A novel PERspective on microTUbule Regulation – APERTuRe

Our goal is to investigate the time scale and localization of lattice exchange in dynamic microtubules using an in vitro setup to gain an understanding on the intrinsic self-regulation mechanisms of the microtubule shaft. To that end we will combine experiments on the lattice dynamics using TIRF fluorescence microscopy with structural data of microtubule lattice conformations obtained by cryo-electron microscopy/tomography and kinetic Monte Carlo simulations to test and validate different mechanisms of lattice turnover.

In a first step we have characterized the spontaneous lattice turnover in the microtubule shaft using a combined experimental and theoretical approach. There, we report that thermal forces are sufficient to remodel the microtubule shaft, despite its apparent stability. Our combined experimental data and numerical simulations on lattice dynamics and structure suggested that dimers can spontaneously leave and be incorporated into the lattice at structural defects. We proposed a model mechanism, where the lattice dynamics is initiated via a passive breathing mechanism at dislocations, which are frequent in rapidly growing microtubules. These results showed that we may need to extend the concept of dissipative dynamics, previously established for microtubule extremities, to the entire shaft, instead of considering it as a passive material.

In a second step we have experimentally characterized the effect of molecular motors on the remodeling of the microtubule shaft. Microtubules function in coordination with kinesin and dynein molecular motors, which use ATP hydrolysis to produce mechanical work and move on microtubules. This raises the possibility that the forces produced by walking motors can break dimer interactions and trigger microtubule disassembly. We tested this hypothesis by studying the interplay between microtubules and moving molecular motors in vitro. Our results show that the mechanical work of molecular motors can remove tubulin dimers from the lattice and rapidly destroy microtubules. Using fluorescently labelled tubulin dimers we found that dimer removal by motors was compensated by the insertion of free tubulin dimers into the microtubule lattice. This self-repair mechanism allows microtubules to survive the damage induced by molecular motors as they move along their tracks. Our study reveals the existence of coupling between the motion of kinesin and dynein motors and the renewal of the microtubule lattice.

Microtubules are essential structures in living cells, which grow and shrink at their extremities. Surprisingly, structural defects in the microtubule lattice, such as dislocation, endow also the shaft with dynamical features, thus opening a new avenue to understand microtubule regulation and stability. Currently we are investigating the role of vacancies and molecular motors in the shaft dynamics.

L. Schaedel, S. Triclin, D. Chrétien, A. Abrieu, C. Aumeier, J. Gaillard, L. Blanchoin, M. Théry, K. John (2019) Lattice defects induce microtubule self-renewal. Nature Physics 15 : 830. doi:10.1038/s41567-019-0542-4.
S. Triclin, I. Daisuke, J. Gaillard, Z.M. Htet, M. De Santis, D. Portran, E. Derivery, C. Aumeier, L. Schaedel, K. John, C. Leterrier, S.L. Reck- Peterson, L.. Blanchoin, M. Thery (2020) Self- repair protects microtubules from their destruction by molecular motors. BioRxiv : 499020. doi:10.1101/499020.

Microtubules are a central structure in living cells, involved in cell division, migration, and intracellular transport. Therefore, they are a primary drug target against severe pathologies, among them neurodegenerative diseases and cancer. A complete understanding of the mechanisms regulating their dynamics and stability is a central issue in cell biology and a key challenge for human health.

Common textbook knowledge states that microtubule lattice dynamics is restricted to elongation and shortening at the microtubule tips. The irreversible hydrolysis of GTP-tubulin at the microtubule tip gives rise to a non-equilibrium phenomenon, called “dynamic instability”. This behavior is crucial for many cellular processes, e.g. spindle positioning during mitosis, where microtubules switch rapidly between elongation and shortening phases. Since the discovery of the dynamic instability more than 30 years ago, research on microtubule regulation has been mainly focused on mechanisms acting on the microtubule tip. However, during interphase, microtubules live much longer and also the shaft lattice becomes a potential regulation point of microtubule stability.

Recently, we discovered that the microtubule shaft lattice shows an unexpected dynamics. Tubulin dimers incorporate directly into the shaft lattice in localized regions. Our preliminary data and model simulations suggest, that structural defects might be the origin of the observed localized tubulin incorporation.

The objective of our project is to characterize microtubule lattice turnover in the shaft using non-taxolated, i.e. dynamic, microtubules in an in vitro setup. We are interested in the spontaneous lattice turnover in response to thermal forces alone, i.e. in the absence of mechanical forces or biochemical factors. We will investigate the temporal and spatial properties of tubulin exchange to gain an understanding on the intrinsic material properties of microtubules.

To that end we will combine experiments on the lattice dynamics using TIRF fluorescence microscopy with cryo-EM imaging of the microtubule lattice structure. Furthermore, we will use kinetic Monte Carlo simulations to elucidate the underlying mechanisms of lattice turnover, which are consistent with our measured data. Our research team brings together the required expertise: Manuel Théry (TIRF fluorescence microscopy, IUH Paris), Denis Chrétien (cryo-EM imaging, IGDR Rennes) and Karin John (model simulations, LIPhy Grenoble).

Potentially, a continuous localized dimer turnover introduces stabilizing GTP-tubulin dimers into the lattice, which could (i) serve as a rescue points to stop microtubule depolymerization and (ii) be recognized by regulatory proteins, which are typically associated with the microtubule tip.