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

Abstract

Key Points 1• The platelet specific -1 tubulin (encoded by TUBB1) is polyglutamylated and polyglycylated in platelet producing iPSC-2 derived megakaryocytes (MKs). 3 • The platelet marginal band is polyglutamylated upon activation. 4 • Polymodification in both MKs and platelets impact motor protein localisation. 5• Patients with C-terminal TUBB1 variants demonstrate macrothrombocytopenia, and the CRISPR mediated knock out of 6 TUBB1 in iPSC-MKs results in a complete loss of proplatelet production. 7• 3 unrelated families with mutations in TTLL10 report moderate to severe bleeding and increased mean platelet volume 8 (MPV), suggesting polyglycylation through TTLL10 is required for healthy platelet production. 9• A system of reversible polymodifications mediated through the graded expression of modifying enzymes (TTLLs and 10 CCPs) throughout MK maturation is required for proplatelet formation and subsequent platelet function. 11 Introduction 12Microtubules are large, cytoskeletal filaments vital to a host of critical functions including cell division, signalling, cargo 13 transport, motility, and function(1-3). Despite their ubiquitous expression and high structural conservation, microtubules also 14 drive unique morphologies and functions in specialist cell types like ciliated cells, spermatozoa, and neurons (4, 5). The question 15 of how filaments expressed in every cell in the body can facilitate complex and highly unique behaviours like neurotransmitter 16 release and retinal organisation has been addressed by the tubulin code. This is a paradigm which accounts for the specialisation 17 of microtubules and their organisation by describing a mechanism in which particular cells express lineage restricted isoforms 18 of tubulin. These cell specific isoforms are then subject to a series of post-translational modifications which alter the mechanical 19 properties of microtubules, and their capacity to recruit accessory proteins (e.g. motor proteins) (1)(2)(3) 5). 20 A host of tubulin post-translational modifications (PTMs) have been reported in a range of cell types, including (but not limited 21 to) tyrosination, acetylation, glutamylation, glycylation, and phosphorylation. In recent years links between the loss of specific 22 tubulin PTMs, either through the aberrant expression of tubulin isoforms or the loss of effecting or reversing enzymes, has 23 emerged (5). The loss of post-translationally modified tubulin has been reported to impact motile and non-motile ciliary 24 function (including respiratory cilia, retinal cells), spermatogenesis, muscular disorders, and neurological development and 25 function (1,(5)(6)(7)(8)(9)(10)(11). Of the many cell systems in which careful regulation of tubulin modification is required for healthy function, 26 the role of the tubulin code in the generation of blood platelets from their progenitors, megakaryocytes (MKs) remains relatively 27 poorly understood. 28 Platelets are the smallest component of peripheral blood, and circulate as anucleate cells with ...

Tubulin in Platelets: When the Shape Matters

“…Thus, upon platelet activation, the marginal band becomes heavily polyglutamylated, which allows the mobilization of motor proteins, such as dynein, to achieve the shape change required during platelet activation [25]. Moreover, in mature megakaryocytes and proplatelets, the polyglutamylation of β1 tubulin-containing microtubules is important in order to induce the formation of the marginal band [56], and it is implicated in proplatelet elongation and in platelet release [57]. A list of the proteins that regulate microtubule organization and dynamics is shown in Table 2.…”

SMIFH2 inhibition of platelets demonstrates a critical role for formin proteins in platelet cytoskeletal dynamics

“…Tubulin PTMs have been shown to be important in regulating both microtubule stability and their interaction with microtubule associated proteins and are part of a tubulin code that regulates cytoskeletal dynamics . Furthermore, tubulin PTMs, including acetylation, polyglutamylation, and polyglycylation have been shown to be important in platelets and megakaryocytes . The changes reported here could affect the platelet microtubule coil in a number of ways: (a) tubulin acetylation is associated with the protection of microtubules from mechanical stress, (b) increased tyrosination is associated with more dynamic microtubules, and (c) increased tyrosination is associated with increased kinesin motor activity .…”

Loss of mDia1 and Fhod1 impacts platelet formation but not platelet function

“…The mechanisms underlying normal proplatelet formation are complex, but it is clear that both the microtubule and actin cytoskeletons are required [ 6 ]. Recent work has identified that both post-translational modifications of tubulin [ 30 , 31 ] and interactions between cytoskeletal motors, the cytoskeleton, and response to the environment (e.g. flow) [ 32 ] modulate platelet formation.…”