The importance of cholesterol for endocytosis has been investigated in HEp-2 and other cell lines by using methyl-␤-cyclodextrin (M␤CD) to selectively extract cholesterol from the plasma membrane. M␤CD treatment strongly inhibited endocytosis of transferrin and EGF, whereas endocytosis of ricin was less affected. The inhibition of transferrin endocytosis was completely reversible. On removal of M␤CD it was restored by continued incubation of the cells even in serum-free medium. The recovery in serum-free medium was inhibited by addition of lovastatin, which prevents cholesterol synthesis, but endocytosis recovered when a water-soluble form of cholesterol was added together with lovastatin. Electron microscopical studies of M␤CD-treated HEp-2 cells revealed that typical invaginated caveolae were no longer present. Moreover, the invagination of clathrin-coated pits was strongly inhibited, resulting in accumulation of shallow coated pits. Quantitative immunogold labeling showed that transferrin receptors were concentrated in coated pits to the same degree (approximately sevenfold) after M␤CD treatment as in control cells. Our results therefore indicate that although clathrin-independent (and caveolae-independent) endocytosis still operates after removal of cholesterol, cholesterol is essential for the formation of clathrin-coated endocytic vesicles.

Background:The mechanism of unconventional secretion of ␣-synuclein is unknown. Results: Autophagy of ␣-synuclein followed by exocytosis of autophagy intermediates (exophagy) are increased by expression of TPPP/p25␣. Conclusion: Exophagy of ␣-synuclein is increased by lysosomal dysfunction and/or altered trafficking of autophagosomes. Significance: Exophagy of ␣-synuclein might represent the first step in inter-neuronal spread of Lewy body disease.

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The superoxide-producing phagocyte NADPH oxidase consists of a membrane-bound flavocytochrome b 558 complex, and cytosolic factors p47phox, p67phox and the small GTPase Rac, which translocate to the membrane to assemble the active complex following cell activation. We here show that insolubility of NADPH oxidase subunits in nonionic detergents TX-100, Brij-58, and Brij-98 is a consequence of inclusion into cholesterol-enriched membrane microdomains (lipid rafts). Thus, flavocytochrome b 558 , in a cholesterol-dependent manner, segregated to the bouyant low-density detergent-resistant membrane (DRM) fraction, and the cytosolic NADPH oxidase factors associated dynamically with low-density DRM. Further, superoxide production following cholesterol depletion was severely compromised in intact cells or in a cell-free reconstituted system, correlating with a reduced translocation of cytosolic phox subunits to the membrane. In analogy with the widely accepted role of lipid rafts as signaling platforms, our data indicate that cholesterol-enriched microdomains act to recruit and/or organize the cytosolic NADPH oxidase factors in the assembly of the active NADPH oxidase.

The urokinase-type plasminogen activator receptor (uPAR) plays an important role on the cell surface in mediating extracellular degradative processes and formation of active TGF-β, and in nonproteolytic events such as cell adhesion, migration, and transmembrane signaling. We have searched for mechanisms that determine the cellular location of uPAR and may participate in its disposal. When using purified receptor preparations, we find that uPAR binds to the cation-independent, mannose 6-phosphate/insulin-like growth factor–II (IGF-II) receptor (CIMPR) with an affinity in the low micromolar range, but not to the 46-kD, cation-dependent, mannose 6-phosphate receptor (CDMPR). The binding is not perturbed by uPA and appears to involve domains DII + DIII of the uPAR protein moiety, but not the glycosylphosphatidylinositol anchor. The binding occurs at site(s) on the CIMPR different from those engaged in binding of mannose 6-phosphate epitopes or IGF-II. To evaluate the significance of the binding, immunofluorescence and immunoelectron microscopy studies were performed in transfected cells, and the results show that wild-type CIMPR, but not CIMPR lacking an intact sorting signal, modulates the subcellular distribution of uPAR and is capable of directing it to lysosomes. We conclude that a site within CIMPR, distinct from its previously known ligand binding sites, binds uPAR and modulates its subcellular distribution.

Dopamine regulates reward, cognition, and locomotor functions. By mediating rapid reuptake of extracellular dopamine, the dopamine transporter is critical for spatiotemporal control of dopaminergic neurotransmission. Here, we use super-resolution imaging to show that the dopamine transporter is dynamically sequestrated into cholesterol-dependent nanodomains in the plasma membrane of presynaptic varicosities and neuronal projections of dopaminergic neurons. Stochastic optical reconstruction microscopy reveals irregular dopamine transporter nanodomains (∼70 nm mean diameter) that were highly sensitive to cholesterol depletion. Live photoactivated localization microscopy shows a similar dopamine transporter membrane organization in live heterologous cells. In neurons, dual-color dSTORM shows that tyrosine hydroxylase and vesicular monoamine transporter-2 are distinctively localized adjacent to, but not overlapping with, the dopamine transporter nanodomains. The molecular organization of the dopamine transporter in nanodomains is reversibly reduced by short-term activation of NMDA-type ionotropic glutamate receptors, implicating dopamine transporter nanodomain distribution as a potential mechanism to modulate dopaminergic neurotransmission in response to excitatory input.

