Flow cytometry gating strategies

The gating strategy for each flow cytometry experiment is described, with accompanying pictures of gates

TorsinA can be expressed in the endoplasmic reticulum of yeast.

<p>(A) Diagram of constructs created for this work (upper panel) and summary of the different forms of torsinA used in these experiments (lower panel). TorsinA was localized to the endoplasmic reticulum (ER) using the signal sequence of the endogenous yeast protein KAR2 and an HDEL sequence. (B) Microscopy of yeast strains used. TorsinA was localized to the contiguous lumen of the nuclear envelope and ER. Some signal was also seen in the vacuole (arrow head), suggesting a portion of the protein was degraded. A representative frame is shown for each strain. Scale bar  =  2 µM. (C) Growth of torsinA-expressing yeast on plates. Each row is a 5-fold dilution of the previous row. Expression of wild type (WT) or mutant torsinA did not impact the growth rate. Uninduced plates included 1 mM methionine and induced plates lacked methionine.</p

[<i>SWI</i>⁺] cells have lower fitness and do not survive dry conditions.

<p>(A) The survival of [<i>SWI</i>⁺] (red) and [<i>swi</i>^<i>−</i>] (blue) cells from overnight cultures (no stress), 6-day–starved cultures (starvation), and antifungal-treated overnight cultures (amphotericin B and caspofungin) were measured using a viability stain and flow cytometry. Error bars indicate standard deviation (<i>n</i> = 3). All numerical data in this figure and the flow cytometry gating strategy are available from the Dryad Digital Repository: <a href="https://doi.org/10.5061/dryad.d5r16" target="_blank">http://dx.doi.org/10.5061/dryad.d5r16</a>. (B) Growth comparison of [<i>swi</i>^<i>−</i>] cells and [<i>SWI</i>⁺] cells in media buffered at pH 7.5. Cell density was measured by absorbance every 15 minutes. (C) Survival of [<i>SWI</i>⁺] and [<i>swi</i>^<i>−</i>] cultures after removal of liquid media followed by drying under blowing air for 24 hours. The survival of [<i>SWI</i>⁺] was below the detection limit using viability stain and flow cytometry (1 in 10,000 cells). Error bars indicate standard deviation (<i>n</i> = 3). (D) Recovery of dried [<i>SWI</i>⁺] and [<i>swi</i>^<i>−</i>] strains in liquid culture in microtiter plates. After rehydration of dried cells into the original volume of the liquid culture, a 25-fold dilution into fresh media was made in microtiter wells. Growth after four days is depicted.</p

Pioneer cells established by the [<i>SWI</i>⁺] prion can promote dispersal and out-crossing in yeast

<div><p>To thrive in an ever-changing environment, microbes must widely distribute their progeny to colonize new territory. Simultaneously, they must evolve and adapt to the stresses of unpredictable surroundings. In both of these regards, diversity is key—if an entire population moved together or responded to the environment in the same way, it could easily go extinct. Here, we show that the epigenetic prion switch [<i>SWI</i>⁺] establishes a specialized subpopulation with a “pioneer” phenotypic program in <i>Saccharomyces cerevisiae</i>. Cells in the pioneer state readily disperse in water, enabling them to migrate and colonize new territory. Pioneers are also more likely to find and mate with genetically diverse partners, as inhibited mating-type switching causes mother cells to shun their own daughters. In the nonprion [<i>swi</i>^<i>−</i>] state, cells instead have a “settler” phenotype, forming protective flocs and tending to remain in their current position. Settler cells are better able to withstand harsh conditions like drought and alkaline pH. We propose that these laboratory observations reveal a strategy employed in the wild to rapidly diversify and grant distinct, useful roles to cellular subpopulations that benefit the population as a whole.</p></div

TorsinA cannot rescue α-synuclein-induced toxicity in yeast.

<p>Assay showing the ability of yeast to grow in the presence or absence of α-synuclein (α-syn) with and without torsinA. Each row is a 5-fold dilution of the previous row. TorsinA cannot rescue α-syn-induced toxicity.</p

Coexpression of torsinA and printor does not uncover a phenotype stemming from torsinA expression.

