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Abstract：
Phosphatidylinositol phosphates (PIPs) are involved in many cellular events as important secondary messengers. In Entamoeba histolytica, a human intestinal protozoan parasite, virulence-associated mechanisms such as cell motility, vesicular traffic, trogo- and phagocytosis are regulated by PIPs. It has been well established that PI3P, PI4P, and PI(3,4,5)P3 play specific roles during amoebic trogo- and phagocytosis. In the present study, we demonstrated the nuclear localization of PI4P in E. histolytica trophozoites in steady state with immunofluorescence imaging and immunoelectron microscopy, using anti-PI4P antibodies and PI4P biosensors [substrate of the Icm/ Dot type IV secretion system (SidM)]. We further showed that the nuclear PI4P decreased after a co-culture with human erythrocytes or Chinese hamster ovary (CHO) cells. However, concomitant changes in the localization and the amount of PI(4,5)P2, which is the expected major metabolized (phosphorylated) product of PI4P, were not observed. This phenomenon was specifically caused by whole or ghost erythrocytes and CHO cells, but not artificial beads. The amount of PIP2 and PIP, biochemically estimated by [32P]-phosphate metabolic labeling and thin layer chromatography, was decreased upon erythrocyte adherence. Altogether, our data indicate for the first time in eukaryotes that erythrocyte attachment leads to the metabolism of nuclear PIPs, and metabolites other than PI(4,5)P2 may be involved in the regulation of downstream cellular events such as cytoskeleton rearrangement or transcriptional regulation.
摘要：
磷脂酰肌醇磷酸酯（PIPs）作为重要的第二信使参与许多细胞事件。在溶组织内阿米巴，人类肠道原生动物寄生虫，毒力相关机制，如细胞运动性，水泡交通,吞噬作用和吞噬作用受PIP调节。众所周知，PI3P，PI4P,PI(3,4,5)P3在阿米巴吞噬作用和吞噬作用中起特定作用。在本研究中,我们通过免疫荧光成像和免疫电子显微镜证明了PI4P在稳定状态下在溶组织大肠杆菌滋养体中的核定位，使用抗PI4P抗体和PI4P生物传感器[Icm/DotIV型分泌系统(SidM)的底物]。我们进一步表明，与人红细胞或中国仓鼠卵巢（CHO）细胞共培养后，核PI4P降低。然而,伴随的位置变化和PI（4,5）P2的量，这是预期的主要代谢（磷酸化）产物PI4P，没有被观察到。这种现象是由完整或幽灵红细胞和CHO细胞引起的，但不是人造珠子。PIP2和PIP的数量，通过[32P]-磷酸盐代谢标记和薄层色谱法进行生化评估，红细胞粘附性降低。总之,我们的数据表明，在真核生物中，红细胞附着导致核PIP的代谢，和除PI(4,5)P2以外的代谢物可能参与下游细胞事件如细胞骨架重排或转录调节的调节。
