Post-translational polymodification of β1 tubulin regulates motor protein localisation in platelet production and function

In specialised cells, the expression of specific tubulin isoforms and their subsequent post-translational modifications drive and coordinate unique morphologies and behaviours. The mechanisms by which β 1 tubulin, the platelet and megakaryocyte (MK) lineage restricted tubulin isoform, drives platelet production and function remains poorly understood. We investigated the roles of two key post-translational polymodifications (polyglutamylation and polyglycylation) on these processes using a cohort of thrombocytopenic patients, human induced pluripotent stem cell (iPSC) derived MKs, and healthy human donor platelets. We find distinct patterns of polymodification in MKs and platelets, mediated by the antagonistic activities of the cell specific expression of Tubulin Tyrosine Ligase Like (TTLLs) and Cytosolic Carboxypeptidae (CCP) enzymes. The resulting microtubule patterning spatially regulates motor proteins to drive proplatelet formation in megakaryocytes, and the cytoskeletal reorganisation required for thrombus formation. This work is the first to show a reversible system of polymodification by which different cell specific functions are achieved. Key Points The platelet specific β 1 tubulin (encoded by TUBB1 ) is polymodified (polyglutamylated and polyglycylated) in platelet producing iPSC-derived megakaryocytes (MKs), this patterning spatially regulates motor proteins, and its disruption in TUBB1 patient variants results in a loss of platelet production. A system of reversible polymodifications mediated through the graded expression of modifying enzymes (TTLLs and CCPs) throughout MK maturation is required for proplatelet formation and subsequent platelet function.