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b1-tubulin role in platelet function
Megakaryocyte and platelet polymodification
is regulated through the expression of both modifying and reversing enzymes
We hypothesized that the expression of cell specific sub- sets of effecting (tubulin tyrosine ligase like [TTLL]) and reversing (cytosolic carboxypeptidase [CCP]) enzymes are required to achieve the distinctive polymodification we observe in MK and platelets. We designed a quantitative real-time polymerase chain reaction (qRT-PCR) panel to interrogate the expression of the 13 known mammalian TTLL and six CCP. We generated RNA from iPSC-MK at different stages of maturation (Figures 6A; Online Supplementary Figure S4A). Day 1 (d1) cells are representa- tive of a pool of hematopoeitic stem cells (HSC) and MK progenitors, while day 5 (d5) cells are comprised of 60% CD41/42b+ cells (Figures 6A; Online Supplementary Figure
AB
S4E). Finally, d5 cells treated with heparin to induce pro- platelet formation (d5+Hep) were used to interrogate whether there is any specific upregulation of TTLL and/or CCP on proplatelet formation (Figures 6A; Online Supplementary Figure S4D).
GAPDH housekeeping controls for each of the three sam- ples (d1, d5, d5+Hep) show equivalent amplification of the housekeeping control, while results for TTLL family pro- teins show a number of enzymes expressed at different lev- els across the maturation of these cells (Figure 6B and C). Candidate TTLL observed in the initial endpoint PCR were taken forward for quantification across replicates generated from multiple differentiations (Figure 6C and D). We find a significantly increased expression of TTLL1, TTLL2, TTLL4, and TTLL10 on proplatelet formation in cells treat- ed with heparin (**P=0.0081, *P=0.0105, *P=0.0260, ***P=
CE
DF
Figure 5. Continued on following page.
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