scholarly journals Detection of membrane cholesterol by filipin in isolated rat liver coated vesicles is dependent upon removal of the clathrin coat.

1984 ◽  
Vol 99 (1) ◽  
pp. 315-319 ◽  
Author(s):  
C J Steer ◽  
M Bisher ◽  
R Blumenthal ◽  
A C Steven
Keyword(s):  
Rat Liver ◽  
Plasma Membranes ◽  
Cholesterol Content ◽  
Coated Vesicles ◽  
Coated Vesicle ◽  
Electron Microscopic ◽  
Coated Pits ◽  
Phospholipid Ratio ◽  
Thin Section Analysis

We investigated the cholesterol content of highly purified populations of coated vesicles from rat liver by biochemical quantitation and by cytochemical electron microscopy using the polyene antibiotic filipin. Failure of this reagent to elicit its typical response for a cholesterol-containing membrane, i.e., a characteristically corrugated or rippled appearance by thin section analysis, had led to the hypothesis (Montesano, R., A. Perrelet, P. Vassalli, and L. Orci, 1979, Proc. Natl. Acad. Sci. USA., 76:6391-6395) that cholesterol is specifically excluded from the plasma membrane domains associated with coated pit regions. The present electron microscopic results showed that although the response of coated vesicle membranes to filipin was also negative, uncoated vesicles whose clathrin coats had been removed in vitro exhibited a strong filipin-positive response. Quantitated biochemically, the cholesterol-to-phospholipid ratio of the coated vesicles was found to be indistinguishable from that of control preparations of plasma membranes isolated from rat liver. Taken together, the results indicate that the filipin-negative response of coated vesicles (and probably also that of coated pits) is due not to abnormally low cholesterol content, but rather to the stabilizing influence of their enveloping clathrin coats which inhibit the characteristic structural expression of the filipin-cholesterol complexes.

scholarly journals Light and electron microscopic immunocytochemical localization of clathrin in rat cerebellum and kidney.

1982 ◽  
Vol 30 (9) ◽  
pp. 853-863 ◽  
Author(s):  
C T Lin ◽  
J Garbern ◽  
J Y Wu
Keyword(s):  
Rat Kidney ◽  
Multivesicular Body ◽  
Coated Vesicles ◽  
Coated Vesicle ◽  
Ground Substance ◽  
Synaptic Membrane ◽  
Reaction Products ◽  
Electron Microscopic ◽  
Coated Pits ◽  
Autophagic Vacuoles

The precise cellular and subcellular locations of coated vesicle protein, clathrin, in rat kidney and cerebellum have been visualized by immunocytochemical techniques. In the renal tubular epithelia, clathrin-positive products were found on both free ribosomes and on those attached to rough endoplasmic reticulum (RER) and the nuclear envelope. No clathrin was observed in the cisternae of RER or the Golgi apparatus. Clathrin-positive reaction products could also be seen on coated pits, coated vesicles, Golgi-associated vesicles, basolateral cell membrane, the ground substance, and in the autophagic vacuoles. In cerebellar Purkinje and granule cell bodies, reaction products were seen localized on coated vesicles, on the budding areas from the Golgi-associated membrane and Golgi-associated vesicles. Furthermore, the membrane of the multivesicular body, the bound-ribosomes, and the ground substance were also stained. In the myelinated axon, the clathrin appeared to be concentrated on certain segments and seemed to fill in the space between neurotubules and some vesicles. In certain synaptic terminals clathrin was often seen attached to presynaptic vesicles, presynaptic membrane, and post-synaptic membrane. However, in most mossy fibers, some synaptic vesicles were not stained. These observations suggest that clathrin is synthesized on bound and free ribosomes and discharged into the cytosol where it becomes associated with a variety of ground substances and assembles on coated pits, coated vesicles, Golgi-associated vesicles, presynaptic vesicles, and pre- and postsynaptic membranes. Clathrin may be finally degraded in autophagic vacuoles.

scholarly journals The Role of Dynamin and Its Binding Partners in Coated Pit Invagination and Scission

