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.     

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.     

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.     

Fruit softening and pectin disassembly: an overview of nanostructural pectin modifications assessed by atomic force microscopy

Background

One of the main factors that reduce fruit quality and lead to economically important losses is oversoftening. Textural changes during fruit ripening are mainly due to the dissolution of the middle lamella, the reduction of cell-to-cell adhesion and the weakening of parenchyma cell walls as a result of the action of cell wall modifying enzymes. Pectins, major components of fruit cell walls, are extensively modified during ripening. These changes include solubilization, depolymerization and the loss of neutral side chains. Recent evidence in strawberry and apple, fruits with a soft or crisp texture at ripening, suggests that pectin disassembly is a key factor in textural changes. In both these fruits, softening was reduced as result of antisense downregulation of polygalacturonase genes. Changes in pectic polymer size, composition and structure have traditionally been studied by conventional techniques, most of them relying on bulk analysis of a population of polysaccharides, and studies focusing on modifications at the nanostructural level are scarce. Atomic force microscopy (AFM) allows the study of individual polymers at high magnification and with minimal sample preparation; however, AFM has rarely been employed to analyse pectin disassembly during fruit ripening.

Scope

In this review, the main features of the pectin disassembly process during fruit ripening are first discussed, and then the nanostructural characterization of fruit pectins by AFM and its relationship with texture and postharvest fruit shelf life is reviewed. In general, fruit pectins are visualized under AFM as linear chains, a few of which show long branches, and aggregates. Number- and weight-average values obtained from these images are in good agreement with chromatographic analyses. Most AFM studies indicate reductions in the length of individual pectin chains and the frequency of aggregates as the fruits ripen. Pectins extracted with sodium carbonate, supposedly located within the primary cell wall, are the most affected.     

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.     

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.     

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.     

The measurement of exonuclease activities by atomic force microscopy

We have applied atomic force microscopy (AFM) to the measurement of BAL 31 nuclease activities. BAL 31 nuclease, a species of exonuclease, is used to remove unwanted sequences from the termini of DNA before cloning. For cutting out only the appropriate sequences, it is important to know the nuclease properties, such as digestion speed and the distribution of the lengths of the digested DNA. AFM was used to obtain accurate measurements on the lengths of DNA fragments before and after BAL 31 nuclease digestion. We analyzed 4 DNAs with known number of base pairs (288, 778, 1818, and 3162 base pairs) for correlating the contour length measured by AFM with the number of base pairs under the deposition conditions used. We used this calibration for analyzing DNA degradation by BAL 31 nuclease from the AFM measurement of contour lengths of digested DNAs. In addition, the distribution of digested DNA could be analyzed in more detail by AFM than by electrophoresis, because digested DNA were measured as a population by electrophoresis, but were measured individually by AFM. These results show that AFM will be a useful new technique for measuring nuclease activities. Received: 8 August 1997 / Accepted: 10 September 1997     

Inactivated enzymes as probes of the structure of arabinoxylans as observed by atomic force microscopy

The complex structures of water-soluble wheat arabinoxylans have been mapped along individual molecules, and within populations, using the visualisation of the binding of inactivated enzymes by atomic force microscopy (AFM). It was demonstrated that site-directed mutagenesis (SDM) can be used to produce inactive enzymes as structural probes. For the SDM mutants AFM has been used to compare the binding of different xylanases to arabinoxylans. Xylanase mutant E386A, derived from the Xyn11A enzyme (Neocallimastrix patriciarium), was shown to bind randomly along arabinoxylan molecules. The xylanase binding was also monitored following Aspergillus niger arabinofuranosidase pre-treatment of samples. It was demonstrated that removal of arabinose side chains significantly altered the binding pattern of the inactivated enzyme. Xylanase mutant E246A, derived from the Xyn10A enzyme (Cellvibrio japonicus), was found to show deviations from random binding to the arabinoxylan chains. It is believed that this is due to the effect of a small residual catalytic activity of the enzyme that alters the binding pattern of the probe. Control procedures were developed and assessed to establish that the interactions between the modified xylanases and the arabinoxylans were specific interactions. The experimental data demonstrates the potential for using inactivated enzymes and AFM to probe the structural heterogeneity of individual polysaccharide molecules.     

