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At least 991 records · Page 55Linked to original sources
PubMed2005

Gradient of rigidity in the lamellipodia of migrating cells revealed by atomic force microscopy.

Changes in mechanical properties of the cytoplasm have been implicated in cell motility, but there is little information about these properties in specific regions of the cell at specific stages of the cell migration process. Fish epidermal keratocytes with their stable shape and steady motion represent an ideal system to elucidate temporal and spatial dynamics of the mechanical state of the cytoplasm. As the shape of the cell does not change during motion and actin network in the lamellipodia is nearly stationary with respect to the substrate, the spatial changes in the direction from the front to the rear of the cell reflect temporal changes in the actin network after its assembly at the leading edge. We have utilized atomic force microscopy to determine the rigidity of fish keratocyte lamellipodia as a function of time/distance from the leading edge. Although vertical thickness remained nearly constant throughout the lamellipodia, the rigidity exhibited a gradual but significant decrease from the front to the rear of the lamellipodia. The rigidity profile resembled closely the actin density profile, suggesting that the dynamics of rigidity are due to actin depolymerization. The decrease of rigidity may play a role in facilitating the contraction of the actin-myosin network at the lamellipodium/cell body transition zone.

Actins↗
PubMed2004

Atomic force microscopy reveals defects within mica supported lipid bilayers induced by the amyloidogenic human amylin peptide.

To date, over 20 peptides or proteins have been identified that can form amyloid fibrils in the body and are thought to cause disease. The mechanism by which amyloid peptides cause the cytotoxicity observed and disease is not understood. However, one of the major hypotheses is that amyloid peptides cause membrane perturbation. Hence, we have studied the interaction between lipid bilayers and the 37 amino acid residue polypeptide amylin, which is the primary constituent of the pancreatic amyloid associated with type 2 diabetes. Using a dye release assay we confirmed that the amyloidogenic human amylin peptide causes membrane disruption; however, time-lapse atomic force microscopy revealed that this did not occur by the formation of defined pores. On the contrary, the peptide induced the formation of small defects spreading over the lipid surface. We also found that rat amylin, which has 84% identity with human amylin but cannot form amyloid fibrils, could also induce similar lesions to supported lipid bilayers. The effect, however, for rat amylin but not human amylin, was inhibited under high ionic conditions. These data provide an alternative theory to pore formation, and how amyloid peptides may cause membrane disruption and possibly cytotoxicity.

Aluminum Silicates↗
PubMed2005

Effects of lactose permease on the phospholipid environment in which it is reconstituted: a fluorescence and atomic force microscopy study.

The membrane transport protein lactose permease (LacY), a member of the major facilitator superfamily containing 12 membrane-spanning segments connected by hydrophilic loops, was reconstituted in liposomes whose composition was 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol in a 3:1 molar ratio. The structural order of the lipid membranes, in the presence and absence of LacY, was assessed using steady-state fluorescence anisotropy. The features of the anisotropy curves obtained with 1,6-phenyl-1,3,5-hexatriene and 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene p-toluenesulfonate suggest a surface effect of LacY on the membranes. Atomic force microscopy imaging of supported planar bilayers (SPBs) deposited onto mica was used to examine the effect of LacY on the nanostructure of the phospholipid matrix. Two separated domains were observed in SPBs formed from pure phospholipid mixture. Protein assemblies segregated from the rest of the matrix were observed after the extension of proteoliposomes. The effect of the protein on the electrostatic surface potential of the bilayer was also examined using a fluorescent pH indicator, 4-heptadecyl-7-hydroxycoumarin. Changes in surface potential were enhanced in the presence of the substrate (i.e., lactose). Taken together the results indicate that LacY is segregated into the phospholipid matrix and has moderate effects on the acyl chain order of the bilayers. The changes in surface electrical properties of the bilayers suggest a role for the phospholipid headgroups in proton transfer to the amino acids involved in substrate translocation.

Amino Acid Sequence↗
PubMedDate not supplied

Atomic force microscopy reveals aggregation of gastric mucin at low pH.

Mammalian gastric mucin, at high concentration, is known to form a gel at low pH, behavior essential to the protection of the stomach from auto-digestion. Atomic force microscopy (AFM) measurements of dilute solutions of porcine gastric mucin in an aqueous environment in the pH range 6-2 provide a direct visualization of extended fiberlike molecules at pH 6 that aggregate at pH 4 and below forming well-defined clusters at pH 2. The clusters consist of 10 or less molecules. AFM images of mucin at high concentration at pH 2 reveal clusters similar to those seen in the dilute solutions at low pH. We also imaged human gastric mucus revealing a network having a "pearl necklace" structure. The "pearls" are similar in size to the clusters found in the purified porcine gastric mucin gels. AFM images of deglycosylated mucin reveal that the deglycosylated portions of the molecule re-fold into compact, globular structures suggesting that the oligosaccharide chains are important in maintaining the extended conformation of mucin. However, the oligosaccharides do not appear to be directly involved in the aggregation at low pH, as clusters of similar size are observed at pH 2 in both native and deglycosylated mucin.

