Detyrosination enrichment on microtubule subsets is established by the interplay between a stochastically-acting enzyme and microtubule stability

Abstract

Microtubules in cells consist of functionally diverse subpopulations carrying distinct post-translational modifications (PTMs). Akin to the histone code, the tubulin code regulates a myriad of microtubule functions ranging from intracellular transport to chromosome segregation. Yet, how individual PTMs only occur on subsets of microtubules to contribute to microtubule specialization is not well understood. In particular, microtubule detyrosination, which is the removal of the C-terminal tyrosine on α-tubulin subunits, marks the stable population of microtubules and modifies how microtubules interact with other microtubule-associated proteins to regulate a wide range of cellular processes. Previously, we found that, in certain cell types, only a small subpopulation of microtubules is highly enriched with the detyrosination mark (~30%) and that detyrosination spans most of the length of a microtubule, often adjacent to a completely tyrosinated microtubule. How the activity of a cytosolic detyrosinase, Vasohibin (VASH) leads to only a small subpopulation of highly detyrosinated microtubules is unclear. Here, using quantitative super-resolution microscopy, we visualized nascent microtubule detyrosination events in cells consisting of 1-3 detyrosinated α-tubulin subunits after Nocodazole washout. Microtubule detyrosination accumulates slowly and in a disperse pattern across the microtubule length. By visualizing single molecules of VASH in live cells, we found that VASH engages with microtubules stochastically on a short time scale suggesting limited removal of tyrosine per interaction, consistent with the super-resolution results. Combining these quantitative imaging results with simulations incorporating parameters from our experiments, we propose a stochastic model for cells to establish a subset of detyrosinated microtubules via a detyrosination-stabilization feedback mechanism.

Macromolecular Crowding Tailors the Microtubule Cytoskeleton Through Tubulin Modifications and Microtubule-Associated Proteins

“…Although both of these modifications are associated with "stable" microtubules [2,13,52] and often occur on the same subset of microtubules [52,53,86,87], we have discovered that specific cellular conditions select for one PTM over the other, indicating that these modifications can be separated and utilized for specific cellular purposes. Our results are consistent with prior studies that show acetylation and detyrosination modifications are not always tightly linked, such as in cancer cells, in migrating cells, and upon microtubule regrowth after depolymerization [52,88,89]. Therefore, although these modifications are not mutually exclusive, we show here that MAP7 association has the ability to promote acetylation and limit detyrosination, thus decoupling them.…”

Computer simulation reveals the effect of severing enzymes on dynamic and stabilized microtubules

“…However, only the attachment and detachment of the severing enzyme were considered in the models for the sake of simplicity. These models can be further extended to include the diffusion of severing enzymes on the MT lattice as studied in a recent model of Vasohibin diffusion on the MT [67].…”

“…This is because the GMPCPP seed helps in MT rescue. In in-vivo experiments, high values of catastrophe and rescue frequencies are reported [61,67]. The in-vivo studies could not be compared with the simulation results due to their high concentrations of free tubulins.…”