (4), (2011).</li><li>Outeiro, T. F., Lindquist, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Yeast+Cells+Provide+Insight+into+Alpha-Synuclein+Biology+and+Pathobiology.">Yeast Cells Provide Insight into Alpha-Synuclein Biology and Pathobiology.</a> Science. 302 (5651), 1772-1775 (2003).</li><li>Forman, M. S., Trojanowski, J. Q., Lee, V. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurodegenerative+diseases:+a+decade+of+discoveries+paves+the+way+for+therapeutic+breakthroughs.">Neurodegenerative diseases: a decade of discoveries paves the way for therapeutic breakthroughs.</a> Nat Med. 10 (10), 1055-1063 (2004).</li><li>Morimoto, R. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stress,+aging,+and+neurodegenerative+disease.">Stress, aging, and neurodegenerative disease.</a> N Engl J Med. 355 (21), 2254-2255 (2006).</li><li>Elden, A. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ataxin-2+intermediate-length+polyglutamine+expansions+are+associated+with+increased+risk+for+ALS.">Ataxin-2 intermediate-length polyglutamine expansions are associated with increased risk for ALS.</a> Nature. 466 (7310), 1069-1075 (2010).</li><li>Cooper, A. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alpha-synuclein+blocks+ER-Golgi+traffic+and+Rab1+rescues+neuron+loss+in+Parkinson's+models.">Alpha-synuclein blocks ER-Golgi traffic and Rab1 rescues neuron loss in Parkinson's models.</a> Science. 313 (5785), 324-328 (2006).</li><li>Gitler, A. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alpha-synuclein+is+part+of+a+diverse+and+highly+conserved+interaction+network+that+includes+PARK9+and+manganese+toxicity.">Alpha-synuclein is part of a diverse and highly conserved interaction network that includes PARK9 and manganese toxicity.</a> Nature genetics. 41 (3), 308-315 (2009).</li><li>Su, L. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Compounds+from+an+unbiased+chemical+screen+reverse+both+ER-to-Golgi+trafficking+defects+and+mitochondrial+dysfunction+in+Parkinson's+disease+models.">Compounds from an unbiased chemical screen reverse both ER-to-Golgi trafficking defects and mitochondrial dysfunction in Parkinson's disease models.</a> Disease models &amp; mechanisms. (3-4), 194-208 (2010).</li><li>Tardiff, D. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Yeast+reveal+a+'druggable'+Rsp5/Nedd4+network+that+ameliorates+alpha-synuclein+toxicity+in+neurons.">Yeast reveal a 'druggable' Rsp5/Nedd4 network that ameliorates alpha-synuclein toxicity in neurons.</a> Science. 342 (6161), 979-983 (2013).</li><li>Tardiff, D. F., Tucci, M. L., Caldwell, K. A., Caldwell, G. A., Lindquist, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Different+8-hydroxyquinolines+protect+models+of+TDP-43+protein,+alpha-synuclein,+and+polyglutamine+proteotoxicity+through+distinct+mechanisms.">Different 8-hydroxyquinolines protect models of TDP-43 protein, alpha-synuclein, and polyglutamine proteotoxicity through distinct mechanisms.</a> The Journal of biological chemistry. 287 (6), 4107-4120 (2012).</li><li>Kim, H. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Therapeutic+modulation+of+eIF2alpha+phosphorylation+rescues+TDP-43+toxicity+in+amyotrophic+lateral+sclerosis+disease+models.">Therapeutic modulation of eIF2alpha phosphorylation rescues TDP-43 toxicity in amyotrophic lateral sclerosis disease models.