Mammalian cells take up extracellular material by a variety of different mechanisms that are collectively termed endocytosis. Endocytic mechanisms serve many important cellular functions including the uptake of extracellular nutrients, regulation of cell-surface receptor expression, maintenance of cell polarity, and antigen presentation. Endocytic pathways are also utilized by viruses, toxins, and symbiotic microorganisms to gain entry into cells. One of the best-characterized endocytic mechanisms is receptor-mediated endocytosis via clathrin-coated pits. This type of endocytosis constitutes the major emphasis of this review, with a brief discussion of other endocytic mechanisms and their comparison with the receptor-mediated pathway. This review describes and evaluates critically current understanding of the mechanisms of entry of plasma membrane components such as the receptor-ligand complexes and membrane lipids as well as the extracellular fluid into cells. The intracellular sorting and trafficking of these molecules upon internalization are also described. The roles of endocytosis in physiological and pathological processes are discussed. These include maintenance of cell polarization, antigen presentation, glucose transport, atherosclerosis, Alzheimer's disease, and the endocytosis of toxins and viruses.

Ferroptosis, a non-apoptotic form of programmed cell death, is triggered by oxidative stress in cancer, heat stress in plants, and hemorrhagic stroke. A homeostatic transcriptional response to ferroptotic stimuli is unknown. We show that neurons respond to ferroptotic stimuli by induction of selenoproteins, including antioxidant glutathione peroxidase 4 (GPX4). Pharmacological selenium (Se) augments GPX4 and other genes in this transcriptional program, the selenome, via coordinated activation of the transcription factors TFAP2c and Sp1 to protect neurons. Remarkably, a single dose of Se delivered into the brain drives antioxidant GPX4 expression, protects neurons, and improves behavior in a hemorrhagic stroke model. Altogether, we show that pharmacological Se supplementation effectively inhibits GPX4-dependent ferroptotic death as well as cell death induced by excitotoxicity or ER stress, which are GPX4 independent. Systemic administration of a brain-penetrant selenopeptide activates homeostatic transcription to inhibit cell death and improves function when delivered after hemorrhagic or ischemic stroke.

Considerable evidence shows that lateral inhomogeneities in lipid composition and physical properties exist in biological membranes. These membrane lipid domains are proposed to play important roles in processes such as signal transduction and membrane traffic. However, there is not at present an adequate description of the nature of these lipid domains in terms of their size, abundance, composition, or dynamics. We discuss the current analyses of the properties and function of membrane domains in cells and compare their properties with chemically simpler model membrane systems that can be understood in greater detail.

Cholesterol is an important constituent of most mammalian cell membranes and its concentration in various cellular membranes is tightly regulated. Although there is much information about cholesterol distribution and trafficking in cells, it is primarily derived from indirect measurements, and the results obtained using different approaches are often conflicting. A cholesterol analog that faithfully mimics the properties of cholesterol and can be followed in living cells would thus be very useful. In this study, we report the fluorescence imaging of such an analog, dehydroergosterol (DHE), in living cells. DHE differs from cholesterol in having three additional double bonds and an extra methyl group. In model systems, DHE closely mimics the behavior of native cholesterol. Using triple-labeling studies, we show that DHE colocalizes extensively with endocytosed transferrin, an endocytic recycling compartment marker, and with a marker for the trans-Golgi network, Tac-TGN38. This distribution of DHE is qualitatively similar to that observed when cells are labeled with the fluorescent cholesterol-binding polyene antibiotic, filipin, although there are differences in apparent proportions of DHE and filipin that are localized at the plasma membrane. Another cholesterol derivative, 25-NBD-cholesterol, has a structure that is compromised by the presence of a bulky NBD group and does not distribute to the same organelles as DHE or filipin. In addition, we show in this manuscript that kinetic processes can be followed in living cells by monitoring recovery of DHE fluorescence in a photobleached region over time. Our observations provide evidence for the presence of a large intracellular cholesterol pool in the endocytic recycling compartment and the trans-Golgi network that might play important roles in the trafficking of lipids, lipid-anchored proteins, and transmembrane proteins that preferentially partition into cholesterol-enriched membrane domains. In addition, this intracellular cholesterol pool might be involved in the maintenance of cellular cholesterol homeostasis.

