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A.O. Khan et al.
These cells are characteristically large, polyploid cells with unique morphological structures (e.g., the invaginated membrane system [IMS]) required to facilitate the produc- tion of thousands of blood platelets and packaged within them the required pro-thrombotic factors.15,16 MK form long, beaded extensions into the lumen of bone marrow sinu- soids - where these proplatelet extensions then experience fission under the flow of sinusoidal blood vessels which results in the release of barbell shaped pre-platelets and platelets into the blood stream.16
Both MK and platelets express a lineage-restricted iso- form of b1-tubulin encoded by the gene TUBB1.17 In humans, TUBB1 mutations have been shown to result in impaired platelet production, with a resulting macrothrom- bocytopenia.18,19 More recently, a C-terminal truncation of b1-tubulin has been shown to cause a macrothrombocy- topenia, suggesting that C-terminal modifications may be drivers of protein function and causative of the disease phe- notype observed.20
While the loss of b1-tubulin is known to result in macrothrombocytopenia, the mechanisms by which this isoform of tubulin effects the dramatically different cytoskeletal behaviors of platelets and MK remains poorly understood. In the context of the tubulin code, MK and platelets present a particularly interesting model. Both cells express b1-tubulin, but undergo markedly different cytoskeletal changes. To date, acetylation and tyrosination have been the PTM primarily reported in MK and platelets, however neither modification is specific to the C-terminal tail encoded by TUBB1. Fiore et al. showed that a C-termi- nal truncation of b1-tubulin phenocopies the complete loss of the protein.20 We, therefore, hypothesize that PTM spe- cific to the C-terminus of TUBB1 are required for the com- plex morphological rearrangements required for both MK and platelet function.
The C-terminal tail of b1 tubulin is particularly rich in glu- tamate residues which are often targeted for two key PTM implicated in human disease. Polyglutamylation and polyg- lycylation are PTM which target glutamate residues on both tubulin subunits (a and b) and result in the addition of glu- tamate or glycine residues respectively.1,2,21 As both PTM tar- get the same residue they are together referred to as poly- modification. Polymodification has been observed in micro- tubules in centrioles, axenomes, neuronal outgrowths, and mitotic spindles.1,2
To date polyglycylation has not been reported in MK or platelets. Recently Van Dijk et al. reported on the polyglu- tamylation of b1 tubulin downstream of the integrin a2bb3 in a CHO cell line engineered to express TUBB1, murine MK, and platelets spread on fibrinogen.22 In our work, we report the effects of the loss of the C-terminus of b1-tubulin in patients with rare TUBB1 variants linked to the low platelet counts. Using induced pluripotent stem cell (iPSC) derived MK and CRISPR knockout (KO), we report a mech- anism of polymodification which distinctly patterns b1- tubulin in MK and platelets to spatially coordinate key motors. We describe the graded, cell specific expression of enzymes which mediate this, and finally report a novel gene, TTLL10, which is associated with excessive bleeding.
Methods
Study approval
Whole blood was obtained for each experiment from healthy
volunteers under the University of Birmingham’s ERN 11-0175 license ’The regulation of activation of platelets’. The Genotyping and Phenotyping of Platelets (GAPP) study was approved by the National Research Ethics Service Committee West Midlands – Edgbaston (REC reference 06/MRE07/36). Participants gave written informed consent in compliance with the Declaration of Helsinki. The GAPP study is included in the National Institute for Health Research Non-Malignant Hematology study portfolio (ID 9858), and registered at ISRCTN (http://www.isrctn.org) as ISRCTN77951167.
Whole exome sequencing
In order to identify the possible causative variants in these families we sequenced the whole exome of the affected individ- uals with the SureSelect human All Exon 50 Mb kit (Agilent Technologies) and sequenced on the HiSeq 2500 (Illumina) with 100 bp paired-end reads.
Stem cell culture and induced pluripotent stem cell megakayocytedifferentiation
Gibco human episomal iPSC line was purchased from Thermo Scientific and cultured on Geltrex basement membrane in StemFlex medium (Thermo Scientific). iPSC differentiation to mature, proplatelet forming MK was performed using a protocol based on work published by Feng et al.23 The IDT Alt-R®RNP system was used to target and knock out TUBB1. iPSC transfec- tion was performed using Lipofectamine Stem (Life Technologies) according to the manufacturer instructions.
TUBB1 homology modeling
Homology models of TUBB1 wild-type (WT) and variants
were made using SWISS MODEL software,24–27 using the solved TUBB3 heterodimer as a template PDB: 5IJ028. TUBB1 and TUBB3 share approximately 80% sequence identity, and the model cre- ated corresponds to residues 1-425 of TUBB1. (Note that the C- terminal tail of TUBB1 is not presented in this model as there is no known or homologous structure available for this highly divergent sequence. The C-terminal portion of this model is used only as a means to visualize the effect of genetic variations on the C-terminal tail of TUBB1).
Statistical analysis
Statistical analysis was performed using GraphPad PRISM 7. Specifics of each test are detailed in the figure legends of the rel- evant figures. P-values below 0.05 were considered significant.
Detailed Materials and Methods can be seen in the Online Supplementary Appendix.
Results
Identification and initial characterization of TUBB1 variants in patients with inherited thrombocytopenia and platelet dysfunction
Using whole exome sequencing and Congenica Clinical genetic variant interpretation software29 of patients recruited to the GAPP study, two C-terminal TUBB1 vari- ants were identified in unrelated families presenting with macrothrombocytopenia (Figure 1A; Online Supplementary Figure S1). Affected individuals in family A were found to be heterozygous for an arginine to tryptophan amino acid substitution (c.1075C>T, p.R359W) in TUBB1. Individuals in this family also carry a GFI1B variant (p.Cys168Phe). Variants in both genes have been linked to thrombocy- topenia, however only individuals A:1 and A:3 (carrying
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haematologica | 2022; 107(1)