</a> Nature. 46 (2), 152-160 (2014).</li><li>Ju, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+yeast+model+of+FUS/TLS-dependent+cytotoxicity.">A yeast model of FUS/TLS-dependent cytotoxicity.</a> PLoS Biol. 9 (4), (2011).</li><li>Shorter, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hsp104:+a+weapon+to+combat+diverse+neurodegenerative+disorders.">Hsp104: a weapon to combat diverse neurodegenerative disorders.</a> Neurosignals. 16 (1), 63-74 (2008).</li><li>Vashist, S., Cushman, M., Shorter, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Applying+Hsp104+to+protein-misfolding+disorders.">Applying Hsp104 to protein-misfolding disorders.</a> Biochem Cell Biol. 88 (1), 1-13 (2010).</li><li>DeSantis, M. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Operational+Plasticity+Enables+Hsp104+to+Disaggregate+Diverse+Amyloid+and+Nonamyloid+Clients.">Operational Plasticity Enables Hsp104 to Disaggregate Diverse Amyloid and Nonamyloid Clients.</a> Cell. 151 (4), 778-793 (2012).</li><li>Jackrel, M. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Potentiated+Hsp104+Variants+Antagonize+Diverse+Proteotoxic+Misfolding+Events.">Potentiated Hsp104 Variants Antagonize Diverse Proteotoxic Misfolding Events.</a> Cell. 156 (1-2), 170-182 (2014).</li><li>Wittrup, K. D., Verdine, G. L., Wittrup, K. D., Verdine, G. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Methods in Enzymology. 503, 13-14 (2012).</li><li>Boeke, J. D., Trueheart, J., Natsoulis, G., Fink, G. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Methods in Enzymology. 154, 164-175 (1987).</li><li>Miyazaki, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Creating+Random+Mutagenesis+Libraries+by+Megaprimer+PCR+of+Whole+Plasmid+(MEGAWHOP).+Directed+Evolution+Library+Creation:+Methods+in+Molecular+Biology.">Creating Random Mutagenesis Libraries by Megaprimer PCR of Whole Plasmid (MEGAWHOP). Directed Evolution Library Creation: Methods in Molecular Biology.</a> eds FrancesH Arnold &amp; George Georgiou. 231, 23-28 (2003).</li><li>Saibil, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chaperone+machines+for+protein+folding,+unfolding+and+disaggregation.">Chaperone machines for protein folding, unfolding and disaggregation.</a> Nat Rev Mol Cell Biol. 14 (10), 630-642 (2013).</li><li>Gietz, R. D., Schiestl, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-efficiency+yeast+transformation+using+the+LiAc/SS+carrier+DNA/PEG+method.">High-efficiency yeast transformation using the LiAc/SS carrier DNA/PEG method.</a> Nat. Protocols. 2 (1), 31-34 (2007).</li><li>Armakola, M., Hart, M. P., Gitler, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TDP-43+toxicity+in+yeast.">TDP-43 toxicity in yeast.</a> Methods. 53 (3), 238-245 (2011).</li><li>Alberti, S., Gitler, A. D., Lindquist, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+suite+of+Gateway&#174;+cloning+vectors+for+high-throughput+genetic+analysis+in+Saccharomyces+cerevisiae.">A suite of Gateway&#174; cloning vectors for high-throughput genetic analysis in Saccharomyces cerevisiae.</a> Yeast. 24 (10), 913-919 (2007).</li></ol>