2001 ◽  
Vol 152 (2) ◽  
pp. 309-324 ◽  
Author(s):  
Elaine Hill ◽  
Jeroen van der Kaay ◽  
C. Peter Downes ◽  
Elizabeth Smythe
Keyword(s):  
Plasma Membrane ◽  
Sh3 Domain ◽  
Coated Vesicles ◽  
Coated Vesicle ◽  
Vesicle Formation ◽  
Coated Pits ◽  
Binding Partners ◽  
Late Stages

Plasma membrane clathrin-coated vesicles form after the directed assembly of clathrin and the adaptor complex, AP2, from the cytosol onto the membrane. In addition to these structural components, several other proteins have been implicated in clathrin-coated vesicle formation. These include the large molecular weight GTPase, dynamin, and several Src homology 3 (SH3) domain–containing proteins which bind to dynamin via interactions with its COOH-terminal proline/arginine-rich domain (PRD). To understand the mechanism of coated vesicle formation, it is essential to determine the hierarchy by which individual components are targeted to and act in coated pit assembly, invagination, and scission. To address the role of dynamin and its binding partners in the early stages of endocytosis, we have used well-established in vitro assays for the late stages of coated pit invagination and coated vesicle scission. Dynamin has previously been shown to have a role in scission of coated vesicles. We show that dynamin is also required for the late stages of invagination of clathrin-coated pits. Furthermore, dynamin must bind and hydrolyze GTP for its role in sequestering ligand into deeply invaginated coated pits. We also demonstrate that the SH3 domain of endophilin, which binds both synaptojanin and dynamin, inhibits both late stages of invagination and also scission in vitro. This inhibition results from a reduction in phosphoinositide 4,5-bisphosphate levels which causes dissociation of AP2, clathrin, and dynamin from the plasma membrane. The dramatic effects of the SH3 domain of endophilin led us to propose a model for the temporal order of addition of endophilin and its binding partner synaptojanin in the coated vesicle cycle.

scholarly journals Reconstitution of clathrin-coated pit budding from plasma membranes.

1991 ◽  
Vol 114 (5) ◽  
pp. 881-891 ◽  
Author(s):  
H C Lin ◽  
M S Moore ◽  
D A Sanan ◽  
R G Anderson
Keyword(s):  
Plasma Membranes ◽  
Ldl Receptor ◽  
Coated Vesicle ◽  
Coated Pits ◽  
Receptor Mediated Endocytosis ◽  
Gold Label ◽  
Cytosolic Factors

Receptor-mediated endocytosis begins with the binding of ligand to receptors in clathrin-coated pits followed by the budding of the pits away from the membrane. We have successfully reconstituted this sequence in vitro. Highly purified plasma membranes labeled with gold were obtained by incubating cells in the presence of anti-LDL receptor IgG-gold at 4 degrees C, attaching the labeled cells to a poly-L-lysine-coated substratum at 4 degrees C and then gently sonicating them to remove everything except the adherent membrane. Initially the gold label was clustered over flat, clathrin-coated pits. After these membranes were warmed to 37 degrees C for 5-10 min in the presence of buffer that contained cytosol extract, Ca2+, and ATP, the coated pits rounded up and budded from the membrane, leaving behind a membrane that was devoid of LDL gold. Simultaneous with the loss of the ligand, the clathrin triskelion and the AP-2 subunits of the coated pit were also lost. These results suggest that the budding of a coated pit to form a coated vesicle occurs in two steps: (a) the spontaneous rounding of the flat lattice into a highly invaginated coated pit at 37 degrees C; (b) the ATP, 150 microM Ca2+, and cytosolic factors(s) dependent fusion of the adjoining membrane segments at the neck of the invaginated pit.

Direct measurement of clathrin-coated vesicle formation using a cell-free assay

1997 ◽  
Vol 110 (24) ◽  
pp. 3105-3115 ◽  
Author(s):  
A. Gilbert ◽  
J.P. Paccaud ◽  
J.L. Carpentier
Keyword(s):  
Direct Measurement ◽  
Plasma Membranes ◽  
Cultured Cells ◽  
Significant Proportion ◽  
Coated Vesicles ◽  
Coated Vesicle ◽  
Vesicle Formation ◽  
Coated Pits ◽  
Direct Estimate ◽  
Free System

