APERTuRe

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.