Macromolecular dynamics of the cell surface during the formation of coated pits is revealed by fracture-flip

1988 ◽  
Vol 91 (1) ◽  
pp. 161-173
Author(s):  
K. Fujimoto ◽  
P. Pinto da Silva
Keyword(s):  
Cell Surface ◽  
Peripheral Region ◽  
Electron Microscopic ◽  
Coated Pits ◽  
Membrane Surfaces ◽  
Pit Formation ◽  
Sequence Of Events ◽  
Macromolecular Dynamics ◽  
Microscopic Studies ◽  
Free Membrane

We report here the macromolecular dynamics of the cell surface of rat alveolar macrophages during spreading on a substratum, a process that involves the formation of numerous coated pits. We used ‘fracture-flip’ to prepare high-resolution platinum-shadowed replicas of membrane surfaces. Our observations show the following sequence of events associated with coated pit formation: at 4 degree C the cell surface of macrophages is covered with a moderate density of particulate components, with most ranging from 10 to 25 nm in diameter. These particles appear to be randomly distributed over the cell surface. Incubation of adherent cells at 37 degree C for 15 min results in the formation of large loose clusters (area 0.5-4 micron2) of particles on the adherent surfaces. After incubation of macrophages for 30 min at 37 degree C, these clusters become tighter and eventually form circular depressions (200–300 nm in diameter), which we interpret as part of a process of invagination. After 60 min, the depressions become much steeper. At this time surface particles can be observed on the intervening non-invaginated regions, and the peripheral region of the adherent membrane, as well as the free membrane. Fracture-flip reveals the presence of structures undetected in previous electron-microscopic studies and provides ultrastructural evidence for the clustering of surface macromolecules that is involved in the formation of coated pits.

scholarly journals Acidification of the cytosol inhibits endocytosis from coated pits.

1987 ◽  
Vol 105 (2) ◽  
pp. 679-689 ◽  
Author(s):  
K Sandvig ◽  
S Olsnes ◽  
O W Petersen ◽  
B van Deurs
Keyword(s):  
Cell Surface ◽  
Lucifer Yellow ◽  
Fluid Phase ◽  
Endocytic Pathway ◽  
Electron Microscopic ◽  
Coated Pits ◽  
Internal Ph ◽  
Microscopic Studies ◽  
Epidermal Growth ◽  
In Cells

Acidification of the cytosol of a number of different cell lines strongly reduced the endocytic uptake of transferrin and epidermal growth factor. The number of transferrin binding sites at the cell surface was increased in acidified cells. Electron microscopic studies showed that the number of coated pits at the cell surface was not reduced in cells with acidified cytosol. Experiments with transferrin-horseradish peroxidase conjugates and a monoclonal anti-transferrin receptor antibody demonstrated that transferrin receptors were present in approximately 75% of the coated pits both in control cells and in cells with acidified cytosol. The data therefore indicate that the reason for the reduced endocytic uptake of transferrin at internal pH less than 6.5 is an inhibition of the pinching off of coated vesicles. In contrast, acidification of the cytosol had only little effect on the uptake of ricin and the fluid phase marker lucifer yellow. Ricin endocytosed by cells with acidified cytosol exhibited full toxic effect on the cells. Although the pathway of this uptake in acidified cells remains uncertain, some coated pits may still be involved. However, the data are also consistent with the possibility that an alternative endocytic pathway involving smooth (uncoated) pits exists.

scholarly journals Surface charge distribution on the endothelial cell of liver sinusoids.

