Phosphoinositides and engulfment

Macropinocytosis is a type of large-scale endocytosis that is triggered by the interaction of receptor proteins and ligands, such as growth factors, cytokines, chemokines, and lipopolysaccharide (LPS). Macropinocytosis ingests the extracellular fluid solutes and conveys them into the lysosome in the context of cell growth and differentiation. Aside from its physiological functions, macropinocytosis has been observed in viral infections. While the infectious mechanism of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is still unknown, recent studies suggest the involvement of macropinocytosis in its cell entry. In this review, we discuss the roles of endocytosis in SARS-CoV/SARS-CoV-2 cell entries and propose a hypothetical role of macropinocytosis in SARS-CoV-2 cell entry.     

Phosphoinositides and phagocytosis

Phosphoinositide 3 kinases (PI3Ks)*Abbreviation used in this paper: PI3K, phosphoinositide 3 kinase. are known as regulators of phagocytosis. Recent results demonstrate that class I and III PI3Ks act consecutively in phagosome formation and maturation, and that their respective products, phosphatidylinositol 3,4,5-trisphosphate (PI[3,4,5]P(3)) and phosphatidylinositol 3-phosphate (PI[3]P), accumulate transiently at different stages. Phagosomes containing Mycobacterium tuberculosis do not acquire the PI(3)P-binding protein EEA1, which is required for phagosome maturation. This suggests a possible mechanism of how this microorganism evades degradation in phagolysosomes.     

Phosphoinositides and vesicular membrane traffic

Phosphoinositide lipids were initially discovered as precursors for specific second messengers involved in signal transduction, but have now taken the center stage in controlling many essential processes at virtually every cellular membrane. In particular, phosphoinositides play a critical role in regulating membrane dynamics and vesicular transport. The unique distribution of certain phosphoinositides at specific intracellular membranes makes these molecules uniquely suited to direct organelle-specific trafficking reactions. In this regulatory role, phosphoinositides cooperate specifically with small GTPases from the Arf and Rab families. This review will summarize recent progress in the study of phosphoinositides in membrane trafficking and organellar organization and highlight the particular relevance of these signaling pathways in disease. This article is part of a Special Issue entitled Lipids and Vesicular Transport.     

Phosphoinositides and the golgi complex

Phosphoinositides act as precursors of second messengers and membrane ligands for protein modules. Specific lipid kinases and phosphatases are located and differentially regulated in cell organelles, generating a non-uniform distribution of phosphoinositides. Although it is not clear whether and how the phosphoinositide pools are integrated, it is certain that they locally control fundamental processes, including membrane trafficking. This applies to the Golgi complex, where a direct, central role of the phosphatidylinositol 4,5-bisphosphate precursor phosphatidylinositol 4-phosphate has recently been reported.     

Phosphoinositides Control Epithelial Development

Epithelial organs consist on layers of cubical cells that separate different compartments. They form a physical barrier that allows the regulated transports of certain molecules ions. To perform this and other functions epithelial cells require to be highly polarized. The molecular mechanisms that integrate cellular polarity with epithelial architecture poorly understood. Using a three-dimensional model of epithelial morphogenesis, have recently reported a molecular mechanism for the formation of the apical membrane and the central lumen1. This molecular pathway is initiated by the membrane segregation of phosphoinositides at the apical domain. Apically localized phosphatidylinositol(bisphosphate [PtdIns(4,5)P2] recruits the scaffolding protein Annexin2 and the GTPase Cdc42 to generate the apical plasma membrane domain and the central lumen.     

Phosphoinositides and the endocytic pathway

Phosphorylation of the phosphatidylinositol headgroup generates seven varieties of phosphoinositide of which PtdIns(4,5)P₂, PtdIns3P and PtdIns (3,5)P₂ have established roles on the endocytic pathway. In this review, we discuss the enzymes responsible for generation and turnover of these lipids, which are keys to determining compartmental identity and the flux of material through the endocytic system. The enzymatic generation of lipids serves as an amplification mechanism through which a wide variety of effector molecules can be recruited.     