The authors have nothing to disclose.

Here we present our approach to isolating potentiated Hsp104 variants that suppress the toxicity of disease-associated substrates using yeast proteinopathy models. Using this approach, large libraries of variants can be screened in high throughput, with the only limitation being the number of variants that pass the 5-FOA secondary screen. By performing these steps in 96 well format, we routinely screen up to 200 hits at a time in the 5-FOA step over the course of 1-2 weeks. The number of hits obtained during the initial ...

Yeast proteinopathy models have been developed for protein-misfolding disorders including amyotrophic lateral sclerosis (ALS) and Parkinson&#8217;s disease (PD)1-3. Expression of the proteins TDP-43 and FUS, which misfold in ALS patients, are toxic and mislocalize to form cytoplasmic aggregates in yeast1,2. Similarly, expression of &#945;-synuclein (&#945;-syn), which is implicated in PD, is toxic and mislocalizes to form cytoplasmic aggregates in yeast3. These features recapitulate phenotypes in patients with these disorders4,5. Thus, yeast models provide a useful platform for screening for proteins or small molecules that prevent or reverse these phenotypes2,6-13. We are interested in the development of proteins that are capable of reversing aggregation and toxicity due to TDP-43, FUS, and &#945;-syn. We focus on Hsp104, an AAA+ protein from yeast that is uniquely capable of disaggregating proteins both from amorphous aggregates and amyloid in yeast, yet it has no human homologue14,15. Hsp104 is finely tuned to disaggregate endogenous yeast prions and has only limited ability to disaggregate substrates implicated in human neurodegenerative diseases, which it never ordinarily encounters16,17. Thus, we aim to engineer enhanced versions of Hsp104 that are able to efficaciously disaggregate these human substrates. To do so, we construct large libraries of Hsp104 variants using error-prone PCR; these libraries can be screened using the yeast proteinopathy models17. We have adopted a domain-targeted approach to constructing and screening libraries, as Hsp104 is very large17. We initially focused on the middle domain (MD) of Hsp10417, though similar approaches can be employed to screen other domains. These models enable screening for disaggregase activity directly, as opposed to alternative techniques such as surface display, which is restricted to use for monitoring binding18.
Our protocol is based on two screening steps (Figure 1). First, Hsp104 variants that suppress the toxicity of the disease substrate in yeast are selected. To do so, the Hsp104 variants and disease-associated substrate are cotransformed into &#8710;hsp104 yeast. We employ &#8710;hsp104 yeast to explore Hsp104 sequence space in the absence of wild-type (WT) Hsp10417. Importantly, deletion of Hsp104 does not affect &#945;-syn, FUS, or TDP-43 toxicity in yeast, and expression of Hsp104WT provides minimal rescue1,13,17. The yeast is then plated on inducing media to induce expression of both proteins. Yeast harboring Hsp104 variants that suppress toxicity of the disease-associated substrate confer growth of the colony. These variants are selected for further analysis, while colonies maintaining variants that do not suppress toxicity die. However, false positives are a substantial problem in this screen. Expression of TDP-43, FUS, and &#945;-syn are highly toxic, which creates a strong selective pressure for the appearance of spontaneous genetic suppressors of toxicity unrelated to the Hsp104 variant being expressed. Thus, we have used a secondary screen that is also relatively high throughput to eliminate these nonspecific toxicity suppressors17. In this secondary screen, selected yeast are treated with 5-Fluorootic Acid (5-FOA) to counter select for the Hsp104 plasmid19. The strains are then assessed for substrate (TDP-43, FUS, or &#945;-syn) toxicity via spotting assay to ensure that the toxicity of the substrate is restored after loss of the Hsp104 plasmid. Thus, yeast in which toxicity is restored in this secondary screen presumably originally displayed toxicity suppression due to the presence of the Hsp104 variant. These yeast are designated as &#8216;hits&#8217; and the Hsp104 plasmid should then be recovered and sequenced to identify the mutations in the Hsp104 gene17 (Figure 1). Any hits should then be reconfirmed by constructing the mutation independently using site-directed mutagenesis and then retesting for toxicity suppression. The potential applications for this protocol are broad. Using these methods, libraries of any type of protein could be screened for variants that suppress toxicity of any substrate protein that is toxic in yeast.

<table border="0" cellpadding="0" cellspacing="0" width="766">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:320px;">Table of Specific Materials/Equipment</td>
			<td style="width:227px;"></td>
			<td style="width:132px;"></td>
			<td style="width:87px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">GeneMorphII EZClone Domain Mutagenesis Kit</td>
			<td>Agilent</td>
			<td>200552</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">150mm Petri dishes</td>
			<td>Falcon</td>
			<td>351058</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">5-Fluorootic Acid</td>
			<td>Research Products International</td>
			<td>f10501-5.0</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">96-DeepWell 2mL Plates</td>
			<td>Eppendorf</td>
			<td>0030 502.302</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">96 bold replicator tool</td>
			<td>V&#38;P Scientific</td>
			<td>vp-404</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">ExoSAP-IT</td>
			<td>Affymetrix</td>
			<td>78200</td>
			<td></td>
		</tr>
	</tbody>
</table>