Microglia are the resident immune cells of the CNS and constitute a self-sustaining population of CNS-adapted tissue macrophages. As mononuclear phagocytic cells, they express high levels of superoxide-producing NADPH oxidases (NOX). The sole function of the members of the NOX family is to generate reactive oxygen species (ROS) that are believed to be important in CNS host defence and in the redox signalling circuits that shape the different activation phenotypes of microglia. NOX are also important in pathological conditions, where over-generation of ROS contributes to neuronal loss via direct oxidative tissue damage or disruption of redox signalling circuits. In this review, we assess the evidence for involvement of NOX in CNS physiopathology, with particular emphasis on the most important surface receptors that lead to generation of NOX-derived ROS. We evaluate the potential significance of the subcellular distribution of NOX isoforms for redox signalling or release of ROS to the extracellular medium. Inhibitory mechanisms that have been reported to restrain NOX activity in microglia and macrophages in vivo are also discussed. We provide a critical appraisal of frequently used and recently developed NOX inhibitors. Finally, we review the recent literature on NOX and other sources of ROS that are involved in activation of the inflammasome and discuss the potential influence of microglia-derived oxidants on neurogenesis, neural differentiation and culling of surplus progenitor cells. The degree to which excessive, badly timed or misplaced NOX activation in microglia may affect neuronal homeostasis in physiological or pathological conditions certainly merits further investigation. LINKED ARTICLESThis article is part of a themed section on Redox Biology and Oxidative Stress in Health and Disease. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v174.12/issuetoc Abbreviations CR3, complement receptor 3; DPI, diphenylene iodonium; HMGB1, high mobility group box 1; JAK, Janus kinase; NAC, N-acetylcysteine; NLRP3, NOD-like receptor family, pyrin domain containing 3; SVZ, subventricular zone; TLR, toll-like receptor IntroductionMicroglia are the tissue-specific macrophages of the CNS, and unlike other brain cells, they derive from yolk sac haematopoietic stem cells, which populate the mouse brain around embryonic day 9 (Alliot et al., 1999;Ginhoux et al., 2010;Kierdorf et al., 2013). Once established in the brain parenchyma, the population of CNS microglia is maintained throughout life by proliferation when needed, which occurs independently of bone marrow-derived precursors (Prinz and Priller, 2014;Ajami et al., 2007;Elmore et al., 2014; Bruttger et al., 2015).Since they were first identified by Pio del Rio-Hortega in 1920, most research has concentrated on the neuropathological associations of microglia (Kettenmann and Verkhratsky, 2011). Many effector functions of microglia are potentially cytotoxic, and a substantial body of evidence links excessive activation of mi...

The role of caveolae in endocytosis is hotly debated. Here, we argue that most caveolae are stable microdomains at the cell surface. Only a small fraction of caveolae is constitutively internalized, leading to a quantitatively minor uptake of ligands and receptors. In addition, we suggest that a more pronounced downregulation of caveolae from the plasma membrane can occur, presumably stimulated by receptor cross-linking and clustering in caveolae. Finally, we propose that future studies dealing with internalization of caveolae should actually document such internalization and include kinetic data. In recent years, increasing attention has been paid to the role of caveolae in various cellular processes, and a plethora of functions have been ascribed to these small, characteristically shaped invaginations of the plasma membrane that are associated with caveolin. However, currently there is still much confusion about the role of caveolae in endocytosis. Supporting a role in endocytosis are several reports on the uptake of cholera toxin (CT) and also studies on SV40 virus internalization (1,2). Against a role in endocytosis are findings from fluorescence recovery after photobleaching (FRAP) studies showing that caveolae are highly immobile structures that do not have a high turnover at the plasma membrane (3). Indeed, caveolin apparently prevents caveolae from pinching off (4,5) and very recent data also show that caveolae are not obligatory for SV40 entry (6). In addition, other recent studies, for instance, on the uptake of prion proteins (7) and CT (8-13) have brought new life to the discussion. A summary of the evidence against caveolae as a major endocytic carrier is shown in Table 1. In this review, we present a unifying working model for caveolar internalization on the basis of available information.