A Phosphorylation‐Induced Micellization Switch in the Low‐Complexity Domain of TDP‐43

Phase separation (PS) of the low-complexity domain (LCD) of TAR DNA-binding protein 43 kDa (TDP-43) is linked to pathogenic aggregates in amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD-TDP). Here, we show that extensive phosphorylation of the LCD C-terminus redirects its self-assembly. Coarse-grained Monte Carlo simulations predicted that 12 Ser phosphorylations partition the 148-residue LCD into a hydrophobic N-terminal and highly charged C-terminal block, favouring finite-sized micellization over macroscopic PS. In vitro, LCD phosphorylated by casein kinase 1 delta (CK1δ; mean of 12 phosphorylations by native mass spectrometry) and phosphomimetic 12D/12DD mutants formed spherical nanoparticles (≈ 20-50 nm) above a low-micromolar critical micelle concentration, whereas the unphosphorylated LCD underwent reversible PS that matured into fibrils. Increasing ionic strength shifted the mutants toward anisotropic morphologies (wormlike 12D micelles and rigid 12DD nanocylinders). Turbidity assays and confocal imaging directly visualized the absence of PS in the phosphorylated form. Negative-stain and cryo-electron microscopy (cryo-EM) confirmed the spherical micellar architecture for the phosphorylated LCD and 12D/12DD mimics. Our data identify phosphorylation as a molecular switch tuning macrophase separation and fibril formation of TDP-43 LCD, providing a framework for an aggregation-protective role through microphase separation into size-limited micelles. Whether these assemblies are stable or kinetically trapped on pathological timescales remains unclear.