Kalasova Ilona, Fáberová Veronika, Kalendová Alžběta, Yildirim Sukriye, Uličná Lívia, Venit Tomáš, Hozák Pavel
Department of Biology of the Cell Nucleus, Institute of Molecular Genetics of the Academy of Sciences of the Czech Republic, v.v.i., Vídeňská 1083, 142 20, Prague, Czech Republic.
Faculty of Science, Charles University in Prague, Albertov 6, 128 43, Prague, Czech Republic.
Histochem Cell Biol. 2016 Apr;145(4):485-96. doi: 10.1007/s00418-016-1409-8. Epub 2016 Feb 4.
Phosphoinositides (PIs) are glycerol-based phospholipids containing hydrophilic inositol ring. The inositol ring is mono-, bis-, or tris-phosphorylated yielding seven PIs members. Ample evidence shows that PIs localize both to the cytoplasm and to the nucleus. However, tools for direct visualization of nuclear PIs are limited and many studies thus employ indirect approaches, such as staining of their metabolic enzymes. Since localization and mobility of PIs differ from their metabolic enzymes, these approaches may result in incomplete data. In this paper, we tested commercially available PIs antibodies by light microscopy on fixed cells, tested their specificity using protein-lipid overlay assay and blocking assay, and compared their staining patterns. Additionally, we prepared recombinant PIs-binding domains and tested them on both fixed and live cells by light microscopy. The results provide a useful overview of usability of the tools tested and stress that the selection of adequate tools is critical. Knowing the localization of individual PIs in various functional compartments should enable us to better understand the roles of PIs in the cell nucleus.
磷酸肌醇（PIs）是一类基于甘油的磷脂，含有亲水性的肌醇环。肌醇环可发生单磷酸化、双磷酸化或三磷酸化，产生七种磷酸肌醇成员。大量证据表明，磷酸肌醇既存在于细胞质中，也存在于细胞核中。然而，直接可视化细胞核内磷酸肌醇的工具有限，因此许多研究采用间接方法，如对其代谢酶进行染色。由于磷酸肌醇的定位和流动性与其代谢酶不同，这些方法可能会导致数据不完整。在本文中，我们通过光学显微镜对固定细胞上的市售磷酸肌醇抗体进行了测试，使用蛋白质-脂质覆盖分析和阻断分析测试了它们的特异性，并比较了它们的染色模式。此外，我们制备了重组磷酸肌醇结合结构域，并通过光学显微镜在固定细胞和活细胞上对其进行了测试。结果提供了对所测试工具可用性的有用概述，并强调选择合适的工具至关重要。了解单个磷酸肌醇在各种功能区室中的定位，应该能使我们更好地理解磷酸肌醇在细胞核中的作用。