isolating potentiated hsp104 variants using yeast proteinopathy models

Many protein-misfolding disorders can be modeled in the budding yeast Saccharomyces cerevisiae. Proteins such as TDP-43 and FUS, implicated in amyotrophic lateral sclerosis, and &#945;-synuclein, implicated in Parkinson&#8217;s disease, are toxic and form cytoplasmic aggregates in yeast. These features recapitulate protein pathologies observed in patients with these disorders. Thus, yeast are an ideal platform for isolating toxicity suppressors from libraries of protein variants. We are interested in applying protein disaggregases to eliminate misfolded toxic protein conformers. Specifically, we are engineering Hsp104, a hexameric AAA+ protein from yeast that is uniquely capable of solubilizing both disordered aggregates and amyloid and returning the proteins to their native conformations. While Hsp104 is highly conserved in eukaryotes and eubacteria, it has no known metazoan homologue. Hsp104 has only limited ability to eliminate disordered aggregates and amyloid fibers implicated in human disease. Thus, we aim to engineer Hsp104 variants to reverse the protein misfolding implicated in neurodegenerative disorders. We have developed methods to screen large libraries of Hsp104 variants for suppression of proteotoxicity in yeast. As yeast are prone to spontaneous nonspecific suppression of toxicity, a two-step screening process has been developed to eliminate false positives. Using these methods, we have identified a series of potentiated Hsp104 variants that potently suppress the toxicity and aggregation of TDP-43, FUS, and &#945;-synuclein. Here, we describe this optimized protocol, which could be adapted to screen libraries constructed using any protein backbone for suppression of toxicity of any protein that is toxic in yeast.

We have constructed a library of Hsp104 variants randomized in the middle domain and screened it for suppression of TDP-43 toxicity. The library was transformed and plated onto glucose and galactose plates (Figure 2) to assess the stringency of the screen. Single colonies were selected and the strains were counter selected using 5-FOA to eliminate the Hsp104 plasmid. These strains were then assessed to confirm that toxicity was due to TDP-43 alone, without the Hsp104 variants. Spotting assays of a subset...

Watch this Scientific Journal Video about Isolating Potentiated Hsp104 Variants Using Yeast Proteinopathy Models at JoVE.com

Isolating Potentiated Hsp104 Variants Using Yeast Proteinopathy Models

Yeast proteinopathy models are valuable tools to assess the toxicity and aggregation of proteins implicated in disease. Here, we present methods for screening Hsp104 variant libraries for toxicity suppressors. This protocol could be adapted to screen any protein library for toxicity suppressors of any protein that is toxic in yeast.

Many protein-misfolding disorders can be modeled in the budding yeast Saccharomyces cerevisiae. Proteins such as TDP-43 and FUS, implicated in amyotrophic ...

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8.1K Views.  Perelman School of Medicine at the University of Pennsylvania. Yeast proteinopathy models are valuable tools to assess the toxicity and aggregation of proteins implicated in disease. Here, we present methods for screening Hsp104 variant libraries for toxicity suppressors. This protocol could be adapted to screen any protein library for toxicity suppressors of any protein that is toxic in yeast.

Video: Isolating Potentiated Hsp104 Variants Using Yeast Proteinopathy Models

A Quantitative Assessment of The Yeast Lipidome using Electrospray Ionization Mass Spectrometry