Identification of TrkA on living PC12 cells by atomic force microscopy

In neural cells, nerve growth factor (NGF) initiates its survival signal through the binding to its cell surface receptor tyrosine kinase A (TrkA). Understanding the pattern of TrkA distribution and association in living cells can provide a fingerprint for the diagnostic comparison with alterations underlying ligand-receptor dysfunction seen in various neurological diseases. In this study, we use the NGF-TrkA-specific interaction as a probe to identify TrkA on living PC12 cell by atomic force microscopy (AFM). An NGF-modified AFM tip was used to perform force volume (FV) imaging, generating a 2D force map to illustrate the distribution and association of TrkA on PC12 cell membrane. It is found that TrkA is highly aggregated at local regions of the cell. This unique protein association may be required to promote its function as a receptor of NGF. The methodology that we developed in this study can be adapted by other systems, thus providing a general tool for investigating protein association in its natural environment.     

The elastic modulus of Matrigel as determined by atomic force microscopy

Recent studies indicate that the biophysical properties of the cellular microenvironment strongly influence a variety of fundamental cell behaviors. The extracellular matrix’s (ECM) response to mechanical force, described mathematically as the elastic modulus, is believed to play a particularly critical role in regulatory and pathological cell behaviors. The basement membrane (BM) is a specialization of the ECM that serves as the immediate interface for many cell types (e.g. all epithelial cells) and through which cells are connected to the underlying stroma. Matrigel is a commercially available BM-like complex and serves as an easily accessible experimental simulant of native BMs. However, the local elastic modulus of Matrigel has not been defined under physiological conditions. Here we present the procedures and results of indentation tests performed on Matrigel with atomic force microscopy (AFM) in an aqueous, temperature controlled environment. The average modulus value was found to be approximately 450 Pa. However, this result is considerably higher than macroscopic shear storage moduli reported in the scientific literature. The reason for this discrepancy is believed to result from differences in test methods and the tendency of Matrigel to soften at temperatures below 37° C.     

Double minute chromosomes in mouse methotrexate-resistant cells studied by atomic force microscopy

Double minute chromosomes (DMs) are acentric, autonomously replicating extra-chromosomes and frequently mediate gene amplification in tumor and drug resistant cells. Atomic force microscopy (AFM) is a powerful tool in microbiology. We used AFM to explore the ultrastructure of DMs in mouse fibroblasts 3T3R500. DMs in various phases of cell cycle were also studied in order to elucidate the mechanisms of their duplication and separation. Metaphase spread and induced premature condensed chromosomes (PCCs) were observed under the AFM. DMs were detected to be composed of two compact spheres linked by fibers. The fibers of DMs directly connected with metaphase chromosomes were observed. Many single-minutes and few DMs were detected in G1 PCCs, while more DMs were detected in S PCCs than in G1 PCCs. Besides, all of the DMs in G2 PCCs were coupled. Our present results suggested that DMs might divide into single-minutes during or before G1-phase, followed by duplication of the single-minutes in S-phase. Moreover, we introduced a new powerful tool to study DMs and got some ideal results.     

Visualization of alkali-denatured supercoiled plasmid DNA by atomic force microscopy

To study the alkali denaturation of supercoiled DNA, plasmid pBR322 was treated with gradient concentrations of NaOH solution. The results of gel electrophoresis showed that the alkali denaturation of the supercoiled DNA occurred in a narrow range of pH value (12.88-12.90). The alkali-denatured supercoiled DNA ran, as a sharp band, faster than the supercoiled DNA. The supercoiled plasmid DNA of pBR322, pACYC184 and pJGX15A were denatured by NaOH, and then visualized by atomic force microscopy. Compared with the supercoiled DNA, the atomic force microscopy images of the alkali-denatured supercoiled DNA showed rough surface with many kinks, bulges on double strands with inhomogeneous diameters. The apparent contour lengths of the denatured DNA were shortened by 16%, 16% and 50% for pBR322, pACYC184 and pJGX15A, respectively. All evidence suggested that the alkali-denatured supercoiled DNA had a stable conformation with unregistered, topologically constrained double strands and intrastrand secondary structure.     

Nanoscale topography of hepatitis B antigen particles by atomic force microscopy

Hepatitis B virus envelope is mainly composed of three forms of the same protein expressed from different start codons of the same open reading frame. The smaller form named S protein corresponds to the C-terminal common region and represents about 80% of the envelope proteins. It is mainly referred as hepatitis B virus surface antigen (HBsAg). Over expressed in the host cell, this protein can be produced as spherical and tubular self-organized particles. Highly immunogenic, these particles are used in licensed hepatitis B vaccines. In this study we have combined transmission electron microscopy and atomic force microscopy to determine the shape and size of HBsAg particles produced from the yeast Hansenula polymorpha. Tapping mode atomic force microscopy in liquid allows structural details of the surface to be delineated with a resolution in the nanometer range. Particles were decorated by closely packed spike-like structures protruding from particle surface. Protrusions appeared uniformly distributed at the surface and an average number of 75 protrusions per particle were calculated. Importantly, we demonstrated that proteins mainly contribute to the topography of the protrusions.     

