An easy method to discover cell membrane antigen with atomic force microscopy

AbstractOver the last decade, nanoneedle-based systems have demonstrated to be extremely useful in cell biology. They can be used as nanotools for drug delivery, biosensing or biomolecular recognition inside cells; or they can be employed to select and sort in parallel a large number of living cells. When using these nanoprobes, the most important requirement is to minimize the cell damage, reducing the forces and indentation lengths needed to penetrate the cell membrane. This is normally achieved by reducing the diameter of the nanoneedles. However, several studies have shown that nanoneedles with a flat tip display lower penetration forces and indentation lengths. In this work, we have tested different nanoneedle shapes and diameters to reduce the force and the indentation length needed to penetrate the cell membrane, demonstrating that ultra-thin and sharp nanoprobes can further reduce them, consequently minimizing the cell damage.

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Improved unroofing protocols for cryo-electron microscopy, atomic force microscopy and freeze-etching electron microscopy and the associated mechanisms

Microscopy ◽

10.1093/jmicro/dfaa028 ◽

2020 ◽

Vol 69 (6) ◽

pp. 350-359

Author(s):

Nobuhiro Morone ◽

Eiji Usukura ◽

Akihiro Narita ◽

Jiro Usukura

Keyword(s):

Electron Microscopy ◽

Atomic Force Microscopy ◽

Cell Membrane ◽

Apical Cell ◽

Force Microscopy ◽

Atomic Force ◽

Cryo Electron Microscopy ◽

Cytoplasmic Surface ◽

A Cell ◽

Freeze Etching

Abstract Unroofing, which is the mechanical shearing of a cell to expose the cytoplasmic surface of the cell membrane, is a unique preparation method that allows membrane cytoskeletons to be observed by cryo-electron microscopy, atomic force microscopy, freeze-etching electron microscopy and other methods. Ultrasound and adhesion have been known to mechanically unroof cells. In this study, unroofing using these two means was denoted sonication unroofing and adhesion unroofing, respectively. We clarified the mechanisms by which cell membranes are removed in these unroofing procedures and established efficient protocols for each based on the mechanisms. In sonication unroofing, fine bubbles generated by sonication adhered electrostatically to apical cell surfaces and then removed the apical (dorsal) cell membrane with the assistance of buoyancy and water flow. The cytoplasmic surface of the ventral cell membrane remaining on the grids became observable by this method. In adhesion unroofing, grids charged positively by coating with Alcian blue were pressed onto the cells, thereby tightly adsorbing the dorsal cell membrane. Subsequently, a part of the cell membrane strongly adhered to the grids was peeled from the cells and transferred onto the grids when the grids were lifted. This method thus allowed the visualization of the cytoplasmic surface of the dorsal cell membrane. This paper describes robust, improved protocols for the two unroofing methods in detail. In addition, micro-unroofing (perforation) likely due to nanobubbles is introduced as a new method to make cells transparent to electron beams.

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Biological Atomic Force Microscopy for Imaging Gold-Labeled Liposomes on Human Coronary Artery Endothelial Cells

Journal of Pharmaceutics ◽

10.1155/2013/875906 ◽

2013 ◽

Vol 2013 ◽

pp. 1-8 ◽

Cited By ~ 2

Author(s):

Ana-María Zaske ◽

Delia Danila ◽

Michael C. Queen ◽

Eva Golunski ◽

Jodie L. Conyers

Keyword(s):

