RT Journal Article SR Electronic T1 Post-translational polymodification of β1 tubulin regulates motor protein localisation in platelet production and function JF bioRxiv FD Cold Spring Harbor Laboratory SP 595868 DO 10.1101/595868 A1 Abdullah O. Khan A1 Alexandre Slater A1 Annabel Maclachlan A1 Phillip L.R. Nicolson A1 Jeremy A. Pike A1 Jasmeet S. Reyat A1 Jack Yule A1 Steven G. Thomas A1 Neil V. Morgan YR 2019 UL http://biorxiv.org/content/early/2019/10/09/595868.abstract AB In specialised cells, the expression of specific tubulin isoforms and their subsequent post-translational modifications drive and co-ordinate unique morphologies and behaviours. The mechanisms by which β1 tubulin, the platelet and megakaryocyte 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 megakaryocytes, and healthy human donor platelets. We find distinct patterns of polymodification in megakaryocytes and platelets, mediated by the cell specific expression of effecting (Tubulin Tyrosine Ligase Like - TTLL) and reversing (Cytosolic Carboxypeptidase - 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 PointsThe platelet specific β1 tubulin (encoded by TUBB1) is polyglutamylated and polyglycylated in platelet producing iPSC-derived megakaryocytes (MKs).The platelet marginal band is polyglutamylated upon activation.Polymodification in both MKs and platelets impact motor protein localisation.Patients with C-terminal TUBB1 variants demonstrate macrothrombocytopenia, and the CRISPR mediated knock out of TUBB1 in iPSC-MKs results in a complete loss of proplatelet production.3 unrelated families with mutations in TTLL10 report moderate to severe bleeding and increased mean platelet volume (MPV), suggesting polyglycylation through TTLL10 is required for healthy 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.