Lipids are one of the major classes of biomolecules and play important roles membrane dynamics, energy storage, and signalling1-4. The budding yeast Saccharomyces cerevisiae, a genetically and biochemically manipulable unicellular eukaryote with annotated genome and very simple lipidome, is a valuable model for studying biological functions of various lipid species in multicellular eukaryotes2,3,5. S. cerevisiae has 10 major classes of lipids with chain lengths mainly of 16 or 18 carbon atoms and either zero or one degree of unsaturation6,7. Existing methods for lipid identification and quantification - such as high performance liquid chromatography, thin-layer chromatography, fluorescence microscopy, and gas chromatography followed by MS - are well established but have low sensitivity, insufficiently separate various molecular forms of lipids, require lipid derivitization prior to analysis, or can be quite time consuming. Here we present a detailed description of our experimental approach to solve these inherent limitations by using survey-scan ESI/MS for the identification and quantification of the entire complement of lipids in yeast cells. The described method does not require chromatographic separation of complex lipid mixtures recovered from yeast cells, thereby greatly accelerating the process of data acquisition. This method enables lipid identification and quantification at the concentrations as low as g/ml and has been successfully applied to assessing lipidomes of whole yeast cells and their purified organelles. Lipids extraction from whole yeast cells for using this method of lipid analysis takes two to three hours. It takes only five to ten minutes to run each sample of extracted and dried lipids on a Q-TOF mass spectrometer equipped with a nano-electrospray source.

We describe a new quantitative lipidomics method for identifying numerous lipid species in yeast using survey-scan electrospray ionization mass spectrometry (ESI/MS). This method exceeds currently available methods for lipid identification and quantification in the ability to resolve various molecular forms of lipids, sensitivity, and speed.

Lipids are one of the major classes of biomolecules and play important roles membrane dynamics, energy storage, and signalling1-4. The budding yeast ...

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 ...

Yeast Colony Embedding Method

Patterning of different cell types in embryos is a key mechanism in metazoan development. Communities of microorganisms, such as colonies and biofilms also display patterns of cell types. For example, in the yeast S. cerevisiae, sporulated cells and pseudohyphal cells are not uniformly distributed in colonies. The functional importance of patterning and the molecular mechanisms that underlie these patterns are still poorly understood.
One challenge with respect to investigating patterns of cell types in fungal colonies is that unlike metazoan tissue, cells in colonies are relatively weakly attached to one another. In particular, fungal colonies do not contain the same extensive level of extracellular matrix found in most tissues . Here we report on a method for embedding and sectioning yeast colonies that reveals the interior patterns of cell types in these colonies. The method can be used to prepare thick sections (0.5 &mu;) useful for light microscopy and thin sections (0.1 &mu;) suitable for transmission electron microscopy. Asci and pseudohyphal cells can easily be distinguished from ovoid yeast cells by light microscopy , while the interior structure of these cells can be visualized by EM.
The method is based on surrounding colonies with agar, infiltrating them with Spurr's medium, and then sectioning. Colonies with a diameter in the range of 1-2 mm are suitable for this protocol. In addition to visualizing the interior of colonies, the method allows visualization of the region of the colony that invades the underlying agar.

A method for embedding yeast colonies allowing sectioning for light and electron microscopy. This protocol allows determination of the distribution of sporulated cells and pseudohyphal cells within colonies providing a new tool toward understanding the organization of cell types within a fungal community.

Patterning of  different cell types in embryos is a key mechanism in metazoan development. Communities of microorganisms, such as colonies and biofilms ...

Principles of Rodent Surgery for the New Surgeon

For both scientific and animal welfare reasons, training in basic surgical concepts and techniques should be undertaken before ever seeking to perform surgery on a rodent. Students, post-doctoral scholars, and others interested in performing surgery on rodents as part of a research protocol may not have had formal surgical training as part of their required coursework. Surgery itself is a technical skill, and one that will improve with practice. The principles of aseptic technique, however, often remain unexplained or untaught. For most new surgeons, this vital information is presented in piecemeal fashion or learned on the job, neither of which is ideal. It may also make learning how to perform a particular surgery difficult, as the new surgeon is learning both a surgical technique and the principles of asepsis at the same time. This article summarizes and makes recommendations for basic surgical skills and techniques necessary for successful rodent surgery. This article is designed to supplement hands-on training by the user's institution.

Before attempting surgery, a new surgeon should have training in basic surgical techniques and concepts. This article will present basic surgical considerations with an emphasis on rodents.

For both scientific and animal welfare reasons, training in basic surgical concepts and techniques should be undertaken before ever seeking to perform ...

