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F R Maxfield

Publications and source records attributed to F R Maxfield.

At least 73 records · Page 4Linked to original sources
PubMed1994

Sequestration of GPI-anchored proteins in caveolae triggered by cross-linking.

Glycosyl-phosphatidylinositol (GPI)-anchored proteins have been reported to reside in clusters collected over small membrane invaginations called caveolae. The detection of different GPI-anchored proteins with fluorescently labeled monoclonal antibodies showed that these proteins are not constitutively concentrated in caveolae; they enter these structures independently after cross-linking with polyclonal secondary antibodies. Analysis of the cell surface distribution of the GPI-anchored folate receptor by electron microscopy confirms these observations. Thus, multimerization of GPI-anchored proteins regulates their sequestration in caveolae, but in the absence of agents that promote clustering they are diffusely distributed over the plasma membrane.

3T3 Cells↗
PubMed1994

Yolk platelets in Xenopus oocytes maintain an acidic internal pH which may be essential for sodium accumulation.

Yolk platelets constitute an embryonic endocytic compartment that stores maternally synthesized nutrients. The pH of Xenopus yolk platelets, measured by photometry on whole oocytes which had endocytosed FITC-vitellogenin, was found to be acidic (around pH 5.6). Experiments on digitonin-permeabilized oocytes showed that acidification was due to the activity of an NEM- and bafilomycin A1-sensitive vacuolar proton-ATPase. Proton pumping required chloride, but was not influenced by potassium or sodium. Passive proton leakage was slow, probably due to the buffer capacity of the yolk, and was dependent on the presence of cytoplasmic monovalent cations. In particular, sodium could drive proton efflux through an amiloride-sensitive Na+/H+ exchanger. 8-Bromo-cyclic-AMP was found to increase acidification, suggesting that pH can be regulated by intracellular second messengers. The moderately acidic pH does not promote degradation of the yolk platelets, which in oocytes are stable for weeks, but it is likely to be required to maintain the integrity of these organelles. Furthermore, the pH gradient created by the proton pump, when coupled with the Na+/H+ exchanger, is probably responsible for the accumulation and storage of sodium into the yolk platelets during oogenesis.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1994

Applications of ratio fluorescence microscopy in the study of cell physiology.

Quantitative fluorescence microscopy is becoming an increasingly important tool in the study of cell biology. Fluorescence microscopy has long been used for qualitative characterizations of subcellular distributions of proteins, lipids, nucleic acids, and ions, but quantifying these distributions is complicated by a variety of optical, biological, and physical factors. Many factors that complicate quantification of fluorescence in cells can be circumvented by analyzing fluorescence ratios derived from pairs of fluorescence images. In this review we will discuss the factors that affect fluorescence quantification, the advantages of quantifying fluorescence as a ratio, and give examples of how fluorescence ratio microscopy is being applied in studies of cell biology.

Animals↗
PubMed1994

Changes in yolk platelet pH during Xenopus laevis development correlate with yolk utilization. A quantitative confocal microscopy study.

The variations of the pH in Xenopus yolk platelets have been estimated by fluorescence confocal microscopy and computer image processing. For pH measurements in vitellogenic oocytes, the pH-sensitive fluorescent dye, DM-NERF, was coupled to vitellogenin, and the DM-NERF-vitellogenin was taken up by oocytes via receptor-mediated endocytosis. Dual emission ratio measurements of internalized DM-NERF-vitellogenin indicated that the mature yolk platelets are mildly acidic (pH 5.6). Their precursors, the primordial yolk platelets, have a similar pH. This pH is probably sufficiently low for the partial cleavage of vitellogenin to yolk proteins, but not for yolk degradation. The yolk platelet pH at various developmental stages was estimated by measuring the accumulation of Acridine Orange, both in isolated yolk platelets and in disaggregated embryonic cells. During oogenesis, the yolk platelets accumulated a constant amount of Acridine Orange, corresponding to a pH of around 5.7. During embryogenesis, however, yolk platelets became progressively much more acidic (pH < 5). Acidification correlated with yolk degradation in the various tissues examined, and yolk utilization was blocked when acidification was inhibited with bafilomycin, an inhibitor of vacuolar H+-ATPase. Bafilomycin also inhibited differentiation of cells isolated from stage 13-15 embryos. These data show that the yolk platelet pH is developmentally regulated and is involved in triggering yolk degradation. Also, yolk acidification and degradation appeared to be associated with cell differentiation and with the formation of the endosomal/lysosomal compartment, typical of adult cells, but absent in early embryos.

