Resolving the molecular structure of microtubules under physiological conditions with scanning force microscopy

We have imaged microtubules, essential structural elements of the cytoskeleton in eukaryotic cells, in physiological conditions by scanning force microscopy. We have achieved molecular resolution without the use of cross-linking and chemical fixation methods. With tip forces below 0.3 nN, protofilaments with ~6 nm separation could be clearly distinguished. Lattice defects in the microtubule wall were directly visible, including point defects and protofilament separations. Higher tip forces destroyed the top half of the microtubules, revealing the inner surface of the substrate-attached protofilaments. Monomers could be resolved on these inner surfaces.Abbreviations APTS (3-aminopropyl)triethoxysilane - DETA N¹-[3-(trimethoxysilyl)propyl]diethylenetriamine - EM electron microscopy - MT microtubule - SFM scanning force microscopy     

Imaging microtubules and kinesin decorated microtubules using tapping mode atomic force microscopy in fluids

The atomic force microscope has been used to investigate microtubules and kinesin decorated microtubules in aqueous solution adsorbed onto a solid substrate. The netto negatively charged microtubules did not adsorb to negatively charged solid surfaces but to glass covalently coated with the highly positively charged silane trimethoxysilylpropyldiethylenetriamine (DETA) or a lipid bilayer of 1,2-dipalmitoyl-3-dimethylammoniumpropane. Using electron beam deposited tips for microtubules adsorbed on DETA, single protofilaments could be observed showing that the resolution is up to 5 nm. Under conditions where the silane coated surfaces are hydrophobic, microtubules opened, presumably at the seam, whose stability is lower than that of the bonds between the other protofilaments. This led to a “sheet” with a width of about 100 nm firmly attached to the surface. Microtubules decorated with a stoichiometric low amount of kinesin molecules in the presence of the non-hydrolyzable ATP-analog 5′-adenylylimidodiphosphate could also be adsorbed onto silane-coated glass. Imaging was very stable and the molecules did not show any scan-induced deformation even after hundreds of scans with a scan frequency of 100 Hz. Received: 23 February 1999 / Revised version: 19 July 1999 / Accepted: 17 August 1999     

Atomic force microscope-based single-molecule force spectroscopy of RNA unfolding

Single-molecule force spectroscopy (SMFS) using the atomic force microscope (AFM) has emerged as an important tool for probing biomolecular interaction and exploring the forces, dynamics, and energy landscapes that underlie function and specificity of molecular interaction. These studies require attaching biomolecules on solid supports and AFM tips to measure unbinding forces between individual binding partners. Herein we describe efficient and robust protocols for probing RNA interaction by AFM and show their value on two well-known RNA regulators, the Rev-responsive element (RRE) from the HIV-1 genome and an adenine-sensing riboswitch. The results show the great potential of AFM–SMFS in the investigation of RNA molecular interactions, which will contribute to the development of bionanodevices sensing single RNA molecules.     

Atomic force microscopy of tomato and sugar beet pectin molecules

Atomic force microscopy has been used to image the structure of pectin molecules isolated from unripe tomato and sugar beet tissue. The tomato pectin molecules were found to be extended stiff chains with a weight average contour length of L_W = 174 nm and a number average contour length of L_N = 132 nm (L_W/L_N = 1.32). A proportion of the pectin molecules (30%) were found to be branched structures. Chemical analysis of the sugar beet pectin extracts showed that the samples contained protein (8.6%). This protein proved difficult to remove and is believed to be covalently attached to the polysaccharide. Imaging of the extracted pectin revealed largely un-aggregated chains: a small fraction (33%) of which were extended stiff polysaccharide chains and a major fraction (67%) of which were of polysaccharide–protein complexes containing a single protein molecule attached to one end of the polysaccharide chains (‘tadpoles’). In addition the sample contained a small number of aggregated structures. The un-aggregated pectin molecules were found to be predominately linear structures with a small fraction (17%) of branched structures. The branched structures were all in the free polysaccharide fraction and no branched pectin chains were observed in the protein–polysaccharide complexes. Alkali treatment was found to remove the protein. For the alkali-treated, un-aggregated structures the average contour lengths were found to be L_W = 137 nm, L_N = 108 nm with L_W/L_N = 1.27. It is proposed that the ‘tadpole’ structures contribute to the unusual emulsifying properties of sugar beet pectin.     

