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)P2 ) 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)P2 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.