engleski

The role of phosphatidylinositol 3-monophosphate (PI3P) in megakaryocyte maturation and platelet production

The maturation of megakaryocytes (MKs) implies the polyploidization of the nucleus and the formation of a demarcation membrane system (DMS), an extensive internal membrane reservoir necessary for platelet production. While the initiation of the DMS starts with plasma membrane invagination and continues in the perinuclear region by the insertion of Golgi-derived vesicles, the membrane source(s) and mechanism for further DMS expansion, cell growth, and proplatelet formation remain elusive. In the present study, we investigated the role of endolysosomal system during MKs maturation and platelet production. Vps34 kinase and its product lipid phosphatidylinositol 3-monophosphate (PI3P) are key components of early and late endosomes where they regulate vesicular trafficking. We demonstrate that, during maturation, Vps34-derived PI3P translocates from the perinuclear early endosomes (EE) in immature, to peripheral late endosomes/lysosomes (LE/Lys) in mature MKs. Moreover, PI3P was found in close contact with PI(4, 5)P2 (plasma membrane/DMS) and LE/Lys in mature MKs. Sequestration of PI3P by expression of 2xFYVE domain, specific pharmacological inhibition of Vps34-mediated PI3P production, or depletion of PI3P by expression of PI3P-phosphatase (MTM1) decreased proplatelet formation. At the same time, inhibition of PI3P production led to the intracellular accumulation of LE/Lys markers preventing their surface expression, suggesting an essential role for PI3P in LE/Lys translocation toward the plasma membrane. Moreover, inhibition of Vps34 at earlier stages of MK maturation caused aberrant DMS development, decreased the size of MKs, and the expression of GPIb, a DMS marker. In line with the above was targeting of downstream events in endosome maturation: overexpression of the dominant-negative late endosomal GTPase Rab7 T22N or pharmacological inhibition of PI3P conversion to PI(3, 5)P2 led to the enlarged LE/Lys, reduced surface levels of LE/Lys markers, and decreased proplatelet formation. In parallel, we investigated the role of early endosomal GTPase Rab5 which regulates endocytic uptake and represents the starting point for endosome maturation. In addition, Rab5 mediates the recruitment of Vps34 on the membrane of EEs, which is a key step during early-to-late endosome maturation. Rab5 mutants expressed from retroviruses showed the opposing effects on EE morphology and the amount of endocytosis in MKs. Active Rab5 Q79L induced the formation of large EEs that accumulated GPIb, increased transferrin internalization, and at the same time increased proplatelet production. In contrast, inactive Rab5 N133L caused EE fragmentation, GPIb retention at the plasma membrane, impaired transferrin endocytosis, and strongly blocked proplatelet formation. Furthermore, active Rab5 Q79L increased membrane labeling by lipophilic dye PKH26 suggesting that the increased levels of the endocytosis might contribute to the internal membrane content in MKs. Together, these results suggest that PI3P-positive LE/Lys contribute to the membrane growth and proplatelet formation by their translocation to the cell periphery and fusion with the plasma membrane, while early endosomal Rab5 regulates GPIb trafficking and represents the limiting step during endocytosis in MKs, the amount of which we believe is a major determinant of membrane growth and proplatelet production.

megakaryocytes, PI3P, Rab GTPases, lysosomes, nucleolus

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