1984 ◽  
Vol 99 (2) ◽  
pp. 639-647 ◽  
Author(s):  
L Ghitescu ◽  
A Fixman
Keyword(s):  
Endothelial Cell ◽  
Cell Surface ◽  
Liver Sinusoid ◽  
Electron Microscopic ◽  
Coated Pits ◽  
Endothelial Cell Surface ◽  
Liver Sinusoids ◽  
Surface Charge Distribution ◽  
Cationic Residues

The topography of the charged residues on the endothelial cell surface of liver sinusoid capillaries was investigated by using electron microscopic tracers of different size and charge. The tracers used were native ferritin (pl 4.2-4.7) and its cationized (pl 8.4) and anionized (pl 3.7) derivatives, BSA coupled to colloidal gold (pl of the complex 5.1), hemeundecapeptide (pl 4.85), and alcian blue (pl greater than 10). The tracers were either injected in vivo or perfused in situ through the portal vein of the mouse liver. In some experiments, two tracers of opposite charge were sequentially perfused with extensive washing in between. The liver was processed for electron microscopy and the binding pattern of the injected markers was recorded. The electrostatic nature of the tracer binding was assessed by perfusion with high ionic strength solutions, by aldehyde quenching of the plasma membrane basic residues, and by substituting the cell surface acidic moieties with positively charged groups. Results indicate that the endothelial cells of the liver sinusoids expose on their surface both cationic and anionic residues. The density distribution of these charged groups on the cell surface is different. While the negative charge is randomly and patchily scattered all over the membrane, the cationic residues seem to be accumulated in coated pits. The charged groups co-exist in the same coated pit and bind the opposite charged macromolecule. It appears that the fixed positive and negative charges of the coated pit glycocalyx are mainly segregated in space. The layer of basic residues is located at 20-30-nm distance of the membrane, while most of the negative charges lie close to the external leaflet of the plasmalemma.

The effects of cytochalasin D and phorbol myristate acetate on the apical endocytosis of ricin in polarised Caco-2 cells

1996 ◽  
Vol 109 (12) ◽  
pp. 2927-2935 ◽  
Author(s):  
W. Shurety ◽  
N.A. Bright ◽  
J.P. Luzio
Keyword(s):  
Cell Surface ◽  
Apical Cell ◽  
Microscopic Investigation ◽  
Electron Microscopic Investigation ◽  
Cytochalasin D ◽  
Apical Surface ◽  
Electron Microscopic ◽  
Coated Pits ◽  
Acid Media ◽  
Kinase C

Apical endocytosis of 125I-ricin in Caco-2 cells was inhibited > 95% by hypertonic and/or acid media, consistent with the major uptake route being clathrin-mediated. The presence of apical cell surface bound ricin-gold in clathrin coated pits and vesicles was observed by electron microscopy. An electron microscopic investigation in which ricin-gold bound to the apical surface was quantitated, showed that cytochalasin D, which inhibits apical but not basolateral endocytosis, prevented movement of ricin-gold along the microvillar surface. This was consistent with an actin bound mechanochemical motor within microvilli driving the movement of membranous components towards the cell body. Cytochalasin D also caused an increase in the number of coated pits observed at the apical cell surface relative to the number observed in untreated cells. Stimulation of apical endocytosis of ricin by phorbol 12-myristate 13-acetate showed the characteristics of being mediated by protein kinase C, was not due to an effect on ricin movement along the microvillar surface, and may be explained by increases in formation and pinching off of clathrin coated pits at the apical cell surface.

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 The interaction of 125I-insulin with cultured 3T3-L1 adipocytes: quantitative analysis by the hypothetical grain method.

1983 ◽  
Vol 31 (7) ◽  
pp. 859-870 ◽  
Author(s):  
J Y Fan ◽  
J L Carpentier ◽  
E Van Obberghen ◽  
N M Blackett ◽  
C Grunfeld ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Multivesicular Bodies ◽  
Electron Microscopic ◽  
Coated Pits ◽  
Sequence Of Events ◽  
Villous Surface ◽  
Preferential Binding ◽  
Em Autoradiography ◽  
Typical Distribution ◽  
Morphologic Analysis

