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Biomedical subjects

Frederick R Maxfield

Publications and source records attributed to Frederick R Maxfield.

At least 19 recordsLinked to original sources
PubMed2007

Degradation of fibrillar forms of Alzheimer's amyloid beta-peptide by macrophages.

Cultured microglia internalize fibrillar amyloid Abeta (fAbeta) and deliver it to lysosomes. Degradation of fAbeta by microglia is incomplete, but macrophages degrade fAbeta efficiently. When mannose-6 phosphorylated lysosomal enzymes were added to the culture medium of microglia, degradation of fAbeta was increased, and the increased degradation was inhibited by excess mannose-6-phosphate, which competes for binding and endocytic uptake. This suggests that low activity of one or more lysosomal enzymes in the microglia was responsible for the poor degradation of fAbeta. To further characterize the degradation of fAbeta in late endosomes and lysosomes, we analyzed fAbeta-derived intracellular degradation products in macrophages and microglia by mass spectrometry. Fragments with truncations in the first 12 N-terminal residues were observed in extracts from both cell types. We also analyzed material released by the cells. Microglia released mainly intact Abeta1-42, whereas macrophages released a variety of N-terminal truncated fragments. These results indicate that initial proteolysis near the N-terminus is similar in both cell types, but microglia are limited in their ability to make further cuts in the fAbeta.

Amyloid beta-Peptides↗
PubMed2006

Sterol, protein and lipid trafficking in Chinese hamster ovary cells with Niemann-Pick type C1 defect.

We studied the trafficking of sterols, lipids and proteins in Niemann-Pick type C (NPC) cells. The NPC is an inherited disorder involving the accumulation of sterol and lipids in modified late-endosome/lysosome-like storage organelles. Most sterol accumulation studies in NPC cells have been carried out using low-density lipoprotein (LDL) as the sterol source, and it has been shown that sterol efflux from late endosomes is impaired in NPC cells. In this study, we used a fluorescent sterol analog, dehydroergosterol, which can be quickly and efficiently delivered to the plasma membrane. Thus, we were able to study the trafficking kinetics of the non-LDL-derived sterol pool, and we found that dehydroergosterol accumulates in the storage organelles over the course of several hours in NPC cells. We also found that dialkylindocarbocyanine lipid-mimetic analogs that recycle efficiently from early endosomes in wild-type cells are targeted to late endosomal organelles in NPC cells, and transferrin receptors recycle slowly and inefficiently in NPC cells. These data are consistent with multiple trafficking defects in both early and late endosomes in NPC cells.

Animals↗
PubMed2006

Modeling the structure of the StART domains of MLN64 and StAR proteins in complex with cholesterol.

Steroidogenic acute regulatory protein-related lipid transfer (StART) domains are ubiquitously involved in intracellular lipid transport and metabolism and other cell-signaling events. In this work, we use a flexible docking algorithm, comparative modeling, and molecular dynamics (MD) simulations to generate plausible three-dimensional atomic models of the StART domains of human metastatic lymph node 64 (MLN64) and steroidogenic acute regulatory protein (StAR) proteins in complex with cholesterol. Our results show that cholesterol can adopt a similar conformation in the binding cavity in both cases and that the main contribution to the protein-ligand interaction energy derives from hydrophobic contacts. However, hydrogen-bonding and water-mediated interactions appear to be important in the fine-tuning of the binding affinity and the position of the ligand. To gain insights into the mechanism of binding, we carried out steered MD simulations in which cholesterol was gradually extracted from within the StAR model. These simulations indicate that a transient opening of loop Omega1 may be sufficient for uptake and release, and they also reveal a pathway of intermediate states involving residues known to be crucial for StAR activity. Based on these observations, we suggest specific mutagenesis targets for binding studies of cholesterol and its derivatives that could improve our understanding of the structural determinants for ligand binding by sterol carrier proteins.