Manual Restraint and Common Compound Administration Routes in Mice and Rats

Being able to safely and effectively restrain mice and rats is an important part of conducting research. Working confidently and humanely with mice and rats requires a basic competency in handling and restraint methods. This article will present the basic principles required to safely handle animals. One-handed, two-handed, and restraint with specially designed restraint objects will be illustrated. Often, another part of the research or testing use of animals is the effective administration of compounds to mice and rats. Although there are a large number of possible administration routes (limited only by the size and organs of the animal), most are not used regularly in research. This video will illustrate several of the more common routes, including intravenous, intramuscular, subcutaneous, and oral gavage. The goal of this article is to expose a viewer unfamiliar with these techniques to basic restraint and substance administration routes. This video does not replace required hands-on training at your facility, but is meant to augment and supplement that training.

Working safely and humanely with research rodents requires a core competency in handling and restraint methods. This article will present the basic principles required to safely handle and effectively administer compounds to mice and rats.

Being able to safely and effectively restrain mice and rats is an important part of conducting research.  Working confidently and humanely with mice and ...

High-throughput Yeast Plasmid Overexpression Screen

The budding yeast, Saccharomyces cerevisiae, is a powerful model system for defining fundamental mechanisms of many important cellular processes, including those with direct relevance to human disease. Because of its short generation time and well-characterized genome, a major experimental advantage of the yeast model system is the ability to perform genetic screens to identify genes and pathways that are involved in a given process. Over the last thirty years such genetic screens have been used to elucidate the cell cycle, secretory pathway, and many more highly conserved aspects of eukaryotic cell biology 1-5. In the last few years, several genomewide libraries of yeast strains and plasmids have been generated 6-10. These collections now allow for the systematic interrogation of gene function using gain- and loss-of-function approaches 11-16. Here we provide a detailed protocol for the use of a high-throughput yeast transformation protocol with a liquid handling robot to perform a plasmid overexpression screen, using an arrayed library of 5,500 yeast plasmids. We have been using these screens to identify genetic modifiers of toxicity associated with the accumulation of aggregation-prone human neurodegenerative disease proteins. The methods presented here are readily adaptable to the study of other cellular phenotypes of interest.

Here we describe a plasmid overexpression screen in Saccharomyces cerevisiae, using an arrayed plasmid library and a high-throughput yeast transformation protocol with a liquid handling robot.

The budding yeast, Saccharomyces cerevisiae, is a powerful model system for defining fundamental mechanisms of many important cellular processes, ...

Purification of Hsp104, a Protein Disaggregase

Hsp104 is a hexameric AAA+ protein1 from yeast, which couples ATP hydrolysis to protein disaggregation2-10 (Fig. 1). This activity imparts two key selective advantages. First, renaturation of disordered aggregates by Hsp104 empowers yeast survival after various protein-misfolding stresses, including heat shock3,5,11,12. Second, remodeling of cross-beta amyloid fibrils by Hsp104 enables yeast to exploit myriad prions (infectious amyloids) as a reservoir of beneficial and heritable phenotypic variation13-22. Remarkably, Hsp104 directly remodels preamyloid oligomers and amyloid fibrils, including those comprised of the yeast prion proteins Sup35 and Ure223-30. This amyloid-remodeling functionality is a specialized facet of yeast Hsp104. The E. coli orthologue, ClpB, fails to remodel preamyloid oligomers or amyloid fibrils26,31,32.
Hsp104 orthologues are found in all kingdoms of life except, perplexingly, animals. Indeed, whether animal cells possess any enzymatic system that couples protein disaggregation to renaturation (rather than degradation) remains unknown33-35. Thus, we and others have proposed that Hsp104 might be developed as a therapeutic agent for various neurodegenerative diseases connected with the misfolding of specific proteins into toxic preamyloid oligomers and amyloid fibrils4,7,23,36-38. There are no treatments that directly target the aggregated species associated with these diseases. Yet, Hsp104 dissolves toxic oligomers and amyloid fibrils composed of alpha-synuclein, which are connected with Parkinson's Disease23 as well as amyloid forms of PrP39. Importantly, Hsp104 reduces protein aggregation and ameliorates neurodegeneration in rodent models of Parkinson's Disease23 and Huntington's disease38. Ideally, to optimize therapy and minimize side effects, Hsp104 would be engineered and potentiated to selectively remodel specific aggregates central to the disease in question4,7. However, the limited structural and mechanistic understanding of how Hsp104 disaggregates such a diverse repertoire of aggregated structures and unrelated proteins frustrates these endeavors30,40-42.
To understand the structure and mechanism of Hsp104, it is essential to study the pure protein and reconstitute its disaggregase activity with minimal components. Hsp104 is a 102kDa protein with a pI of ~5.3, which hexamerizes in the presence of ADP or ATP, or at high protein concentrations in the absence of nucleotide43-46. Here, we describe an optimized protocol for the purification of highly active, stable Hsp104 from E. coli. The use of E. coli allows simplified large-scale production and our method can be performed quickly and reliably for numerous Hsp104 variants. Our protocol increases Hsp104 purity and simplifies His6-tag removal compared to a previous purification method from E. coli47. Moreover, our protocol is more facile and convenient than two more recent protocols26,48.

