Early Hetero-Oligomerization of Tau Fragments Redirects Cofactor-Free Tau Assembly and Fibril Morphology.

Early tau assembly intermediates are increasingly recognized as bioactive species that govern downstream aggregation and cellular dysfunction, yet it remains unresolved whether distinct tau fragments directly interact during these early stages to redirect pathway selection. Here, we use a cofactor-free two-peptide model, TauPHF43 and jR2R3P301L, to test whether fragments that share the PHF6 amyloidogenic motif but differ in sequence context coassemble before mature fibrils emerge. Recent cryo-EM work established heparin-induced jR2R3P301L fibrils as a minimal 4R tau-prion model; our study instead probes the early cofactor-free assembly landscape of this peptide and its remodeling by TauPHF43. We find that jR2R3P301L rapidly forms heterogeneous higher-order oligomers and short, bundled fibrils that reduce the CCK-8 signal in HEK293 and HMC3 cells. Experimental data and candidate models support compact strand-loop-strand-like assembly states, including a putative conversion-limited hexamer. TauPHF43 is largely monomeric and nonfibrillogenic in isolation but forms jR2R3P301L-rich hetero-oligomers. A candidate [2 + 8] model qualitatively suggests that peripheral TauPHF43 association may favor an extended jR2R3P301L scaffold, consistent with the longer, thinner fibrils observed in the mixture. Coassembly also modifies CCK-8 responses in a cell-type-dependent manner. These findings support early hetero-oligomerization as one mechanism by which local sequence context and cross-fragment interactions can reshape tau assembly pathways and generate structural and biological heterogeneity.