Animals↗
PubMed2001

Population analysis of subsaturated 172-12 nucleosomal arrays by atomic force microscopy detects nonrandom behavior that is favored by histone acetylation and short repeat length.

Concatameric 5 S rDNA templates reconstituted in vitro into nucleosomal arrays provide very popular chromatin models for many kinds of studies. Here, atomic force microscopy is used to determine the population distributions for one such nucleosomal array, the 172-12, reconstituted to various subsaturated levels with nonacetylated or hyperacetylated HeLa histones. This array is a model for short linker length genomes and transcriptionally active and newly replicated chromatins. The analysis shows that as input histone levels increase, template occupation increases progressively as discrete population distributions. The distributions are random at low (n(av) < 4) and high (n(av) > 8) loadings but display specific nonrandom features, such as a deficit of molecules with one nucleosome more or less than the peak species in the distribution and enhanced distribution breadths, in the mid-range (n(av) = 4-8). Thus, the mid-range of occupation on polynucleosomal arrays may be a special range for chromatin structure and/or assembly. The mid-range nonrandom features are enhanced in distributions from short repeat (172-12) arrays, particularly for unacetylated chromatin, and in distributions from hyperacetylated chromatin, particularly for long repeat (208-12) arrays. Thus, short repeat length and acetylation can affect basic chromatin properties, like population tendencies, in very similar ways and therefore may cause similar changes in chromatin structure. Some possible effects are suggested. The data also indicate that it is thermodynamically more difficult for hyperacetylated nucleosomes to assemble onto the 172-12 templates, a result having implications for in vivo chromatin assembly.

Acetylation↗
PubMed1997

Interaction of DNA-dependent protein kinase with DNA and with Ku: biochemical and atomic-force microscopy studies.

DNA-dependent protein kinase (DNA-PK or the scid factor) and Ku are critical for DNA end-joining in V(D)J recombination and in general non-homologous double-strand break repair. One model for the function of DNA-PK is that it forms a complex with Ku70/86, and this complex then binds to DNA ends, with Ku serving as the DNA-binding subunit. We find that DNA-PK can itself bind to linear DNA fragments ranging in size from 18 to 841 bp double-stranded (ds) DNA, as indicated by: (i) mobility shifts; (ii) crosslinking between the DNA and DNA-PK; and (iii) atomic-force microscopy. Binding of the 18 bp ds DNA to DNA-PK activates it for phosphorylation of protein targets, and this level of activation is not increased by addition of purified Ku70/86. Ku can stimulate DNA-PK activity beyond this level only when the DNA fragments are long enough for the independent binding to the DNA of both DNA-PK and Ku. Atomic-force microscopy indicates that under such conditions, the DNA-PK binds at the DNA termini, and Ku70/86 assumes a position along the ds DNA that is adjacent to the DNA-PK.

Antigens, Nuclear↗
PubMed2003

Self-assembly of drug-polymer complexes: a spontaneous nanoencapsulation process monitored by atomic force microscopy.

Since hydrophilic matrices were proposed for controlled drug delivery, many polymeric excipients have been studied to make drug release fit the desired profiles. It has been pointed out that lambda-carrageenan, a sulfated polymer from algae, can suitably control the release rate of basic drugs from hydrophilic matrices. Furthermore, the relevance of hydrophobic interactions in drug-polymer aqueous systems has already been demonstrated, although no references to morphological features as well as to the kinetics of the interaction complexes formation have been published to date. In this work, we propose a method to monitor the topographical evolution of the interaction between lambda-carrageenan and dexchlorpheniramine maleate, in order to determine how the release profiles can be so easily controlled. For this purpose, solutions of both polymer and drug were prepared at very low concentration. Solutions were mixed and small volumes were taken every hour for over a period of 24 h and subsequently analyzed. The characterization technique used, atomic force microscopy, provides a high resolution, allowing plotting of three-dimensional images of the sample morphology within the nanometric scale. The results demonstrate that lambda-carrageenan is able to nanoencapsulate spontaneously dexchlorpheniramine maleate molecules, which offers the possibility of controlling the release rate of the drug with no need of complex technological processes. Moreover, this work demonstrates the suitability of atomic force microscopy for the specific case of the on-time monitoring of interaction processes that occur in pharmaceutical systems.