Here, we describe a protocol for the purification of highly active Hsp104, a hexameric AAA+ protein from yeast, which couples ATP hydrolysis to protein disaggregation. This scheme exploits a His6-tagged construct for affinity purification from E. coli followed by anion-exchange chromatography, His6-tag removal with TEV protease, and size-exclusion chromatography.

Hsp104 is a hexameric AAA+ protein1 from yeast, which couples ATP hydrolysis to protein disaggregation2-10 (Fig. 1). This activity imparts two key ...

Mouse Sperm Cryopreservation and Recovery using the I&middot;Cryo Kit

Thousands of new genetically modified (GM) strains of mice have been created since the advent of transgenesis and knockout technologies. Many of these valuable animals exist only as live animals, with no backup plan in case of emergency. Cryopreservation of embryos can provide this backup, but is costly, can be a lengthy procedure, and generally requires a large number of animals for success. Since the discovery that mouse sperm can be successfully cryopreserved with a basic cryoprotective agent (CPA) consisting of 18&#37; raffinose and 3&#37; skim milk, sperm cryopreservation has become an acceptable and cost-effective procedure for archiving, distributing and recovery of these valuable strains.
 Here we demonstrate a newly developed I&bull;Cryo kit for mouse sperm cryopreservation. Sperm from five commonly-used strains of inbred mice were frozen using this kit and then recovered. Higher protection ratios of sperm motility (&gt; 60&#37;) and rapid progressive motility (&gt; 45&#37;) compared to the control (basic CPA) were seen for sperm frozen with this kit in 5 inbred mouse strains. Two cell stage embryo development after IVF with the recovered sperm was improved consistently in all 5 mouse strains examined. Over a 1.5 year period, 49 GM mouse lines were archived by sperm cryopreservation with the I&bull;Cryo kit and later recovered by IVF.

Here we demonstrate the newly developed I&#x2022;Cryo kit for mouse sperm cryopreservation. Two-cell stage embryo development with frozen-thawed sperm was improved consistently in 5 mouse strains with the use of this kit. Over a 1.5 year period, 49 genetically modified mouse lines were archived by sperm cryopreservation with the I&#x2022;Cryo kit and later successfully recovered by IVF.

Thousands of new genetically modified (GM) strains of mice have been created since the advent of transgenesis and knockout technologies.  Many of these ...