Factors controlling the last stages of clathrin-coated vesicle formation were investigated using an assay allowing direct measurement of the detachment of these vesicles from the plasma membrane. Plasma membranes from cultured cells surface-labelled with 125I-alpha2-macroglobulin (a ligand that preferentially associates with clathrin-coated pits) were isolated by sonication of cells attached to a poly-L-lysine-coated substratum and incubated in the presence of nucleotide(s) +/− cytosol. A significant proportion of the membrane-associated radioactivity was released into the incubation medium in sedimentable form (14x10(6)g). The nucleotide and ligand specificities of this process together with the results of a series of biochemical, morphological and gradient analyses, led to the conclusion that measurement of the released sedimentable radioactivity provides a direct estimate of the formation of clathrin-coated vesicles from clathrin-coated pits. A morphological analysis of quick-frozen replicas of these membranes indicated that only the last stages of clathrin-coated vesicle formation were studied in the assay. Taking advantage of this cell-free system, we demonstrate that membrane-associated cytosolic factors and GTP-binding proteins, noteably dynamin, play a crucial role. Moreover, although these events can occur in the absence of ATP and Ca2+, optimal conditions for the formation of clathrin-coated vesicles require the presence of ATP, GTP and cytosol.

scholarly journals Myosin VI: cellular functions and motor properties

2004 ◽  
Vol 359 (1452) ◽  
pp. 1931-1944 ◽  
Author(s):  
K. C. Holmes ◽  
D. R. Trentham ◽  
R. Simmons ◽  
Rhys Roberts ◽  
Ida Lister ◽  
...  
Keyword(s):  
Optical Tweezers ◽  
Leading Edge ◽  
Force Transducer ◽  
Myosin Vi ◽  
Cellular Functions ◽  
Electron Microscopic ◽  
Coated Pits ◽  
Microscopic Studies ◽  
Golgi Network

Myosin VI has been localized in membrane ruffles at the leading edge of cells, at the trans–Golgi network compartment of the Golgi complex and in clathrin–coated pits or vesicles, indicating that it functions in a wide variety of intracellular processes. Myosin VI moves along actin filaments towards their minus end, which is the opposite direction to all of the other myosins so far studied (to our knowledge), and is therefore thought to have unique properties and functions. To investigate the cellular roles of myosin VI, we identified various myosin VI binding partners and are currently characterizing their interactions within the cell. As an alternative approach, we have expressed and purified full–length myosin VI and studied its in vitro properties. Previous studies assumed that myosin VI was a dimer, but our biochemical, biophysical and electron microscopic studies reveal that myosin VI can exist as a stable monomer. We observed, using an optical tweezers force transducer, that monomeric myosin VI is a non–processive motor which, despite a relatively short lever arm, generates a large working stroke of 18 nm. Whether monomer and/or dimer forms of myosin VI exist in cells and their possible functions will be discussed.

scholarly journals Platelet Satellitism

Blood ◽  
1974 ◽  
Vol 43 (6) ◽  
pp. 831-836 ◽  
Author(s):  
Carl R. Kjeldsberg ◽  
John Swanson
Keyword(s):  
Polymorphonuclear Leukocytes ◽  
Plasma Membranes ◽  
Clinical Importance ◽  
Electron Microscopic ◽  
Normal Platelet ◽  
Microscopic Studies ◽  
Platelet Satellitism ◽  
Platelet Functions

Abstract Platelet adherence to polymorphonuclear leukocytes, or so-called platelet satellitism, has, to our knowledge, been reported in only four patients. We had the opportunity to study this phenomenon in two patients. Platelet satellitism was only seen in EDTA anticoagulated blood, and the platelets were seen to surround polymorphonuclear leukocytes only. Electron microscopic studies demonstrated focally opposed regions of platelet and neutrophil plasma membranes. Phagocytosis of platelets was also observed. In vivo and in vitro platelet functions were normal. Platelet satellitism is an in vitro phenomenon, the cause of which is unknown. We are unable to relate it to functional abnormalitles of the blood, the clinical condition of the patient, or to drugs. This phenomenon has some clinical importance in that it causes spurious thrombocytopenia.

scholarly journals Gangliosides and sialosylglycoproteins in coated vesicles from bovine brain

