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Frederick R Maxfield

Publications and source records attributed to Frederick R Maxfield.

31 records · Page 2Linked to original sources
PubMed2003

Different transport routes for high density lipoprotein and its associated free sterol in polarized hepatic cells.

We analyzed the intracellular transport of HDL and its associated free sterol in polarized human hepatoma HepG2 cells. Using pulse-chase protocols, we demonstrated that HDL labeled with Alexa 488 at the apolipoprotein (Alexa 488-HDL) was internalized by a scavenger receptor class B type I (SR-BI)-dependent process at the basolateral membrane and became enriched in a subapical/apical recycling compartment. Most Alexa 488-HDL was rapidly recycled to the basolateral cell surface and released from cells. Within 30 min of chase at 37 degrees C, approximately 3% of the initial cell-associated Alexa 488-HDL accumulated in the biliary canaliculus (BC) formed at the apical pole of polarized HepG2 cells. Even less Alexa 488-HDL was transported to late endosomes or lysosomes. The fluorescent cholesterol analog dehydroergosterol (DHE) incorporated into Alexa 488-HDL was delivered to the BC within a few minutes, independent of the labeled apolipoprotein. This transport did not require metabolic energy and could be blocked by antibodies against SR-BI. The fraction of cell-associated DHE transported to the BC was comparable when cells were incubated with either Alexa 488-HDL containing DHE or with DHE bound to methyl-beta-cyclodextrin. We conclude that rapid, nonvesicular transport of sterol to the BC and efficient recycling of HDL particles underlies the selective sorting of sterol from HDLs in hepatocytes.

Adenosine Triphosphate↗
PubMed2003

Endocytosed cation-independent mannose 6-phosphate receptor traffics via the endocytic recycling compartment en route to the trans-Golgi network and a subpopulation of late endosomes.

Although the distribution of the cation-independent mannose 6-phosphate receptor (CI-MPR) has been well studied, its intracellular itinerary and trafficking kinetics remain uncertain. In this report, we describe the endocytic trafficking and steady-state localization of a chimeric form of the CI-MPR containing the ecto-domain of the bovine CI-MPR and the murine transmembrane and cytoplasmic domains expressed in a CHO cell line. Detailed confocal microscopy analysis revealed that internalized chimeric CI-MPR overlaps almost completely with the endogenous CI-MPR but only partially with individual markers for the trans-Golgi network or other endosomal compartments. After endocytosis, the chimeric receptor first enters sorting endosomes, and it then accumulates in the endocytic recycling compartment. A large fraction of the receptors return to the plasma membrane, but some are delivered to the trans-Golgi network and/or late endosomes. Over the course of an hour, the endocytosed receptors achieve their steady-state distribution. Importantly, the receptor does not start to colocalize with late endosomal markers until after it has passed through the endocytic recycling compartment. In CHO cells, only a small fraction of the receptor is ever detected in endosomes bearing substrates destined for lysosomes (kinetically defined late endosomes). These data demonstrate that CI-MPR takes a complex route that involves multiple sorting steps in both early and late endosomes.

Animals↗
PubMed2003

The cytoplasmic domain of the low density lipoprotein (LDL) receptor-related protein, but not that of the LDL receptor, triggers phagocytosis.

The macrophage LDL receptor and LDL receptor-related protein (LRP, CD91) mediate the phagocytic-like uptake of atherogenic lipoproteins and apoptotic cells, yet the structural basis of their phagocytic functions is not known. To address this issue, we transfected macrophages with chimeric proteins containing the cytoplasmic tails and transmembrane regions of the LDL receptor or LRP and the ectodomain of CD2, which can bind non-opsonized sheep red blood cells (SRBCs). Macrophages expressing receptors containing the LDL receptor domains were able to bind but not internalize SRBCs. In contrast, macrophages expressing receptors containing the cytoplasmic tail of LRP were able to bind and internalize SRBCs. Chimeras in which the LRP cytoplasmic tail was mutated in two di-leucine motifs and a tyrosine in an NPXYXXL motif were able to endocytose anti-CD2 antibody and bind SRBCs, but SRBC phagocytosis was decreased by 70%. Thus, the phagocytic-like functions of LRP, but not those of the LDL receptor, can be explained by the ability of the LRP cytoplasmic tail to trigger phagocytosis. These findings have important implications for atherogenesis and apoptotic cell clearance and for a fundamental cell biological understanding of how the LDL receptor and LRP function in internalization processes.