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     

Deformability and adhesive force of artificial platelets measured by atomic force microscopy

Platelet glycoprotein GPIaIIa is an adhesive protein that recognizes collagen. We have investigated polymerized albumin particles conjugated with recombinant GPIaIIa (rGPIaIIa-poly Alb) for their platelet-like function. To evaluate the feasibility of these particles to achieve the hemostatic process, we measured the deformability (Young’s modulus and spring constant) and the adhesive force of the particles using atomic force microscopy, which can measure the mechanical properties of individual cells. Our results showed that the Young’s modulus of these particles was 2.3-fold larger than that of natural platelets and 12-fold larger than that of human red blood cells. The Young’s modulus of the particles may have been determined by the properties of the polymerized albumin particle, although the glycoprotein of the platelet surface also contributed to the higher modulus. Our results also showed that the adhesive force of the rGPIaIIa-poly Alb with the collagen ligand was 52% of that of natural platelets. These two mechanical properties (deformability and adhesive force) of cells or particles, such as rGPIaIIa-poly Alb, are important specifications for the optimum design of platelet substitutes.     

Characterisation of heterogeneous arabinoxylans by direct imaging of individual molecules by atomic force microscopy

Atomic force microscopy has been used to characterise populations of extracted water-soluble wheat endosperm arabinoxylans. The adsorbed molecules are extended structures with an estimated Kuhn statistical segment length of 128 nm, suggesting that they adopt an ordered helical structure. However, estimates of the molecular weight distribution, coupled with size exclusion data, suggest that, in solution, the polysaccharides behave as semi-flexible coils, with a Kuhn length of 16 nm. These data imply that adsorption of the arabinoxylan structures onto mica promotes formation of the helical structure. Adoption of this ordered structure is fortunate because it has permitted characterisation of branching observed in a small proportion (approximately 15%) of the population of otherwise linear molecules. The degree of branching has been found to increase with the contour length of the molecules. Degradation of the polysaccharides with xylanase has been used to confirm that both the backbone and branches are based on beta-(1-->4) linked D-xylopyranosyl residues.     

In situ atomic force microscopy of partially demineralized human dentin collagen fibrils

Dentin collagen fibrils were studied in situ by atomic force microscopy (AFM). New data on size distribution and the axial repeat distance of hydrated and dehydrated collagen type I fibrils are presented. Polished dentin disks from third molars were partially demineralized with citric acid, leaving proteins and the collagen matrix. At this stage collagen fibrils were not resolved by AFM, but after exposure to NaOCl(aq) for 100-240 s, and presumably due to the removal of noncollagenous proteins, individual collagen fibrils and the fibril network of dentin connected to the mineralized substrate were revealed. High-aspect-ratio silicon tips in tapping mode were used to image the soft fibril network. Hydrated fibrils showed three distinct groups of diameters: 100, 91, and 83 nm and a narrow distribution of the axial repeat distance at 67 nm. Dehydration resulted in a broad distribution of the fibril diameters between 75 and 105 nm and a division of the axial repeat distance into three groups at 67, 62, and 57 nm. Subfibrillar features (4 nm) were observed on hydrated and dehydrated fibrils. The gap depth between the thick and thin repeating segments of the fibrils varied from 3 to 7 nm. Phase mode revealed mineral particles on the transition from the gap to the overlap zone of the fibrils. This method appears to be a powerful tool for the analysis of fibrillar collagen structures in calcified tissues and may aid in understanding the differences in collagen affected by chemical treatments or by diseases.     

Detection of coccoid forms of Sulfitobacter mediterraneus using atomic force microscopy

The adhesion of the marine alpha-Proteobacteria Sulfitobacter pontiacus, Sulfitobacter mediterraneus, Sulfitobacter brevis, and Staleya guttiformis to a poly(tert-butyl methacrylate) (PtBMA) polymeric surface generates unusual cell morphological peculiarities following attachment. While the type strains S. pontiacus and S. brevis failed to attach to PtBMA, the vegetative cells of type strain S. mediterraneus underwent morphological conversion into coccoid forms during the attachment over an incubation period of 24-72 h. Type strain St. guttiformis cells formed a multilayered biofilm on the PtBMA surface, presumably facilitated by bacterial production of extracellular polysaccharides. The attachment behavior and fine structure of these coccoid forms have been described using atomic force microscopy. The impact of polymeric surfaces of defined hydrophobicity on the formation of coccoid bodies is discussed.