Variability of the topography of low-density lipoprotein (LDL) receptors in the plasma membrane of cultured human skin fibroblasts as revealed by gold-LDL conjugates in conjunction with the surface replication technique

Robenek, Horst; Hesz, A; Rassat, J

doi:10.1016/s0022-5320(83)90049-7

Cited by 19 publications

(12 citation statements)

References 29 publications

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“…The values in Table 1 are consistent with, although at the upper end of, the electron micoscopy data Most electron microscopy experiments indicate that recently inserted LDL receptors are randomized in the plasma membrane. Robenek and co-workers have presented conflicting evidence that indicates that LDL receptors are inserted into the plasma membrane as clusters that are not dispersed before their entrapment in coated pits (Robenek et al-, 1991; Robenek and Hesz, 1983). Our data, which indicate a high proportion of single LDL particles, support the random distribution model.…”

Section: Nm Diameter Particle) a Calcu-supporting

confidence: 73%

Analysis of receptor clustering on cell surfaces by imaging fluorescent particles

Morrison¹,

Anderson²,

Georgiou³

et al. 1994

Biophysical Journal

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Fluorescently labeled low density lipoproteins (LDL) and influenza virus particles were bound to the surface of human fibroblasts and imaged with a cooled slow-scan CCD camera attached to a fluorescence microscope. Particles were also imaged after attachment to polylysine-coated microscope slides. The digital images were analyzed by fitting data points in the region of fluorescent spots by a two-dimensional Gaussian function, thus obtaining a measure of spot intensity with correction for local background. The intensity distributions for particles bound to polylysine slides were mainly accounted for by particle size distributions as determined by electron microscopy. In the case of LDL, the intensity distributions for particles bound to fibroblasts were considerably broadened, indicative of clustering. The on-cell intensity distributions were deconvolved into 1-particle, 2-particle, 3-particle, etc. components using the data obtained with LDL bound to polylysine-coated slides as an empirical measure of the single particle intensity distribution. This procedure yielded a reasonably accurate measure of the proportion of single particles, but large errors were encountered in the proportions of larger cluster sizes. The possibility of studying the dynamics of clustering was investigated by binding LDL to cells at 4 degrees C and observing changes in the intensity distribution with time after warming to 20 degrees C.

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Section: Nm Diameter Particle) a Calcu-supporting

confidence: 73%

Analysis of receptor clustering on cell surfaces by imaging fluorescent particles

Morrison¹,

Anderson²,

Georgiou³

et al. 1994

Biophysical Journal

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“…Since the LDL receptor diffuses much more slowly than most other receptors, random diffusion as the mechanism for receptors getting to coated pits can explain the observed rapid rates of receptormediated endocytosis. We cannot rule out convective flows of receptors to coated pits or preferential inserting of receptors near coated pits; indeed there is recent evidence for the latter process (Robenck and Hesz, 1983), but random diffusion. suffices to explain the observed rates of capture of receptors by coated pits.…”

Section: The Interaction Of Receptors With Coated Pitsmentioning

confidence: 80%

Mathematical Modeling of Receptor-Mediated Endocytosis

et al. 1985

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“…Drying of cultured fibroblasts (Robenek et al, 1983), macrophages (Robenek et al, 1984) and platelets (Isenberg et al, 1987) using the critical point method with the subsequent preparation of surface replicas has permitted the visualization of a number of cell surface proteins over extended areas of plasma membrane surfaces using the transmission electron microscope. To determine whether activation of endothelial cells might cause changes in the surface distribution of PAI-1 not appreciated in conventional thin sections, we studied surface replicas of immunostained unstimulated and TNFct-stimulated endothelial cells.…”

Section: Surface Replicasmentioning

confidence: 99%

Immunoelectron microscopic localization of type 1 plasminogen activator inhibitor on the surface of activated endothelial cells.

Schleef

Loskutoff

Podor

1991

The Journal of Cell Biology

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Abstract. Immunogold EM was employed to compare the distribution of type 1 plasminogen activator inhibitor (PAId) on the surface of agonist-activated human umbilical vein endothelial cells (HUVECs) with that of control, unactivated cells. As previously observed, (Schleef, R. R., T. J. Podor, E. Durme, J. Mimuro, and D. J. , analysis of cross-sections of nonpermeabilized control HUVEC monolayers stained first with affinity-purified rabbit antibodies to PAId and then with goldconjugated goat anti-rabbit IgG, revealed the presence of relatively few gold particles (<1-2 % of the total) on the apical cell surface. The majority of gold particles were detected primarily in the extracellular matrix between the culture substratum and the cell membrane. In contrast, treatment of HUVECs with tumor necrosis factor ot (TNFot; 200 U/ml, 24 h) or with lipopolysaccharide (LPS; 10 #g/ml, 24 h) resulted in an increased staining of PAId not only in the extracellular matrix, but also on the apical cell surface (10-fold increase). Immunoabsorption of the rabbit anti-PAid with purified PAId, or treatment of HUVECs with tissue-type plasminogen activator (2.5/zg/ml, 2 h, 4°C) reduced the amount of staining both on the apical surface and in the extracellular matrix of agonistactivated HUVECs by 80-95 %. The topographical location of PAId on the cell surface was examined further by coupling immunogold staining with high resolution surface replication. Transmission EM of surface replicas from TNFo~-or LPS-activated HUVECs revealed a general increase in PAId staining both on planar regions and within indentations of the apical cell surface. Nonactivated HUVECs revealed PAid-specific immunogold particles only in areas of exposed extracellular matrix between the cells and occasionally at regions of cell-cell contacts. Analysis of activated bovine aortic endothelial cells by immunoelectron microscopy, immunologic assays, and flow cytometry revealed similar increases in surface PAId. These increases in surface PAId could be detected by 3 h and continued over a 24-h period. The expression of PAId on the luminal surface of endothelial cells during immune or inflammatory reactions could reduce endothelial fibrinolytic activity, thus, promoting the localized, pathologic formation of intravascular thrombi.

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Variability of the topography of low-density lipoprotein (LDL) receptors in the plasma membrane of cultured human skin fibroblasts as revealed by gold-LDL conjugates in conjunction with the surface replication technique

Cited by 19 publications

References 29 publications

Analysis of receptor clustering on cell surfaces by imaging fluorescent particles

Analysis of receptor clustering on cell surfaces by imaging fluorescent particles

Mathematical Modeling of Receptor-Mediated Endocytosis

Immunoelectron microscopic localization of type 1 plasminogen activator inhibitor on the surface of activated endothelial cells.

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