Cell-surface receptors and proteins on platelet membranes imaged by scanning force microscopy using immunogold contrast enhancement

Eppell, Steven J.; Simmons, Scott R.; Albrecht, Ralph M.; Marchant, Roger

doi:10.1016/s0006-3495(95)80228-6

Cited by 47 publications

(26 citation statements)

References 28 publications

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“…It could be useful in identifying specific proteins in native membranes with "immuno-AFM" [16,17]. Using the oocyte as an expression system for foreign proteins in combination with specific antibodies it should be feasable to identify individual members of a protein family forming a functional cluster in the plasma membrane as previously proposed [18].…”

Section: Discussionmentioning

confidence: 97%

Plasma Membrane Plasticity of Xenopus laevis Oocyte Imaged with Atomic Force Microscopy

Schillers

Danker²,

Schnittler³

et al. 2000

Cell Physiol Biochem

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Proteins are known to form functional clusters in plasma membranes. In order to identify individual proteins within clusters we developed a method to visualize by atomic force microscopy (AFM) the cytoplasmic surface of native plasma membrane, excised from Xenopus laevis oocyte and spread on poly-L-lysine coated glass. After removal of the vitelline membrane intact oocytes were brought in contact with coated glass and then rolled off. Inside-out oriented plasma membrane patches left at the glass surface were first identified with the lipid fluorescent marker FM1-43 and then scanned by AFM. Membrane patches exhibiting the typical phospholipid bilayer height of 5 nm showed multiple proteins, protruding from the inner surface of the membrane, with heights of 5 to 20 nm. Modelling plasma membrane proteins as spherical structures embedded in the lipid bilayer and protruding into the cytoplasm allowed an estimation of the respective molecular masses. Proteins ranged from 35 to 2,000 kDa with a peak value of 280 kDa. The most frequently found membrane protein structure (40/μm²) had a total height of 10 nm and an estimated molecular mass of 280 kDa. Membrane proteins were found firmly attached to the poly-L-lysine coated glass surface while the lipid bilayer was found highly mobile. We detected protein structures with distinguishable subunits of still unknown identity. Since X. laevis oocyte is a generally accepted expression system for foreign proteins, this method could turn out to be useful to structurally identify specific proteins in their native environment at the molecular level.

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Section: Discussionmentioning

confidence: 97%

Plasma Membrane Plasticity of Xenopus laevis Oocyte Imaged with Atomic Force Microscopy

Schillers

Danker²,

Schnittler³

et al. 2000

Cell Physiol Biochem

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“…This difficulty can be overcome by immunogold labeling of VWF, as carefully chosen size gold markers can be unequivocally identified by AFM, whereas they have enough lateral resolution for quantitative work, even on corrugated surfaces like whole cells. [12][13][14][15] Immunogold was already successfully applied to label VWF either for electron microscopy, 16,17 or for AFM. 10 In our case, a specific immunogold procedure had to be elaborated that permits high-efficiency labeling with acceptable level of nonspecific background.…”

Section: Discussionmentioning

confidence: 99%

Shear-dependent morphology of von Willebrand factor bound to immobilized collagen

Novák

Deckmyn

Damjanovich

et al. 2002

Blood

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We have developed an immunogold von Willebrand factor (VWF) detection method that permits almost complete coverage of individual VWF molecules, and by this unequivocal localization and morphologic analysis of collagen-bound VWF by atomic force microscopy (AFM). Perfusion of gel filtration-purified VWF in parallel plate perfusion chambers over glass coverslips coated with calf skin collagen, followed by AFM imaging in air, enabled us to assess possible morphologic differences between VWF bound at low (0.07 N/m 2 ‫؍‬ 0.7 dynes/ cm 2 ) and high (4.55 N/m 2 ‫؍‬ 45.5 dynes/ cm 2 ) shear stresses. No significant differences in VWF morphology were found, the molecules were oriented almost randomly, and there were no clear signs of VWF "uncoiling" either at a high or at a low shear regime. After perfusing 1 g/mL VWF for 5 minutes, surface coverage at high shear was almost twice the one seen at low shear, and some larger and more irregularly shaped VWF molecules could be seen at high shear. This difference disappeared, however, at 15 minutes of perfusion and was probably caused by diffusion kinetics. Moreover, the presence of 68 ؋ 10 9 /L washed fixed platelets in the perfusate did not have any visible effect on VWF morphology at high versus low shear stress. These findings suggest that shear stress does not influence significantly the overall molecular morphology of VWF during its binding to collagen-coated surface and are consistent with a constitutively expressed affinity of collagen-bound VWF for gly-

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“…It has been previously reported the potential use of AFM in order to localize and identify of membrane receptors (Eppell et al, 1995) and ion channels (Smith et al, 1997). However, these studies have to rely on the use of immunogold particles or fluorescent labeling that needs a combination of AFM with fluorescence microscopy.…”

Section: Resultsmentioning

confidence: 99%

AFM functional imaging on vascular endothelial cells

Chtcheglova

Wildling

Waschke

et al. 2010

J of Molecular Recognition

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a Vascular endothelial (VE)-cadherin is predominantly responsible for the mechanical linkage between endothelial cells, where VE-cadherin molecules are clustered and linked through their cytoplasmic domain to the actin-based cytoskeleton. Clustering and linkage of VE-cadherin to actin filaments is a dynamic process and changes according to the functional state of the cells. Here nano-mapping of VE-cadherin was performed using simultaneous topography and recognition imaging (TREC) technique onto microvascular endothelial cells from mouse myocardium (MyEnd). The recognition maps revealed prominent 'dark' spots (domains or clusters) with the sizes from 10 to 250 nm. These spots arose from a decrease of oscillation amplitude during specific binding between VE-cadherin cis-dimers. They were assigned to characteristic structures of the topography images. After treatment with nocodazole so as to depolymerize microtubules, VE-cadherin domains with a typical ellipsoidal form were still found to be collocalized with cytoskeletal filaments supporting the hypothesis that VE-cadherin is linked to actin filaments. Compared to other conventional techniques such as immunochemistry or single molecule optical microscopy, TREC represents an alternative method to quickly obtain the local distribution of receptors on cell surface with an unprecedented lateral resolution of several nanometers.

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Cell-surface receptors and proteins on platelet membranes imaged by scanning force microscopy using immunogold contrast enhancement

Cited by 47 publications

References 28 publications

Plasma Membrane Plasticity of Xenopus laevis Oocyte Imaged with Atomic Force Microscopy

Plasma Membrane Plasticity of Xenopus laevis Oocyte Imaged with Atomic Force Microscopy

Shear-dependent morphology of von Willebrand factor bound to immobilized collagen

AFM functional imaging on vascular endothelial cells

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