Department of Biotechnology (I.B., R.K., A.J.B), University of Rijeka, Croatia.
Faculty of Medicine (H.M.L), University of Rijeka, Croatia.
Arterioscler Thromb Vasc Biol. 2022 Jan;42(1):e10-e26. doi: 10.1161/ATVBAHA.121.316552. Epub 2021 Nov 4.
Maturation of megakaryocytes culminates with extensive membrane rearrangements necessary for proplatelet formation. Mechanisms required for proplatelet extension and origin of membranes are still poorly understood. GTPase Rab5 (Ras-related protein in brain 5) regulates endocytic uptake and homotypic fusion of early endosomes and regulates phosphatidylinositol 3-monophosphate production important for binding of effector proteins during early-to-late endosomal/lysosomal maturation. Approach and Results: To investigate the role of Rab5 in megakaryocytes, we expressed GFP (green fluorescent protein)-coupled Rab5 wild type and its point mutants Q79L (active) and N133L (inactive) in primary murine fetal liver-derived megakaryocytes. Active Rab5 Q79L induced the formation of enlarged early endosomes, while inactive Rab5 N133L caused endosomal fragmentation. Consistently, an increased amount of transferrin internalization in Rab5 Q79L was impaired in Rab5 N133L expressing megakaryocytes, when compared with GFP or Rab5 wild type. Moreover, trafficking of GPIbβ (glycoprotein Ib subunit beta), a subunit of major megakaryocytes receptor and membrane marker, was found to be mediated by Rab5 activity. While GPIbβ was mostly present along the plasma membrane, and within cytoplasmic vesicles in Rab5 wild type megakaryocytes, it accumulated in the majority of Rab5 Q79L enlarged endosomes. Conversely, Rab5 N133L caused mostly GPIbβ plasma membrane retention. Furthermore, Rab5 Q79L expression increased incorporation of the membrane dye (PKH26), indicating higher membrane content. Finally, while Rab5 Q79L increased proplatelet production, inactive Rab5 N133L strongly inhibited it and was coupled with a decrease in late endosomes/lysosomes. Localization of GPIbβ in enlarged endosomes was phosphatidylinositol 3-monophosphate dependent.
Taken together, our results demonstrate that Rab5-dependent endocytosis plays an important role in megakaryocytes receptor trafficking, membrane formation, and thrombopoiesis.
巨核细胞的成熟伴随着广泛的膜重排，这对于前血小板的形成是必要的。前血小板延伸的机制和膜的起源仍然知之甚少。GTPase Rab5（脑 5 相关 Ras 蛋白）调节早期内体的内吞作用和同源融合，并调节磷脂酰肌醇 3-单磷酸的产生，该磷酸对于早期到晚期内体/溶酶体成熟过程中效应蛋白的结合很重要。方法和结果：为了研究 Rab5 在巨核细胞中的作用，我们在原代小鼠胎肝来源的巨核细胞中表达 GFP（绿色荧光蛋白）偶联的 Rab5 野生型及其点突变体 Q79L（活性）和 N133L（非活性）。活性 Rab5 Q79L 诱导形成扩大的早期内体，而无活性 Rab5 N133L 导致内体碎片化。一致地，与 GFP 或 Rab5 野生型相比，在表达 Rab5 N133L 的巨核细胞中，转铁蛋白内化量增加，但 Rab5 Q79L 表达的巨核细胞中却受到损害。此外，GPIbβ（糖蛋白 Ib 亚单位β）的转运，GPIbβ 是巨核细胞主要受体和膜标记物的一个亚单位，被发现由 Rab5 活性介导。虽然 GPIbβ 主要存在于质膜上，并存在于 Rab5 野生型巨核细胞中的细胞质小泡中，但它在大多数 Rab5 Q79L 扩大的内体中积累。相反，Rab5 N133L 导致 GPIbβ 主要保留在质膜上。此外，Rab5 Q79L 的表达增加了膜染料（PKH26）的掺入，表明膜含量更高。最后，虽然 Rab5 Q79L 增加了前血小板的产生，但失活的 Rab5 N133L 强烈抑制了它，并与晚期内体/溶酶体的减少有关。GPIbβ 在扩大的内体中的定位依赖于磷脂酰肌醇 3-单磷酸。结论：总之，我们的结果表明，Rab5 依赖性内吞作用在巨核细胞受体转运、膜形成和血小板生成中发挥重要作用。
