Frederick R. Maxfield

Role of cholesterol and lipid organization in disease

Membrane lipids are essential for biological functions ranging from membrane trafficking to signal transduction. The composition of lipid membranes influences their organization and properties, so it is not surprising that disorders in lipid metabolism and transport have a role in human disease. Significant recent progress has enhanced our understanding of the molecular and cellular basis of lipid-associated disorders such as Tangier disease, Niemann–Pick disease type C and atherosclerosis. These insights have also led to improved understanding of normal physiology.

Membrane transport in the endocytic pathway.

Despite controversies and debates, some fundamental properties of endosomes become apparent when comparing results from in vivo and in vitro strategies used to study endosomal membrane traffic. In addition, recent studies are starting to unravel the complex organization of early endosomes, in particular along the route followed by recycling receptors.

Microglial Cells Internalize Aggregates of the Alzheimer's Disease Amyloid β-Protein Via a Scavenger Receptor

Microglia are immune system cells associated with Alzheimers disease plaques containing beta-amyloid (A beta). Murine microglia internalize microaggregates of fluorescently labeled or radioiodinated A beta peptide 1-42. Uptake was confirmed using aggregates of unlabeled A beta detected by immunofluorescence. Uptake of A beta was reduced by coincubation with excess acetyl-low density lipoprotein (Ac-LDL) or other scavenger receptor (SR) ligands, and Dil-labeled Ac-LDL uptake by microglia was blocked by excess A beta. CHO cells transfected with class A or B SRs showed significantly enhanced uptake of A beta. These results show that microglia express SRs that may play a significant role in the clearance of A beta plaques. Binding to SRs could activate inflammation responses that contribute to the pathology of Alzheimers disease.

Rapid acidification of endocytic vesicles containing α2-macroglobulin

Abstract We have used fluorescein-labeled α 2 -macroglobulin (F- α 2 M) to measure pH changes in the microenvironment of internalized ligands following receptor-mediated endocytosis. Fluorescence intensities of single BALB/c 3T3 mouse fibroblasts were measured by using a microscope spectrofluorometer with narrow bandpass excitation filters. The pH was determined from the ratio of fluorescein fluorescence intensities with 450 nm and 490 nm excitation. A standard pH curve was obtained by incubating cells with F- α 2 M for 30 min at 37°C followed by fixation and incubation in buffers of varying pH. To measure the pH of endocytic vesicles, cells were incubated with F- α 2 M for 15 min at 37°C. Fluorescence intensities were measured on living cells within 5 min of rinsing. Under these conditions, the pH of the F- α 2 M microenvironment was 5.0 ± 0.2. Using colloidal gold- α 2 M for electron microscopic localizations we have verified that, under these conditions, α 2 M is predominantly in uncoated vesicles that are negative for acid phosphatase activity. With further incubation for 12 hr, we obtained a pH of 5.0 ± 0.2 for the F- α 2 M. Using fluorescein dextran, we obtained a lysosomal pH of 4.6 ± 0.2. These results indicate that endocytic vesicles become acidic prior to fusion with lysosomes.

Segregation of transferrin to a mildly acidic (pH 6.5) para-golgi compartment in the recycling pathway

To study the intracellular sorting of internalized ligands and receptors, we examined the pathways of two ligands: transferrin, which is recycled, and alpha 2-macroglobulin (alpha 2M), which is degraded. In CHO cells the two ligands rapidly segregate into different intracellular compartments. Within 5 min fluorescein-labeled transferrin (F-Tf) is found in a large round juxtanuclear structure. Rhodamine-labeled alpha 2M is found in a punctate pattern. Ultra-structural localization studies demonstrate that colloidal gold-alpha 2M is found predominantly in endocytic vesicles, while ferritin-transferrin is found in small vesicles and tubular structures in a region adjacent to the Golgi complex. Using image intensified fluorescence microscopy and digital image analysis, we determined that the F-Tf containing structure has a pH of 6.4 +/- 0.2, while endocytic vesicles containing F-alpha 2M have a pH of 5.4 +/- 0.1. Our study defines a mildly acidic compartment, distinct from endocytic vesicles, that is involved in the recycling of internalized components back to the cell surface.

Collection of insulin, EGF and α2-Macroglobulin in the same patches on the surface of cultured fibroblasts and common internalization

We have used video intensification microscopy to observe fluorescent derivatives of insulin, epidermal growth factor and alpha2-macroglobulin added to Swiss 3T3-4 cells. At 4 degrees C, each of these polypeptides binds diffusely to specific receptors on the cell surface. When the cells are warmed to 23 or 37 degrees C, the bound insulin epidermal growth factor or alpha2-macroglobulin rapidly forms patches on the cell surface and is internalized. Using fluorescein-labeled alpha2-macroglobulin and rhodamine-labeled derivatives of insulin and epidermal growth factor, we show that all three polypeptides are internalized within the same vesicles by a common pathway. The mechanism for the internalization of these molecules is discussed.

Cholesterol-dependent retention of GPI-anchored proteins in endosomes

Several cell surface eukaryotic proteins have a glycosylphosphatidylinositol (GPI) modification at the Cterminal end that serves as their sole means of membrane anchoring. Using fluorescently labeled ligands and digital fluorescence microscopy, we show that contrary to the potocytosis model, GPI‐anchored proteins are internalized into endosomes that contain markers for both receptor‐mediated uptake (e.g. transferrin) and fluid phase endocytosis (e.g. dextrans). This was confirmed by immunogold electron microscopy and the observation that a fluorescent folate derivative bound to the GPI‐anchored folate receptor is internalized into the same compartment as co‐internalized horseradish peroxidase–transferrin; the folate fluorescence was quenched when cells subsequently were incubated with diaminobenzidine and H2O2. Most of the GPI‐anchored proteins are recycled back to the plasma membrane but at a rate that is at least 3‐fold slower than C6‐NBD‐sphingomyelin or recycling receptors. This endocytic retention is regulated by the level of cholesterol in cell membranes; GPI‐anchored proteins are recycled back to the cell surface at the same rate as recycling transferrin receptors and C6‐NBD‐sphingomyelin in cholesterol‐depleted cells. Cholesterol‐dependent endocytic sorting of GPI‐anchored proteins is consistent with the involvement of specialized lipid domains or ‘rafts’ in endocytic sorting. These results provide an alternative explanation for GPI‐requiring functions of some GPI‐anchored proteins.

Cholesterol Distribution in Living Cells: Fluorescence Imaging Using Dehydroergosterol as a Fluorescent Cholesterol Analog

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.

Cholesterol depletion induces large scale domain segregation in living cell membranes

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.

Intracellular cholesterol transport

Cholesterol is the single most abundant lipid species in mammalian cells. More than 2×109 years of evolution designed this molecule to perfectly fit into phospholipid bilayers regulating the fluidity, permeability and bending stiffness of biological membranes. Cholesterol also serves as a precursor of steroid hormones, bile acids and oxysterols, and its cellular synthesis is regulated by a complex machinery. While the molecular mechanisms underlying cholesterol synthesis are known in great detail, knowledge is rather sparse about the inter-compartment transport of cholesterol, including trafficking modes and kinetics, as well as control of endomembrane cholesterol content. This chapter provides an overview of our recent understanding of intracellular transport of cholesterol. It is aimed to create a link between the well characterized biophysical properties of cholesterol in model membranes and its behavior in living cells.