Background: Microtubule functions are controlled via the precise and timely regulation of their building blocks, the tubulin isotypes, in combination with several post-translational modifications (PTMs). While some tubulin isotypes are known to be crucial for platelet biogenesis (TUBB1, TUBA4), the temporal regulation and biological significance of their PTMs remain mostly unknown. Acetylation, detyrosination, polyglutamylation and polyglycylation are currently considered as the main tubulin PTMs of functional relevance.

Aims: This project aims to characterize the evolution of tubulin PTMs in platelet biogenesis and to identify those that could be functionally relevant for this process.  

Methods: The profiles of tubulin PTMs were analyzed by Western Blot in human CD34⁺-derived cultured megakaryocytes and platelets, and human blood-derived platelets. For reference, they were compared to those in brain and testis. In addition, the transcripts of the enzymes involved in their establishment and removal was followed by qRT-PCR.

Results: Tubulin acetylation remained stable along megakaryocyte maturation, despite a concomitant rise in the levels of the deacetylase HDAC6 transcript. Tubulin detyrosination progressively increased as megakaryocytes differentiated, in line with the increasing levels of the detyrosinase VASH1 transcript. Interestingly, polyglutamylation of α-tubulins drastically accumulated and reached its highest expression in platelets. Polyglutamylation of β-tubulins, in contrast, followed the inverse trend and became undetectable in platelets. This pattern of polyglutamylation could be related to the high transcript expression found for TTLL5, the α-tubulin polyglutamylase, and for the CCP1 and CCP5 deglutamylases. Tubulin polyglycylation remained weak during platelet biogenesis, in agreement with the low expression of the polyglycylase transcripts. Comparison with brain and testis showed that detyrosination was expressed at similar levels, while acetylation and polymodifications were much lower in the platelet lineage.

Conclusions: These results provide the first comprehensive characterization of the tubulin PTM repertoire in the platelet lineage, thus laying the ground for insightful evaluation of their respective role in platelet formation.

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ISTH Congress Abstracts - https://abstracts.isth.org/abstract/evolution-of-the-tubulin-post-translational-modification-repertoire-during-megakaryopoiesis/