Algorithms↗
PubMed2006

Intracellular sterol transport and distribution.

Sterols are important components of many biological membranes, and changes in sterol levels can have dramatic effects on membrane properties. Sterols are transported rapidly between cellular organelles by vesicular and nonvesicular processes. Recent studies have identified transmembrane proteins that facilitate the removal of sterols from membranes as well as soluble cytoplasmic proteins that play a role in their movement through the cytoplasm. The mechanisms by which these proteins work are generally not well understood. Cells maintain large differences in the sterol:phospholipid ratio in different organelles. Recent theoretical and experimental studies indicate ways in which the lipid environment can alter the chemical potential of sterols, which may help to explain aspects of their transport kinetics and distribution.

Animals↗
PubMed2006

Endocytic recycling compartments altered in cisplatin-resistant cancer cells.

The clinical utility of cisplatin to treat human malignancies is often limited by the development of drug resistance. We have previously shown that cisplatin-resistant human KB adenocarcinoma cells that are cross-resistant to methotrexate and heavy metals have altered endocytic recycling. In this work, we tracked lipids in the endocytic recycling compartment (ERC) and found that the distribution of the ERC is altered in KB-CP.5 cells compared with parental KB-3-1 cells. A tightly clustered ERC is located near the nucleus in parental KB-3-1 cells but it appears loosely arranged and widely dispersed throughout the cytoplasm in KB-CP.5 cells. The altered distribution of the ERC in KB-CP.5 cells is related to the amount and distribution of stable detyrosinated microtubules (Glu-alpha-tubulin), as previously shown in Chinese hamster ovary B104-5 cells that carry a temperature-sensitive Glu-alpha-tubulin allele. In addition, B104-5 cells with a dispersed ERC under nonpermissive conditions were more resistant to cisplatin compared with B104-5 cells with a clustered ERC under permissive conditions. We conclude that resistance to cisplatin might be due, in part, to reduced uptake of cisplatin resulting from an endocytic defect reflecting defective formation of the ERC, possibly related to a shift in the relative amounts and distributions of stable microtubules.

Animals↗
PubMed2006

Parallel analysis of v-Src mutant protein function using reverse transfection cell arrays.

The conversion of the genomic information produced by the recent sequencing projects into a comprehensive understanding of the human proteome has yet to occur. A new technology that represents a potential bridge between genomics and proteomics is reverse transfection. Reverse transfection cell microarrays are produced by overlaying cDNA arrays with mammalian cells, generating localized clusters of transfected cells with each cluster overexpressing a unique protein. This miniaturized cell-based microarray format affords parallel functional analysis of thousands of cDNA constructs in a high throughput format. In this report we document the development of a co-transfection methodology for reverse transfection applications. The demonstrated high co-transfection efficiency with a "marker" plasmid encoding for GFP enables the identification of transfected cells and eliminates the need for epitope-tagged constructs in cell-based high throughput screening applications using reverse transfection. This co-transfection method was used to study in parallel the structure/function of multiple versions of the v-Src protein using automated fluorescence microscopy. The wild-type v-Src protein and four mutants having insertions or deletions in the SH2 or SH3 domains displayed high levels of tyrosine kinase activity in HEK293T cells. Three other mutated v-Src proteins, including a kinase-dead version, were shown to be defective for tyrosine kinase activity. This reverse co-transfection approach is applicable for high throughput screening of both cDNA libraries and positional scanning recombinant protein libraries.

Cell Line↗
PubMed2005

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.

Animals↗
PubMed2005

Elevated plasma membrane cholesterol content alters macrophage signaling and function.