Detection of Rare Genomic Variants from Pooled Sequencing Using SPLINTER

As DNA sequencing technology has markedly advanced in recent years2, it has become increasingly evident that the amount of genetic variation between any two individuals is greater than previously thought3. In contrast, array-based genotyping has failed to identify a significant contribution of common sequence variants to the phenotypic variability of common disease4,5. Taken together, these observations have led to the evolution of the Common Disease / Rare Variant hypothesis suggesting that the majority of the "missing heritability" in common and complex phenotypes is instead due to an individual's personal profile of rare or private DNA variants6-8. However, characterizing how rare variation impacts complex phenotypes requires the analysis of many affected individuals at many genomic loci, and is ideally compared to a similar survey in an unaffected cohort. Despite the sequencing power offered by today's platforms, a population-based survey of many genomic loci and the subsequent computational analysis required remains prohibitive for many investigators.
To address this need, we have developed a pooled sequencing approach1,9 and a novel software package1 for highly accurate rare variant detection from the resulting data. The ability to pool genomes from entire populations of affected individuals and survey the degree of genetic variation at multiple targeted regions in a single sequencing library provides excellent cost and time savings to traditional single-sample sequencing methodology. With a mean sequencing coverage per allele of 25-fold, our custom algorithm, SPLINTER, uses an internal variant calling control strategy to call insertions, deletions and substitutions up to four base pairs in length with high sensitivity and specificity from pools of up to 1 mutant allele in 500 individuals. Here we describe the method for preparing the pooled sequencing library followed by step-by-step instructions on how to use the SPLINTER package for pooled sequencing analysis (<a href="http://www.ibridgenetwork.org/wustl/splinter" target="_blank">http://www.ibridgenetwork.org/wustl/splinter</a>). We show a comparison between pooled sequencing of 947 individuals, all of whom also underwent genome-wide array, at over 20kb of sequencing per person. Concordance between genotyping of tagged and novel variants called in the pooled sample were excellent. This method can be easily scaled up to any number of genomic loci and any number of individuals. By incorporating the internal positive and negative amplicon controls at ratios that mimic the population under study, the algorithm can be calibrated for optimal performance. This strategy can also be modified for use with hybridization capture or individual-specific barcodes and can be applied to the sequencing of naturally heterogeneous samples, such as tumor DNA.

Pooled DNA sequencing is a fast and cost-effective strategy to detect rare variants associated with complex phenotypes in large cohorts. Here we describe the computational analysis of pooled, next-generation sequencing of 32 cancer-related genes using the SPLINTER software package. This method is scalable, and applicable to any phenotype of interest.

As DNA sequencing technology has markedly advanced in recent years2, it has become increasingly evident that the amount of genetic variation between any ...

Screening for Functional Non-coding Genetic Variants Using Electrophoretic Mobility Shift Assay (EMSA) and DNA-affinity Precipitation Assay (DAPA)

Population and family-based genetic studies typically result in the identification of genetic variants that are statistically associated with a clinical disease or phenotype. For many diseases and traits, most variants are non-coding, and are thus likely to act by impacting subtle, comparatively hard to predict mechanisms controlling gene expression. Here, we describe a general strategic approach to prioritize non-coding variants, and screen them for their function. This approach involves computational prioritization using functional genomic databases followed by experimental analysis of differential binding of transcription factors (TFs) to risk and non-risk alleles. For both electrophoretic mobility shift assay (EMSA) and DNA affinity precipitation assay (DAPA) analysis of genetic variants, a synthetic DNA oligonucleotide (oligo) is used to identify factors in the nuclear lysate of disease or phenotype-relevant cells. For EMSA, the oligonucleotides with or without bound nuclear factors (often TFs) are analyzed by non-denaturing electrophoresis on a tris-borate-EDTA (TBE) polyacrylamide gel. For DAPA, the oligonucleotides are bound to a magnetic column and the nuclear factors that specifically bind the DNA sequence are eluted and analyzed through mass spectrometry or with a reducing sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) followed by Western blot analysis. This general approach can be widely used to study the function of non-coding genetic variants associated with any disease, trait, or phenotype.

Screening for Functional Non-coding Genetic Variants Using Electrophoretic Mobility Shift Assay EMSA and DNA-affinity Precipitation Assay DAPA

We present a strategic plan and protocol for identifying non-coding genetic variants affecting transcription factor (TF) DNA binding. A detailed experimental protocol is provided for electrophoretic mobility shift assay (EMSA) and DNA affinity precipitation assay (DAPA) analysis of genotype-dependent TF DNA binding.

Population and family-based genetic studies typically result in the identification of genetic variants that are statistically associated with a clinical ...