The murine 3T3-L1 fibroblast under appropriate incubation conditions differentiates into an adipocyte phenotype. This 3T3-L1 adipocyte exhibits many of the morphologic, biochemical, and insulin-responsive features of the normal rodent adipocyte. Using quantitative electron microscopic (EM) autoradiography we find that, when 125I-insulin is incubated with 3T3-L1 adipocytes, the ligand at early times of incubation localizes to the plasma membrane of the cell preferentially to microvilli and coated pits. When the incubation is continued at 37 degrees C, 125I-insulin is internalized by the cells and preferential binding to the villous surface is lost. With the internalization of the ligand, two intracellular structures become labeled, as determined by the method of hypothetical grain analysis. These include large clear, presumably endocytotic, vesicles and multivesicular bodies. Over the first hour of incubation the labeling of these structures increases in parallel, but in the second hour they diverge: the labeling of multivesicular bodies and other lysosomal forms continuing to increase and the labeling of large clear vesicles decreasing. At 3 hours limited but significant labeling occurs in small Golgi-related vesicles that have the typical distribution of GERL. The distinct morphologic features of this cell make it ideal for a quantitative morphologic analysis and allow for an unambiguous view of the sequence of events involved in receptor-mediated endocytosis of a polypeptide hormone. These events are likely to be representative of the processing of insulin by the mature rodent adipocyte.

Cell-cell channel formation and lectins

1991 ◽  
Vol 261 (6) ◽  
pp. C1025-C1032 ◽  
Author(s):  
E. Levine ◽  
R. Werner ◽  
G. Dahl
Keyword(s):  
Membrane Surface ◽  
Significant Inhibition ◽  
Carbohydrate Binding ◽  
Binding Affinities ◽  
Channel Formation ◽  
Electron Microscopic ◽  
Lectin Receptors ◽  
Microscopic Studies ◽  
Free Membrane ◽  
Cell Cell

The oocyte cell-cell channel assay was used to investigate determinants of the rate of channel formation. After injection of connexin-specific mRNA, oocytes accumulate a pool of precursors from which cell-cell channels can form after oocytes are paired. Channel formation was found to be increased if oocytes are pretreated with lectins before pairing. Several lectins differing in their carbohydrate binding affinities can exert this effect. Lectin-specific sugars suppress the effect on cell-cell channel formation only if the sugar is mixed with the lectin before application to the oocyte. If the lectin is first bound to the oocyte and then the sugar is added, no significant inhibition is seen. The promotion of channel formation by lectins is enhanced by adding an incubation period in regular medium after lectin treatment, before pairing of the oocytes. Electron microscopic studies with gold-conjugated lectins show that the lectin receptors are clustered on the free membrane surface and are taken up in endocytotic vesicles. These data suggest that the observed acceleration of cell-cell channel formation by lectins can be attributed to the removal of steric hindrance, which is a consequence of clustering of the bulky glycoprotein lectin receptors as well as of the removal from the surface by endocytosis.

scholarly journals Comparison of rapid changes in surface morphology and coated pit formation of PC12 cells in response to nerve growth factor, epidermal growth factor, and dibutyryl cyclic AMP.

1984 ◽  
Vol 98 (2) ◽  
pp. 457-465 ◽  
Author(s):  
J L Connolly ◽  
S A Green ◽  
L A Greene
Keyword(s):  
Nerve Growth Factor ◽  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Cyclic Amp ◽  
Cell Surface ◽  
Dibutyryl Cyclic Amp ◽  
Coated Pits ◽  
Pit Formation ◽  
Epidermal Growth ◽  
Surface Changes

Scanning and transmission electron microscopic studies were carried out on the rapid cell surface response of PC12 pheochromocytoma cells to treatment with nerve growth factor (NGF), epidermal growth factor (EGF), and dibutyryl cyclic AMP. EGF induced a rapidly initiated series of surface changes identical to those previously observed with NGF. Ruffles appear over the dorsal surface of the cells by 30 s, are prominent at 3 min, and are absent by 7 min. Microvilli disappear as dorsal ruffles become prominent. Peripheral ruffles are seen by 3 min, are prominent on most of the cells by 7 min, and are virtually absent by 15 min. Large blebs are present on 50% of the cells by 2 h and are markedly decreased by 4 h. Within 30 s after NGF or EGF addition, an increase in the density of 60-130-nm coated pits per unit membrane is detectable. This reaches a maximum of two- to threefold in from 1 to 3 min and gradually decreases. Combined treatment with NGF and EGF increases surface ruffling and, after an early peak in coated pits which at 3 min is similar in magnitude to that observed for the separately administered factors, maintains a greater number of pits per unit area than either treatment alone. 3-d pretreatment with NGF greatly reduces the response of the cells to EGF both with respect to surface ruffling and coated pit formation while 4-h NGF pretreatment has no effect on the EGF response. Dibutyryl cyclic AMP induced none of the rapidly onsetting changes caused by NGF or EGF, and therefore it seems unlikely that cyclic AMP mediates these surface changes. Changes in cell surface architecture induced by NGF and EGF on PC12 cells and by NGF in normal sympathetic neurons (as previously described) indicates that such responses may be a widespread phenomenon associated with the interaction of at least some peptide growth factors/hormones with their receptors. These responses may represent or reflect primary events in the mechanism by which these factors act.

