An essential role for α4A-tubulin in platelet biogenesis

Abstract

During platelet biogenesis, microtubules (MTs) are arranged into submembranous structures (the marginal band) that encircle the cell in a single plane. This unique MT array has no equivalent in any other mammalian cell, and the mechanisms responsible for this particular mode of assembly are not fully understood. One possibility is that platelet MTs are composed of a particular set of tubulin isotypes that carry specific posttranslational modifications. Although β1-tubulin is known to be essential, no equivalent roles of α-tubulin isotypes in platelet formation or function have so far been reported. Here, we identify α4A-tubulin as a predominant α-tubulin isotype in platelets. Similar to β1-tubulin, α4A-tubulin expression is up-regulated during the late stages of megakaryocyte differentiation. Missense mutations in the α4A-tubulin gene cause macrothrombocytopenia in mice and humans. Defects in α4A-tubulin lead to changes in tubulin tyrosination status of the platelet tubulin pool. Ultrastructural defects include reduced numbers and misarranged MT coils in the platelet marginal band. We further observed defects in megakaryocyte maturation and proplatelet formation in Tuba4a-mutant mice. We have, thus, discovered an α-tubulin isotype with specific and essential roles in platelet biogenesis.

Figure 1.. Expression of α-tubulin isotypes in platelets and during megakaryopoiesis.

(A) 2D-gel electrophoresis of tubulins purified from human platelets and list of identified α-isotypes. The isotypes were identified by LC-MS/MS analysis of the color-marked spots (blue, red, and double labelling correspond to spots containing α-, β-, and αβ isotypes, respectively). (B) Evolution of α1-, α4A-, α8-, and β1-tubulin transcripts at different stages of MK differentiation. RT-PCR amplification of RNA isolated from MKs differentiated from human CD34⁺ progenitor cells at days 0 to 12 of culture. Bands were quantified on gels and intensity normalized to 18S (mean ± SEM, n = 3). (C) SRM-MS quantification of the α4A- and α8-tubulin isotypes in tubulin purified from human platelets. The content normalized to the total α-tubulin content was calculated as described in the methods from six separate tubulin preparations with analyses performed in triplicate. (D) Western blot analysis of α4A-tubulin levels in tubulin purified from platelets, the brain, and HeLa cells. (Upper panel) Quantification of α4A-tubulin levels in HeLa and brain tubulin relative to platelet tubulin after normalization to the total α-tubulin content. (Lower panel) Representative blot where equivalent amounts of purified tubulin (300 ng) were separated on a 10% gel and probed using pAb7621 polyclonal Ab specific for α4A-tubulin and DM1a mouse mAb recognizing all the α-tubulin isotypes.

Figure 2.. An ENU-induced mutation of Tuba4a causes macrothrombocytopenia and abnormal marginal band formation.

(A) Decreased platelet counts in Plt68 mice. The number of circulating platelets in wild-type and Plt68 mice is represented for each individual mouse (1,199 ± 183 versus 943 ± 207 × 10³ platelets/μl, respectively; mean ± SEM n = 21 wild type and n = 33 Plt68; ***P = 0.0026; t test). (B) Increased platelet volume in Plt68 mice. The mean platelet volume in wild-type and Plt68 mice is represented for each individual mouse (4.9 ± 0.34 versus 7.0 ± 0.17 fL respectively; mean ± SEM n = 21 and n = 33; ***P < 0.0001; t test). (C) Schematic representation of the V260E mutation: A mutation was identified in the gene encoding α4A-tubulin (Tuba4a) in the Plt68 strain resulting in a Val-to-Glu transition at position 260 of the protein (P68366). (D) Representative scanning electron microscopy images of wild-type and Tuba4a^V260E/V260E platelets. (E) Representative transmission electron microscopy images of wild-type and Tuba4a^V260E/V260E platelet suspensions (upper panels) and close-up views of individual platelets in cross section (lower panels).

Figure S1.. Ultrastructure, platelet counts and mean platelet volume in Tubb1 KO mice.

(A). Representative transmission electron microscopy images of WT and β1-tubulin deficient platelet suspensions. (B-C) Platelet counts and mean platelet volume of WT and Tubb1 KO mice of 18 days or 8 weeks of age.

Figure 3.. Naturally occurring mutations of TUBA4A in an individual with mild macrothrombocytopenia.

(A) A double substitution was identified in TUBA4A in the patient, resulting in p.Val181Met and p.Glu183Gln changes in α4A-tubulin. (B) Representative scanning electron microscopy images of control and patient’s platelets. (C) Representative transmission electron microscopy images of control and patient’s platelet suspensions (upper panels) and close-up views of individual platelets in cross section (lower panels).

Figure 4.. Effect of Tuba4a mutations on α4A-tubulin expression and on tyrosination of the α-tubulin pool.

