FIGURE 4.
The effect of ALS-linked TDP-43 mutations on toxicity. A, schematic indicating disease-associated TDP-43 mutations shown above. Color code of mutations indicates toxicity compared with WT (red = considerably more toxic than WT; green = slightly more toxic than WT; black = as toxic as WT). B, spotting assay to compare the toxicity of WT and mutant TDP-43. Serial dilutions of yeast cells transformed with galactose-inducible YFP, WT, or mutant TDP-43-YFP constructs. Transformants were spotted on glucose- (non-inducing) or galactose- (inducing) containing agar plates, and growth assessed after 48–72 h. C, spotting assay to compare the toxicity of WT and mutant TDP-43. Serial dilutions of yeast cells transformed with galactose-inducible untagged WT or mutant TDP-43 constructs. Transformants were spotted on glucose- (non-inducing) or galactose- (inducing) containing agar plates, and growth was assessed after 48–72 h. D, survivorship curve during TDP-43 induction, using the high copy 2μ vector. After induction of empty vector, TDP-43 WT, G294A, Q331K, or M337V, survivorship was determined at the indicated time points by harvesting cells at A600 nm = 1, diluting 1:1000, and plating 300 μl of these cells onto synthetic media containing 2% glucose (represses TDP-43 expression). Plates were incubated at 30 °C, and colony forming units were determined after 2 days. E, TDP-43 expression causes cell death. TDP-43 expression was induced for 6 h, and cells were stained with propidium iodide (PI) to assess viability. WT and mutant TDP-43-expressing cells were positive for PI staining (indicating cell death), whereas empty vector-containing cells were negative for PI staining (indicating viability).