<p>(A) The UPR of the indicated yeast strains was monitored by flow cytometry to detect expression from the UPRE-GFP construct upon stress with CPY* or 1.5 mM DTT. Coexpression of printor does not allow WT torsinA to reduce UPR-related stress. A 1-tailed Student's t-test was used to compare relative fluorescence of torsinA strains to the vector control. * = p<0.01. N = 6 independent trials per each sample. (B) The impact of torsinA with and without printor on trafficking of invertase. Cells were spotted on plates containing the pH sensitive dye, BCP. Simultaneous expression of torsinA and printor does not impact the rate of secretion.</p

Data graphed in Newby and Lindquist 2017

The data used to prepare graphs is given in a Microsoft Excel file. Each worksheet name indicates the figure that was generated from the data given

The [<i>SWI</i>⁺] prion enhances the migration of cells with the flow of water.

<p>(A) A diagram of the experimental procedures used to test the migration of cells on solid media. Pregrown spots of yeast colonies experience “rainfall” as drops of water are pipetted over them. The plates are tilted to allow the water to flow across the surface. After drying and incubating for yeast growth, colonies established by the migrated cells appear. (B) Photograph showing the migration of [<i>swi</i>^<i>−</i>] and [<i>SWI</i>⁺] cells on an agar plate. (C) Quantification of total cell number after the migration experiment. Error bars indicate standard deviation (<i>n</i> = 3). Numerical data are available from the Dryad Digital Repository: <a href="https://doi.org/10.5061/dryad.d5r16" target="_blank">http://dx.doi.org/10.5061/dryad.d5r16</a>. (D) Left: Photograph showing the migration of cells on an agar plate after [<i>SWI</i>⁺] and [<i>swi</i>^<i>−</i>] cells were mixed in the indicated proportions. Arrows indicate large colonies where small flocs of [<i>swi</i>^<i>−</i>] cells have migrated. Center: The same image collected in a green fluorescence channel. Due to the yTRAP sensor [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2003476#pbio.2003476.ref015" target="_blank">15</a>] in both strains, [<i>swi</i>^<i>−</i>] colonies are brightly fluorescent. Right: Migration experiment from panel B photographed in the green fluorescence channel for comparison. yTRAP, yeast transcriptional reporting of aggregating proteins.</p

The [<i>SWI</i>⁺] prion encourages diverse mating partners.

<p>(A) A diagram showing the mating tendencies of [<i>SWI</i>⁺] and [<i>swi</i>^<i>−</i>] haploids (budding yellow cell). Left: A [<i>swi</i>^<i>−</i>] cell will readily switch its mating type after dividing and mate with its own daughter. The resulting diploid cannot out-cross with diverse mating partners (orange cell). Right: In the [<i>SWI</i>⁺] pioneering state, mating-type switching is inhibited, and thus mother cells cannot mate with their own daughters. This increases the likelihood that they will mate with genetically diverse partners. Note that after normal mating-type switching, an additional generation may occur before mating, after which the four haploids involved can mate in pairs (not depicted). (B) Relative out-crossing efficiencies of [<i>swi</i>^<i>−</i>] cells (blue) and [<i>SWI</i>⁺] cells (red). Elimination of the prion from the [<i>SWI</i>⁺] strain (“cured”) returns its out-cross ratio to the low state. The ratio was calculated by normalizing to the mating efficiency of <i>ho</i>^<i>−</i> control cells (<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2003476#pbio.2003476.s002" target="_blank">S2A Fig</a>). Error bars indicate standard deviation (<i>n</i> = 3). Numerical data and the flow cytometry gating strategy are available from the Dryad Digital Repository: <a href="https://doi.org/10.5061/dryad.d5r16" target="_blank">http://dx.doi.org/10.5061/dryad.d5r16</a>.</p

TorsinA does not impact the unfolded protein response or trafficking in yeast.

<p>(A) A construct containing GFP driven by a unfolded protein response (UPR) sensitive promoter (UPRE-GFP) was used to monitor levels of the unfolded protein response (UPR) upon stress with 1.5 mM dithiothreitol (DTT) or mutant carboxypeptidase Y (CPY*). ERO1 served as a positive control. TorsinA is not able to reduce UPR levels caused by either stressor. Statistical analysis was conducted in comparison to the vector control strain with a 1-tailed Student's t-test. * = p<0.05, # = p<0.005, & = p<0.001. N = 6 independent trials per sample. (B)Growth of ero1-1 in the presence and absence of torsinA at 37°C. Each row is a 5-fold dilution of the previous row. TorsinA is not able to rescue the growth defect by the <i>ero1-1</i> mutation. Uninduced plates included 1 mM methionine and induced plates lacked methionine. (C) Trafficking of invertase, as monitored by halos produced by growth of torsinA-expressing strains on plates containing bromocresol purple (BCP). TorsinA does not impact the rate of secretion.</p