Quantitative characterization of single-cell adhesion properties by atomic force microscopy using protein-functionalized microbeads.

A method was developed to characterize the adhesion properties of single cells by using protein-functionalized atomic force microscopy (AFM) probes. The quantification by force spectroscopy of the mean detachment force between cells and a gelatin-functionalized colloidal tip reveals differences in cell adhesion properties that are not within reach of a traditional bulk technique, the washing assay. In this latter method, experiments yield semiquantitative and average adhesion properties of a large population of cells. They are also limited to stringent conditions and cannot highlight disparities in adhesion in the subset of adherent cells. In contrast, this AFM-based method allows for a reproducible and quantitative investigation of the adhesive properties of individual cells in common cell culture conditions and allows for the detection of adhesive subpopulations of cells. These characteristics meet the critical requirements of many fields, such as the study of cancer cell migratory abilities.

A gentle approach to investigate the influence of LRP-1 silencing on the migratory behavior of breast cancer cells by atomic force microscopy and dynamic cell studies.

The aim of the study was to get more insight into the role of LRP-1 in the mechanism of tumor progression in triple negative breast cancer. Atomic force microscopy, videomicroscopy, confocal microscopy and Rho-GTPAse activity assay were used on MDA-MB-231 and LRP-1-silenced cells. Silencing of LRP-1 in MDA-MB-231 cells was shown to led to a dramatic increase in the Young's modulus in parallel to a spectacular drop in membrane extension dynamics as well as a decrease in the cells migration abilities on both collagen I and fibronectin substrates. These results were perfectly correlated to a corresponding change in cell morphology and spreading capacity as well as in Rho-GTPases activity. By a multi-technique approach, it was demonstrated that LRP-1 played a crucial role in the migration of MDA-MB-231 cells by modulating the membrane extension dynamic. The originality of this AFM investigation lies in the non-invasive aspect of the measurements.

Dynamized ultra-low dilution of Ruta graveolens disrupts plasma membrane organization and decreases migration of melanoma cancer cell.

Cutaneous melanoma is a cancer with a very poor prognosis mainly because of metastatic dissemination and therefore a deregulation of cell migration. Current therapies can benefit from complementary medicines as supportive care in oncology. In our study, we show that a dynamized ultra-low dilution of Ruta Graveolens leads to an in vitro inhibition of migration on fibronectin of B16F10 melanoma cells, as well as a decrease in metastatic dissemination in vivo. These effects appear to be due to a disruption of plasma membrane organization, with a change in cell and membrane stiffness, associated with a disorganization of the actin cytoskeleton and a modification of the lipid composition of the plasma membrane. Together, these results demonstrate, in in vitro and in vivo models of cutaneous melanoma, an anti-cancer and anti-metastatic activity of ultra-low dynamized dilution of Ruta graveolens and reinforce its interest as complementary medicine in oncology.

Glyphosate-induced stiffening of HaCaT keratinocytes, a Peak Force Tapping study on living cells.

The skin is the first physiological barrier, with a complex constitution, that provides defensive functions against multiple physical and chemical aggressions. Glyphosate is an extensively used herbicide that has been shown to increase the risk of cancer. Moreover there is increasing evidence suggesting that the mechanical phenotype plays an important role in malignant transformation. Atomic force microscopy (AFM) has emerged within the last decade as a powerful tool for providing a nanometer-scale resolution imaging of biological samples. Peak Force Tapping (PFT) is a newly released AFM-based investigation technique allowing extraction of chemical and mechanical properties from a wide range of samples at a relatively high speed and a high resolution. The present work uses the PFT technology to investigate HaCaT keratinocytes, a human epidermal cell line, and offers an original approach to study chemically-induced changes in the cellular mechanical properties under near-physiological conditions. These experiments indicate glyphosate induces cell membrane stiffening, and the appearance of cytoskeleton structures at a subcellular level, for low cytotoxic concentrations whereas cells exposed to IC50 (inhibitory concentration 50%) treatment exhibit control-like mechanical behavior despite obvious membrane damages. Quercetin, a well-known antioxidant, reverses the glyphosate-induced mechanical phenotype.

