Mutations in Human Tubulin Proximal to the Kinesin-Binding Site Alter Dynamic Instability at Microtubule Plus- and Minus-Ends

Shih-Chieh Ti,Melissa C Pamula,Stuart C Howes,Christian Duellberg,Nicholas I Cade,Ralph E Kleiner,Scott Forth,Thomas Surrey,Eva Nogales,Tarun M Kapoor

doi:10.1016/j.devcel.2016.03.003

Abstract

The assembly of microtubule-based cellular structures depends on regulated tubulin polymerization and directional transport. Here, we purify and characterize tubulin heterodimers that have human β-tubulin isotype III (TUBB3), as well as heterodimers with one of two β-tubulin mutations (D417H or R262H). Both point mutations are proximal to the kinesin-binding site and have been linked to an ocular motility disorder in humans. Compared to wild-type, microtubules with these mutations have decreased catastrophe frequencies and increased average lifetimes of plus- and minus-end-stabilizing caps. Importantly, the D417H mutation does not alter microtubule lattice structure or Mal3 binding to growing filaments. Instead, this mutation reduces the affinity of tubulin for TOG domains and colchicine, suggesting that the distribution of tubulin heterodimer conformations is changed. Together, our findings reveal how residues on the surface of microtubules, distal from the GTP-hydrolysis site and inter-subunit contacts, can alter polymerization dynamics at the plus- and minus-ends of microtubules.

Highlights

Heterodimers of a/b-tubulin undergo guanosine triphosphate (GTP)-dependent polymerization to form microtubules, cytoskeletal filaments essential for diverse cellular processes including neuronal transport, cell migration, and cell division (Desai and Mitchison, 1997; Heald and Khodjakov, 2015; Kapitein and Hoogenraad, 2015)
The proper organization of microtubules depends on dynamic instability, the stochastic transitions of the microtubule between growth and shrinkage, and microtubule-associated proteins (MAPs) that can step along these filaments to transport cargoes or regulate filament polymerization dynamics (Heald and Khodjakov, 2015; Nogales and Zhang, 2016)
MAPs such as the end-binding (EB) proteins that regulate microtubule polymerization dynamics bind tubulin subunits at sites proximal to the GTP-binding pocket of tubulin to sense and modulate changes in nucleotide states that directly contribute to polymerization dynamics (Akhmanova and Steinmetz, 2010; Maurer et al, 2012)

Summary

Introduction

Heterodimers of a/b-tubulin undergo guanosine triphosphate (GTP)-dependent polymerization to form microtubules, cytoskeletal filaments essential for diverse cellular processes including neuronal transport, cell migration, and cell division (Desai and Mitchison, 1997; Heald and Khodjakov, 2015; Kapitein and Hoogenraad, 2015). The proper organization of microtubules depends on dynamic instability, the stochastic transitions of the microtubule between growth and shrinkage, and microtubule-associated proteins (MAPs) that can step along these filaments to transport cargoes or regulate filament polymerization dynamics (Heald and Khodjakov, 2015; Nogales and Zhang, 2016) Consistent with these basic functions, mutations in tubulin or MAPs that disrupt MAP-microtubule interactions can lead to defective cytoskeletal architectures and have been linked to disease (Hirokawa et al, 2009; Niwa et al, 2013; Tischfield et al, 2011). The effect of mutating residues proximal to the kinesin-binding site on parameters of dynamic instability or on tubulin and microtubule conformation is not known

Results

Discussion

Conclusion