OBJECTIVE: During atherogenesis, macrophages migrate into the subendothelial space where they ingest deposited lipoproteins, accumulate lipids, and transform into foam cells. It is unclear why these macrophages do not remove their lipid loads from the region. This study was aimed at testing the hypothesis that macrophage behavior is altered when membrane cholesterol levels are elevated, as might be the case for cells in contact with lipoproteins within atherosclerotic lesions. METHODS AND RESULTS: We examined the effects of elevating membrane cholesterol on macrophage behavior. J774 macrophages were treated with either acetylated low-density lipoprotein (ac-LDL) and ACAT inhibitor or cholesterol-chelated methyl-beta-cyclodextrin (chol-MbetaCD) to increase membrane cholesterol levels. Our results show that elevating the membrane cholesterol of J774 macrophages induced dramatic ruffling, stimulated cell spreading, and affected F-actin organization. Cellular adhesion was required for these effects, and Rac-mediated signaling pathways were involved. Additionally, 3-dimensional transwell chemotaxis assays showed that migration of J774 macrophages was significantly inhibited when membrane cholesterol levels were raised. CONCLUSIONS: These findings indicate that increased membrane cholesterol causes dramatic effects on macrophage cellular functions related to the actin cytoskeleton. They should provide new insights into the early steps of atherogenesis.

Acetyl-CoA C-Acetyltransferase↗
PubMed2005

Automated microscopy screening for compounds that partially revert cholesterol accumulation in Niemann-Pick C cells.

Niemann-Pick disease type C (NPC) is an autosomal recessive genetic disorder manifested by abnormal accumulation of unesterified cholesterol and other lipids. We screened combinatorially synthesized chemical libraries to identify compounds that would partially revert cholesterol accumulation. Cultured CHO cells with NPC phenotypes (CT60 and CT43) were used for screening along with normal CHO cells as a control. We developed an automated microscopy assay based on imaging of filipin fluorescence for estimating cholesterol accumulation in lysosomal storage organelles. Our primary screen of 14,956 compounds identified 14 hit compounds that caused significant reduction in cellular cholesterol accumulation at 10 microM. We then screened a secondary library of 3,962 compounds selected based on chemical similarity to the initial hits and identified 7 compounds that demonstrated greater efficacy and lower toxicity than the original hits. These compounds are effective at concentrations of 123 nM to 3 microM in reducing the cholesterol accumulation in cells with a NPC1 phenotype.

Androstenes↗
PubMed2005

Direct observation of rapid internalization and intracellular transport of sterol by macrophage foam cells.

Transport of the fluorescent cholesterol analog dehydroergosterol (DHE) from the plasma membrane was studied in J774 macrophages (Mphis) with normal and elevated cholesterol content. Cells were labeled with DHE bound to methyl-beta-cyclodextrin. In J774, Mphis with normal cholesterol, intracellular DHE became enriched in recycling endosomes, but was not highly concentrated in the trans-Golgi network or late endosomes and lysosomes. After raising cellular cholesterol by incubation with acetylated low-density lipoprotein (AcLDL), DHE was transported to lipid droplets, and less sterol was found in recycling endosomes. Transport of DHE to droplets was very rapid (t1/2 = 1.5 min after photobleaching) and did not require metabolic energy. In cholesterol-loaded J774 Mphis, the initial fraction of DHE in the plasma membrane was reduced, and rapid DHE efflux from the plasma membrane to intracellular organelles was observed. This rapid sterol transport was not related to plasma membrane vesiculation, as DHE did not become enriched in endocytic vesicles formed after sphingomyelinase C treatment of cells. When cells were incubated with DHE ester incorporated into AcLDL, fluorescence of the sterol was first found in punctate endosomes. After a chase, this DHE colocalized with transferrin in a distribution similar to cells labeled with DHE delivered by methyl-beta-cyclodextrin. Our results indicate that elevation of sterol levels in Mphis enhances transport of sterol from the plasma membrane by a non-vesicular pathway.

Biological Transport↗
PubMed2004

Lipid and cholesterol trafficking in NPC.