Transfer on hydrophobic substrates and AFM imaging of membrane proteins reconstituted in planar lipid bilayers.

The lipid-layer technique allows reconstituting transmembrane proteins at a high density in microns size planar membranes and suspended to a lipid monolayer at the air/water interface. In this paper, we transferred these membranes onto two hydrophobic substrates for further structural analysis of reconstituted proteins by Atomic Force Microscopy (AFM). We used a mica sheet covered by a lipid monolayer or a sheet of highly oriented pyrolytic graphite (HOPG) to trap the lipid monolayer at the interface and the suspended membranes. In both cases, we succeeded in the transfer of large membrane patches containing densely packed or 2D-crystallized proteins. As a proof of concept, we transferred and imaged the soluble Shiga toxin bound to its lipid ligand and the ATP-binding cassette (ABC) transporter BmrA reconstituted into a planar bilayer. AFM imaging with a lateral resolution in the nanometer range was achieved. Potential applications of this technique in structural biology and nanobiotechnology are discussed.

Multimodal Approach for Cancer Cell Investigation.

Atomic force microscopy (AFM) enables the characterization of a wide range of samples including live cells. It is generally admitted that cancer cells are significantly softer than their normal counterparts, but imaging live cells by AFM using traditional modes can be at the cost of time or resolution. We describe how this tool can be used to estimate the motility of cancer versus normal cells, based on topographical and mechanical approaches, and coupled to optical imaging.

The detrimental invasiveness of glioma cells controlled by gadolinium chelate-coated gold nanoparticles.

Glioblastoma are characterized by an invasive phenotype, which is thought to be responsible for recurrences and the short overall survival of patients. In the last decade, the promising potential of ultrasmall gadolinium chelate-coated gold nanoparticles (namely Au@DTDTPA(Gd)) was evidenced for image-guided radiotherapy in brain tumors. Considering the threat posed by invasiveness properties of glioma cells, we were interested in further investigating the biological effects of Au@DTDTPA(Gd) by examining their impact on GBM cell migration and invasion. In our work, exposure of U251 glioma cells to Au@DTDTPA(Gd) led to high accumulation of gold nanoparticles, that were mainly diffusely distributed in the cytoplasm of the tumor cells. Experiments pointed out a significant decrease in glioma cell invasiveness when exposed to nanoparticles. As the proteolysis activities were not directly affected by the intracytoplasmic accumulation of Au@DTDTPA(Gd), the anti-invasive effect cannot be attributed to matrix remodeling impairment. Rather, Au@DTDTPA(Gd) nanoparticles affected the intrinsic biomechanical properties of U251 glioma cells, such as cell stiffness, adhesion and generated traction forces, and significantly reduced the formation of protrusions, thus exerting an inhibitory effect on their migration capacities. Consistently, analysis of talin-1 expression and membrane expression of beta 1 integrin evoke the stabilization of focal adhesion plaques in the presence of nanoparticles. Taken together, our results highlight the interest in Au@DTDTPA(Gd) nanoparticles for the therapeutic management of astrocytic tumors, not only as a radio-enhancing agent but also by reducing the invasive potential of glioma cells.

Carbamylation of elastic fibers is a molecular substratum of aortic stiffness.

Because of their long lifespan, matrix proteins of the vascular wall, such as elastin, are subjected to molecular aging characterized by non-enzymatic post-translational modifications, like carbamylation which results from the binding of cyanate (mainly derived from the dissociation of urea) to protein amino groups. While several studies have demonstrated a relationship between increased plasma concentrations of carbamylated proteins and the development of cardiovascular diseases, molecular mechanisms explaining the involvement of protein carbamylation in these pathological contexts remain to be fully elucidated. The aim of this work was to determine whether vascular elastic fibers could be carbamylated, and if so, what impact this phenomenon would have on the mechanical properties of the vascular wall. Our experiments showed that vascular elastin was carbamylated in vivo. Fiber morphology was unchanged after in vitro carbamylation, as well as its sensitivity to elastase degradation. In mice fed with cyanate-supplemented water in order to increase protein carbamylation within the aortic wall, an increased stiffness in elastic fibers was evidenced by atomic force microscopy, whereas no fragmentation of elastic fiber was observed. In addition, this increased stiffness was also associated with an increase in aortic pulse wave velocity in ApoE-/- mice. These results provide evidence for the carbamylation of elastic fibers which results in an increase in their stiffness at the molecular level. These alterations of vessel wall mechanical properties may contribute to aortic stiffness, suggesting a new role for carbamylation in cardiovascular diseases.