1985 ◽  
Vol 225 (3) ◽  
pp. 713-721 ◽  
Author(s):  
D Gravotta ◽  
H J F Maccioni
Keyword(s):  
Rat Brain ◽  
Plasma Membranes ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Bovine Brain ◽  
Coated Vesicles ◽  
Molar Ratio ◽  
Coated Vesicle ◽  
Synaptic Plasma Membranes ◽  
Morphological Criteria

The content of gangliosides and sialosylglycoproteins was investigated in a coated-vesicle-enriched fraction prepared from bovine brain by the method of Pearse [(1975) J. Mol. Biol. 97, 93-98] and further purified by g.p.c. (glass-permeation chromatography) [Pfeffer & Kelly (1981) J. Cell Biol. 91, 385-391]. From morphological criteria and from the analysis of the polypeptide pattern on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis the coated-vesicle fraction (CV-fraction) appeared more than 95% pure. The ganglioside-NeuAc (N-acetylneuraminate), glycoprotein-NeuAc, phospholipid and cholesterol contents of CV-fraction were compared with those of bovine brain synaptic plasma membranes (SPM). The cholesterol to phospholipid molar ratio was 0.47 +/- 0.07 in CV-fraction and 1.06 +/- 0.08 in SPM. The ganglioside-NeuAc and glycoprotein-NeuAc to phospholipid molar ratios were 0.047 and 0.020 respectively in CV-fraction and 0.039 and 0.016 respectively in SPM. The (Na+ + K+)-dependent ATPase activity sensitive to ouabain (in mumol of Pi/h per nmol of phospholipid) was 1.04 in CV-fraction and 0.63 in SPM; the ratio between this activity and the activity resistant to ouabain was 2 in CV-fraction and 1.4 in SPM. A t.l.c. analysis of the ganglioside fractions showed that most of the ganglioside species present in SPM were present in CV-fraction. In a rat brain coated-vesicle preparation not subjected to g.p.c., the activities [as sugar-radioactivity (c.p.m.) transferred/h per mumol of phospholipid] of the enzymes CMP-NeuAc:sialosyl-lactosylceramide (GM3) sialosyl-, UDP-Gal:N-acetylgalactosaminyl(sialosyl)lactosylceramide (GM2) galactosyl- and UDP-GalNAc:sialosyl-lactosylceramide (GM3) N-acetylgalactosaminyl-transferases, which were considered Golgi-apparatus markers, were about 19, 16 and 10% respectively of those determined in rat brain neuronal perikaryon-enriched fractions. Taken together, the results indicate that most of the major gangliosides are constituents of coated vesicles.

Multivalent interaction between asialofetuin and plasma membrane preparations from the rat liver

1980 ◽  
Vol 58 (12) ◽  
pp. 1414-1420 ◽  
Author(s):  
Maria T. Debanne ◽  
Erwin Regoeczi ◽  
Mark W. C. Hatton
Keyword(s):  
Rat Liver ◽  
Plasma Membranes ◽  
Binding Capacity ◽  
Theoretical Models ◽  
Underlying Mechanism ◽  
Marker Enzyme ◽  
Electron Microscopic ◽  
Microscopic Appearance ◽  
Wide Range ◽  
Electron Microscopic Appearance

Binding of bovine asialofetuin by rat liver plasma membranes was studied using different techniques for the separation of the free and bound forms of the glycoprotein and also different approaches to measure nonspecific binding. The membrane preparations had the electron microscopic appearance of a mixture of lamellae and vesicles and their lipid:protein ratios and marker enzyme profiles fell within the range of values available from the literature. The binding capacity was approximately 15 pmol of asialofetuin per milligram of membrane protein.Scatchard plots of the values obtained over a wide range of concentrations (4.8–12.6 μg asialofetuin per 30 μg membrane protein) after incubation at 22 °C showed pronounced non-linearity which, in combination with evaluations according to other theoretical models, was referable to heterogeneity of binding. In sharp contrast, after incubation at 4 °C the Scatchard plot was linear. This difference is interpreted as the expression of a functional, rather than a chemical, heterogeneity in asialofetuin binding. The underlying mechanism is thought to be competition of galactose groups for binding sites with the result that the number of bonds varies between the galactose groups of a bound asialofetuin molecule and the hepatic lectin, depending on the concentration of the glycoprotein in the incubation mixture.

scholarly journals EFFECT OF ACTIVE ACCUMULATION OF CALCIUM AND PHOSPHATE IONS ON THE STRUCTURE OF RAT LIVER MITOCHONDRIA