Animals↗
PubMed2003

Targeted recycling of PECAM from endothelial surface-connected compartments during diapedesis.

Leukocytes enter sites of inflammation by squeezing through the borders between endothelial cells that line postcapillary venules at that site. This rapid process, called transendothelial migration (TEM) or diapedesis, is completed within 90 s after a leukocyte arrests on the endothelial surface. In this time, the leukocyte moves in ameboid fashion across the endothelial borders, which remain tightly apposed to it during transit. It is not known how the endothelial cell changes its borders rapidly and reversibly to accommodate the migrating leukocyte. Here we show that there is a membrane network just below the plasmalemma at the cell borders that is connected at intervals to the junctional surface. PECAM-1, an integral membrane protein with an essential role in TEM, is found in this compartment and constitutively recycles evenly along endothelial cell borders. During TEM, however, recycling PECAM is targeted to segments of the junction across which monocytes are in the act of migration. In addition, blockade of TEM with antibodies against PECAM specifically blocks the recruitment of this membrane to the zones of leukocyte migration, without affecting the constitutive membrane trafficking.

Cell Adhesion↗
PubMed2003

Membrane lipid organization is critical for human neutrophil polarization.

In response to chemoattractants neutrophils extend an actin-rich pseudopod, which imparts morphological polarity and is required for migration. Even when stimulated by an isotropic bath of chemoattractant, neutrophils exhibit persistent polarization and continued lamellipod formation at the front, suggesting that the cells establish an internal polarity. In this report, we show that perturbing lipid organization by depleting plasma membrane cholesterol levels reversibly inhibits cell polarization and migration. Among other receptor-mediated responses, beta(2) integrin up-regulation was unaffected, and initial calcium mobilization was only partially reduced by cholesterol depletion, indicating that this treatment did not abrogate initial receptor-mediated signal transduction. Interestingly, cholesterol depletion did not prevent initial activation of the GTPase Rac or an initial burst of actin polymerization, but rather it inhibited prolonged activation of Rac and sustained actin polymerization. Collectively, these findings support a model in which the plasma membrane is organized into domains that aid in amplifying the chemoattractant gradient and maintaining cell polarization.

Actins↗
PubMed2003

Ratio imaging instrumentation.

Using ratio imaging to obtain quantitative information from microscope images is a powerful tool that has been used successfully in numerous studies. Although ratio imaging reduces the effects of many parameters that can interfere with accurate measurements, it is not a panacea. In designing a ratio imaging experiment, all of the potential problems discussed in this chapter must be considered. Undoubtedly, other problems that were not discussed can also interfere with accurate and meaningful measurements. Many of the problems discussed here were observed in the authors' laboratories. In our experience there are no standard routines or methods that can foresee every problem before it has been encountered. Good experimental design can minimize problems, but the investigator must continue to be alert. Progress in instrumentation continues to overcome some of the difficulties encountered in ratio imaging. CCD cameras with 12- to 14-bit pixel depth are being used more frequently, and several confocal microscope manufacturers are now also using 12-bit digitization. The dramatic increase in the use of confocal microscopes over the past decade is now causing microscope manufacturers to more critically evaluate the effect of axial chromatic aberration in objectives, and recent designs to minimize this problem are being implemented. Other developments such as the use of AOTFs to attenuate laser lines extend the applicability of ratio imaging. Ratio imaging is clearly applicable to a wide range of cell biological problems beyond its widespread use for measuring ion concentrations. Imaginative but careful use of this technique should continue to provide novel insights into the properties of cells.

Animals↗
PubMed2002

Stearoyl-CoA desaturase inhibits ATP-binding cassette transporter A1-mediated cholesterol efflux and modulates membrane domain structure.

