Post-translational polymodification of the C-terminal tail of TUBB1 regulates motor protein processivity in platelet production and function

Microtubules are ubiquitously expressed cytoskeletal structures responsible for a host of cellular processes from division to cargo transport. In specialised cells, the expression of specific isoforms of tubulin and their subsequent post-translational modifications are thought to drive and co-ordinate unique morphologies and behaviours. The mechanisms by which β-1 tubulin (encoded by TUBB1 ), the platelet and megakaryocytic specific β-tubulin isoform required for platelet production and function, drives these processes remains poorly understood. We investigate the effects of two key tubulin post-translational polymodifications (polyglutamylation and polyglycylation) on the glutamate rich C-terminus of β-1 tubulin using a cohort of thrombocytopenic patients, human induced pluripotent stem cell (iPSC) derived megakaryocytes, and healthy human donor platelets. We find that while megakaryocytes (MKs) are positive for both polymodifications, polyglycylation is substantially reduced on platelets. On platelet activation, the marginal band becomes heavily polyglutamylated, which drives the mobilisation of motor proteins, including axonemal dynein, to achieve the shape change required for the haemostatic role of platelets. Finally, we show that a number of modifying enzymes (Tubulin Tyrosine Like Ligases (TTLLs) and Cytosolic Carboxypeptidases (CCPs)) are up-regulated through MK maturation. In platelets, a single polyglutamylase (TTLL7) is expressed to mediate the polyglutamylation of the marginal band required for shape change on activation. Finally, we report a novel disease causing gene in multiple families (TTLL10) resulting in bleeding despite normal platelet production and function. This work highlights the importance of a complex regulatory mechanism driven by both cell specific tubulin isoform expression and differential post-translational modification to drive specialist function, the loss of which results in disease states.