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b1-tubulin role in platelet function
amount of polyglutamylated tubulin (Figure 4C). An increase in the co-localization between b1-tubulin and polyglutamylated residues suggests that while the total polyglutamylation of platelets remains unchanged through activation, the distribution of these residues is altered (Figure 4D). We confirmed the polyglutamylation (and loss of polyglycylation) in microthrombi generated by CRP stimulation (Figure 4E to G). This data shows that while polyglutamylation and polyglycylation are evident in platelet producing iPSC-MK, polyglycylation is com- pletely lost in mature platelets. This is the first evidence of a system whereby these competitive polymodifications are dramatically altered between the ’parent’ and terminal cell, suggesting markedly different roles for these residues in mediating the function and activity of b1-tubulin. Acetylation and tyrosination have been previously report- ed in platelets, however, their role in maintaining the mar- ginal band and/or driving morphological change on
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platelet activation remains unclear.13 In order to determine whether the polyglutamylation of the marginal band we observe thus far coincides with these PTM, we performed a time course of spreading on fibrinogen to determine whether there is an equivalent increase in either acetyla- tion or tyrosination of the marginal band. Interestingly, we found a significant decrease in acetylation and tyrosi- nation over time (between 0 and 10 minutes), while a notable polyglutamylation of the marginal band is evident from the earliest time point (10 minutes spreading on fib- rinogen) and does not decrease (Figure 4H and I).
Platelet and megakaryocyte polymodifications regulate motor protein localization to drive both proplatelet formation and platelet shape change on activation
Polyglutamylation has been reported as a means by which motor protein processivity is regulated, and like in neuronal cells, MK proplatelet formation is known to be
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Figure 4 Continued on following page.
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