Liver X receptor/retinoid X receptor (LXR/RXR) transcription factors have been found to induce a number of genes involved in the regulation of cellular cholesterol efflux, including the ATP-binding cassette transporter A1 (ABCA1), which mediates the active efflux of cellular cholesterol and phospholipids to extracellular acceptors, such as apolipoprotein A-I (apoA-I). In a screen for macrophage LXR/RXR target genes, we identified stearoyl-CoA desaturases 1 and 2 (Scd1 and Scd2), and subsequently tested the hypothesis that SCD activity might modulate cellular cholesterol efflux. In HEK 293 cells co-transfection of ABCA1 with either SCD1 or SCD2 inhibited ABCA1-mediated cholesterol efflux but not phospholipid efflux. In Chinese hamster ovary (CHO) cells with moderate stable overexpression of SCD1, cholesterol efflux to apoA-I was inhibited by 73%, whereas phospholipid efflux and ABCA1 protein levels were unchanged. In contrast, cholesterol efflux to HDL(2), which is not dependent on ABCA1, was increased 2-fold in CHO-SCD1 cells. The effect of SCD on cholesterol efflux to apoA-I was independent of acyl-CoA:cholesterol acyltransferase (ACAT) activity. SCD activity led to an increased content of plasma membrane monounsaturated fatty acids (18:1) at the expense of saturated fatty acids (18:0). As shown by confocal microscopy, SCD overexpression led to a decrease of Triton X-100-resistant domains in the plasma membrane, indicating a decrease in membrane-ordered regions. The data suggest that SCD changes membrane organization and depletes a specific pool of membrane cholesterol supporting ABCA1-mediated efflux, whereas increasing availability of cholesterol for passive efflux by HDL(2). ABCA1-mediated cholesterol and phospholipid efflux may be uncoupled in pathological states associated with high SCD activity, as in hyperinsulinemic obese mice, or in animals treated with LXR activators.

ATP-Binding Cassette Transporters↗
PubMed2002

Rapid nonvesicular transport of sterol between the plasma membrane domains of polarized hepatic cells.

We studied the transport of the fluorescent cholesterol analog dehydroergosterol (DHE) in polarized HepG2 human hepatoma cells. DHE delivered via methyl-beta-cyclodextrin was delivered to both the apical and basolateral membranes and became concentrated in the apical membrane within 1 min. Intracellular DHE was targeted mainly to vesicles of the subapical compartment or apical recycling compartment (SAC/ARC), where it colocalized with fluorescent transferrin and fluorescent analogs of phosphatidylcholine and sphingomyelin. In contrast, transport of DHE from the plasma membrane to the trans-Golgi network was found to be very low. Vesicles containing DHE traversed the cells in both directions, but vesicular export of DHE from the SAC/ARC to the plasma membrane domains was low. Disruption of the microtubule cytoskeleton disturbed vesicular transport of DHE but not its enrichment in the apical (canalicular) membrane. Transport of DHE to the canalicular membrane after photobleaching was very rapid (t(12) = 1.6 min) and was largely ATP-independent in contrast to enrichment of DHE in the SAC/ARC. Release of DHE from the canalicular membrane was also ATP-independent but slower than the enrichment of sterol in the biliary canaliculus (t(12) = 5.4 min). Canalicular DHE could completely redistribute to the basolateral plasma membrane but could not transfer from one cell to the other cell of an HepG2 couplet. We conclude that sterol shuttles rapidly among the plasma membrane domains and other membrane organelles and that this nonvesicular pathway includes fast transbilayer migration.

Adenosine Triphosphate↗
PubMed2002

Plasma membrane microdomains.

Several lines of evidence indicate that the lipids in the plasma membrane of animal cells are inhomogeneously distributed, and that various types of specialized lipid domains play an important role in many biological processes. The characteristics of these domains, such as size, composition and dynamics, are currently under active investigation. It appears that there are many different types of membrane domains in the plasma membrane, and perhaps the entire membrane should be viewed as a mosaic of microdomains.

Actins↗
PubMed2002

Export from pericentriolar endocytic recycling compartment to cell surface depends on stable, detyrosinated (glu) microtubules and kinesin.

