BPS2026 – Dissecting cytoskeletal dynamic instability with PhuZ: A viral tubulin homolog as a model system

My objective is to establish the bacteriophage-derived tubulin homolog PhuZ as a tractable system for dissecting cytoskeletal polymer dynamics, focusing on dynamic instability (DI) and spontaneous branching. Unlike canonical tubulin which require complex chaperones, PhuZ can be expressed in E. coli yet undergoes GTP-GDP hydrolysis-dependent polymerization and three proto-filament assembly, making it ideal for mechanistic studies. I first characterized critical concentrations (CCs) of wild-type (WT) PhuZ and mutants with altered lateral and longitudinal subunit interfaces. In the Q297N mutant, where glutamine-297 is replaced by asparagine, sedimentation assays confirmed GTP-dependent polymerization and steady-state absorbance revealed a CC Netassembly of 1.6 μM, lower than the 2.5 μM of WT. Transmission electron microscopy (TEM) showed Q297N forms shorter, more numerous filaments, consistent with increased nucleation (filament formation) frequency thus explaining the lower CC Netassembly . Both WT and Q297N filaments displayed spontaneous branching without accessory proteins, prompting expansion to other lateral-bond mutants (R290K and D303E). Next, I examined GMPCPP-stabilized seeds (short filaments stabilized with a non-hydrolyzable GTP analog). Both intact and sonicated seeds supported filament growth and branching when mixed with GTP and subunits at low concentration, demonstrating that branching can initiate from nascent or pre-formed filaments even at low free subunit levels. Finally, I applied the statistical tool for automated dynamic instability analysis (STADIA) to TIRF microscopy generated kymographs (length history plots) of fluorescent PhuZ. STADIA’s machine-learning framework detects subtle growth phases beyond classical two-state models. Preliminary analyses indicate PhuZ filaments mainly undergo sustained growth with limited catastrophes. Collectively, these biochemical, imaging, and computational approaches reveal how specific subunit interfaces regulate CCs, filament morphology, and branching, advancing PhuZ as a powerful model for understanding DI and self-organized branching in cytoskeletal polymers.