Mechanism of the Initial Tubulin Nucleation Phase
2Institute of Life Sciences and the Center for Nanoscience and Nanotechnolog, The Hebrew University of Jersuaelm, Israel
3Institute for Drug Research, School of Pharmacy, The Hebrew University of Jersuaelm, Israel
Microtubules (MTs) are essential in shaping cellular structure, organelle transport, and the mitotic spindle.
To serve these multi-tasks efficiently, MTs are highly dynamic; while some MTs may nucleate and elongate, others may rapidly disassemble. Tubulin nucleation is a highly frequent event, but one of the least understood steps in MT dynamics.
We characterized the structural changes during the initial nucleation phase of dynamic tubulin.
Using SEC-eluted tubulin dimers in an assembly buffer solution free of glycerol and tubulin aggregates enabled us to start from a well-defined initial thermodynamic ensemble of isolated dynamic tubulin dimers and short oligomers. Following a temperature increase, time-resolved X-ray scattering and cryo-TEM during the initial nucleation phase revealed an isodesmic assembly mechanism of 1D tubulin oligomers (where dimers were added and/or removed one at a time), leading to sufficiently stable two-dimensional dynamic nanostructures, required for MT assembly.
A substantial amount of tubulin octamers accumulated before two-dimensional lattices appeared.
In subcritical assembly conditions, we observed a slower isodesmic assembly mechanism, but the concentration of 1D oligomers was insufficient to form the multistranded 2D nucleus required for MT formation.