Role of ICAM-1 polymorphisms (G241R,K469E) in mediating its single-molecule binding ability: Atomic force microscopy measurements on living cells

Atherosclerosis (As) is characterized by chronic inflammation and is a major cause of human mortality. ICAM-1-mediated adhesion of leukocytes in vessel walls plays an important role in the pathogenesis of atherosclerosis. Two single nucleotide polymorphisms (SNPs) of human intercellular adhesion molecule-1 (ICAM-1), G241R and K469E, are associated with a number of inflammatory diseases. SNP induced changes in ICAM-1 function rely not only on the expression level but also on the single-molecule binding ability which may be affected by single molecule conformation variations such as protein splicing and folding. Previous studies have shown associations between G241R/K469E polymorphisms and ICAM-1 gene expression. Nevertheless, few studies have been done that focus on the single-molecule forces of the above SNPs and their ligands.     

Atomic force microscopy to investigate spatial patterns of response to interleukin-1beta in engineered cartilage tissue elasticity

Atomic force microscopy (AFM) has been proposed as a tool to evaluate the structural and mechanical properties of cartilage tissue. Here, we aimed at assessing whether AFM can be employed to quantify spatially resolved elastic response of tissue engineered cartilage (TEC) to short exposure to IL-1β, thus mimicking the initially inflammatory implantation site.     

Tau protein binding forms a 1 nm thick layer along protofilaments without affecting the radial elasticity of microtubules

Tau is one of the most abundant microtubule-associated proteins involved in kinetic stabilization and bundling of axonal microtubules. Although intense research has revealed much about tau function and its involvement in Alzheimer's disease during the past years, it still remains unclear how exactly tau binds on microtubules and if the kinetic stabilization of microtubules by tau is accompanied, at least in part, by a mechanical reinforcement of microtubules. In this paper, we have used atomic force microscopy to address both aspects by visualizing and mechanically analyzing microtubules in the presence of native tau isoforms. We could show that tau at saturating concentrations forms a 1 nm thick layer around the microtubule, but leaves the protofilament structure well visible. The latter observation argues for tau binding mainly along and not across the protofilaments. The radial elasticity of microtubules was almost unaffected by tau, consistent with tau binding along the tops of the protofilaments. Tau did increase the resistance of microtubules against rupture. Finite-element calculations confirmed our findings.     

Atomic force microscopy study of the antigen‐antibody binding force on patient cancer cells based on ROR1 fluorescence recognition

Knowledge of drug–target interaction is critical to our understanding of drug action and can help design better drugs. Due to the lack of adequate single‐molecule techniques, the information of individual interactions between ligand‐receptors is scarce until the advent of atomic force microscopy (AFM) that can be used to directly measure the individual ligand‐receptor forces under near‐physiological conditions by linking ligands onto the surface of the AFM tip and then obtaining force curves on cells. Most of the current AFM single‐molecule force spectroscopy experiments were performed on cells grown in vitro (cell lines) that are quite different from the human cells in vivo. From the view of clinical practice, investigating the drug–target interactions directly on the patient cancer cells will bring more valuable knowledge that may potentially serve as an important parameter in personalized treatment. Here, we demonstrate the capability of AFM to measure the binding force between target (CD20) and drug (rituximab, an anti‐CD20 monoclonal antibody targeted drug) directly on lymphoma patient cancer cells under the assistance of ROR1 fluorescence recognition. ROR1 is a receptor expressed on some B‐cell lymphomas but not on normal cells. First, B‐cell lymphoma Raji cells (a cell line) were used for ROR1 fluorescence labeling and subsequent measurement of CD20‐rituximab binding force. The results showed that Raji cells expressed ROR1, and the labeling of ROR1 did not influence the measurement of CD20‐rituximab binding force. Then the established experimental procedures were performed on the pathological samples prepared from the bone marrow of a follicular lymphoma patient. Cancer cells were recognized by ROR1 fluorescence. Under the guidance of fluorescence, with the use of a rituximab‐conjugated tip, the cellular topography was visualized by using AFM imaging and the CD20‐Rituximab binding force was measured by single‐molecule force spectroscopy. Copyright © 2013 John Wiley & Sons, Ltd.     