Carrageenan↗
PubMed1994

Molecular resolution atomic force microscopy of soluble proteins in solution.

We introduce a simple specimen preparatory method for atomic force microscopy of soluble proteins in aqueous solutions. It is demonstrated that the mica surface is suitable for direct adsorption of macromolecules that are sufficiently stable to withstand the disturbance of the probe for reproducible imaging at high resolution. It is also shown that the main problem impeding successful imaging is the excessive adsorption of macromolecules, as loosely bound macromolecules readily stick to the tip and produce various imaging artifacts.

Animals↗
PubMed1992

Cisplatin induced alterations in oriented fibers of DNA studied by atomic force microscopy.

Oriented fibers of DNA, prepared by the wet spin method, were imaged by atomic force microscopy. It was found that an oriented fiber's substructure is an array of assemblages of DNA. For native DNA, the assemblages exhibit a characteristic width of 76 nm, a thickness of 20 nm, and appear to carry a right handed twist. Treatment with the anticancer drug cisplatin prior to wet spinning induces geometric irregularities, in the form of kinks and width distortions, into the assemblages of DNA.

Cisplatin↗
PubMed1993

Direct visualization of polypeptide shell of ferritin molecule by atomic force microscopy.

The polypeptide shell of the ferritin molecule has been imaged in water by atomic force microscopy (AFM). The central dip and the quaternary structure could be observed on the surface of the ferritin molecule anchored inhomogeneously in two dimensions. These structures observed in the AFM images are quite similar to the electron density map near the top of the apoferritin viewed down from a 4-fold axis structure reported previously (S. H. Banyard, D. K. Stammers, and P. M. Harrison, 1978. Nature (Lond.). 271:282-284). It has been achieved by introducing a "self-screening effect" of the surface charges of the AFM sample (S. Ohnishi, M. Hara, T. Furuno, and H. Sasabe. 1992. Biophys. J. 63:1425-1431) and the specially sharpened stylus of AFM cantilever.

Animals↗
PubMed2002

Mapping of lateral vibration of the tip in atomic force microscopy at the torsional resonance of the cantilever.

Lateral vibration of the tip in atomic force microscopy was mapped at the torsional resonance of the cantilever by attaching a shear piezo element at the base of the cantilever or under the sample. Fixed frequency excitation and self-excitation of torsional motion were implemented. The lateral vibration utilized as measured by an optical lever was in the order of 10 pm to 3 nm, and its frequency approximately 450 kHz for a contact-mode silicon nitride cantilever. The amplitude and phase of the torsional motion of the cantilever was measured by a lock-in-amplifier or a rectifier and plotted in x and y as the sample was raster scanned. The imaging technique gave contrast between graphite terraces, self-assembled monolayer domains, silicon and silicon dioxide, graphite and mica. Changing contrast was observed as silicon islands oxidized in atmosphere, showing that the imaging technique can detect change in lateral tip mobility due to changes occurring near the surface. Torsional self-excitation showed nanometric features of self-assembled monolayer islands due to different lateral dissipation. Dependence of torsional resonance frequency on excitation amplitude, and contrast change due to driving frequency around resonance were observed.

Journal Article↗
PubMed2006

Atomic-scale roughness effect on capillary force in atomic force microscopy.

We study the capillary force in atomic force microscopy by using Monte Carlo simulations. Adopting a lattice gas model for water, we simulated water menisci that form between a rough silicon-nitride tip and a mica surface. Unlike its macroscopic counterpart, the water meniscus at the nanoscale gives rise to a capillary force that responds sensitively to the tip roughness. With only a slight change in tip shape, the pull-off force significantly changes its qualitative variation with humidity.

Journal Article↗
PubMed2005

Atomic force microscopy investigation of the mechanism of calcite microcrystal growth under Kitano conditions.

A combined atomic force microscopy (AFM)-inverted optical microscopy technique has been used to image the surface of calcite single microcrystals, with dimensions of 10-20 microm, at high resolution. The microcrystals were grown on a glass substrate using the Kitano method, a process that involves the outgassing of carbon dioxide from a saturated solution of calcium carbonate. The resulting increase in the supersaturation of the solution, with respect to calcium carbonate, induces crystallization. It is demonstrated, for the first time, that calcite microcrystals formed in this way exhibit a single spiral growth hillock on the (104) surface, as evidenced by a spiral step pattern, indicating that growth occurs at steps arising from an individual screw dislocation. The subsequent reactivity of these crystals under Kitano conditions has been followed in situ using AFM imaging.