Niemann-Pick type C, or NPC for short, is an early childhood disease exhibiting progressive neurological degeneration, associated with hepatosplenomegaly in some cases. The disease, at the cellular level, is a result of improper trafficking of lipids such as cholesterol and glycosphingolipids (GSLs) to lysosome-like storage organelles (LSOs), which become engorged with these lipids. It is believed that the initial defect in trafficking, whether of cholesterol or a GSL, results in an eventual traffic jam in these LSOs. This leads to the retention of not only other lipids, but also of transmembrane proteins that transiently associate with the late endosomes (LE) in normal cells, on their way to other cellular destinations such as the trans-Golgi network (TGN). In this review, we discuss the biophysical properties of lipids and cholesterol that might determine their intracellular itineraries, and how these itineraries are altered in NPC cells, which have defects in the proteins NPC1 or NPC2. We also discuss some potential therapeutic directions being suggested by recent research.

Animals↗
PubMed2004

Enrichment of endoplasmic reticulum with cholesterol inhibits sarcoplasmic-endoplasmic reticulum calcium ATPase-2b activity in parallel with increased order of membrane lipids: implications for depletion of endoplasmic reticulum calcium stores and apoptosis in cholesterol-loaded macrophages.

Macrophages in advanced atherosclerotic lesions accumulate large amounts of unesterified, or "free," cholesterol (FC). FC accumulation induces macrophage apoptosis, which likely contributes to plaque destabilization. Apoptosis is triggered by the enrichment of the endoplasmic reticulum (ER) with FC, resulting in depletion of ER calcium stores, and induction of the unfolded protein response. To explain the mechanism of ER calcium depletion, we hypothesized that FC enrichment of the normally cholesterol-poor ER membrane inhibits the macrophage ER calcium pump, sarcoplasmic-endoplasmic reticulum calcium ATPase-2b (SERCA2b). FC enrichment of ER membranes to a level similar to that occurring in vivo inhibited both the ATPase activity and calcium sequestration function of SERCA2b. Enrichment of ER with ent-cholesterol or 14:0-18:0 phosphatidylcholine, which possess the membrane-ordering properties of cholesterol, also inhibited SERCA2b. Moreover, at various levels of FC enrichment of ER membranes, there was a very close correlation between increasing membrane lipid order, as monitored by 16-doxyl-phosphatidycholine electron spin resonance, and SERCA2b inhibition. In view of these data, we speculate that SERCA2b, a conformationally active protein with 11 membrane-spanning regions, loses function due to decreased conformational freedom in FC-ordered membranes. This biophysical model may underlie the critical connection between excess cholesterol, unfolded protein response induction, macrophage death, and plaque destabilization in advanced atherosclerosis.

Animals↗
PubMed2004

Role of cytoplasmic domain serines in intracellular trafficking of furin.

Furin is a transmembrane protein that cycles between the plasma membrane, endosomes, and the trans-Golgi network, maintaining a predominant distribution in the latter. It has been shown previously that Tac-furin, a chimeric protein expressing the extracellular and transmembrane domains of the interleukin-2 receptor alpha chain (Tac) and the cytoplasmic domain of furin, is delivered from the plasma membrane to the TGN through late endosomes, bypassing the endocytic recycling compartment. Tac-furin also recycles in a loop between the TGN and late endosomes. Localization of furin to the TGN is modulated by a six-amino acid acidic cluster that contains two phosphorylatable serines (SDSEED). We investigated the role of these serines in the trafficking of Tac-furin by using a mutant chimera in which the SDS sequence was replaced by the nonphosphorylatable sequence ADA (Tac-furin/ADA). Although the mutant construct is internalized and delivered to the TGN, both the postendocytic trafficking and the steady-state distribution were found to differ from the wild-type. In contrast with Tac-furin, Tac-furin/ADA does not enter late endosomes after being internalized. Instead, it traffics with transferrin to the endocytic recycling compartment, and from there it is delivered to the TGN. As with Tac-furin, Tac-furin/ADA is sorted from the TGN into late endosomes at steady state, but its retrieval from the late endosomes to the TGN is inhibited. These results suggest that serine phosphorylation plays an important role in at least two steps of Tac-furin trafficking, acting as an active sorting signal that mediates the selective sorting of Tac-furin into late endosomes after internalization, as well as its retrieval from late endosomes back to the TGN.

