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Abstract
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)P2] and phosphatidylinositol-4-monophosphate (PI4P). The localization and roles of both PI(4,5)P2 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)P2 and PI4P in resting and activated PLTs by antibody staining. Here, we show that the intracellular pools of PI(4,5)P2 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)P2 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)P2 and PI4P at PM in activated PLTs, which could also be modulated by OCRL and PI4KIIIα inhibitors. We conclude that PI(4,5)P2 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.
Highlights
Platelets (PLTs) are the smallest blood cells (2–3 μm in diameter) that form from the biggest cells in the bone marrow, megakaryocytes (MKs)
Antibodies were obtained from the following resources: monoclonal mouse antiPI(4,5)P2 (Z-P045) and anti-PI4P (Z-P004), both IgM, were from Echelon Biosciences, polyclonal rabbit CD42b/anti-glycoprotein Ibα (GPIbα) was from Novus Biological (NBP2-89128), phalloidin (A12379) conjugated with Alexa Fluor (AF)-488 was from Invitrogen, monoclonal mouse IgM (MAB1326, R&D Systems„ Abigdon, UK) was a kind gift from Dr Jelena Ban, (Laboratory of Molecular Neurobiology, Department of Biotechnology, University of Rijeka), polyclonal rabbit α-tubulin (SAB4500087, Sigma Aldrich, Taufkirchen, Germany) was a kind gift from Dr Iva Tolic (Laboratory of Cell Biophysics, Ruder Boškovic Institute), and DAPI (D9542) was from Sigma Aldrich
We wanted to determine the localization of PI(4,5)P2 and PI4P in different cell lines to confirm previously established protocols for the subcellular distribution of these lipids [15]
Summary
Platelets (PLTs) are the smallest blood cells (2–3 μm in diameter) that form from the biggest cells in the bone marrow, megakaryocytes (MKs). PLTs detect vessel wall injury, change shape, adhere to the site of injury, and aggregate to form a clot. In addition to this primary function in hemostasis and thrombosis, PLTs are a part of innate immunity and have a role in inflammatory reaction, inflammatory diseases, and the regulation of angiogenesis [1,2,3]. In their resting state, they have a discoid shape. The surface of the PLTs flattens, lamellae are formed, granules and organelles are moved to the center of the cell, and the cells form the so-called “fried egg” appearance [4]
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