Acids↗
PubMed1994

Quantification of low density lipoprotein and transferrin endocytic sorting HEp2 cells using confocal microscopy.

Numerous experiments on CHO cells have shown that endosomes are composed of separate vesicular and tubulovesicular compartments, such as the sorting endosome, the recycling compartment, and the late endosome. However, Hopkins et al. (Nature 346, 335-339, 1990) have reported that endosomes in HEp2 human carcinoma cells form an extensive tubular reticulum. To resolve their observations with previous results from CHO and other cells, we examined the sorting and intracellular transport of endocytosed macromolecules in HEp2 cells, using low density lipoprotein (LDL) and transferrin (Tf) to probe the lysosomally directed and recycling pathways, respectively. Fluorescent LDL and Tf were observed with laser scanning confocal microscopy to visualize simultaneously both probes' sorting and subsequent post-sorting behavior in HEp2 cells. Quantifying the 3-dimensional cellular distributions of fluorescent LDL and Tf, after a variety of pulsechase schemes, gave the ligands' trafficking rates. Initially, both ligands appear in the same punctate sorting endosomes, and fingers of Tf start extending from these sorting endosomes. Tf rapidly leaves dual-labeled sorting endosomes (t1/2 approximately 2.5 minutes) and enters a post-sorting recycling compartment from which it is recycled out of the cell (t1/2 approximately 7 minutes). We present both morphological and kinetic data supporting the existence of these two separate compartments along the recycling pathway in HEp2 cells. LDL remains in punctate sorting endosomes that eventually lose the ability to receive newly endocytosed LDL, and mature into late endosomes. The trafficking and sorting of Tf and LDL in HEp2 cells follow the same general scheme as in CHO cells, indicating that the tubular endosomes previously seen may be the tubular parts of the sorting endosomes and recycling compartments in these cells. We propose that the endosomes in the recycling pathway of HEp2 cells, as in CHO cells, are composed of short-lived sorting endosomes, accessible to both Tf and LDL, and long-lived post-sorting recycling compartments, which contain Tf and recycling receptors but not LDL.

Biological Transport↗
PubMed1993

Isolation of a temperature-sensitive variant Chinese hamster ovary cell line with a morphologically altered endocytic recycling compartment.

We have enriched a mutagenized population of Chinese hamster ovary (CHO) cells for those defective in endocytosis by selection for survival to treatment with transferrin (Tf)-ricin and Tf-diphtheria toxin conjugates. Surviving cells were screened with a fluorescently labeled Tf uptake assay to identify cells with morphologically aberrant endocytic phenotypes. One of the cell lines identified, B104-5, has a striking temperature-induced alteration in the morphology of its endocytic receptor recycling compartment. In parental cells the tightly clustered endocytic recycling compartment is located near the Golgi complex. In the mutant cells, following incubation at 40 degrees C, this compartment appears fragmented and widely dispersed. Surprisingly, this alteration in the morphology of the recycling compartment has no effect on the kinetics of Tf internationalization and recycling. The wild-type endocytic compartment is closely aligned with the microtubule-organizing center and the Golgi apparatus, and like the Golgi, its clustered appearance is dependent upon intact microtubules. Although the disruption of the B104-5 receptor recycling compartment morphology can be phenocopied in wild-type cells by microtubule depolymerizing drugs, the microtubule cytoskeleton in B104-5 cells appears normal in immunofluorescent staining. B104-5 cells, unlike the parental cells, do not proliferate at 40 degrees C. The mutation in B104-5 cells is recessive, as fusion with wild-type cells results in a reversion of the B104-5 phenotype. The finding that the morphology of the recycling compartment in CHO cells can be altered without affecting recycling of endocytosed Tf is consistent with the variety of recycling compartment morphologies observed among different cell lines. An interpretation of this result is that the lesion in B104-5 cells is in a gene that is involved in determining the endocytic compartment morphologies observed in different cell lines.

