Structure, Assembly, and Disassembly of Tubulin Single Rings

Abstract

Single and double tubulin rings were studied under a range of conditions and during microtubule (MT) assembly and disassembly. Here, tubulin was purified from porcine brain and used without any further modifications or additives that promote ring assembly. The structure of single GDP-rich tubulin rings was determined by cryo-transmission electron microscopy and synchrotron solution X-ray scattering. The scattering curves were fitted to atomic models, using our state-of-the-art analysis software, . We found that there is a critical concentration for ring formation, which increased with GTP concentration with temperature. MT assembly or disassembly, induced by changes in temperature, was analyzed by time-resolved small-angle X-ray scattering. During MT assembly, the fraction of rings and unassembled dimers simultaneously decreased. During MT disassembly, the mass fraction of dimers increased. The increase in the concentration of rings was delayed until the fraction of dimers was sufficiently high. We verified that pure dimers, eluted via size-exclusion chromatography, could also form rings. Interestingly, X-ray radiation triggered tubulin ring disassembly. The concentration of disassembled rings versus exposure time followed a first-order kinetics. The disassembly rate constant and initial concentration were determined. X-ray radiation-triggered disassembly was used to determine the concentration of rings. We confirmed that following a temperature jump, the mass fraction of rings decreased and then stabilized at a constant value during the first stage of the MT assembly kinetics. This study sheds light on the most basic assembly and disassembly conditions for in vitro single GDP-rich tubulin rings and their relation to MT kinetics.

Hierarchical Assembly Pathways of Spermine Induced Tubulin Conical-Spiral Architectures

“…Sample preparation. Stored tubulin was thawed on ice, after which three polymerization/depolymerization cycles (at 36°C and 0 °C, 30 min each) were performed to obtain GDP-rich tubulin (about 80% of the exchangeable sites contained GDP, determined using published protocols 28,39 ). The tubulin was then centrifuged for 30 min at 15,700 g and 4 °C.…”

“…. The different time-resolved data sets of each reaction showed that repeated exposure to the X-ray beam triggered disassembly of relatively small tubulin structures 28 (tubulin oligomers, THPs or TCSs subunits), which slowed down the assembly kinetics. As at least two identical time sets (delays and intervals) were used in all of the reaction conditions (4 mg/ml tubulin with 1, 5, or 20 mM spermine), we were able to compare the reaction rates.…”

Synchrotron Scattering Methods for Nanomaterials and Soft Matter Research

“…A well-known example is the study of muscle contraction along the pathway of physiological activation [79]. During this decade, TR-SAXS enabled probing in depth the self-assembly of virus particles [80,81], tubulin single rings [82], etc. from their constituting subunits.…”

Nano-particles carried by multiple dynein motors: A Self-Regulating Nano-Machine

“…Microtubules preparation. Black tubulin from porcine brain, purified via three polymerization/depolymerization cycles (65), was kindly provided by Uri Raviv (Hebrew University). Purified tubulin was flash-frozen in liquid nitrogen and kept at -80˚C until used.…”