Mesoscale organization of domains in the plasma membrane - beyond the lipid raft.

The plasma membrane is compartmentalized into several distinct regions or domains, which show a broad diversity in both size and lifetime. The segregation of lipids and membrane proteins is thought to be driven by the lipid composition itself, lipid-protein interactions and diffusional barriers. With regards to the lipid composition, the immiscibility of certain classes of lipids underlies the "lipid raft" concept of plasmalemmal compartmentalization. Historically, lipid rafts have been described as cholesterol and (glyco)sphingolipid-rich regions of the plasma membrane that exist as a liquid-ordered phase that are resistant to extraction with non-ionic detergents. Over the years the interest in lipid rafts grew as did the challenges with studying these nanodomains. The term lipid raft has fallen out of favor with many scientists and instead the terms "membrane raft" or "membrane nanodomain" are preferred as they connote the heterogeneity and dynamic nature of the lipid-protein assemblies. In this article, we will discuss the classical lipid raft hypothesis and its limitations. This review will also discuss alternative models of lipid-protein interactions, annular lipid shells, and larger membrane clusters. We will also discuss the mesoscale organization of plasmalemmal domains including visible structures such as clathrin-coated pits and caveolae.

7-Ketocholesterol impairs phagocytosis and efferocytosis via dysregulation of phosphatidylinositol 4,5-bisphosphate.

The plasma membrane is inhomogeneously organized containing both highly ordered and disordered nanodomains. 7-Ketocholesterol (7KC), an oxysterol formed from the nonenzymatic oxidation of cholesterol, is a potent disruptor of membrane order. Importantly, 7KC is a component of oxidized low-density lipoprotein and accumulates in macrophage and foam cells found in arterial plaques. Using a murine macrophage cell line, J774, we report that both IgG-mediated and phosphatidylserine-mediated phagocytic pathways are inhibited by the accumulation of 7KC. Examination of the well-studied Fcγ receptor pathway revealed that the cell surface receptor abundance and ligand binding are unaltered while downstream signaling and activation of spleen tyrosine kinase is not affected. However, while the recruitment of phospholipase Cγ1 was unaffected its apparent enzymatic activity was compromised resulting in sustained phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2 ] levels and polymerized actin at the base of the phagocytic cup. Additionally, we found that 7KC prevented the activation of PLCß downstream of the P2Y6 G-protein coupled receptor and that 7KC impaired PLCγ activity in response to a direct elevation of cytosolic calcium induced by ionomycin. Finally, we demonstrate that 7KC partly attenuates the activity of rapamycin recruitable constitutively active PLCß3. Together, our results demonstrate that the accumulation of 7KC impairs macrophage function by altering PtdIns(4,5)P2 catabolism and, thus, impairing actin depolymerization required for the completion of phagocytosis.

Quantitative Live-Cell Fluorescence Microscopy During Phagocytosis.

Phagocytosis is a receptor-mediated process whereby professional phagocytes internalize invading pathogens and apoptotic bodies into an intracellular vacuole or phagosome, leading to their degradation. During the formation and maturation of the phagosome, several lipids undergo changes and effector proteins are recruited on the nascent phagosome in a concerted manner. These highly localized, dynamic, and transient processes can only be studied by methods capable of high spatial and temporal resolution. The use of genetically encoded chimeric constructs coupled with fluorescence confocal microscopy enables the continuous, noninvasive analysis of the distribution and metabolism of lipids and effector proteins during phagocytosis. Here, we describe a method where the mouse macrophage cell line, RAW 264.7, and primary macrophages are transiently transfected with fluorescent chimeric probes to analyze and quantify phagocytosis of immunoglobulin-opsonized particles, using confocal microscopy.

Membrane curvature induced by proximity of anionic phospholipids can initiate endocytosis.

The plasma membrane is uniquely enriched in phosphatidylserine (PtdSer). This anionic phospholipid is restricted almost exclusively to the inner leaflet of the plasmalemma. Because of their high density, the headgroups of anionic lipids experience electrostatic repulsion that, being exerted asymmetrically, is predicted to favor membrane curvature. We demonstrate that cholesterol limits this repulsion and tendency to curve. Removal of cholesterol or insertion of excess PtdSer increases the charge density of the inner leaflet, generating foci of enhanced charge and curvature where endophilin and synaptojanin are recruited. From these sites emerge tubules that undergo fragmentation, resulting in marked endocytosis of PtdSer. Shielding or reduction of the surface charge or imposition of outward membrane tension minimized invagination and PtdSer endocytosis. We propose that cholesterol associates with PtdSer to form nanodomains where the headgroups of PtdSer are maintained sufficiently separated to limit spontaneous curvature while sheltering the hydrophobic sterol from the aqueous medium.