1964 ◽  
Vol 23 (1) ◽  
pp. 21-38 ◽  
Author(s):  
John W. Greenawalt ◽  
Carlo S. Rossi ◽  
Albert L. Lehninger
Keyword(s):  
Specific Gravity ◽  
Rat Liver ◽  
Cesium Chloride ◽  
Liver Mitochondria ◽  
Rat Liver Mitochondria ◽  
Electron Microscopic ◽  
Dense Granules ◽  
Phosphate Ions ◽  
Microscopic Studies

Rat liver mitochondria allowed to accumulate maximal amounts of Ca++ and HPO4= ions from the suspending medium in vitro during respiration have a considerably higher specific gravity than normal mitochondria and may be easily separated from the latter by isopycnic centrifugation in density gradients of sucrose or cesium chloride. When the mitochondria are allowed to accumulate less than maximal amounts of Ca++ and HPO4= from the medium, they have intermediate specific gravities which are roughly proportional to their content of calcium phosphate. Maximally "loaded" mitochondria are relatively homogeneous with respect to specific gravity. Correlated biochemical and electron microscopic studies show that Ca++-loaded mitochondria contain numerous dense granules, of which some 85 per cent are over 500 A in diameter. These granules are electron-opaque not only following fixation and staining with heavy metal reagents, but also following fixation with formaldehyde, demonstrating that the characteristic granules in Ca++-loaded mitochondria have intrinsic electron-opacity. The dense granules are almost always located within the inner compartment of the mitochondria and not in the space between the inner and outer membranes. They are frequently located at or near the cristae and they often show electron-transparent "cores." Such granules appear to be made up of clusters of smaller dense particles, but preliminary x-ray diffraction analysis and electron diffraction studies have revealed no evidence of crystallinity in the deposits. The electron-opaque granules decrease in number when the Ca++-loaded mitochondria are incubated with 2,4-dinitrophenol; simultaneously there is discharge of Ca++ and phosphate from the mitochondria into the medium.

scholarly journals Immunofluorescent localization of 100K coated vesicle proteins.

1986 ◽  
Vol 102 (1) ◽  
pp. 48-54 ◽  
Author(s):  
M S Robinson ◽  
B M Pearse
Keyword(s):  
Plasma Membrane ◽  
Golgi Apparatus ◽  
Limited Proteolysis ◽  
Bovine Brain ◽  
Coated Vesicles ◽  
Cell Protein ◽  
Coated Vesicle ◽  
Coated Pits ◽  
Double Labeling ◽  
Vesicle Proteins

A family of coated vesicle proteins, with molecular weights of approximately 100,000 and designated 100K, has been implicated in both coat assembly and the attachment of clathrin to the vesicle membrane. These proteins were purified from extracts of bovine brain coated vesicles by gel filtration, hydroxylapatite chromatography, and preparative SDS PAGE. Peptide mapping by limited proteolysis indicated that the polypeptides making up the three major 100K bands have distinct amino acid sequences. When four rats were immunized with total 100K protein, each rat responded differently to the different bands, although all four antisera cross-reacted with the 100K proteins of human placental coated vesicles. After affinity purification, two of the antisera were able to detect a 100K band on blots of whole 3T3 cell protein and were used for immunofluorescence, double labeling the cells with either rabbit anti-clathrin or with wheat germ lectin as a Golgi apparatus marker. Both antisera gave staining that was coincident with anti-clathrin, with punctate labeling of the plasma membrane and perinuclear Golgi apparatus labeling. Thus, the 100K proteins are present on endocytic as well as Golgi-derived coated pits and vesicles. The punctate patterns were nearly identical with anti-100K and anti-clathrin, indicating that when vesicles become uncoated, the 100K proteins are removed as well as clathrin. One of the two antisera gave stronger plasma membrane labeling than Golgi apparatus labeling when compared with the anti-clathrin antiserum. The other antiserum gave stronger Golgi apparatus labeling. Although we have as yet no evidence that these two antisera label different proteins on blots of 3T3 cells, they do show differences on blots of bovine brain 100K proteins. This result, although preliminary, raises the possibility that different 100K proteins may be associated with different pathways of membrane traffic.

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