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A.O. Khan et al.
is analogous to the PTM of ciliated cells, and so we rea- soned that axonemal dynein, an isoform of the motor exclusive to axonemes, may play a role in both platelet formation and activation.6,37,38 We find evidence of axone- mal dynein on both proplatelet extensions and at the leading edge of spreading platelets. To our knowledge this is the first evidence of a functional role of axonemal dynein outside of classical ciliated structures. In our TUBB1 KO MK, we observe a decrease in the co-localiza- tion of dynein to polyglutamyulated tubulin, suggesting that the loss of proplatelet formation observed in these cells is due to a dysregulation of the dynein-mediated microtubule sliding known to drive the elongation of the proplatelet shaft.39 The similarities between flagella and MK proplatelet extensions have been discussed previous- ly, Italiano et al. observe structures similar to flagella in taxol treated MK, an idea which led to the microtubule telescoping experiment reported by Patel et al.39,40
Our data suggests a tightly regulated, reversible system of polymodification which must be mediated by the cell specific expression of TTLL and CCP. Our expression profiles show a number of TTLL and CCP are expressed by MK, while only the polyglutamylase TTLL7 is expressed by platelets consistent with the different pat- terns of polymodification oberved in these cells. In MK we find expression of two TTLLs known to be involved in glycylation - the initiase TTLL3 and the elongase TTLL10, with a significant increase in the expression of TTLL10 on platelet production. Our findings support a role of TTLL10 as a polyglycylase on co-expression with TTLL3 as reported by Ikegami et al. in cell lines through co-transfection experiments (Figure 7).
Finally, we report a novel gene, TTLL10, in three unre- lated families with excessive bleeding. We identify three unrelated families with TTLL10 variants which result in an increase in an established history of bleeding which provides an invaluable insight to the potential role of polyglycylation in the context of platelet production and function. Our data shows that both TTLL3 and TTLL10 are expressed in platelet producing MK. Our patient cohort do not lose TTLL3 function, and as such the action of TTLL3 as an initiase will occupy glutamate residues which would otherwise be polyglutamylated. In these patients we likely see a loss of polyglycylation, but no coincident increase in polyglutamylation due to the nor- mal function of the initiase (TTLL3). As polyglutamyla- tion and monoglycylation are unaffected, platelet counts (and production) are normal, however, affected individu- als appear to have an increased platelet volume and bleeding, suggesting a role for the extended glycine tail in regulating platelet size, with a downstream effect on the ability of platelets to prevent bleeding.
Patel et al. describe a system by which continuous poly- merization is key to the dynein-mediated sliding required for platelet production.39 This work, alongside reports of tyrosination, acetylation, and polyglutamylation in differ- ent MK models, suggests that tubulin PTM are likely part of a highly dynamic system in which polymerizing microtubules are subject to modifications which coordi- nate platelet production and packaging. Future work will interrogate the interplay between different PTM, and how they fit into a highly dynamic landscape of MT polymerization and function. Indeed, in our KO TUBB1 cells we do not observe a complete loss of polymodifica- tion, suggesting that compensatory mechanisms which target other isoforms of tubulin may come into play. There is likely a more extensive effect on the expression of tubulins and their interplay with other cytoskeletal proteins which should be a focus of future work. Similarly, the role of these PTMs will need to be further validated in primary human CD34-derived MK.
This work supports the paradigm of a ’tubulin code’ and the importance of microtubule patterning in healthy, and thus diseased, MK and platelets. We provide novel insights into the mechanisms by which b1-tubulin functions in these unique cells.
Disclosures
No conflicts of interest to disclose.
Contributions
AOK and NVM devised and performed experiments; AOK and NVM wrote the manuscript; AS performed homology model- ing, cloning, transfection, and platelet spreading experiments; AM performed platelet spreading and analysis of patient sequencing data; PLRN performed platelet preparations and reviewed the manuscript; JAP developed and applied image analysis workflows; JSR assisted with western blotting; JY contributed to platelet prepa- rations; all authors reviewed the manuscript.
Acknowledgments
We thank the families for providing samples and our clinical and laboratory colleagues for their help. The authors would like to thank the TechHub and COMPARE Core facilities at the University of Birmingham. AOK is a Wellcome funded Sir Henry Wellcome Fellow (218649/Z/19/Z). We thank Professor Steve Watson for his ongoing support and invaluable mentorship. The authors do not have any conflict of interest.
Funding
This work was supported by the British Heart Foundation (PG/13/36/30275; FS/13/70/30521; FS/15/18/31317; PG/16/103/32650; IG/18/2/33544).This research was funded in whole or in part by the Wellcome Trust [218649/Z/19/Z].
References
1.WadeRH.Onandaroundmicrotubules:an overview. Mol Biotechnol. 2009;43(2):177- 191.
2. Gadadhar S, Dadi H, Bodakuntla S, et al. Tubulin glycylation controls primary cilia length. J Cell Biol. 2017; 216(9):2701-2713.
3. Ludueña RF. A hypothesis on the origin and evolution of tubulin. Int Rev Cell Mol Biol. 2013;302:41-185.
4. Magiera Maria M, Singh Puja, Janke Carsten.
SnapShot: functions of tubulin posttransla- tional modifications. Cell. 2018;173(6):1552- 1552.e1.
8. Bosch Grau Montserrat, Masson Christel, Gadadhar Sudarshan, et al. Alterations in the balance of tubulin glycylation and glutamy- lation in photoreceptors leads to retinal degeneration. J Cell Sci. 2017;130(5):938-
5.Gadadhar S, Bodakuntla S, Natarajan K,
Janke C. The tubulin code at a glance. J Cell
Sci. 2017;130(8):1347-1353. 949.
6. Wloga D, Joachimiak E, Louka P, Gaertig J. Posttranslational modifications of tubulin and cilia. Cold Spring Harb Perspect Biol. 2017;9(6):a028159.
7. Reiter JF, Leroux MR. Genes and molecular pathways underpinning ciliopathies. Nat Rev Mol Cell Biol. 2017;18(9):533-547.
9. Ikegami K, Sato S, Nakamura K, Ostrowski LE, Setou M. Tubulin polyglutamylation is essential for airway ciliary function through the regulation of beating asymmetry. Proc Natl Acad Sci U S A. 2010;107(23):10490- 10495.
10. Wu HY, Wei P, Morgan JI. Role of cytosolic
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