Animals↗
PubMed1993

Regulation of leukocyte locomotion by Ca2+.

Neutrophils migrate towards sites of inflammation and infection by chemotaxis. Their motility is dependent on the actin cytoskeleton and on adhesion to extracellular substrates, but how these are regulated in response to stimuli is not clear. This review focuses on the potential role of Ca(2+) as a second messenger in neutrophil motility. Several effects of Ca(2+) and Ca(2+)-binding proteins on the stability and crosslinking of actin polymers have been demonstrated in vitro. Nevertheless, the complex mechanism by which Ca(2+) regulates actin in neutrophils is not fully understood. In addition, intracellular Ca(2+) regulates the intergin-mediated adhesion of neutrophils to extracellular matrix.

Journal Article↗
PubMed1993

Sorting of membrane components from endosomes and subsequent recycling to the cell surface occurs by a bulk flow process.

A central question in the endocytic process concerns the mechanism for sorting of recycling components (such as transferrin or low density lipoprotein receptors) from lysosomally directed components; membrane-associated molecules including receptors are generally directed towards the recycling pathway while the luminal content of sorting endosomes, consisting of the acid-released ligands, are lysosomally targeted. However, it is not known whether recycling membrane receptors follow bulk membrane flow or if these proteins are actively sorted from lysosomally directed material because of specific protein sequences and/or structural features. Using quantitative fluorescence microscopy we have determined the endocytic route and kinetics of traffic of the bulk carrier, membrane lipids, to address this issue directly. We show that N-[N-(7-nitro-2,1,3-benzoxadiazol-4-yl)-epsilon-aminohexanoyl]- sphingosylphosphorylcholine (C6-NBD-SM) in endocytosed as bulk membrane, and it transits the endocytic system kinetically and morphologically identically to fluorescently labeled transferrin in a CHO cell line. With indistinguishable kinetics, the two labeled markers sort from lysosomally destined molecules in peripherally located sorting endosomes, accumulate in a peri-centriolar recycling compartment, and finally exit the cell. Other fluorescently labeled lipids, C6-NBD-phosphatidylcholine and galactosylceramide also traverse the same pathway. The constitutive nature of sorting of bulk membrane towards the recycling pathway and the lysosomal direction of fluid phase implies a geometric basis of sorting.

4-Chloro-7-nitrobenzofurazan↗
PubMed1993

The End2 mutation in CHO cells slows the exit of transferrin receptors from the recycling compartment but bulk membrane recycling is unaffected.

We have characterized a new CHO cell line (12-4) derived from a parental line, TRVb-1, that expresses the human transferrin receptor. This mutant belongs to the end2 complementation group of endocytosis mutants. Like other end2 mutants, the endosomes in 12-4 cells show a partial acidification defect. These cells internalize LDL and transferrin at 70% of the rate of parental cells and externalize transferrin at 55% of the parental rate (Johnson, L. S., J. F. Presley, J. C. Park, and T. E. McGraw. J. Cell Physiol. 1993). In this report, we have used fluorescence microscopy to determine which step in receptor trafficking is affected in the mutants. Transferrin is sorted from LDL and is delivered to a peri-centriolar recycling compartment at rates similar to parental cells. However, the rate constant for exit of transferrin from the recycling compartment in mutant cells is 0.025 min-1 vs 0.062 min-1 in the parental line. We also measured the trafficking of a bulk membrane marker, 6-[N-[7-nitrobenzo-2-oxa-1,3-diazol-4-yl]-amino]hexanoyl- sphingosylphosphorylcholine (C6-NBD-SM) that labels the exofacial side of the plasma membrane. C6-NBD-SM enters the same recycling compartment as transferrin, and it exits the recycling compartment at a rate of 0.060-0.065 min-1 in both parental and 12-4 cells. We conclude that bulk membrane flow in the recycling pathway of 12-4 cells is normal, but exit of transferrin from the recycling compartment is slowed due to retention in this compartment. Thus, in the mutant cell line the recycling compartment carries out a sorting function, retaining transferrin over bulk membrane.