Revealing the elasticity of an individual aortic fiber during ageing at nanoscale by in situ atomic force microscopy.

Arterial stiffness is a complex process affecting the aortic tree that significantly contributes to cardiovascular diseases (systolic hypertension, coronary artery disease, heart failure or stroke). This process involves a large extracellular matrix remodeling mainly associated with elastin content decrease and collagen content increase. Additionally, various chemical modifications that accumulate with ageing have been shown to affect long-lived assemblies, such as elastic fibers, that could affect their elasticity. To precisely characterize the fiber changes and the evolution of its elasticity with ageing, high resolution and multimodal techniques are needed for precise insight into the behavior of a single fiber and its surrounding medium. In this study, the latest developments in atomic force microscopy and the related nanomechanical modes are used to investigate the evolution and in a near-physiological environment, the morphology and elasticity of aorta cross sections obtained from mice of different ages with an unprecedented resolution. In correlation with more classical approaches such as pulse wave velocity and fluorescence imaging, we demonstrate that the relative Young's moduli of elastic fibers, as well as those of the surrounding areas, significantly increase with ageing. This nanoscale characterization presents a new view on the stiffness process, showing that, besides the elastin and collagen content changes, elasticity is impaired at the molecular level, allowing a deeper understanding of the ageing process. Such nanomechanical AFM measurements of mouse tissue could easily be applied to studies of diseases in which elastic fibers suffer pathologies such as atherosclerosis and diabetes, where the precise quantification of fiber elasticity could better follow the fiber remodeling and predict plaque rupture.

Low-diluted Phenacetinum disrupted the melanoma cancer cell migration.

Dynamic and reciprocal interactions generated by the communication between tumor cells and their matrix microenvironment, play a major role in the progression of a tumor. Indeed, the adhesion of specific sites to matrix components, associated with the repeated and coordinated formation of membrane protrusions, allow tumor cells to move along a determined pathway. Our study analyzed the mechanism of action of low-diluted Phenacetinum on murine cutaneous melanoma process in a fibronectin matrix environment. We demonstrated a reduction of dispersed cell migration, early and for as long as 24 h, by altering the formation of cell protrusions. Moreover, low-diluted Phenacetinum decreased cell stiffness highly on peripheral areas, due to a disruption of actin filaments located just under the plasma membrane. Finally, it modified the structure of the plasma membrane by accumulating large ordered lipid domains and disrupted B16 cell migration by a likely shift in the balance between ordered and disordered lipid phases. Whereas the correlation between the excess of lipid raft and cytoskeleton disrupting is not as yet established, it is clear that low-diluted Phenacetinum acts on the actin cytoskeleton organization, as confirmed by a decrease of cell stiffness affecting ultimately the establishment of an effective migration process.

Biocompatibility of Gd-Loaded Chitosan-Hyaluronic Acid Nanogels as Contrast Agents for Magnetic Resonance Cancer Imaging.

Although the research on nanogels incorporating Gd chelates for theranostic applications has grown exponentially in recent years, knowledge about their biocompatibility is limited. We compared the biocompatibility of Gd-loaded hyaluronic acid-chitosan-based nanogels (GdCA⊂CS-TPP/HA) with two chitosan concentrations (2.5 and 1.5 mg·mL-1 respectively) using SVEC4-10 murine lymph node endothelial cells. The sulforhodamine B method and released lactate dehydrogenase (LDH) activity were used as cell viability tests. Reactive oxygen species (ROS), reduced glutathione (GSH) and malondialdehyde (MDA) were measured by spectrophotometric and fluorimetric methods. Nrf-2 protein expression was evaluated by Western blot analysis and genotoxicity by alkaline comet assay. After 24 h, the cells viability was not affected by all types and doses of nanogels. The increase of ROS induced a low decrease of GSH concentration and a time-dependent raise of MDA one was produced by citric GdDOTA⊂CS-TPP/HA with a chitosan concentration of 1.5 mg·mL-1, at the highest dose applied. None of the tested nanogels induced changes in Nrf-2 protein expression. A slight but significant genotoxic effect was caused only by citric GdDOTA⊂CS-TPP/HA where CS concentration was 1.5 mg·mL-1. Our results showed a better biocompatibility with lymph node endothelial cells for Gd-loaded hyaluronic acid-chitosan based nanogels with a concentration in chitosan of 2.5 mg·mL-1.