A significant fraction of internalized transferrin (Tf) concentrates in the endocytic recycling compartment (ERC), which is near the microtubule-organizing center in many cell types. Tf then recycles back to the cell surface. The mechanisms controlling the localization, morphology, and function of the ERC are not fully understood. We examined the relationship of Tf trafficking with microtubules (MTs), specifically the subset of stable, detyrosinated Glu MTs. We found some correlation between the level of stable Glu MTs and the distribution of the ERC; in cells with low levels of Glu MTs concentrated near to the centriole, the ERC was often tightly clustered, whereas in cells with higher levels of Glu MTs throughout the cell, the ERC was more dispersed. The clustered ERC in Chinese hamster ovary cells became dispersed when the level of Glu MTs was increased with taxol treatment. Furthermore, in a temperature-sensitive Chinese hamster ovary cell line (B104-5), the cells had more Glu MTs when the ERC became dispersed at elevated temperature. Microinjecting purified anti-Glu tubulin antibody into B104-5 cells at elevated temperature induced the redistribution of the ERC to a tight cluster. Microinjection of anti-Glu tubulin antibody slowed recycling of Tf to the cell surface without affecting Tf internalization or delivery to the ERC. Similar inhibition of Tf recycling was caused by microinjecting anti-kinesin antibody. These results suggest that stable Glu MTs and kinesin play a role in the organization of the ERC and in facilitating movement of vesicles from the ERC to the cell surface.

Animals↗
PubMed2002

Microtubule asymmetry during neutrophil polarization and migration.

The development of cell polarity in response to chemoattractant stimulation in human polymorphonuclear neutrophils (PMNs) is characterized by the rapid conversion from round to polarized morphology with a leading lamellipod at the front and a uropod at the rear. During PMN polarization, the microtubule (MT) array undergoes a dramatic reorientation toward the uropod that is maintained during motility and does not require large-scale MT disassembly or cell adhesion to the substratum. MTs are excluded from the leading lamella during polarization and motility, but treatment with a myosin light chain kinase inhibitor (ML-7) or the actin-disrupting drug cytochalasin D causes an expansion of the MT array and penetration of MTs into the lamellipod. Depolymerization of the MT array before stimulation caused 10% of the cells to lose their polarity by extending two opposing lateral lamellipodia. These multipolar cells showed altered localization of a leading lamella-specific marker, talin, and a uropod-specific marker, CD44. In summary, these results indicate that F-actin- and myosin II-dependent forces lead to the development and maintenance of MT asymmetry that may act to reinforce cell polarity during PMN migration.

Azepines↗
PubMed2001

Vesicular and non-vesicular sterol transport in living cells. The endocytic recycling compartment is a major sterol storage organelle.

We examined the intracellular transport of sterol in living cells using a naturally fluorescent cholesterol analog, dehydroergosterol (DHE), which has been shown to mimic many of the properties of cholesterol. By using DHE loaded on methyl-beta-cyclodextrin, we followed this cholesterol analog in pulse-chase studies. At steady state, DHE co-localizes extensively with transferrin (Tf), a marker for the endocytic recycling compartment (ERC), and redistributes with Tf in cells with altered ERC morphology. Expression of a dominant-negative mutation of an ERC-associated protein, mRme-1 (G429R), results in the slowing of both DHE and Tf receptor return to the cell surface. [3H]Cholesterol is found in the same fraction as 125I-Tf on sucrose density gradients, and this fraction can be specifically shifted to a higher density based on the presence of horseradish peroxidase-conjugated Tf in the same organelle. Whereas vesicular transport of Tf and efflux of DHE from the ERC are entirely blocked in energy-depleted cells, delivery of DHE to the ERC from the plasma membrane is only slightly affected. Biochemical studies performed using [3H]cholesterol show that the energy dependence of cholesterol transport to and from the ERC is similar to DHE transport. We propose that a large portion of intracellular cholesterol is localized in the ERC, and this pool might be important in maintaining cellular cholesterol homeostasis.

Animals↗