4-Chloro-7-nitrobenzofurazan↗
PubMed1993

Beta-very low density lipoprotein is sequestered in surface-connected tubules in mouse peritoneal macrophages.

beta-very low density lipoprotein (VLDL) is a large lipoprotein with multiple apoprotein E (apoE) molecules that bind to the LDL receptors on mouse macrophages. Even though they bind to the same receptor, the endocytic processing of beta-VLDL differs from low density lipoprotein (LDL). LDL is rapidly delivered to perinuclear lysosomes and degraded, but much of the beta-VLDL is retained in peripheral compartments for several minutes. We have investigated the properties of these peripheral compartments. Measurement of the pH was made using FITC-phosphatidylethanolamine incorporated into the beta-VLDL, and we found that the peripheral compartments were near neutral in pH. These peripheral, beta-VLDL containing compartments were poorly accessible to antibodies, but a low molecular weight fluorescence quencher (trypan blue) entered the compartments within a few seconds. Intermediate voltage EM of cells labeled with colloidal-gold-beta-VLDL revealed that the peripheral compartments are tubular, surface-connected invaginations. Kinetic studies with fluorescent beta-VLDL showed that the compartments become fully sealed with a half-time of 6 min, and the beta-VLDL is then delivered rapidly to perinuclear lysosomes. By monitoring fluorescence energy transfer between lipid analogs incorporated into the beta-VLDL, some processing of the lipoprotein in the peripheral tubular compartments is demonstrated. The novel mode of uptake of beta-VLDL may account for the high cholesterol ester accumulation induced by this lipoprotein.

Animals↗
PubMed1993

Regulation of neutrophil motility and adhesion by intracellular calcium transients.

Neutrophils are guided to the sites of infection or inflammation by gradients of chemoattractants. Chemoattractants stimulate rapid and repeated changes in neutrophil intracellular calcium, [Ca2+]i, which correlate with cell spreading, pseudopod extension, motility, change of direction and phagocytosis. However, blocking the [Ca2+]i transients has little effect on cell spreading, polarization or pseudopod extension. Thus, either the [Ca2+]i transients are not required for cell spreading, polarization or pseudopod extension or other redundant mechanisms are present that allow the cells to perform these functions in vitro. In contrast, cell motility is [Ca2+]i dependent when the cells are examined on physiological substrates such as fibronectin or vitronectin. Calcium-buffered cells appear to make repeated attempts to move but are unable to detach from a fibronectin or vitronectin substrate. Motility can be restored to [Ca2+]i buffered cells by blocking substrate attachment with RGD peptides or by using a less adherent substrate such as albumin. A similar inhibition of motility on vitronectin could be induced by inhibitors of the calcium/calmodulin-dependent phosphatase, calcineurin. Thus, the periodic increases in [Ca2+]i apparently activate the phosphatase calcineurin to initiate a cycle of detachment from the vitronectin substratum. These data suggest that the [Ca2+]i transients regulate motility by coordinating a series of substrate-specific attachment/detachment events.

Calcineurin↗
PubMed1993

Properties of acidified compartments in hippocampal neurons.

