A Plethora of Functions Condensed into Tiny Phospholipids: The Story of PI4P and PI(4,5)P2.

Phosphoinositides (PIs) are small, phosphorylated lipids that serve many functions in the cell. They regulate endo- and exocytosis, vesicular trafficking, actin reorganization, and cell mobility, and they act as signaling molecules. The most abundant PIs in the cell are phosphatidylinositol-4-monophosphate (PI4P) and phosphatidylinositol-4,5-bisphosphate [PI(4,5)P2]. PI4P is mostly localized at the Golgi apparatus where it regulates the anterograde trafficking from the Golgi apparatus to the plasma membrane (PM), but it also localizes at the PM. On the other hand, the main localization site of PI(4,5)P2 is the PM where it regulates the formation of endocytic vesicles. The levels of PIs are regulated by many kinases and phosphatases. Four main kinases phosphorylate the precursor molecule phosphatidylinositol into PI4P, divided into two classes (PI4KIIα, PI4KIIß, PI4KIIIα, and PI4KIIIß), and three main kinases phosphorylate PI4P to form PI(4,5)P2 (PI4P5KIα, PI4P5KIß, and PI4P5KIγ). In this review, we discuss the localization and function of the kinases that produce PI4P and PI(4,5)P2, as well as the localization and function of their product molecules with an overview of tools for the detection of these PIs.

The intracellular and plasma membrane pools of phosphatidylinositol-4-monophosphate control megakaryocyte maturation and proplatelet formation.

Background: Megakaryocytes (MKs) develop from hematopoietic stem cells after stimulation by the cytokine thrombopoietin. During megakaryopoiesis, MKs enlarge, undergo the process of endomitosis, and develop intracellular membranes (demarcation membrane system, DMS). During DMS formation, there is active transport of proteins, lipids, and membranes from the Golgi apparatus to the DMS. The most important phosphoinositide that controls anterograde transport from the Golgi apparatus to the plasma membrane (PM) is phosphatidylinositol-4-monophosphate (PI4P), whose levels are controlled by suppressor of actin mutations 1-like protein (Sac1) phosphatase at the Golgi and endoplasmic reticulum. Objectives: Here we investigated the role of Sac1 and PI4P in megakaryopoiesis. Methods: We analyzed Sac1 and PI4P localization in primary MKs derived from fetal liver or bone marrow and in the DAMI cell line by immunofluorescence. The intracellular and PM pools of PI4P in primary MKs were modulated by expression of Sac1 constructs from retroviral vector and inhibition of PI4 kinase IIIα, respectively. Results: We showed that in primary mouse MKs, PI4P is mostly found in the Golgi apparatus and the PM in immature MKs, while in mature MKs, it is found in the cell periphery and at the PM. The exogenous expression of wild-type but not C389S mutant (catalytically dead) Sac1 results in the perinuclear retention of the Golgi apparatus resembling immature MKs, with decreased ability to form proplatelets. The pharmacologic inhibition of PI4P production specifically at the PM also resulted in a significant decrease in MKs that form proplatelets. Conclusion: These results indicate that both intracellular and PM pools of PI4P mediate MK maturation and proplatelet formation.

Oculocerebrorenal syndrome of Lowe protein controls cytoskeletal reorganisation during human platelet spreading.

Lowe syndrome (LS) is a rare, X-linked disorder characterised by numerous symptoms affecting the brain, the eyes, and the kidneys. It is caused by mutations in the oculocerebrorenal syndrome of Lowe (OCRL) protein, a 5-phosphatase localised in different cellular compartments that dephosphorylates phosphatidylinositol-4,5-bisphosphate into phosphatidylinositol-4-monophosphate. Some patients with LS also have bleeding disorders, with normal to low platelet (PLT) count and impaired PLT function. However, the mechanism of PLT dysfunction in patients with LS is not completely understood. The main function of PLTs is to activate upon vessel wall injury and stop the bleeding by clot formation. PLT activation is accompanied by a shape change that is a result of massive cytoskeletal rearrangements. Here, we show that OCRL-inhibited human PLTs do not fully spread, form mostly filopodia, and accumulate actin nodules. These nodules co-localise with ARP2/3 subunit p34, vinculin, and sorting nexin 9. Furthermore, OCRL-inhibited PLTs have a retained microtubular coil with high levels of acetylated tubulin. Also, myosin light chain phosphorylation is decreased upon OCRL inhibition, without impaired degranulation or integrin activation. Taken together, these results suggest that OCRL contributes to cytoskeletal rearrangements during PLT activation that could explain mild bleeding problems in patients with LS.

Developmental differences of in vitro cultured murine bone marrow- and fetal liver-derived megakaryocytes.

Multiple lines of evidence support differences in the megakaryopoiesis during development. Murine in vitro models to study megakaryopoiesis employ cultured megakaryocytes MKs derived from adult bone marrow (BM) or fetal livers (FL) of mouse embryos. Mouse models allow to study the molecular basis for cellular changes utilizing conditional or knock-out models and permit further in vitro genetic or pharmacological manipulations. Despite being extensively used, MKs cultured from these two sources have not been systematically compared. In the present study, we compared BM- and FL-derived MKs, assessing their size, proplatelet production capacity, expression of common MK markers (αIIb, ß3, GPIb α, ß) and cytoskeletal proteins (filamin A, ß1-tubulin, actin), the subcellular appearance of α-granules (VWF), membranes (GPIbß) and cytoskeleton (F-actin) throughout in vitro development. We demonstrate that FL MKs although smaller in size, spontaneously produce more proplatelets than BM MKs and at earlier stages express more ß1-tubulin. In addition, early FL MKs show increased internal GPIbß staining and present higher GPIbß (early and late) and VWF (late stages) total fluorescence intensity (TFI)/cell size than BM MKs. BM MKs have up-regulated TPO signaling corresponding to their bigger size and ploidy, without changes in c-Mpl. Expressing endogenous ß1-tubulin or the presence of heparin improves BM MKs ability to produce proplatelets. These data suggest that FL MKs undergo cytoplasmic maturation earlier than BM MKs and that this, in addition to higher ß1-tubulin levels and GPIb, supported with an extensive F-actin network, could contribute to more efficient proplatelet formation in vitro.

Imaging of Intracellular and Plasma Membrane Pools of PI(4,5)P2 and PI4P in Human Platelets.

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.