Phosphoinositides in cell architecture

Inositol phospholipids have been implicated in almost all aspects of cellular physiology including spatiotemporal regulation of cellular signaling, acquisition of cellular polarity, specification of membrane identity, cytoskeletal dynamics, and regulation of cellular adhesion, motility, and cytokinesis. In this review, we examine the critical role phosphoinositides play in these processes to execute the establishment and maintenance of cellular architecture. Epithelial tissues perform essential barrier and transport functions in almost all major organs. Key to their development and function is the establishment of epithelial cell polarity. We place a special emphasis on highlighting recent studies demonstrating phosphoinositide regulation of epithelial cell polarity and how individual cells use phosphoinositides to further organize into epithelial tissues.     

Phosphoinositides in phagolysosome and autophagosome biogenesis

Interconversions of phosphoinositides play a pivotal role during phagocytosis and at the subsequent stages of phagosomal maturation into the phagolysosome. Several model systems have been used to study the role of phosphoinositides in phagosomal membrane remodelling. These include phagosomes formed by inanimate objects such as latex beads, or pathogenic bacteria, e.g. Mycobacterium tuberculosis. The latter category provides naturally occurring tools to dissect membrane trafficking processes governing phagolysosome biogenesis. M. tuberculosis persists in infected macrophages by blocking Rab conversion and affecting Rab effectors. One of the major Rab effectors involved in this process is the type III phosphatidylinositol 3-kinase hVPS34. The lipid kinase hVPS34 and its enzymatic product PtdIns3P are critical for the default pathway of phagosomal maturation into phagolysosomes. Mycobacteria block PtdIns3P production and thus arrest phagosomal maturation. PtdIns3P is also critical for the process of autophagy, recently recognized as an effector of innate immunity defenses. Induction of autophagy by pharmacological, physiological, or immunological means, overcomes mycobacterial phagosome maturation block in a PtdIns3P generation dependent manner and eliminates intracellular M. tuberculosis. PtdIns3P and PtdIns3P-dependent processes represent an important cellular nexus where fundamental trafficking processes, disease causing host-pathogen interactions, and innate and adaptive immunity defense mechanisms meet.     

Phosphoinositides alter lipid bilayer properties

Phosphatidylinositol-4,5-bisphosphate (PIP₂), which constitutes ∼1% of the plasma membrane phospholipid, plays a key role in membrane-delimited signaling. PIP₂ regulates structurally and functionally diverse membrane proteins, including voltage- and ligand-gated ion channels, inwardly rectifying ion channels, transporters, and receptors. In some cases, the regulation is known to involve specific lipid–protein interactions, but the mechanisms by which PIP₂ regulates many of its various targets remain to be fully elucidated. Because many PIP₂ targets are membrane-spanning proteins, we explored whether the phosphoinositides might alter bilayer physical properties such as curvature and elasticity, which would alter the equilibrium between membrane protein conformational states—and thereby protein function. Taking advantage of the gramicidin A (gA) channels’ sensitivity to changes in lipid bilayer properties, we used gA-based fluorescence quenching and single-channel assays to examine the effects of long-chain PIP₂s (brain PIP₂, which is predominantly 1-stearyl-2-arachidonyl-PIP₂, and dioleoyl-PIP₂) on bilayer properties. When premixed with dioleoyl-phosphocholine at 2 mol %, both long-chain PIP₂s produced similar changes in gA channel function (bilayer properties); when applied through the aqueous solution, however, brain PIP₂ was a more potent modifier than dioleoyl-PIP₂. Given the widespread use of short-chain dioctanoyl-phosphoinositides, we also examined the effects of diC₈-phosphoinositol (PI), PI(4,5)P₂, PI(3,5)P₂, PI(3,4)P₂, and PI(3,4,5)P₃. The diC₈ phosphoinositides, except for PI(3,5)P₂, altered bilayer properties with potencies that decreased with increasing head group charge. Nonphosphoinositide diC₈ phospholipids generally were more potent bilayer modifiers than the polyphosphoinositides. These results show that physiological increases or decreases in plasma membrane PIP₂ levels, as a result of activation of PI kinases or phosphatases, are likely to alter lipid bilayer properties, in addition to any other effects they may have. The results further show that exogenous PIP₂, as well as structural analogues that differ in acyl chain length or phosphorylation state, alters lipid bilayer properties at the concentrations used in many cell physiological experiments.     