Structural characterization and in vivo pro-tumor properties of a highly conserved matrikine.

Elastin-derived peptides (EDPs) exert protumor activities by increasing tumor growth, migration and invasion. A number of studies have highlighted the potential of VGVAPG consensus sequence-derived elastin-like polypeptides whose physicochemical properties and biocompatibility are particularly suitable for in vivo applications, such as drug delivery and tissue engineering. However, among the EDPs, the influence of elastin-derived nonapeptides (xGxPGxGxG consensus sequence) remains unknown. Here, we show that the AGVPGLGVG elastin peptide (AG-9) present in domain-26 of tropoelastin is more conserved than the VGVAPG elastin peptide (VG-6) from domain-24 in mammals. The results demonstrate that the structural features of AG-9 and VG-6 peptides are similar. CD, NMR and FTIR spectroscopies show that AG-9 and VG-6 present the same conformation, which includes a mixture of random coils and ß-turn structures. On the other hand, the supraorganization differs between peptides, as demonstrated by AFM. The VG-6 peptide gathers in spots, whereas the AG-9 peptide aggregates into short amyloid-like fibrils. An in vivo study showed that AG-9 peptides promote tumor progression to a greater extent than do VG-6 peptides. These results were confirmed by in vitro studies such as 2D and 3D proliferation assays, migration assays, adhesion assays, proteinase secretion studies and pseudotube formation assays to investigate angiogenesis. Our findings suggest the possibility that the AG-9 peptide present in patient sera may dramatically influence cancer progression and could be used in the design of new, innovative antitumor therapies.

Influence of calcium on direct incorporation of membrane proteins into in-plane lipid bilayer.

Reconstitution of transmembrane proteins by direct incorporation into supported lipid bilayers (SLBs) is a new method to provide suitable samples for high-resolution atomic force microscopy (AFM) analysis of membrane proteins. First experiments have reported successful incorporation of proteins into detergent-destabilized SLBs. Here, we analyzed by AFM the incorporation of membrane proteins in the presence of calcium, a divalent cation functionally important for several membrane proteins. Using lipid-phase-separated membranes, we first show that calcium strongly stabilizes the SLBs decreasing the insertion of low cmc detergents, dodecyl-beta-maltoside, dodecyl-beta-thiomaltoside, and N-hexadecylphosphocholine (Fos-Choline-16) and further insertion of proteins. However, high yield of protein insertion is recovered in the presence of calcium by increasing the detergent concentration in the solution. These data revealed the importance of the calcium in the structure of SLBs and provided new insights into the mechanism of protein insertion into these model membranes.

Subcellular and supracellular mechanical stress prescribes cytoskeleton behavior in Arabidopsis cotyledon pavement cells.

Although it is a central question in biology, how cell shape controls intracellular dynamics largely remains an open question. Here, we show that the shape of Arabidopsis pavement cells creates a stress pattern that controls microtubule orientation, which then guides cell wall reinforcement. Live-imaging, combined with modeling of cell mechanics, shows that microtubules align along the maximal tensile stress direction within the cells, and atomic force microscopy demonstrates that this leads to reinforcement of the cell wall parallel to the microtubules. This feedback loop is regulated: cell-shape derived stresses could be overridden by imposed tissue level stresses, showing how competition between subcellular and supracellular cues control microtubule behavior. Furthermore, at the microtubule level, we identified an amplification mechanism in which mechanical stress promotes the microtubule response to stress by increasing severing activity. These multiscale feedbacks likely contribute to the robustness of microtubule behavior in plant epidermis. DOI: http://dx.doi.org/10.7554/eLife.01967.001.

