Rie Ayukawa

Overexpression, purification, and functional analysis of recombinant human tubulin dimer.

Microtubules consisting of tubulin dimers play essential roles in various cellular functions. Investigating the structure–function relationship of tubulin dimers requires a method to prepare sufficient quantities of recombinant tubulin. To this end, we simultaneously expressed human α1‐ and β3‐tubulin using a baculovirus‐insect cell expression system that enabled the purification of 5 mg recombinant tubulin per litre of cell culture. The purified recombinant human tubulin could be polymerized into microtubules that glide on a kinesin‐coated glass surface. The method provides a powerful tool for in vitro functional analyses of microtubules.

A flipped ion pair at the dynein-microtubule interface is critical for dynein motility and ATPase activation

Salt bridges at the dynein–microtubule interface couple microtubule binding to ATPase activation and thereby control the directional movement of dynein

KIF1A Repeats Cycle of ‘FREE Diffusion’ and ‘SPECIFIC Binding’ during Weak Binding State

The nature of intermolecular interaction between motor and cytoskeletal filament during the weak binding state is not fully understood. In the case of kinesin, while structural analyses revealed that kinesin binds to a specific binding site on tubulin, motility data suggested that kinesin undergoes diffusion, searching for its next binding site. To understand how specific binding and diffusion are compatible in a single ADP state, we analyzed the motion of the single-headed kinesin KIF1A on various mutant microtubules (MTs) in the presence of ADP, using the single molecule motility assay.We prepared two series of mutant MTs. The first is a series with increased/decreased negative charges at the C-terminal tails (CTTs) of tubulin, reported to be indispensable for the weak binding of KIF1A to the MT (Okada et al., 2000). The second is a series of charged-to-alanine mutants in the H11-12 loop and H12 of tubulin (α-E415, -E416, -E418, -E421 and β-E410, -D417), found to be critical for kinesin motility and ATPase (Uchimura et al., 2010). The analyses of KIF1A movement showed that a reduction of negative charges in CTTs leads to a reduction in both the duration of interaction and the diffusion length of KIF1A, yet the diffusion constant was not greatly changed. In contrast, in most of the charged-to-alanine tubulin mutants, the diffusion constant of KIF1A increased and the duration shortened, but the diffusion length was unaffected. These results indicate that KIF1A-MT interaction in the ADP state can be modeled as an equilibrium between two substates: a dynamic ‘diffusion state’ and a static ‘binding state’. While CTTs stabilize the former, the critical residues in the H11-12 loop and H12 of tubulin stabilize the latter. This model is applicable to dimeric kinesin.