TDP-43 amyloid conversion in cells tracks the net charge of a polyampholyte motif, not condensate solidification

Abstract

Liquid-liquid phase separation of TDP-43 is implicated in ALS and FTLD, yet how its condensates convert into pathological amyloid in living cells remains poorly defined. Here we identify a polyampholyte motif (PAM) in the linker between RRM2 and the amyloid-forming low-complexity domain as a charge-tunable determinant of fibrillization that operates independently of condensate solidification. In budding yeast expressing GFP-TDP-43{Delta}NLS, charge reversal of three PAM arginines (R268E/R272E/R275E; 3R-E) converted liquid puncta into ProteoStat-positive fibrils. Unexpectedly, fluorescence recovery after photobleaching revealed that 3R-E condensates were more mobile than wild-type condensates, whereas the least mobile condensates, in both yeast and human U2OS cells, converted least. Across 24 PAM charge variants, the proportion of fibril-bearing cells scaled linearly with net charge independently of residue identity, and charge neutralization delayed rather than abolished conversion. Phosphomimetic substitution of the PAM serines alone induced fibrils, implicating phosphorylation as a potential physiological modulator of PAM charge.