Structure of human chromosomes studied by atomic force microscopy

In this work human chromosomes have been treated with RNase and pepsin to remove the layer of cellular material that covers the standard preparations on glass slides. This allows characterization of the topography of chromosomes at nanometer scale in air and in physiological solution by atomic force microscopy. Imaging of the dehydrated structure in air indicates radial arrangement of chromatin loops as the last level of DNA packing. However, imaging in liquid reveals a last level of organization consisting of a hierarchy of bands and coils. Additionally force curves between the tip and the chromosome in liquid are consistent with radial chromatin loops. These results and previous electron microscopy studies are analyzed, and a model is proposed for the chromosome structure in which radial loops and helical coils coexist.     

Structure of adsorbed fibrinogen obtained by scanning force microscopy

It is shown that scanning force microscopy (SFM), operated in the attractive mode, can be used to obtain high resolution pictures of adsorbed fibrinogen molecules on solid surfaces, without the need for staining or special microscope grids. SFM also reveals the three-dimensional structure of the adsorbed molecules. Two forms of adsorbed fibrinogen are demonstrated on hydrophobic silicone dioxide surfaces; a trinodular about 60 nm long and a globular with about a 40 nm diameter. Polymeric networks formed after storage of the surface with adsorbed fibrinogen in PBS for 11 days are also shown. The SFM-results for the trinodular structure suggest the existence of loops or peptide chains extending outside the basic structure of the fibrinogen molecule.     

Size distribution measurement of vesicles by atomic force microscopy

Vesicles have been utilized as nanoscale vehicles for reagents including potential drug delivery systems. When used to deliver drugs, vesicle size and the size distribution are important factors in the determination of the dosage, cell specificity, and rate of clearance from the body. Current size measurement techniques for vesicles are electron microscopy and dynamic light scattering, but their results are not equal. Therefore atomic force microscopy was attempted as another size measurement technique. After adsorption of the vesicles from a low-concentration solution of vesicles on mica substrate, each vesicle is generally found as a flattened structure. The diameters of vesicles in these solutions and their distribution have been successfully estimated from the surface area of the flattened structure of each vesicle. At higher concentrations, we have found a monolayer crammed with dome-shaped vesicles on the substrate. The diameters of vesicles in these solutions have also been successfully estimated from the surface area of the dome-shaped structure of each vesicle. Diameters of vesicles in solution estimated from two different vesicle concentrations are not close to those reported by electron microscope studies but are close to those reported by dynamic light scattering studies.     

Atomic force microscopy captures the initiation of methyl-directed DNA mismatch repair

In Escherichia coli, errors in newly-replicated DNA, such as the incorporation of a nucleotide with a mis-paired base or an accidental insertion or deletion of nucleotides, are corrected by a methyl-directed mismatch repair (MMR) pathway. While the enzymology of MMR has long been established, many fundamental aspects of its mechanisms remain elusive, such as the structures, compositions, and orientations of complexes of MutS, MutL, and MutH as they initiate repair. Using atomic force microscopy, we—for the first time—record the structures and locations of individual complexes of MutS, MutL and MutH bound to DNA molecules during the initial stages of mismatch repair. This technique reveals a number of striking and unexpected structures, such as the growth and disassembly of large multimeric complexes at mismatched sites, complexes of MutS and MutL anchoring latent MutH onto hemi-methylated d(GATC) sites or bound themselves at nicks in the DNA, and complexes directly bridging mismatched and hemi-methylated d(GATC) sites by looping the DNA. The observations from these single-molecule studies provide new opportunities to resolve some of the long-standing controversies in the field and underscore the dynamic heterogeneity and versatility of MutSLH complexes in the repair process.     