We have studied cultured rat hippocampal neurons using electron microscopic procedures based on the accumulation of DAMP (dinitroanilino-aminomethyl-dipropylamine) to identify acidified locales, fluorescence procedures to provide information about the pHs within certain endocytic compartments, and the uptake of horseradish peroxidase to evaluate effects of altering pH on membrane cycling in the axonal varicosities. We find that the endocytic compartments related to the lysosome-endosome system in the cell bodies and dendrites of these neurons maintain pHs in the range of about 5 to 6.5. This is the range that would be expected for structures participating in lysosomal digestion and for such functions as the endosomal dissociation of ligands from receptors. We also find that, as judged by uptake of horseradish peroxidase, exposure of the preparations to weak bases that neutralize intracellular compartments does not abolish the endocytic labeling of synaptic vesicles in the axonal varicosities. This suggests that passage through a markedly acidified compartment or stage is not obligatory for the endocytic phase of the cycling of synaptic vesicles.

Acids↗
PubMed1992

Inhibition of neutrophil chemokinesis on vitronectin by inhibitors of calcineurin.

Migration of human polymorphonuclear neutrophils on vitronectin is dependent on repeated transient increases in the concentration of intracellular free calcium ([Ca2+]i). A specific peptide inhibitor of the Ca(2+)-calmodulin-dependent phosphatase calcineurin was introduced into the cytoplasm of neutrophils. The peptide inhibited neutrophil migration on vitronectin by interfering with the release of the cells from sites of attachment. A similar reduction in motility on vitronectin occurred when cells were treated with the immunosuppressant FK506, which also inhibits calcineurin when bound to its binding protein, FKBP. These results indicate that a rise in [Ca2+]i reduces integrin-mediated adhesion to vitronectin by a mechanism that requires calcineurin activity.

Amino Acid Sequence↗
PubMed1992

Delivery of ligands from sorting endosomes to late endosomes occurs by maturation of sorting endosomes.

After endocytosis, lysosomally targeted ligands pass through a series of endosomal compartments. The endocytic apparatus that accomplishes this passage may be considered to take one of two forms: (a) a system in which lysosomally targeted ligands pass through preexisting, long-lived early sorting endosomes and are then selectively transported to long-lived late endosomes in carrier vesicles, or (b) a system in which lysosomally targeted ligands are delivered to early sorting endosomes which themselves mature into late endosomes. We have previously shown that sorting endosomes in CHO cells fuse with newly formed endocytic vesicles (Dunn, K. W., T. E. McGraw, and F. R. Maxfield. 1989. J. Cell Biol. 109:3303-3314) and that previously endocytosed ligands lose their accessibility to fusion with a half-time of approximately 8 min (Salzman, N. H., and F. R. Maxfield. 1989. J. Cell Biol. 109:2097-2104). Here we have studied the properties of individual endosomes by digital image analysis to distinguish between the two mechanisms for entry of ligands into late endosomes. We incubated TRVb-1 cells (derived from CHO cells) with diO-LDL followed, after a variable chase, by diI-LDL, and measured the diO content of diI-containing endosomes. As the chase period was lengthened, an increasing percentage of the endosomes containing diO-LDL from the initial incubation had no detectable diI-LDL from the second incubation, but those endosomes that contained both probes showed no decrease in the amount of diO-LDL per endosomes. These results indicate that (a) a pulse of fluorescent LDL is retained by individual sorting endosomes, and (b) with time sorting endosomes lose the ability to fuse with primary endocytic vesicles. These data are inconsistent with a preexisting compartment model which predicts that the concentration of ligand in sorting endosomes will decline during a chase interval, but that the ability of the stable sorting endosome to receive newly endocytosed ligands will remain high. These data are consistent with a maturation mechanism in which the sorting endosome retains and accumulates lysosomally directed ligands until it loses its ability to fuse with newly formed endocytic vesicles and matures into a late endosome. We also find that, as expected according to the maturation model, new sorting endosomes are increasingly labeled during the chase period indicating that new sorting endosomes are continuously formed to replace those that have matured into late endosomes.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1992

Polarized binding of lipoprotein lipase to endothelial cells. Implications for its physiological actions.

