Structure and Dynamics of Single-isoform Recombinant Neuronal Human Tubulin

Abstract

Microtubules are polymers that cycle stochastically between polymerization and depolymerization, i.e. they exhibit “dynamic instability.” This behavior is crucial for cell division, motility, and differentiation. Although studies in the last decade have made fundamental breakthroughs in our understanding of how cellular effectors modulate microtubule dynamics, analysis of the relationship between tubulin sequence, structure, and dynamics has been held back by a lack of dynamics measurements with and structural characterization of homogeneous isotypically pure engineered tubulin. Here, we report for the first time the cryo-EM structure and in vitro dynamics parameters of recombinant isotypically pure human tubulin. α1A/βIII is a purely neuronal tubulin isoform. The 4.2-Å structure of post-translationally unmodified human α1A/βIII microtubules shows overall similarity to that of heterogeneous brain microtubules, but it is distinguished by subtle differences at polymerization interfaces, which are hot spots for sequence divergence between tubulin isoforms. In vitro dynamics assays show that, like mosaic brain microtubules, recombinant homogeneous microtubules undergo dynamic instability, but they polymerize slower and have fewer catastrophes. Interestingly, we find that epitaxial growth of α1A/βIII microtubules from heterogeneous brain seeds is inefficient but can be fully rescued by incorporating as little as 5% of brain tubulin into the homogeneous α1A/βIII lattice. Our study establishes a system to examine the structure and dynamics of mammalian microtubules with well defined tubulin species and is a first and necessary step toward uncovering how tubulin genetic and chemical diversity is exploited to modulate intrinsic microtubule dynamics.

The tubulin code at a glance

“…recombinant tubulin with controlled PTMs -in order to study the mechanisms of the tubulin code in vitro. The first exciting advances in this direction have recently been made (Barisic et al, 2015;Minoura et al, 2013;Pamula et al, 2016;Sirajuddin et al, 2014;Vemu et al, 2016).…”

Tubulin acetylation protects long-lived microtubules against mechanical ageing

“…Since αK40 buttresses one of the two loops that form side-to-side contacts 27 , it has been proposed that an electrostatic bond involving αK40 alters the strength of α-α interactions 9,23 . This hypothesis has however eluded structural investigations as the nine amino acids flanking αK40 remain the last unsolved part of the α/β-tubulin core 19 , even in a 4.2 Å structure of microtubules assembled from non-acetylated recombinant tubulin 28 . Together, these data suggest that the αK40 loop is flexible and that the lateral contact whose strength is reduced by αK40 acetylation is itself dynamic.…”

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

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

“…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.…”