The Structure and Dynamics of C. elegans Tubulin Reveals the Mechanistic Basis of Microtubule Growth

Abstract

The dynamic instability of microtubules is a conserved and fundamental mechanism in eukaryotes. Yet microtubules from different species diverge in their growth rates, lattice structures, and responses to GTP hydrolysis. Therefore, we do not know what limits microtubule growth, what determines microtubule structure, or whether the mechanisms of dynamic instability are universal. Here, we studied microtubules from the nematode C. elegans, which have strikingly fast growth rates and non-canonical lattices in vivo. Using a reconstitution approach, we discovered that C. elegans microtubules combine intrinsically fast growth with very frequent catastrophes. We solved the structure of C. elegans microtubules to 4.8 Å and discovered sequence divergence in the lateral contact loops, one of which is ordered in C. elegans but unresolved in other species. We provide direct evidence that C. elegans tubulin has a higher free energy in solution and propose a model wherein the ordering of lateral contact loops activates tubulin for growth.

Multi-scale Computational Modeling of Tubulin-Tubulin Lateral Interaction

“…S3 and Table S1). As shown in Figure 5A, we observe that there is no energy barrier found between the bound and unbound states in any of our PMF replicates and is unlikely to exist because there is no significant desolvation of the binding interface, in contrast to the assumption made in some previous BD studies (39,40). For comparison, the PMF obtained by MD is plotted along with previously published BD estimates of the lateral bond strength (38,39) (see Fig.…”

“…Since the M-loop is relatively ordered in the laterally bonded state, compared to a single dimer in solution, we conclude that the decreased entropy upon binding acts to destabilize the lateral bond (40). Besides, the decreased fluctutations due to lateral bond is more amplified in the M-loop in β-tubulin, highlighting a key lateral role for the M-loop in βtubulin compared to α-tubulin (62).…”

“…For instance, catastrophe, microtubule's switching from growth to shortening, has historically been described as stemming from loss of the stabilizing GTPcap due to hydrolysis (37,(42)(43)(44), while recent Brownian dynamics studies (39,45) suggested that stochastic variations in the number of curled protofilaments is responsible for causing catastrophe, albeit while using a different shape and strength for potential of interactions of tubulins from previous models (41,46). In addition, lateral bond formation has been identified as the limiting step in forming the microtubule lattice with a large entropic component in another study (40), but no direct calculations have been reported for the lateral bond strength or its nucleotide-state dependency even though it is critical for assembly and potentially for dynamic instability as well.…”

“…By contrast, coarse-grained Brownian dynamics (BD) with ~ms time scales at a cost of atomistic detail, and thermo-kinetic (TK) simulations with less detail allowing access to ~100s time scales, together enable recapitulation of the kinetic rates and microtubule tip structures consistent with those found in vitro and in vivo (35)(36)(37)(38). The interactions of particles in the BD simulations, here being the tubulin dimers, are modeled using an input potential, which can be dissimilar for tubulin as a function of its nucleotide state in different studies (7,(38)(39)(40)(41). However, the interaction energy profiles have been adjusted in these models to match experimental observations of MT tip structure, dynamic assembly rates, and microtubule stiffness.…”

Crystal structure of human PACRG in complex with MEIG1

“…Microtubules were polymerized from α-and β-tubulin isolated from cow brain, using a protocol adapted from Ashford et al [59] and described recently [60]. All reactions were set up in PCR tubes containing 20 μM tubulin, 20 μM WT/mutant MEIG1:PACRG, 4 mM MgCl2, 1 mM GTP, and BRB80 buffer (80 mM PIPES, 1 mM MgCl2, 1 mM EGTA, pH 6.8).…”