Lipoprotein lipase (LPL) that is associated with the luminal surface of capillary endothelial cells hydrolyzes circulating lipoprotein triglyceride molecules. Because LPL is synthesized by cells on the abluminal side of endothelial cells, LPL must contact both the abluminal as well as the luminal sides of the endothelium. To determine whether LPL interacts identically with apical (luminal) and basolateral (abluminal) sides of endothelial cells, we investigated binding, transport, and cellular uptake of LPL presented to each side of bovine aortic endothelial cell monolayers grown on semipermeable filters. When LPL was included in the medium on either the apical or basolateral side of the cells, a similar amount of LPL was found in the medium on the opposite side of the cells. Heat-inactivated LPL crossed the monolayers more rapidly in both directions. When cell surface LPL was assessed, more LPL bound to the apical than the basolateral endothelial cell surface. Release of cell surface-associated LPL was assessed with the use of heparin. Less heparin was required to dissociate apical-surface LPL. When LPL (4 micrograms/ml) was in contact with the apical surface for 1 hour, 32.8 +/- 4.9 ng LPL per 24-mm filter were internalized by the cells. If the LPL was in the basolateral medium, only 6 +/- 1.8 ng LPL were found inside the cells. Heat inactivation decreased LPL binding to cell surfaces and internalization by the cells. LPL interactions with the cells were also studied morphologically by using Texas Red (TR)-labeled LPL and confocal microscopy. More TR-LPL was associated with and internalized by the apical endothelial surface. Incubation of cells with TR-LPL in the basolateral medium led to accumulation of LPL on the apical surface, suggesting that the LPL was transported across the cells. Inclusion of TR-LPL on the apical surface did not lead to appreciable accumulation of LPL on the basolateral cell surface. Therefore, endothelial cells are polarized to accumulate LPL on the apical surface. In addition, more LPL is internalized from this side of the cells. We postulate that the polarity of endothelial cells allows LPL to collect at its physiological site of action, i.e., on the luminal surface.

Animals↗
PubMed1991

Clearance of chylomicron remnants by the low density lipoprotein receptor-related protein/alpha 2-macroglobulin receptor.

The involvement of the low density lipoprotein receptor-related protein (LRP) in chylomicron remnant (CR) catabolism was investigated. Ligand blot analyses demonstrated that beta-very low density lipoproteins (beta-VLDL) incubated with apolipoprotein E (beta-VLDL+E) bound to the LRP and low density lipoprotein receptors, whereas active (receptor-binding) alpha 2-macroglobulin (alpha 2M) bound only to LRP partially purified from rat liver membranes. Iodinated beta-VLDL+E and active alpha 2M showed high affinity binding to the LRP/alpha 2M receptor of low density lipoprotein receptor-negative fibroblasts. The binding and degradation of radiolabeled alpha 2M by these cells were partially inhibited by beta-VLDL+E. Furthermore, alpha 2M interfered with the internalization of beta-VLDL+E and subsequent induction in the cholesterol esterification by these cells. These studies suggested that remnant lipoproteins and active alpha 2M compete for binding to the LRP/alpha 2M receptor. Next, we examined whether the LRP/alpha 2M receptor plays a role, in the presence of low density lipoprotein receptors, in the in vivo catabolism of CR in mice. In vivo studies demonstrated that the unlabeled active, but not the native, alpha 2M partially inhibited the plasma clearance and hepatic uptake of radiolabeled CR or apoE-enriched radiolabled CR. Likewise, apoE-enriched CR retarded the plasma clearance and hepatic uptake of radiolabeled active alpha 2M. These studies provide physiological evidence that the LRP/alpha 2M receptor may function as a CR receptor that removes CR from the plasma.

Animals↗