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

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), and 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.

Human β-Tubulin Isotypes Can Regulate Microtubule Protofilament Number and Stability

“…To purify recombinant forms of different human tubulin isotypes we modified the purification strategy we previously developed to obtain recombinant human β-tubulin, which purified as heterodimers with a mixture of human and insect α-tubulin (Ti et al, 2016). We found that inclusion of an affinity tag on human α-tubulin, similar to the one we used for β-tubulin, substantially reduced protein yield.…”

Challenges and opportunities in the high-resolution cryo-EM visualization of microtubules and their binding partners

“…With the development of new purification strategies [24] and expression systems [25,26], it has recently become possible to study more pure tubulin samples from alternative sources. A number of recent structural studies have now used recombinant human tubulin [25,27] and purified yeast tubulin [28] (which is significantly simpler in terms of tubulin isoforms). The latter opens the possibility to carry out parallel in vitro and in vivo studies in the characterization of tubulin mutants, as well as to explore, given the evolutionary distance between yeast and mammalian tubulin, the conservation (or not) of different structural properties between these two MT systems.…”

Sequence diversity of tubulin isotypes in regulation of the mitochondrial voltage-dependent anion channel

“…The results of these studies (3,(32)(33)(34)(35) demonstrate that recombinant tubulins are reliable means to study tubulin isotype-specific effects in vitro and to dissect the role of individual tubulin's CTTs.…”

“…fully functional and able to polymerize and form microtubules suitable for motor protein motility and processivity (3,(33)(34)(35). The recombinant human homogeneous microtubules showed different rates of polymerization compared with wild type (32,35).…”

“…The recombinant human homogeneous microtubules showed different rates of polymerization compared with wild type (32,35).…”