To understand the mechanisms for endocytic sorting of lipids, we investigated the trafficking of three lipid-mimetic dialkylindocarbocyanine (DiI) derivatives, DiIC16(3) (1,1′-dihexadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate), DiIC12(3) (1,1′- didodecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate), and FAST DiI (1,1′-dilinoleyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate), in CHO cells by quantitative fluorescence microscopy. All three DiIs have the same head group, but differ in their alkyl tail length or unsaturation; these differences are expected to affect their distribution in membrane domains of varying fluidity or curvature. All three DiIs initially enter sorting endosomes containing endocytosed transferrin. DiIC16(3), with two long 16-carbon saturated tails is then delivered to late endosomes, whereas FAST DiI, with two cis double bonds in each tail, and DiIC12(3), with saturated but shorter (12-carbon) tails, are mainly found in the endocytic recycling compartment. We also find that DiOC16(3) (3,3′- dihexadecyloxacarbocyanine perchlorate) and FAST DiO (3,3′-dilinoleyloxacarbocyanine perchlorate) behave similarly to their DiI counterparts. Furthermore, whereas a phosphatidylcholine analogue with a BODIPY (4,4-difluoro-4-bora-3a,4a-diaza-s-indacene) fluorophore attached at the end of a 5-carbon acyl chain is delivered efficiently to the endocytic recycling compartment, a significant fraction of another derivative with BODIPY attached to a 12-carbon acyl chain entered late endosomes. Our results thus suggest that endocytic organelles can sort membrane components efficiently based on their preference for association with domains of varying characteristics.

Local inhomogeneities in lipid composition play a crucial role in regulation of signal transduction and membrane traffic. Nevertheless, most evidence for microdomains in cells remains indirect, and the nature of membrane inhomogeneities has been difficult to characterize. We used lipid analogs and lipid-anchored proteins with varying fluidity preferences to examine the effect of modulating cellular cholesterol on domain formation. We show that lowering cholesterol levels induces formation of visible micrometer-scale domains in the plasma membrane of several mammalian cell types with complementary distributions of fluorescent lipid analogs with preferences for fluid or ordered domains. A uniform distribution is restored by cholesterol repletion. Unexpectedly, cholesterol depletion does not visibly alter the distribution of a crosslinked or uncrosslinked glycosylphosphatidylinositol-anchored protein (the folate receptor). We also examined the effect of varying cholesterol content on the cold Triton X-100 solubility of several membrane constituents. Although a cholesterol analog, dehydroergosterol, and a glycosylphosphatidylinositol-anchored protein are largely retained after extraction, a lipid analog with saturated 16-carbon acyl chains is largely removed when the cellular cholesterol level is lowered. This result indicates that after cholesterol depletion molecules in the more ordered domains can be extracted differentially by cold nonionic detergents.

