Bura Ana, Jurak Begonja Antonija
Department of Biotechnology, University of Rijeka, 51000 Rijeka, Croatia.
Life (Basel). 2021 Dec 1;11(12):1331. doi: 10.3390/life11121331.
Phosphoinositides (PIs) are phosphorylated membrane lipids that have a plethora of roles in the cell, including vesicle trafficking, signaling, and actin reorganization. The most abundant PIs in the cell are phosphatidylinositol-4,5-bisphosphate [PI(4,5)P] and phosphatidylinositol-4-monophosphate (PI4P). The localization and roles of both PI(4,5)P and PI4P are well established, is the broadly accepted methodological approach for their immunocytochemical visualization in different cell compartments in several cell lines. However, not much is known about these PIs in platelets (PLTs), the smallest blood cells that detect vessel wall injury, activate, and stop the bleeding. Therefore, we sought to investigate the localization of PI(4,5)P and PI4P in resting and activated PLTs by antibody staining. Here, we show that the intracellular pools of PI(4,5)P and PI4P can be detected by the established staining protocol, and these pools can be modulated by inhibitors of OCRL phosphatase and PI4KIIIα kinase. However, although resting PLTs readily stain for the plasma membrane (PM) pools of PI(4,5)P and PI4P, just a few activated cells were stained with the established protocol. We show that optimized protocol allows for the visualization of PI(4,5)P and PI4P at PM in activated PLTs, which could also be modulated by OCRL and PI4KIIIα inhibitors. We conclude that PI(4,5)P and PI4P are more sensitive to lipid extraction by permeabilizing agents in activated than in resting human PLTs, which suggests their different roles during PLT activation.
磷酸肌醇（PIs）是磷酸化的膜脂，在细胞中具有多种作用，包括囊泡运输、信号传导和肌动蛋白重组。细胞中最丰富的PIs是磷脂酰肌醇-4,5-二磷酸[PI(4,5)P]和磷脂酰肌醇-4-单磷酸（PI4P）。PI(4,5)P和PI4P的定位和作用已得到充分证实，这是在几种细胞系的不同细胞区室中对它们进行免疫细胞化学可视化的广泛接受的方法。然而，对于血小板（PLTs）中这些PIs的了解并不多，血小板是检测血管壁损伤、激活并止血的最小血细胞。因此，我们试图通过抗体染色研究PI(4,5)P和PI4P在静息和活化血小板中的定位。在此，我们表明可以通过既定的染色方案检测到PI(4,5)P和PI4P的细胞内池，并且这些池可以被OCRL磷酸酶和PI4KIIIα激酶的抑制剂调节。然而，尽管静息血小板很容易对PI(4,5)P和PI4P的质膜（PM）池进行染色，但按照既定方案只有少数活化细胞被染色。我们表明优化后的方案可以使活化血小板中PM处的PI(4,5)P和PI4P可视化，它们也可以被OCRL和PI4KIIIα抑制剂调节。我们得出结论，在活化的人血小板中，PI(4,5)P和PI4P比静息血小板中的对通透剂介导的脂质提取更敏感，这表明它们在血小板活化过程中具有不同的作用。