Atomic Force Microscopy ◽

Endothelial Cells ◽

Coronary Artery ◽

Cell Membrane ◽

Fluorescent Microscopy ◽

Human Coronary Artery ◽

Force Microscopy ◽

Cell Internalization ◽

Atomic Force

Although atomic force microscopy (AFM) has been used extensively to characterize cell membrane structure and cellular processes such as endocytosis and exocytosis, the corrugated surface of the cell membrane hinders the visualization of extracellular entities, such as liposomes, that may interact with the cell. To overcome this barrier, we used 90 nm nanogold particles to label FITC liposomes and monitor their endocytosis on human coronary artery endothelial cells (HCAECs) in vitro. We were able to study the internalization process of gold-coupled liposomes on endothelial cells, by using AFM. We found that the gold-liposomes attached to the HCAEC cell membrane during the first 15–30 min of incubation, liposome cell internalization occurred from 30 to 60 min, and most of the gold-labeled liposomes had invaginated after 2 hr of incubation. Liposomal uptake took place most commonly at the periphery of the nuclear zone. Dynasore monohydrate, an inhibitor of endocytosis, obstructed the internalization of the gold-liposomes. This study showed the versatility of the AFM technique, combined with fluorescent microscopy, for investigating liposome uptake by endothelial cells. The 90 nm colloidal gold nanoparticles proved to be a noninvasive contrast agent that efficiently improves AFM imaging during the investigation of biological nanoprocesses.

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2P250 Mechanics of cell membrane and lipid layer by Atomic Force Microscopy

Seibutsu Butsuri ◽

10.2142/biophys.45.s182_2 ◽

2005 ◽

Vol 45 (supplement) ◽

pp. S182

Author(s):

M. Oka ◽

M. Yasuda ◽

H. Uehara ◽

Afrin Rehana ◽

H. Sekiguchi ◽

...

Keyword(s):

Atomic Force Microscopy ◽

Cell Membrane ◽

Lipid Layer ◽

Force Microscopy ◽

Atomic Force

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Imidazolium-Based Ionic Liquids Affect Morphology and Rigidity of Living Cells: an Atomic Force Microscopy Study

10.26434/chemrxiv.6216383 ◽

2018 ◽

Author(s):

Massimiliano Galluzzi ◽

Carsten Schulte ◽

Paolo Milani ◽

Alessandro Podestà

Keyword(s):

Atomic Force Microscopy ◽

Ionic Liquids ◽

Cell Membrane ◽

Molecular Mechanisms ◽

Single Cells ◽

Metastatic Cancer ◽

Toxic Effects ◽

Force Microscopy ◽

Atomic Force ◽

The Impact

The study of the toxicity, biocompatibility, and environmental sustainability of room-temperature Ionic Liquids (ILs) is still in its infancy. Understanding the impact of ILs on living organisms, especially from the aquatic ecosystem, is urgent, since on one side large amounts of these substances are widely employed as solvents in industrial chemical processes, and on the other side evidences of toxic effects of ILs on microorganisms and single cells have been observed. To date, the toxicity of ILs have been investigated by means of macroscopic assays aimed at characterizing the effective concentrations (like the EC50) that cause the dead of a significant fraction of the population of microorganisms and cells. These studies allowed to identify the cell membrane as the first target of the IL interaction, whose effectiveness was correlated to the lipophilicity of the cation, i.e. to the length of the lateral alkyl chain. Our study aimed at characterizing the molecular mechanisms of the toxicity of ILs. To this purpose, we carried out a combined topographic and mechanical analysis by Atomic Force Microscopy of living breast metastatic cancer cells (MDA-MB-231) upon interaction with imidazolium-based ILs. We showed that ILs are able to induce modifications of the overall rigidity (effective Young modulus) and morphology of the cells. Our results demonstrate that ILs act on the physical properties of the cell membrane, and possibly induce cytoskeletal reorganization, already at concentrations below the EC50. These potentially toxic effects are stronger at higher IL concentrations, as well as with longer lateral chains in the cation.<br>

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Changes in red blood cell membrane structure in type 2 diabetes: a scanning electron and atomic force microscopy study

Cardiovascular Diabetology ◽

10.1186/1475-2840-12-25 ◽

2013 ◽

Vol 12 (1) ◽

pp. 25 ◽

Cited By ~ 98

Author(s):

Antoinette V Buys ◽

Mia-Jean Van Rooy ◽

Prashilla Soma ◽

Dirk Van Papendorp ◽

Boguslaw Lipinski ◽

...