The transbilayer distribution of many lipids in the plasma membrane and in endocytic compartments is asymmetric, and this has important consequences for signaling and membrane physical properties. The transbilayer distribution of cholesterol in these membranes is not properly established. Using the fluorescent sterols, dehydroergosterol and cholestatrienol, and a variety of fluorescence quenchers, we studied the transbilayer distribution of sterols in the plasma membrane (PM) and the endocytic recycling compartment (ERC) of a CHO cell line. A membrane impermeant quencher, 2,4,6-trinitrobenzene sulfonic acid, or lipid-based quenchers that are restricted to the exofacial leaflet of the plasma membrane only reduce the fluorescence intensity of these sterols in the plasma membrane by 15-32%. When the same quenchers have access to both leaflets, they quench 70 -80% of the sterol fluorescence. Sterol fluorescence in the ERC is also quenched efficiently in the permeabilized cells. In microinjection experiments, delivery of quenchers into the cytosol efficiently quenched the fluorescent sterols associated with the PM and with the ERC. Quantitative analysis indicates that 60 -70% of the PM sterol is in the cytoplasmic leaflet. This means that cholesterol constitutes ϳ40 mol% of cytoplasmic leaflet lipids, which may have important implications for intracellular cholesterol transport and membrane domain formation. INTRODUCTIONBiological membranes often have a pronounced asymmetry in the transbilayer distribution of lipids, and this can have important functional consequences. In mammalian cell plasma membranes (PM), the cytoplasmic leaflets are enriched in phosphatidylethanolamine (PE) and phosphatidylserine (PS), whereas the exofacial leaflets is enriched in sphingomyelin (SM; Pomorski and Menon, 2006;van Meer et al., 2008). The negative electrical charge on the cytoplasmic leaflet of the PM, which is mainly created by PS and phosphoinositides, permits strong electrostatic interactions with peripheral cationic proteins (Yeung et al., 2008). The elevated concentration of SM in the outer leaflet of the PM, combined with a high level of cholesterol, would promote formation of a liquid-ordered (l o ) phase.Although cholesterol is one of the most abundant lipid molecules in the PM and plays important regulatory roles in biological processes, there is great uncertainty about its transbilayer distribution. The reported values for the amount of cholesterol in the outer leaflet ranges from as little as 13% in a study of mouse synaptic membranes (Hayashi et al., 2002), to as much as 67% in human erythrocytes (Fisher, 1976). A major reason for these disparities has been the lack of suitable techniques to study transbilayer sterol distribution in living cells. In one of the earliest reports on this subject, cholesterol was reported to be enriched in the outer leaflet of human erythrocytes based on freeze-fracture studies (Fisher, 1976). Though elegant, some steps in this analysis could potentially perturb the transbilayer distribution...

A shift in the wavelength of maximum fluorescence emission toward higher wavelengths, caused by a shift in the excitation wavelength toward the red edge of the absorption band, is termed the Red Edge Excitation Shift (REES). This effect is mostly observed with polar fluorophores in motionally restricted media such as very viscous solutions or condensed phases. In this paper, we report the red edge excitation shift of a membrane-bound phospholipid molecule whose headgroup is covalently labeled with a 7-nitrobenz-2-oxa-1,3-diazol-4-y1 (NBD) moiety. When incorporated into model membranes of dioleoyl-sn-glycero-3-phosphocholine (DOPC), the NBD-labeled phospholipid (NBD-PE), exhibits a red edge excitation shift of 10 nm. In addition, fluorescence polarization of NBD-PE in membranes shows both excitation and emission wavelength dependence. The nonpolar membrane probe 1,6-diphenyl-1,3,5-hexatriene (DPH) does not show red edge excitation shift in model membranes. The lifetime of NBD-PE in DOPC vesicles was found to be dependent on both excitation and emission wavelengths. These wavelength-dependent lifetimes are correlated to the reorientation of solvent dipoles around the excited-state dipole of the NBD moiety in the membrane. The magnitude of the red shift in the emission maximum for NBD-PE was found to be independent of temperature, between 12 and 54 O C , and of the physical state (gel or fluid) of the membrane. Taken together, these observations are indicative of the motional restriction experienced by this fluorophore in the membrane. Red edge excitation shift promises to be a powerful tool in probing membrane organization and dynamics.Organized molecular assemblies such as membranes can be considered as large cooperative units with characteristics very different from the individual structural units which constitute them. Fluorescence spectroscopy has been one of the principal techniques to study organization and dynamics of biological and model membranes because of its suitable time scale, noninvasive nature, and intrinsic sensitivity (Radda, 1975;Lakowicz, 1980Lakowicz, , 1981. For a fluorophore in a bulk nonviscous solvent, the dipolar relaxation of the solvent molecules around the fluorophore in the excited state is much faster than the fluorescence lifetime. The fluorescence decay rates and the wavelength of maximum emission, in such a case, are therefore usually independent of the excitation wavelength. However, these parameters do show excitation wavelength dependence if the dipolar relaxation of the solvent molecules is slow in the excited state, such that the relaxation time is comparable to or longer than the fluorescence lifetime (Chen, 1967;Fletcher, 1968;Galley & Purkey, 1970;Rubinov & Tomin, 1970;Castelli & Forster, 1973;Itoh & Azumi, 1975;Demchenko, 1982; Lakowicz & Keating-Nakamoto, 1984). Such a shift in the wavelength of maximum emission toward higher wavelengths, caused by a shift in the excitation wavelength toward the red edge of the absorption band, is termed the red edge excitatio...