Vps34 derived phosphatidylinositol 3-monophosphate modulates megakaryocyte maturation and proplatelet production through late endosomes/lysosomes

Background Development of platelet precursor cells, megakaryocytes (MKs), implies an increase in their size; formation of the elaborate demarcation membrane system (DMS); and extension of branched cytoplasmic structures, proplatelets, that will release platelets. The membrane source(s) for MK expansion and proplatelet formation have remained elusive. Objective We hypothesized that traffic of membranes regulated by phosphatidylinositol 3-monophosphate (PI3P) contributes to MK maturation and proplatelet formation. Results In immature MKs, PI3P produced by the lipid kinase Vps34 is confined to perinuclear early endosomes (EE), while in mature MKs PI3P shifts to late endosomes and lysosomes (LE/Lys). PI3P partially colocalized with the plasma membrane marker phosphatidylinositol 4,5-bisphosphate (PI(4,5)P-2) and with LE/Lys in mature MKs, suggests that PI3P-containing LE/Lys membranes contribute to MK expansion and proplatelet formation. Consistently, we found that sequestration of PI3P, specific pharmacological inhibition of Vps34-mediated PI3P production, or depletion of PI3P by PI3-phosphatase (MTM1)-mediated hydrolysis potently blocked proplatelet formation. Moreover, Vps34 inhibition led to the intracellular accumulation of enlarged LE/Lys, and decreased expression of surface LE/Lys markers. Inhibiting Vps34 at earlier MK stages caused aberrant DMS development. Finally, inhibition of LE/Lys membrane fusion by a dominant negative mutant of the small GTPase Rab7 or pharmacological inhibition of PI3P conversion into PI(3,5)P(2)led to enlarged LE/Lys, reduced surface levels of LE/Lys markers, and decreased proplatelet formation. Conclusion Our results suggest that PI3P-positive LE/Lys contribute to the membrane growth and proplatelet formation in MKs by their translocation to the cell periphery and fusion with the plasma membrane.