Role of Tubulin Detyrosination in Platelet Biogenesis

Introduction. Microtubules are hollow cylindrical polymers that are made of α/β-tubulin heterodimers. They are involved in a broad range of essential functions such as cell division, intracellular trafficking or cell motility. Their functional diversity is thought to be regulated through the expression of distinct α- and β-tubulin isotypes together with several post-translational modifications (PTMs); a concept known as tubulin code. In the megakaryocytic lineage, while tubulin isotypes are well-known regulators of platelet biogenesis (TUBB1, TUBA4A, TUBA8)¹, the temporal regulation and biological significance of tubulin PTMs remain mostly unknown.

Aims. This project aims to characterize the kinetics and to evaluate the functional importance of tubulin detyrosination, one of the main α-tubulin PTM, in platelet biogenesis.

Methods. Analyzes were performed in megakaryocytes and platelets obtained from cultured human CD34⁺ hematopoietic stem and progenitor cells. To characterize the kinetics of tubulin detyrosination, the PTM and its modifying enzymes were monitored at the proteomic (Western Blot) and transcriptional (qRT-PCR) levels, at several defined stages of megakaryocyte differentiation. To evaluate the function of tubulin detyrosination, differentiation of CD34⁺ hematopoietic stem and progenitor cells was performed in the presence of the SVBP/VASH-detyrosinase inhibitor EpoY² or following CRISPR-mediated inactivation of the VASH detyrosinase co-factor SVBP.

Results. Tubulin detyrosination levels progressively increased as megakaryocytes matured. This was accompanied by a gradual increase in the transcript levels of the detyrosinase VASH1, while those of its cofactor SVBP remained stable. Transcripts of the newly identified detyrosinase MATCAP³ mirrored those of VASH1. Together, these results suggest that tubulin detyrosination occurring during platelet biogenesis could be mediated by both the SVBP-VASH1 complex and/or MATCAP, and prompt the question of their functional importance in the process. To evaluate this, we first focused on the SVBP-VASH1 complex with the inhibitor EpoY. Interestingly, while this compound had no impact on early megakaryocyte differentiation, the final stage of platelet biogenesis, proplatelet formation, was perturbed. In particular, proplatelets obtained in the presence of EpoY showed morphological alterations with thinner unbranched shafts, suggesting potential repercussions on platelet production. Preliminary data obtained with SVBP-inactivated megakaryocytes confirmed these observations. Follow-up work will aim to evaluate the downstream consequences on platelet production as well as the relative contribution of MATCAP.

Conclusion. These results suggest that tubulin detyrosination could act as a potential regulator of terminal megakaryocyte maturation, and further highlight the importance of the microtubule cytoskeleton and the tubulin code in platelet biogenesis.

References.

1. Kimmerlin, Q., Strassel, C., Eckly, A., and Lanza, F. (2023). The tubulin code in platelet biogenesis. Semin Cell Dev Biol 137, 63-73. 10.1016/j.semcdb.2022.01.010.
2. Hotta, T., Haynes, S.E., Blasius, T.L., Gebbie, M., Eberhardt, E.L., Sept, D., Cianfrocco, M., Verhey, K.J., Nesvizhskii, A.I., and Ohi, R. (2021). Parthenolide Destabilizes Microtubules by Covalently Modifying Tubulin. Curr Biol 31, 900-907 e906. 10.1016/j.cub.2020.11.055.
3. Landskron, L., Bak, J., Adamopoulos, A., Kaplani, K., Moraiti, M., van den Hengel, L.G., Song, J.Y., Bleijerveld, O.B., Nieuwenhuis, J., Heidebrecht, T., et al. (2022). Posttranslational modification of microtubules by the MATCAP detyrosinase. Science 376, eabn6020. 10.1126/science.abn6020.