Dynamic response of HPV16/anti-HPV16 pairs with unbinding events studied by atomic force microscopy

This paper proposes an effective approach to distinguish whether samples include Human Papilloma virus type-16 (HPV16) by Atomic force microscopy (AFM). AFM is an important instrument in nanobiotechnology field. At first we identified the HPV16 by Polymerase chain reaction (PCR) analysis and Western blotting from specimen of the HPV patient (E12) and the normal (C2), and then we used an AFM to observe the surface ultrastructure by tapping mode and to measure the unbinding force between HPV16 coupled to an AFM tip and anti-HPV16 L1 coated on the substrate surface by contact mode. The experimental results by tapping mode show that the size of a single HPV viron was similar to its SEM image from the previous literatures; moreover, based on the purposed methods and the analysis, two obvious findings that we can determine whether or not the subject is a HPV patient can be derived from the results; one is based on the distribution of unbinding forces, and the other is based on the distribution of the stiffness. Furthermore, the proposed method could be a useful technique for further investigating the potential role among subtypes of HPVs in the oncogenesis of human cervical cancer.     

Probing elasticity and adhesion of live cells by atomic force microscopy indentation

Atomic force microscopy (AFM) indentation has become an important technique for quantifying the mechanical properties of live cells at nanoscale. However, determination of cell elasticity modulus from the force–displacement curves measured in the AFM indentations is not a trivial task. The present work shows that these force–displacement curves are affected by indenter-cell adhesion force, while the use of an appropriate indentation model may provide information on the cell elasticity and the work of adhesion of the cell membrane to the surface of the AFM probes. A recently proposed indentation model (Sirghi, Rossi in Appl Phys Lett 89:243118, 2006), which accounts for the effect of the adhesion force in nanoscale indentation, is applied to the AFM indentation experiments performed on live cells with pyramidal indenters. The model considers that the indentation force equilibrates the elastic force of the cell cytoskeleton and the adhesion force of the cell membrane. It is assumed that the indenter-cell contact area and the adhesion force decrease continuously during the unloading part of the indentation (peeling model). Force–displacement curves measured in indentation experiments performed with silicon nitride AFM probes with pyramidal tips on live cells (mouse fibroblast Balb/c3T3 clone A31-1-1) in physiological medium at 37°C agree well with the theoretical prediction and are used to determine the cell elasticity modulus and indenter-cell work of adhesion. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Actin microridges characterized by laser scanning confocal and atomic force microscopy

We have characterized the cell surface of zebrafish stratified epithelium using a combined approach of light and atomic force microscopy under conditions which simulate wound healing. Microridges rise on average 100 nm above the surface of living epithelial cells, which correlate to bundles of cytochalasin B-insensitive actin filaments. Time-lapse microscopy revealed the bundles to form a highly dynamic network on the cell surface, in which bundles and junctions were severed and annealed on a time scale of minutes. Atomic force microscopy topographs further indicated that actin bundle junctions identified were of two types: overlaps and integrated end to side T- and Y-junctions. The surface bundle network is found only on the topmost cell layer of the explant, and never on individual locomoting cells. Possible functions of these actin bundles include cell compartmentalization of the cell surface, resistance to mechanical stress, and F-actin storage.     