Amino Acid Substitution↗
PubMed2004

Effects of cholesterol depletion and increased lipid unsaturation on the properties of endocytic membranes.

Lipid analogs with dialkylindocarbocyanine (DiI) head groups and short or unsaturated hydrocarbon chains (e.g. DiIC(12) and FAST DiI) enter the endocytic recycling compartment efficiently, whereas lipid analogs with long, saturated tails (e.g. DiIC(16) and DiIC(18)) are sorted out of this pathway and targeted to the late endosomes/lysosomes (Mukherjee, S., Soe, T. T., and Maxfield, F. R. (1999) J. Cell Biol. 144, 1271-1284). This differential trafficking of lipid analogs with the same polar head group was interpreted to result from differential partitioning to different types of domains with varying membrane order and/or curvature. Here we investigate the system further by monitoring the trafficking behavior of these lipid analogs under conditions that alter domain properties. There was a marked effect of cholesterol depletion on the cell-surface distribution and degree of internalization of the lipid probes. Furthermore, instead of going to the late endosomes/lysosomes as in control cells, long chain DiI analogs, such as DiIC(16), were sorted to the recycling pathway in cholesterol-depleted cells. We confirmed that this difference was due to a change in overall membrane properties, and not cholesterol levels per se, by utilizing a Chinese hamster ovary cell line that overexpressed transfected stearoyl-CoA desaturase 1, a rate-limiting enzyme in the production of monounsaturated fatty acids. These cells have a decrease in membrane order because they contain a much larger fraction of unsaturated fatty acids. These cells showed alteration of DiI trafficking very similar to cholesterol-depleted cells. By using cold Triton X-100 extractability of different lipids as a criterion to determine the membrane properties of intracellular organelles, we found that the endocytic recycling compartment has abundant detergent-resistant membranes, in contrast to the late endosomes and lysosomes.

Animals↗
PubMed2004

Oxalic acid alters intracellular calcium in endothelial cells.

Patients with chronic renal failure (CRF) who undergo hemodialysis experience accelerated atherosclerosis and premature death. While the cause of uremic atherogenesis is unknown, we reported that uremic levels of oxalate, an excretory metabolite, severely inhibit proliferation and migration of human endothelial cells (EC) without affecting other cell types. Since the physical, cellular and molecular events of endothelial injury are clearly established as key factors in the development of plaque, and since inhibition of proliferation and migration would enhance endothelial injury, we have proposed that oxalate is an atherogenic toxin of uremia. In the current study, we used in situ cell counting and total DNA measurement to show that the inhibitory effect of oxalate on proliferation is exclusive to endothelial cells among human cell lines tested (endothelial cells, fibroblasts, aortic smooth muscle cells (SMC), glioblastoma and embryonic kidney cells). Using the fluorescent calcium indicators fura-2 and fluo-3, we correlated the inhibition of proliferation with a prolonged elevation in intracellular free calcium levels. We also demonstrated that all cells tested internalize 14C-oxalic acid. We conclude that plasma oxalate exerts its atherogenic effects by elevating intracellular calcium exclusively in endothelial cells and preventing re-endothelialization.

Biological Transport, Active↗
PubMed2004

Endocytic recycling.

After endocytosis, most membrane proteins and lipids return to the cell surface, but some membrane components are delivered to late endosomes or the Golgi. We now understand that the pathways taken by internalized molecules that eventually recycle to the cell surface can be surprisingly complex and can involve a series of sorting events that occur in several organelles. The molecular basis for many of these sorting processes is only partly understood.

Animals↗
PubMed2004

Membrane domains.

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.

Lipid Metabolism↗