Characterisation of tissue factor-bearing extracellular vesicles with AFM: comparison of air-tapping-mode AFM and liquid Peak Force AFM.

INTRODUCTION: Extracellular vesicles (EVs) are shed from cells and carry markers of the parent cells. Vesicles derived from cancer cells reach the bloodstream and locally influence important physiological processes. It has been previously shown that procoagulant vesicles are circulating in patients' fluids. These EVs are therefore considered as promising biomarkers for the thrombotic risk. Because of their small size, classical methods such as flow cytometry suffer from limitation for their characterisation. Atomic force microscopy (AFM) has been proposed as a promising complementary method for the characterisation of EVs. OBJECTIVES: THE OBJECTIVES OF THIS STUDY ARE: (a) to develop and validate AFM with specific antibodies (anti-TF) and (b) to compare air and liquid modes for EVs' size and number determination as potential biomarkers of the prothrombotic risk. METHODS: AFM multimode nanoscope III was used for air tapping mode (TM). AFM catalyst was used for liquid Peak Force Tapping (PFT) mode. Vesicles are generated according to Davila et al.'s protocol. Substrates are coated with various concentrations of antibodies, thanks to ethanolamine and glutaraldehyde. RESULTS: Vesicles were immobilised on antibody-coated surfaces to select tissue factor (TF)-positive vesicles. The size range of vesicles observed in liquid PFT mode is 6-10 times higher than in air mode. This corresponds to the data found in the literature. CONCLUSION: We recommend liquid PFT mode to analyse vesicles on 5 µg/ml antibody-coated substrates.

Inhibition of mitochondrial aconitase by succination in fumarate hydratase deficiency.

The gene encoding the Krebs cycle enzyme fumarate hydratase (FH) is mutated in hereditary leiomyomatosis and renal cell cancer (HLRCC). Loss of FH activity causes accumulation of intracellular fumarate, which can directly modify cysteine residues to form 2-succinocysteine through succination. We undertook a proteomic-based screen in cells and renal cysts from Fh1 (murine FH)-deficient mice and identified 94 protein succination targets. Notably, we identified the succination of three cysteine residues in mitochondrial Aconitase2 (ACO2) crucial for iron-sulfur cluster binding. We show that fumarate exerts a dose-dependent inhibition of ACO2 activity, which correlates with increased succination as determined by mass spectrometry, possibly by interfering with iron chelation. Importantly, we show that aconitase activity is impaired in FH-deficient cells. Our data provide evidence that succination, resulting from FH deficiency, targets and potentially alters the function of multiple proteins and may contribute to the dysregulated metabolism observed in HLRCC.

QUANTITATIVE NANOMECHANICAL MAPPING OF MARINE DIATOM IN SEAWATER USING PEAK FORCE TAPPING ATOMIC FORCE MICROSCOPY(1).

It is generally accepted that a diatom cell wall is characterized by a siliceous skeleton covered by an organic envelope essentially composed of polysaccharides and proteins. Understanding of how the organic component is associated with the silica structure provides an important insight into the biomineralization process and patterning on the cellular level. Using a novel atomic force microscopy (AFM) imaging technique (Peak Force Tapping), we characterized nanomechanical properties (elasticity and deformation) of a weakly silicified marine diatom Cylindrotheca closterium (Ehrenb.) Reimann et J. C. Lewin (strain CCNA1). The nanomechanical properties were measured over the entire cell surface in seawater at a resolution that was not achieved previously. The fibulae were the stiffest (200 MPa) and the least deformable (only 1 nm). Girdle band region appeared as a series of parallel stripes characterized by two sets of values of Young's modulus and deformation: one for silica stripes (43.7 Mpa, 3.7 nm) and the other between the stripes (21.3 MPa, 13.4 nm). The valve region was complex with average values of Young's modulus (29.8 MPa) and deformation (10.2 nm) with high standard deviations. After acid treatment, we identified 15 nm sized silica spheres in the valve region connecting raphe with the girdle bands. The silica spheres were neither fused together nor forming a nanopattern. A cell wall model is proposed with individual silica nanoparticles incorporated in an organic matrix. Such organization of girdle band and valve regions enables the high flexibility needed for movement and adaptation to different environments while maintaining the integrity of the cell.