Atomic force microscopy study of the antimicrobial action of Sushi peptides on Gram negative bacteria

The antibacterial effect of the endotoxin-binding Sushi peptides against Gram-negative bacteria (GNB) is investigated in this study. Similar characteristics observed for Atomic force microscopy (AFM) images of peptide-treated Escherichia coli and Pseudomonas aeruginosa suggest that the Sushi peptides (S3) evoke comparable mechanism of action against different strains of GNB. The results also indicate that the Sushi peptides appear to act in three stages: damage of the bacterial outer membrane, permeabilization of the inner membrane and disintegration of both membranes. The AFM approach has provided vivid and detailed close-up images of the GNB undergoing various stages of antimicrobial peptide actions at the nanometer scale. The AFM results support our hypothesis that the S3 peptide perturbs the GNB membrane via the “carpet-model” and thus, provide important insights into their antimicrobial mechanisms.     

How to orient the functional GroEL-SR1 mutant for atomic force microscopy investigations

We present high-resolution atomic force microscopy (AFM) imaging of the single-ring mutant of the chaperonin GroEL (SR-EL) from Escherichia coli in buffer solution. The native GroEL is generally unsuitable for AFM scanning as it is easily being bisected by forces exerted by the AFM tip. The single-ring mutant of GroEL with its simplified composition, but unaltered capability of binding substrates and the co-chaperone GroES, is a more suited system for AFM studies. We worked out a scheme to systematically investigate both the apical and the equatorial faces of SR-EL, as it binds in a preferred orientation to hydrophilic mica and hydrophobic highly ordered pyrolytic graphite. High-resolution topographical imaging and the interaction of the co-chaperone GroES were used to assign the orientations of SR-EL in comparison with the physically bisected GroEL. The usage of SR-EL facilitates single molecule studies on the folding cycle of the GroE system using AFM.     

Assessing the Cytoskeletal System and its Elements in C6 Glioma Cells and Astrocytes by Atomic Force Microscopy

Object To investigate how the characteristic structure of the cytoskeleton in glioma cells is associated with invasiveness. Methods The whole cytoskeletal system was characterized by atomic force microscopy (AFM), while single cytoskeletal elements were exhibited by AFM and using cytoskeletal protein inhibitors to inhibit microfilaments or/and microtubules and displayed by immunofluorescence microscopy. The fluorescence intensity of F-actin was measured by flow cytometry and the structural difference between C6 glioma cells and astrocytes was studied. Results Cytoskeletons in both cells presented network structures, however, the C6 glioma cells showed an irregular edge root and their microfilaments were creber and dense. Intermediate filaments were extensive network structure with non-polarized multipoint connections. The microtubules were relatively big and long and formed tight bundles with close connections between bundles. Astrocytes had a regular and smooth edge, with sparse microfilaments, while the intermediate filaments were dense and interwoven and the microtubules were dense bundled, but only loosely connected each other. Besides, the fluorescence intensity of F-actin was significantly higher in C6 glioma cells (202.54 ± 11.06) than in the astrocytes (62.64 ± 10.23), P < 0.01. Conclusion Whole cytoskeleton and its elements of C6 cells were disclosed of characteristic structures associated with invasiveness. Meanwhile, the content of F-actin could be used as a parameter for measuring cell invasiveness.     

Atomic force microscopy differentiates discrete size distributions between membrane protein containing and empty nanolipoprotein particles

To better understand the incorporation of membrane proteins into discoidal nanolipoprotein particles (NLPs) we have used atomic force microscopy (AFM) to image and analyze NLPs assembled in the presence of bacteriorhodopsin (bR), lipoprotein E4 n-terminal 22k fragment scaffold and DMPC lipid. The self-assembly process produced two distinct NLP populations: those containing inserted bR (bR-NLPs) and those that did not (empty-NLPs). The bR-NLPs were distinguishable from empty-NLPs by an average increase in height of 1.0 nm as measured by AFM. Streptavidin binding to biotinylated bR confirmed that the original 1.0 nm height increase corresponds to br-NLP incorporation. AFM and ion mobility spectrometry (IMS) measurements suggest that NLP size did not vary around a single mean but instead there were several subpopulations, which were separated by discrete diameters. Interestingly, when bR was present during assembly the diameter distribution was shifted to larger particles and the larger particles had a greater likelihood of containing bR than smaller particles, suggesting that membrane proteins alter the mechanism of NLP assembly.