Keyword(s):

Type 2 Diabetes ◽

Atomic Force Microscopy ◽

Cell Membrane ◽

Membrane Structure ◽

Microscopy Study ◽

Atomic Force Microscopy Study ◽

Force Microscopy ◽

Atomic Force ◽

Scanning Electron

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DISEASED RED BLOOD CELLS STUDIED BY ATOMIC FORCE MICROSCOPY

International Journal of Nanoscience ◽

10.1142/s0219581x02000899 ◽

2002 ◽

Vol 01 (05n06) ◽

pp. 683-688 ◽

Cited By ~ 5

Author(s):

YONG CHEN ◽

JIYE CAI ◽

JINGXIAN ZHAO

Keyword(s):

Lung Cancer ◽

Atomic Force Microscopy ◽

Cell Membrane ◽

Erythrocyte Membrane ◽

Mammalian Cells ◽

Membrane Surface ◽

Force Microscopy ◽

Membrane Surfaces ◽

Atomic Force ◽

Mean Width

In recent years, many mammalian cells, especially erythrocytes because of simpleness of their membrane surfaces, were widely studied by atomic force microscopy. In our study, diseased erythrocytes were taken from patients of lung cancer, myelodisplastic syndrome (MDS), and so on. We obtained many clear topographical images of numerous erythrocytes, single erythrocyte, and ultramicrostructure of erythrocyte membrane surfaces from normal persons and patients. By studying the red cells of lung cancer patients, we found that many erythrocytes of lung cancer patient have changed into echinocytes. One erythrocyte has 10–20 short projections, most of which, with a mean width of 589.0 nm and a length of 646.7 nm, are on the edge of cell. The projections in the center of echinocytes are lodged and embedded, but in conventional model of echinocytes, the projections in the center stretch outside cell membrane, so a novel model of erythrocytes was designed in our paper. After observation of microstructure of MDS patient's erythrocyte membrane surface, we found that many apertures with different diameters of tens to hundreds nanometers appeared on the surface of cell membrane. It can be concluded that AFM may be widely applied in clinic pathological inspection.

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Evidence for a Role for the Plasma Membrane in the Nanomechanical Properties of the Cell Wall as Revealed by an Atomic Force Microscopy Study of the Response of Saccharomyces cerevisiae to Ethanol Stress

Applied and Environmental Microbiology ◽

10.1128/aem.01213-16 ◽

2016 ◽

Vol 82 (15) ◽

pp. 4789-4801 ◽

Cited By ~ 21

Author(s):

Marion Schiavone ◽

Cécile Formosa-Dague ◽

Carolina Elsztein ◽

Marie-Ange Teste ◽

Helene Martin-Yken ◽

...

Keyword(s):

Saccharomyces Cerevisiae ◽

Atomic Force Microscopy ◽

Cell Wall ◽

Cell Membrane ◽

Yeast Cells ◽

Ethanol Stress ◽

Wild Type ◽

Force Microscopy ◽

Nanomechanical Properties ◽

Atomic Force

ABSTRACTA wealth of biochemical and molecular data have been reported regarding ethanol toxicity in the yeastSaccharomyces cerevisiae. However, direct physical data on the effects of ethanol stress on yeast cells are almost nonexistent. This lack of information can now be addressed by using atomic force microscopy (AFM) technology. In this report, we show that the stiffness of glucose-grown yeast cells challenged with 9% (vol/vol) ethanol for 5 h was dramatically reduced, as shown by a 5-fold drop of Young's modulus. Quite unexpectedly, a mutant deficient in the Msn2/Msn4 transcription factor, which is known to mediate the ethanol stress response, exhibited a low level of stiffness similar to that of ethanol-treated wild-type cells. Reciprocally, the stiffness of yeast cells overexpressingMSN2was about 35% higher than that of the wild type but was nevertheless reduced 3- to 4-fold upon exposure to ethanol. Based on these and other data presented herein, we postulated that the effect of ethanol on cell stiffness may not be mediated through Msn2/Msn4, even though this transcription factor appears to be a determinant in the nanomechanical properties of the cell wall. On the other hand, we found that as with ethanol, the treatment of yeast with the antifungal amphotericin B caused a significant reduction of cell wall stiffness. Since both this drug and ethanol are known to alter, albeit by different means, the fluidity and structure of the plasma membrane, these data led to the proposition that the cell membrane contributes to the biophysical properties of yeast cells.IMPORTANCEEthanol is the main product of yeast fermentation but is also a toxic compound for this process. Understanding the mechanism of this toxicity is of great importance for industrial applications. While most research has focused on genomic studies of ethanol tolerance, we investigated the effects of ethanol at the biophysical level and found that ethanol causes a strong reduction of the cell wall rigidity (or stiffness). We ascribed this effect to the action of ethanol perturbing the cell membrane integrity and hence proposed that the cell membrane contributes to the cell wall nanomechanical properties.

