On Yeast And ALS (Part 2)
- 3 minsBack in April I wrote a post describing the currently available yeast models for ALS and mentioned I would be doing yeast work in the summer.
My goal for the summer is to do drug screens in yeast models of ALS. Initially the idea was to use the TDP43 and FUS models from Aaron Gitler’s lab at Stanford and generate a SOD1 model with the G93A mutation, which is found in patients and is also the mutation the mouse model contains. We decided to not generate the SOD1 G93A model after reading a 20-year-old paper (older than I am!) in which they transformed yeast lacking their own SOD1 gene with WT human SOD1 and human SOD1 containing one of three mutations (including G93A). They saw there wasn’t a growth defect in the yeast SOD1 knockout mutant strains compared to the strain containing WT human SOD1 and the strain containing WT yeast SOD1.
Current state of the project
We got e. coli containing plasmids with WT TDP43, WT FUS and TDP43 M337V from the Gitler lab. We plated the e. coli, picked 3 colonies per plasmid and grew them overnight at 37 degrees celsius. We then used Zymo’s miniprep kit to extract the DNA. We got a final concentration of 47.3 ng/ul for the TPD43 WT, 69.1 ng/ul for the TDP43 M337V and 199.5 ng/ul for the FUS WT.
We plated both BY4741 and BY4742 yeast in YEPD agar plates and left them in a 30 degrees celsius incubator for 48h. We then picked a colony of each and grew them for 14h (again at 30 degrees).
Next steps
We’ll transform the yeast with the plasmid DNA using lithium acetate.
For spotting assays the yeast will be grown overnight at 30 degrees celsius in synthetic media lacking uracil and containing 2% raffinose until they reach log or mid-long phase. Cultures will then be normalized for A600 nm serially diluted and spotted onto synthetic solid media containing glucose (SD/-Ura) or galactose (SGal/-Ura) lacking uracil and grown at 30 °C for 2–3 days.
For the survivorship assays briefly after the induction of empty vector, TDP43 WT, TDP43 M337V or FUS WT in 2% galactose, survivorship is determined at certain time points by harvesting cells at an A600 nm of 1, diluting them 1:1000, and plating 300 μl of these cells on synthetic media containing 2% glucose. Plates are incubated at 30 °C for 2 days. Colony forming units are then determined. We would expect that with the TDP43 models fewer than 10% of cells will be able to form colonies following 12h of expression, and by 24h fewer than 2% will still alive.
To assess aggregation the yeast is grown as described above, then normalized to A600 nm = 0.2 prior to galactose induction, After 6h of induction cells with >3 foci under the YFP channel are considered as cells with aggregation.
References
Rabizadeh, S et al. Mutations associated with amyotrophic lateral sclerosis convert superoxide dismutase from an antiapoptotic gene to a proapoptotic gene: Studies in yeast and neural cells. Proc. Natl. Acad. Sci. 92, 3024-3028 (1995)
Cooper, AA et al. α-Synuclein Blocks ER-Golgi Traffic and Rab1
Rescues Neuron Loss in Parkinson’s Models Science. 313(5785) 324-328 (2006)
Johnson, BS et al. A yeast TDP-43 proteinopathy model: Exploring the molecular determinants of TDP-43 aggregation and cellular toxicity. PNAS, 105(17) 6439–6444 (2008)
Sun, Z et al. Molecular Determinants and Genetic Modifiers of Aggregation and Toxicity for the ALS Disease Protein FUS/TLS. PLoS Biology, E1000614-E1000614 (2010)
Jamie Cayley
Grad Student & TA at Missouri State University