Richard G.W. Anderson

Monensin Interrupts the Recycling of Low Density Lipoprotein Receptors in Human Fibroblasts

In cultured human fibroblasts, each LDL receptor mediates the internalization of approximately 100 particles of LDL every 20 hr. We provide evidence that this reutilization of LDL receptors involves the recycling of receptors into and out of the cell and that the carboxylic ionophore monensin blocks the return of the receptors to the surface. In the presence of monensin and LDL, 75% of the receptors disappeared from the cell surface within 15 min and more than 90% disappeared within 60 min. The receptors that left the surface were trapped intracellularly within perinuclear vacuoles, as visualized by indirect immunofluorescence with the use of LDL, monensin caused about 50% of the receptors to be trapped intracellularly within 15 min. The receptors that remained on the surface after monensin treatment could be trapped within the cell if LDL was added subsequently in the continued presence of monensin. Monensin did not decrease surface LDL receptors in fibroblasts from a patient (J.D.) with the internalization-defective form of familial hypercholesterolemia. In these mutant cells, LDL receptors are not localized to coated pits. The current data are interpreted to indicate that: in normal fibroblasts about 50% of surface LDL receptors absence of LDL; the remaining 50% of surface receptors can be induced to recycle by the presence of LDL; and monensin interrupts this recycling by preventing the receptor from returning to the surface, thereby causing the receptors to accumulate within the cell.

Depletion of intracellular potassium arrests coated pit formation and receptor-mediated endocytosis in fibroblasts

Depletion of intracellular potassium (K+) caused a marked reduction in the rate of endocytosis of receptor-bound low density lipoprotein (LDL) and epidermal growth factor (EGF) in human fibroblasts. K+ could be depleted slowly by a 3-hr incubation of cells in isotonic K+-free buffer. Rapid K+ depletion was induced by incubation of cells for 5 min with hypotonic medium, followed by transfer to isotonic K+-free buffer. Within 30 min of this treatment, cellular K+ levels fell by more than 60%. When the K+ level fell below a threshold of 40% of normal, the number of coated pits declined by 80% and the rate of endocytosis of 125I-LDL decreased by 70 to 95% despite normal to increased receptor binding. Similar results were obtained with 125I-epidermal growth factor. Addition of KCl to the culture medium up to 2 hr after K+ depletion restored cellular K+ levels and returned endocytosis of 125I-LDL promptly to normal. RbCl was as effective as KCl, but CsCl, LiCl, and (CH3)4NCl had no effect. Restoration by KCl was blocked by ouabain, indicating that uptake via the Na+/K+ ATPase was required. These data demonstrate that depletion of intracellular K+ reversibly arrests coated pit formation and receptor-mediated endocytosis in human fibroblasts.

The J. D. mutation in familial hypercholesterolemia: Amino acid substitution in cytoplasmic domain impedes internalization of LDL receptors

Genomic DNA encompassing the terminal exons of the gene for the low density lipoprotein (LDL) receptor was isolated from J.D., a patient with familial hypercholesterolemia whose receptor fails to cluster in coated pits. The DNA sequence revealed a substitution of a cysteine codon for a tyrosine codon at residue 807 in the cytoplasmic domain of the receptor. We reproduced this substitution through oligonucleotide-directed mutagenesis of the normal human receptor cDNA. Upon transfection into receptor-deficient hamster cells, the cDNA specified a receptor that bound LDL normally, but entered the cell slowly. Electron microscopy showed that this receptor was distributed diffusely over the cell surface, whereas the receptor produced by the normal cDNA was concentrated in coated pits. These results support the hypothesis that cytoplasmic domains direct receptors to coated pits, thereby determining the high rate of receptor internalization in animal cells.

Increase in membrane cholesterol: A possible trigger for degradation of HMG CoA reductase and crystalloid endoplasmic reticulum in UT-1 cells

The crystalloid endoplasmic reticulum (ER) houses large amounts of HMG CoA reductase, the rate-controlling enzyme in cholesterol synthesis. The crystalloid ER appears in UT-1 cells, a line of Chinese hamster ovary cells that has been chronically starved of cholesterol as a result of growth in the presence of compactin, an inhibitor of reductase. When cholesterol was provided to UT-1 cells in the form of low density lipoprotein (LDL), the reductase and crystalloid ER were destroyed. This destruction was preceded by an increase in the cholesterol content of crystalloid ER membranes, as judged by a 4- to 8-fold increase in their ability to form complexes with filipin, a cholesterol-binding compound that can be visualized in freeze-fracture electron micrographs. Filipin binding to other membranes was unchanged. Thus insertion of cholesterol into the crystalloid ER membrane may trigger the degradation of reductase and the membrane itself.