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Changes in red blood cell membrane structure in G6PD deficiency: An atomic force microscopy study

Clinica Chimica Acta ◽

10.1016/j.cca.2015.02.042 ◽

2015 ◽

Vol 444 ◽

pp. 264-270 ◽

Cited By ~ 14

Author(s):

Jia Tang ◽

Chengrui Jiang ◽

Xiao Xiao ◽

Zishui Fang ◽

Lei Li ◽

...

Keyword(s):

Atomic Force Microscopy ◽

Cell Membrane ◽

Blood Cell ◽

Red Blood Cell ◽

G6pd Deficiency ◽

Membrane Structure ◽

Microscopy Study ◽

Atomic Force Microscopy Study ◽

Force Microscopy ◽

Atomic Force

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Topological Structures and Membrane Nanostructures of Erythrocytes after Splenectomy in Hereditary Spherocytosis Patients via Atomic Force Microscopy

Cell Biochemistry and Biophysics ◽

10.1007/s12013-016-0755-4 ◽

2016 ◽

Vol 74 (3) ◽

pp. 365-371 ◽

Cited By ~ 4

Author(s):

Ying LI ◽

Liyuan Lu ◽

Juan LI

Keyword(s):

Atomic Force Microscopy ◽

Cell Membrane ◽

Hereditary Spherocytosis ◽

Structural Characteristics ◽

Cytoskeletal Proteins ◽

Clinical Practices ◽

Average Cell ◽

Force Microscopy ◽

Atomic Force ◽

Membrane Ultrastructure

Abstract Hereditary spherocytosis is an inherited red blood cell membrane disorder resulting from mutations of genes encoding erythrocyte membrane and cytoskeletal proteins. Few equipments can observe the structural characteristics of hereditary spherocytosis directly expect for atomic force microscopy In our study, we proved atomic force microscopy is a powerful and sensitive instrument to describe the characteristics of hereditary spherocytosis. Erythrocytes from hereditary spherocytosis patients were small spheroidal, lacking a well-organized lattice on the cell membrane, with smaller cell surface particles and had reduced valley to peak distance and average cell membrane roughness vs. those from healthy individuals. These observations indicated defects in the certain cell membrane structural proteins such as α- and β-spectrin, ankyrin, etc. Until now, splenectomy is still the most effective treatment for symptoms relief for hereditary spherocytosis. In this study, we further solved the mysteries of membrane nanostructure changes of erythrocytes before and after splenectomy in hereditary spherocytosis by atomic force microscopy. After splenectomy, the cells were larger, but still spheroidal-shaped. The membrane ultrastructure was disorganized and characterized by a reduced surface particle size and lower than normal Ra values. These observations indicated that although splenectomy can effectively relieve the symptoms of hereditary spherocytosis, it has little effect on correction of cytoskeletal membrane defects of hereditary spherocytosis. We concluded that atomic force microscopy is a powerful tool to investigate the pathophysiological mechanisms of hereditary spherocytosis and to monitor treatment efficacy in clinical practices. To the best of our knowledge, this is the first report to study hereditary spherocytosis with atomic force microscopy and offers important mechanistic insight into the underlying role of splenectomy.

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