(A) α4A-tubulin is not detected in platelets from Tuba4a^V260E/V260E mice and the α-tubulin pool is hypertyrosinated. Platelet lysates from wild-type and Tuba4a^V260E/V260E mice were separated by SDS-PAGE and probed with pAb7621 against α4A-tubulin, DM1a recognizing all the α-tubulin isotypes, pAb5274 against β1-tubulin, 1.A2 against tyrosinated α-tubulin, or 1D5 against detyrosinated α-tubulin. Blots were also probed for GAPDH as a loading control. Representative of four separate experiments. (B) α4A-tubulin is decreased in platelets from the patient and the α-tubulin pool is hypertyrosinated. Platelet lysates from a control individual and the patient were processed as in (A). (C, D) Decreased α4A-tubulin labelling in the marginal band of platelets from Tuba4a^V260E/V260E mice (C) and the patient (D). Platelets were fixed in PFA-Triton X-100, captured on poly-L lysine–coated slides, incubated with pAb7620 or pAb7621 against α4A-tubulin and revealed with GAR-Alexa 488.

Figure S2.. qRT-PCR analysis of tubulin transcripts in wild type and Tuba4a^V260E/V260E megakaryocytes.

(A) qRT-PCR analysis of transcripts for Tuba and Tubb transcripts in WT (white bars) and Tuba4aV260E (black bars) megakaryocytes cultured from bone marrow Lin- cells. Mean values from three separate cultures. Values were normalized using Tbp (TATA Binding Protein) as a reference using the ΔΔct method. (B) qRT-PCR analysis of Tuba4a transcripts in WT Lin- cells (D0) and differentiated megakaryocytes (D3). Mean values from three separate cultures. Values were normalized using Tbp (TATA Binding Protein) as a reference using the ΔΔct method.

Figure S3.. 3-D modelling of the V260E mutation in α4A tubulin.

(A) Electrostatic analysis. (left) TUBA4A wild type (right) V260E mutation. (B) Molecular modelling. (right) V260E mutant interactions. The red lines show the pseudo bonds created due to steric clashes of E260 residues with the neighboring residues around the mutated site.

Figure S4.

Serial dilutions of platelet lysates from WT and Tuba4a^V260E/V260E mice were separated by SDS-PAGE, probed with the antibody against acetylated α-tubulin (6-11B-1) (left panel) or DM1a recognizing all the α-tubulin isotypes (right panel) and revealed with GAM-HRP.

Figure 5.. Abnormal ultrastructure of stage III bone marrow MKs in Tuba4a^V260E/V260E mice.

(A) Distribution of bone marrow MKs according to their stage of differentiation (I–III). MKs were staged in wild-type and Tuba4a^V260E/V260E mice according to morphological criteria (described in the Materials and Methods section) from transmission electron microscopy examination of bone marrow sections. Data expressed as the percentage of each stage correspond to 80 and 99 cells analyzed, respectively. Stage III* corresponds to MKs with an abnormal ultrastructure as represented in panel B. (**P < 0.001; two-way ANOVA with Bonferroni posttest). (B) Representative transmission electron microscopy images of stage III MKs. A proportion of stage III MKs exhibited abnormal ultrastructural features in Tuba4a^V260E/V260E mice as compared with wild-type mice, characterized by a smaller size, a more condensed/compact demarcation membrane system, and lack of peripheral zone.

Figure 6.. Impaired proplatelet formation in Tuba4a^V260E/V260E mice.

(A) Proplatelet extensions in bone marrow explants. (left) Representative DIC microscopy images of MKs observed at the edge of a bone marrow slice showing well-developed proplatelets in the wild-type and lack of pseudopodial extensions in Tuba4a^V260E/V260E mice. (right) The graph represents the percentage of MKs displaying proplatelets (wild type: 36.8 ± 8.1% and Tuba4a^V260E/V260E: 2.1 ± 0.7%; N = 494 and 495 MKs, respectively; *P = 0.0112, t test). (B) Proplatelet extensions of MKs cultured from Lin⁻ progenitors. (left) Representative DIC microscopy images of MKs cultured for 4 d from Lin⁻progenitors showing MKs with abnormal shapes, displaying thick protrusions, in Tuba4a^V260E/V260E mice instead of well-developed proplatelets as seen in the WT. (right) The graph represents the percentage of MKs displaying proplatelets and thick protrusions (WT: 44.3 ± 11.5% and 10.1 ± 1.1%, respectively; Tuba4a^V260E/V260E: 29.3 ± 3.1% and 35.9 ± 2.1%, respectively; values from 213 WT and 162 Tuba4a^V260E/V260E MKs). (**P < 0.001; two-way ANOVA with Bonferroni posttest).