Occurrence of low density lipoprotein receptors within large pits on the surface of human fibroblasts as demonstrated by freeze-etching

Abstract The surface distribution of specific receptor sites for plasma low density lipoprotein (LDL) in cultured human fibroblasts was studied with the technique of freeze-fracturing/deep-etching. Through the use of LDL covalently linked to ferritin as a visual probe, the receptor sites were observed to be concentrated within large pits on the cell surface. These large pits corresponded to the coated pits previously identified as the sites of LDL-ferritin binding by transmission electron microscopy (TEM). On the true surface of the cell, these large pits appeared as shallow depressions of irregular contour. These large pits were also identified within the fracture plane of the membrane where they could be distinguished from a population of smaller pits that corresponded to small flask-shaped invaginations seen in TEM. The membrane that composed the large pits appeared to differ from the remainder of the plasma membrane in that it contained twice the number of intramembrane particles per μm2 of surface and in that these intramembrane particles were of larger mean diameter. The current data lend support to the hypothesis that the large coated pits of the plasma membrane represent discrete regions where the membrane is structurally adapted to carry out the adsorptive endocytosis of receptor-bound macromolecules.

Binding and internalization of 125I-LDL in normal and mutant human fibroblasts. A quantitative autoradiographic study.

Abstract Using a quantitative EM autoradiographic technique, we have visualized the membrane binding and receptor-mediated uptake of low density lipoprotein (LDL) in human fibroblasts. The initial binding was restricted to the plasma membrane (2 h of incubation at 4 °C) and approx. 62% of the grains could be localized to coated pits in the plasma membrane. When the incubations were carried out at 37 °C, 125I radioactivity was found both on the membrane and within the cell and predominantly localized on or within lysosomes. In cells from the patient J. D., a familial hypercholesterolemic homozygote with an internalization defect, initial binding of 125I-LDL was restricted to the plasma membrane but not preferentially localized to coated segments of the plasma membrane. After incubation for 30 min at 37 °C, the membrane bound 125I-LDL in J. D. cells was not internalized. These data confirm results obtained with ferritin-labeled LDL and illustrate the complementary application of two different morphologic probes, each of which offers special advantages for special problems.

Estrogen dependent ciliogenesis in the chick oviduct.

SummaryBoth the hormone dependency and the morphological details of estrogen-dependent ciliogenesis in the shell gland of the chick oviduct were investigated. Ciliogenesis was initiated on day 3 of estrogen treatment, and progressively more cells became differentiated until, on day 10, ∼ 55% ciliation occurred with 17β-estradiol (1 mg/day) and ∼75% ciliation occurred with diethylstilbestrol (1 mg/day). Simultaneous administration of progesterone with diethylstilbestrol (1 mg each/day for 10 days) caused a 50% depression in the number of ciliated cells on day 10. The rate of ciliogenesis was found to be affected by progesterone and the type of estrogen administered. The minimum stimulatory dose of estradiol was found to be between 0.01 mg/day and 0.05 mg/day. Ciliogenic cells were first recognized by the appearance of pro-basal bodies in the apical portion of the cell. Pro-basal body maturation and cilium formation were the same as those described for the chick trachea. Ciliogenesis in the chick was found to be homologous to estrogen-dependent ciliogenesis in various mammalian oviducts.

RECEPTOR-MEDIATED UPTAKE OF MACROMOLECULES AND THEIR DELIVERY TO LYSOSOMES IN HUMAN FIBROBLASTS

Efficient mechanisms exist in human fibroblasts for the cellular uptake and delivery to lysosomes of specific macromolecules. The uptake of a variety of macromolecules, including low-density lipoprotein (the cholesterol transport protein in plasma), transcobalamin II (the vitamin B12 transport protein in plasma), epidermal growth factor (a growth-promoting polypeptide hormone), and lysosomal enzymes, is facilitated by an initial binding of the macromolecule to a distinct class of cell surface receptors. In the case of plasma low-density lipoprotein, the coupling of receptor binding, cellular uptake, and delivery to lysosomes is achieved through the localization of the low-density lipoprotein receptors in coated pits on the cell surface. Invagination of these coated pits occurs with such rapidity that the receptor-bound lipoprotein particle is quickly delivered to lysosomes, where it can be degraded so that its cholesterol component can become available for cellular nutrition.

Receptor-mediated delivery of photoprotective agents by low-density lipoprotein.

Low density lipoprotein (LDL) has been used to deliver toxic molecules to cells by receptor-mediated endocytosis. In these studies, the cholesteryl ester core of LDL was replaced with a lipophilic, toxic molecule. We now report that photoprotective azo dyes can be stably incorporated into LDL, and that this reconstituted LDL protects cells from the photosensitizing action of pyrene methanol (PM) in a receptor-dependent process. The photoprotective action of the azo dye is due to its ability to scavenge singlet oxygen that is produced by the photosensitive agent in response to UV light.

The biogenesis of cell structures and the expression of assembly information

Abstract The assembly of cellular structures is considered to be a linear process that begins with the synthesis of structural molecules. At various points during assembly, additional genetic information may be required for proper assembly. Based on the location of genetic information expression during assembly, structure biogenesis can be grouped into four categories: (1) those which require only information for the synthesis of structural macromolecules; (2) those which require information for the post-translational modification of precursor structural macromolecules; (3) those which require genetic information for the actual assembly step; and (4) those which require information for post-assembly modification of the structure. Examples are given to illustrate and document each of these types of assembly reactions. Further, the usefulness of this scheme for understanding intracellular and extracellular assembly processes is discussed.