Journal Article↗
PubMed2005

Noncontact atomic force microscopy studies of ultrathin films of amorphous solid water deposited on Au(111).

Noncontact atomic force microscopy was used to study the morphological changes of an ultrathin amorphous solid water (ASW) film as a function of deposition temperature, annealing temperature, and annealing time. ASW deposited at 80 or 108 K on Au(111) formed truncated hemispherical clusters of increasing size during annealing at 134 K; these clusters were inferred to be crystalline. The number of nuclei present at the outer surface of the film after deposition was greater for higher deposition temperature. For lower cluster densities, depletion of the ASW film around the clusters was observed when the clusters became larger and dendritic growth was observed when the apparent cluster footprint radius exceeded 100 nm.

Journal Article↗
PubMed1993

Atomic force microscopy of long DNA: imaging in air and under water.

We have obtained striking atomic force microscopy images of the intact lambda bacteriophage genome and of several lambda restriction fragments both in air and under water. The DNA is unstained and the images are stable under continuous scanning for up to 30 min. Measured contour lengths of fully imaged restriction fragments and intact lambda DNA are accurate to within a few percent. The key to this development is the use of a process for binding unmodified double-stranded DNA to chemically treated mica surfaces. This procedure leads to strong DNA attachment and yields high-quality images that are stable under repeated scanning, even with the sample submerged in water. This allows normal hydration conditions to be maintained during scanning and in addition leads to a general improvement of image quality. Both the lateral resolution and the contrast increase by a factor of approximately 3 under water.

Bacteriophage lambda↗
PubMed1993

Atomic force microscopy of bacteriophage T4 and its tube-baseplate complex.

Bacteriophage T4 was imaged by atomic force microscopy with the finest resolution to date with a clear image of tail fibers of an estimated diameter of 2-3 nm. T4 phages were spread on a clean surface of silicon wafer and dried under air before observation with an atomic force microscope. The head, tail and tail fibers were routinely imaged with somewhat distorted dimensions. The ease of imaging isolated phage particles with a good resolution raised our expectation for the further use of AFM in biomedical applications.

Bacteriophage T4↗
PubMed2006

Polymorphism and ultrastructural organization of prion protein amyloid fibrils: an insight from high resolution atomic force microscopy.

Amyloid fibrils were produced from the full-length mouse prion protein (PrP) under solvent conditions similar to those used for the generation of synthetic prions from PrP 89-230. Analysis of the ultrastructure by atomic force microscopy revealed extremely broad polymorphism in fibrils formed under a single growth condition. Fibrils varied with respect to the number of constitutive filaments and the manner in which the filaments were assembled. PrP polymerization was found to show several peculiar features: (i) the higher-order fibrils/ribbons were formed through a highly hierarchical mechanism of assembly of lower-order fibrils/ribbons; (ii) the lateral assembly proceeded stepwise; at each step, a semi-stable fibrillar species were generated, which were then able to enter the next level of assembly; (iii) the assembly of lower into higher-order fibrils occurred predominantly in a vertical dimension via stacking of ribbons on top of each other; (iv) alternative modes of lateral association co-existed under a single growth condition; (iv) the fibrillar morphology changed even within individual fibrils, illustrating that alternative modes of filament assembly are inter-convertible and thermodynamically equivalent. The most predominant fibrillar types were classified into five groups according to their height, each of which was divided in up to three subgroups according to their width. Detailed analysis of ultrastructure revealed that the fibrils of the major subtype (height 3.61(+/-0.28)nm, width 31.1(+/-2.0)nm) were composed of two ribbons, each of which was composed of two filaments. The molecular volume calculations indicated that a single PrP molecule occupied a distance of approximately 1.2 nm within a single filament. High polymorphism in fibrils generated in vitro is reminiscent of high morphological diversity of scrapie-associated fibrils isolated from scrapie brains, suggesting that polymorphism is peculiar for polymerization of PrP regardless of whether fibrils are formed in vitro or under pathological conditions in vivo.

Amyloid↗
PubMed2006

Localization of linker histone in chromatosomes by cryo-atomic force microscopy.

Linker histones play a fundamental role in determining higher order chromatin structure as a consequence of their association with nucelosomal DNA. Yet the locations and structural consequences of linker histone binding are still enigmatic. Here, using cryo-atomic force microscopy, we show that the linker histone H5 in native chromatin and in chromatosomes reconstituted on the 5S rDNA template is located at the dyad of the nucleosome core particle, within the "stem" structure. Direct measurement also indicates that the length of free linker DNA between chromatosomes in native chromatin is approximately 30 bp, slightly shorter than that estimated from nuclease digestion assays.

Binding Sites↗