RESEARCH POSTER PRESENTATION DESIGN © 2011

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Methods

Future Directions

• AmFRET experimentation is ongoing and will be necessary in determining

which proteins form aggregates.

• Develop a more selective gTOW plasmid (Figure 5) that necessitates

plasmid retention by way of inclusion of a degron tag to URA3.

• Apply the results of gTOW and AmFRET studies toward understanding how

cells compensate for aneuploidy.

Halfmann R. (2016). A glass menagerie of low complexity sequences. Current

Opinion in Structural Biology. 38 9-16

Wolff S, Weissman J.S., and Dillin A. (2014). Differential Scales of Protein

Quality Control. Cell. 157(1) 52-64

Makanae K, Kintaka R, Makino T, Kitano H, and Moriya H. (2013).

Identification of dosage-sensitive genes in Saccharomyces cerevisiae using

the genetic tug-of-war method. Genome Research. 23(2) 300-311

Sopko R, Huang D, Preston N, Chua G, Papp B, Kafadar K, Snyder M, Oliver

SG, Cyert M, Hughes TR, Boone C, and Andrews B. (2006). Mapping

pathways and phenotypes by systematic gene overexpression. Molecular

Cell. 21(3) 319-330

Acknowledgements

We would like to thank Tarique Khan, Ellen Bruner, and the rest of the

Halfmann lab for valuable discussion about this work as well as their

experimental support. Our work was greatly assisted by the Stowers Core

Facilities, especially Cytometry, Molecular Biology, and Media Preparation.

Finally, we are exceptionally grateful to the Stowers Foundation for

making this work possible.

Maintaining proteostasis through aggregationMason Wilkinson,1,2 Shriram Venkatesan,1 and Randal Halfmann1,3

1Stowers Institute for Medical Research, Kansas City, MO 641102Department of Molecular Biology, University of Kansas, Lawrence, KS 66045

3Department of Molecular and Integrative Physiology, University of Kansas Medical Center, Kansas City, KS 66160

Aggregation Compensates for

Dosage:

Live Cell

No

Aggregation:

Dead Cell

Toxic

Protein

Discussion

• gTOW experiments were largely successful. From twelve toxic gene

candidates, three showed clear non-Gaussian tendencies akin to MOT3

and six show need for further study.

• Bimodal distribution is only suggestive of aggregation. It is not a

guarantee. Non-Gaussian distribution may occur for several reasons.

• Trace bimodality may be observed in cases of zero toxicity.

• Overexpression may result in gain of function. This is unlikely, but

not unheard of.

• Uncertain distribution exhibited by samples may be due to noise or

insufficient gene expression.

• Less likely, perhaps insufficient selective pressure is exerted by

the gTOW plasmid to surmount the effects of the toxic protein.

• More likely, cells may accumulate URA3p prior to SGal-Ura

induction, then eject the plasmid and survive briefly on their

stock.

Figure 1: Overexpression of aggregating and non-aggregating toxic proteins.

Normally resulting in cell death, aggregation may save cells from proteotoxicity

given high expression.

Genetic Tug-of-War (gTOW):

• Two opposing selective pressures (Figure 2A) establish a median plasmid

copy number in a population of Saccharomyces cerevisiae.

• URA3-1 Auxotrophy: High copy number required to grow.

• Toxic Gene Minimal copy number for minimal toxicity.

• Green fluorescence from a mEos3.1 tag (Figure 2B) indicates expression.

• No Aggregation: Gaussian Distribution

• Aggregation: Non-Gaussian Distribution

Results

Figure 2: A) Structure gTOW/AmFRET plasmid. GAL1 promoter constitutively

expresses toxic gene. truncURA promoter necessitates high plasmid copy number

for survival in –Ura. B) Green fluorescence intensity in cells expressing different

genes measured by flow cytometry. Peak from 0-1e3 is likely noise. MOT3 shows

bimodal distribution typical of a toxic, aggregating protein. Conversely, MOT3

(ΔNLS) is toxic as evidenced by a high-intensity Gaussian distribution.

Many proteins are toxic when their native stoichiometry is disturbed, but

several mechanisms exist to maintain proteostasis. We hypothesize that

protein aggregation may be another such means. Consider that when

overexpressed, Saccharomyces cerevisiae prion MOT3p is toxic as a

monomer, but not as an aggregate. Using gTOW and AmFRET, we analyzed

eleven other toxic genes in this yeast to investigate if aggregation is used

to compensate for their dosage during overexpression (Figure 1). This

research will be useful in understanding the cellular implications of

protein aggregation as well as the effects of aneuploidy and gene dosage.

Abstract

Figure 4: Results of gTOw experimentation in SGal-Ura media as recorded

by imaging flow cytometry. Samples exhibiting non-Gaussian distribution

are highlighted in green boxes. Uncertain distribution is indicated by

yellow boxes. Negatives (Gaussian) are unboxed.

Amphifluoric Förster Resonance Energy Transfer (AmFRET)(Figure 4)

• An excited Donor Fluorophore transfers its energy to an Acceptor

Fluorophore, resulting in emission from the acceptor (Figure 3A).

• Photo-convertible mEos3.1 tag Donor (green) and acceptor (red)

fluorophores are the same (Figure 3B), hence “Amphifluoric” FRET.

• Requires close proximity (<10nm) Indicative of aggregation.

Green Fluorescence Intensity

MOT3 MOT3 (ΔNLS)

Figure 3: Basis of AmFRET. Green mEos emission falls in the absorption spectrum

of red mEos. This emission energy can be transferred to the red counterpart,

resulting in red emission. FRET levels are divided by baseline red fluorescence to

yield “Ratiometric FRET,” which establishes low and high FRET populations.

mEos3.1 mEos3.1405 nm

A B

A B

Wavelength (nm)

References

mEos3.1

SFP1(Supposed Prion)

NUP53ROX1

KSP1 RFG1

MSS11

WHI4

AKR1

YCK1

TPK2FIS1

SGN1

Note: Twelve different toxic genes were subjected to gTOW analysis.

MOT3 and mEos3.1 were used as positive and negative controls

respectively. AmFRET experimentation is ongoing, and as such, only

gTOW results are displayed here. All genes are from Saccharomyces

cerevisiae with the exception of RFG1 from Candida albicans.

The dark peak around 1e3 intensity is likely noise.

gTOW / AmFRET Vector

GAL1 Toxic Gene mEos3.1

URA3truncURA

Improved gTOW / AmFRET Vector

GAL1 Toxic Gene mEos3.1

URA3Full URA LEU 2A 5-6D

Figure 5: Potential improvements to the current gTOW vector. Cells will be grown

in –Leu-Ura. 2A is an autocatalytic polypeptide cleavage site that will allows LEU

and URA 3 to act independently. 5-6D is a degron tag that marks URA3 for

proteasome degradation. In this way URA3 must constantly be produced to

survive –Ura conditions, requiring the plasmid not be expelled.

MOT3

(Positive Control)

MOT3 (ΔNLS)

(Negative Control)