Phosphoinositides in membrane traffic.

Phosphoinositides serve as direct local modulators or recruiters of the protein machineries that control membrane trafficking. In the past year, examples of phosphoinositide effectors include regulators of small GTPases in coat assembly, dynamin in clathrin coated vesicle formation and FYVE finger proteins in endocytic membrane traffic. A novel phosphoinositide appears to regulate effectors involved in the formation of multivesicular endosomes.     

Phosphoinositides and their functions in apicomplexan parasites

Phosphoinositides are the phosphorylated derivatives of the structural membrane phospholipid phosphatidylinositol. Single or combined phosphorylation at the 3, 4 and 5 positions of the inositol ring gives rise to the seven different species of phosphoinositides. All are quantitatively minor components of cellular membranes but have been shown to have important functions in multiple cellular processes. Here we describe our current knowledge of phosphoinositide metabolism and functions in apicomplexan parasites, mainly focusing on Toxoplasma gondii and Plasmodium spp. Even though our understanding is still rudimentary, phosphoinositides have already shown their importance in parasite biology and revealed some very particular and parasite-specific functions. Not surprisingly, there is a strong potential for phosphoinositide synthesis to be exploited for future anti-parasitic drug development.     

Two-step engulfment of apoptotic cells

Apoptotic cells expose phosphatidylserine on their surface as an "eat me" signal, and macrophages respond by engulfing them. Although several molecules that specifically bind phosphatidylserine have been identified, the molecular mechanism that triggers engulfment remains elusive. Here, using a mouse pro-B cell line, Ba/F3, that grows in suspension, we reconstituted the engulfment of apoptotic cells. The parental Ba/F3 cells did not engulf apoptotic cells. Ba/F3 transformants expressing T cell immunoglobulin- and mucin-domain-containing molecule 4 (Tim4), a type I membrane protein that specifically binds phosphatidylserine, efficiently bound apoptotic cells in a phosphatidylserine-dependent manner but did not engulf them. However, Ba/F3 transformants expressing both Tim4 and the integrin α(v)β(3) complex bound to and engulfed apoptotic cells in the presence of milk fat globule epidermal growth factor factor VIII (MFG-E8), a secreted protein that can bind phosphatidylserine and integrin α(v)β(3). These results indicate that the engulfment of apoptotic cells proceeds in two steps: Tim4 tethers apoptotic cells, and the integrin α(v)β(3) complex mediates engulfment in coordination with MFG-E8. A similar two-step engulfment of apoptotic cells was observed with mouse resident peritoneal macrophages. Furthermore, the Tim4/integrin-mediated engulfment by the Ba/F3 cells was enhanced in cells expressing Rac1 and Rab5, suggesting that this system well reproduces the engulfment of apoptotic cells by macrophages.     

Phosphoinositides of sheep platelets plasma membranes.

Phospholipid composition of sheep blood platelets and its various plasma membrane fractions have been analyzed. Based on their flotation rates in discontinuous sucrose density gradient centrifugation, three membrane fractions were isolated. 5'-Nucelotidase and alkaline phosphatase were distributed nearly equally in all the three membrane fractions. However these membrane fractions showed differences in the distribution of phosphatidyl ethanolamine, phosphatidyl choline and phosphoinositides. Phosphatidyl ethanolamine was predominant in fraction I (11.05 micrograms PLP/mg protein) while phosphatidyl choline was predominant in fractions II and III (110.10 and 68.30 micrograms PLP/mg protein respectively). Phosphatidyl inositol (Ptd-InsP) was equally distributed in all three membrane fractions. However, both Ptd-InsP and phosphatidyl inositol 4,5-bisphosphate were about 4-fold higher in fraction II (73.55 and 89.89 micrograms PLP/mg protein respectively).