Voltage-dependent anion channel transports calcium ions through biomimetic membranes.

The mitochondrial outer membrane channel (VDAC), a central player in mitochondria and cell death, was reconstituted in polymer-supported phospholipid bilayers. Highly purified VDAC was first reconstituted in vesicles; channel properties and NADH-ferricyanide reductase activity were ascertained before deposition onto solid substrates. 1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine/1,2-distearoyl-sn-glycero-3-phosphoethanolamine-poly(ethylene glycol)-N-hydroxysuccinimide mixed vesicles containing VDAC were linked onto amine-grafted surfaces (glass and gold) and disrupted to form a VDAC-containing polymer-tethered planar bilayer. Surface plasmon spectroscopy, fluorescence microscopy, and atomic force microscopy measurements ascertained the membrane thickness, fluidity, and continuity. VDAC reconstituted in bilayers efficiently transported calcium ions and was modulable by two channel blockers, 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid and l-glutamate. The novel setup may allow the study of the assembly of a polyprotein complex centered on VDAC and its role in mitochondrial biology, calcium fluxes, and apoptosis.

High-resolution AFM of membrane proteins directly incorporated at high density in planar lipid bilayer.

The heterologous expression and purification of membrane proteins represent major limitations for their functional and structural analysis. Here we describe a new method of incorporation of transmembrane proteins in planar lipid bilayer starting from 1 pmol of solubilized proteins. The principle relies on the direct incorporation of solubilized proteins into a preformed planar lipid bilayer destabilized by dodecyl-beta-maltoside or dodecyl-beta-thiomaltoside, two detergents widely used in membrane biochemistry. Successful incorporations are reported at 20 degrees C and at 4 degrees C with three bacterial photosynthetic multi-subunit membrane proteins. Height measurements by atomic force microscopy (AFM) of the extramembraneous domains protruding from the bilayer demonstrate that proteins are unidirectionally incorporated within the lipid bilayer through their more hydrophobic domains. Proteins are incorporated at high density into the bilayer and on incubation diffuse and segregate into protein close-packing areas. The high protein density allows high-resolution AFM topographs to be recorded and protein subunits organization delineated. This approach provides an alternative experimental platform to the classical methods of two-dimensional crystallization of membrane proteins for the structural analysis by AFM. Furthermore, the versatility and simplicity of the method are important intrinsic properties for the conception of biosensors and nanobiomaterials involving membrane proteins.

Antigen binding forces of single antilysozyme Fv fragments explored by atomic force microscopy.

We used atomic force microscopy (AFM) to explore the antigen binding forces of individual Fv fragments of antilysozyme antibodies (Fv). To detect single molecular recognition events, genetically engineered histidine-tagged Fv fragments were coupled onto AFM tips modified with mixed self-assembled monolayers (SAMs) of nitrilotriacetic acid- and tri(ethylene glycol)-terminated alkanethiols while lysozyme (Lyso) was covalently immobilized onto mixed SAMs of carboxyl- and hydroxyl-terminated alkanethiols. The quality of the functionalization procedure was validated using X-ray photoelectron spectroscopy (surface chemical composition), AFM imaging (surface morphology in aqueous solution), and surface plasmon resonance (SPR, specific binding in aqueous solution). AFM force-distance curves recorded at a loading rate of 5000 pN/s between Fv- and Lyso-modified surfaces yielded a distribution of unbinding forces composed of integer multiples of an elementary force quantum of approximately 50 pN that we attribute to the rupture of a single antibody-antigen pair. Injection of a solution containing free Lyso caused a dramatic reduction of adhesion probability, indicating that the measured 50 pN unbinding forces are due to the specific antibody-antigen interaction. To investigate the dynamics of the interaction, force-distance curves were recorded at various loading rates. Plots of unbinding force vs log(loading rate) revealed two distinct linear regimes with ascending slopes, indicating multiple barriers were present in the energy landscape. The kinetic off-rate constant of dissociation (k(off) approximately = 1 x 10(-3) s(-1)) obtained by extrapolating the data of the low-strength regime to zero force was in the range of the k(off) estimated by SPR.