scholarly journals Pit formation and rapid changes in surface morphology of sympathetic neurons in response to nerve growth factor.

1981 ◽  
Vol 90 (1) ◽  
pp. 176-180 ◽  
Author(s):  
J L Connolly ◽  
S A Green ◽  
L A Greene
Keyword(s):  
Nerve Growth Factor ◽  
Growth Factor ◽  
Cell Surface ◽  
Sympathetic Neurons ◽  
Cell Types ◽  
Coated Pits ◽  
Nerve Growth ◽  
Pheochromocytoma Cells ◽  
Pit Formation ◽  
Transmission Electron

Scanning and transmission electron microscope studies were carried out on the rapid cell surface responses of cultured newborn rat sympathetic neurons to nerve growth factor (NGF), a substance that promotes their survival and differentiation. The somas of sympathetic neurons continuously exposed to NGF or deprived of the factor for 4-5 h have a very smooth surface. After readdition of NGF to the latter type of cultures, there is rapidly initiated a transient, sequential change in the cell surface. Microvilli and small ruffles appear within 30 s and are most prominent by 1 min. By 3 min of exposure, the microvilli and ruffles decrease in prominence, and by 7 min the somal surface is again smooth. By 30 s after NGF readdition, as increase in the number of 60- tp 130-nm coated pits is also detectable. This increase reaches a maximum of about threefold from 0.5 to 3 min and then gradually decreases. Alterations in the surface did not occur on the nonneuronal cell types present in the cultures and were not observed in response to another basic protein (cytochrome c) or to physical manipulation. Changes in cell surface architecture induced by NGF in normal sympathetic neurons and, as previously described, in PC12 pheochromocytoma cells indicate that such responses may present or reflect primary events in the mechanism of the factor's action.

scholarly journals Simultaneous visualization of LDL receptor distribution and clathrin lattices on membranes torn from the upper surface of cultured cells.

1991 ◽  
Vol 39 (8) ◽  
pp. 1017-1024 ◽  
Author(s):  
D A Sanan ◽  
R G Anderson
Keyword(s):  
Electron Microscope ◽  
Cell Surface ◽  
Cultured Cells ◽  
Membrane Filter ◽  
Membrane Surface ◽  
Cellulose Acetate Membrane ◽  
Electron Microscopic ◽  
Coated Pits ◽  
Extensive Experience ◽  
Simultaneous Visualization

We have developed a method for simultaneous visualization by electron microscopy of both the distribution of cell surface receptors and architectural features of the inner membrane surface, such as clathrin-coated pits. Electron microscope grids were covered with formvar and coated with poly-L-lysine. These grids were then placed on a piece of buffer-impregnated cellulose acetate membrane filter maintained at 4 degrees C on an ice bath. Cells of interest were grown on glass coverslips and incubated with either a ligand-gold or an antibody-gold conjugate specific for the membrane determinant of interest. The coverslip with gold-labeled cells was then overlaid on the grids and pressure was applied. When the grid was removed, large areas of the upper cell surface, which had labeled determinants, remained adherent to the formvar support. With the proper staining, both the gold particles and internal membrane features could be seen at the same time in the electron microscope. This method is rapid, does not require extensive experience with electron microscopic technique, and permits viewing of membrane samples that are large enough to perform quantitative analysis of gold distribution in relation to membrane specializations.

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