共查询到20条相似文献,搜索用时 15 毫秒
1.
Resolving the molecular structure of microtubules under physiological conditions with scanning force microscopy 总被引:3,自引:0,他引:3
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 相似文献
2.
Tamayo J 《Journal of structural biology》2003,141(3):198-207
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. 相似文献
3.
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. 相似文献
4.
Elasticity of normal and cancerous human bladder cells studied by scanning force microscopy 总被引:5,自引:0,他引:5
M. Lekka P. Laidler D. Gil J. Lekki Z. Stachura A. Z. Hrynkiewicz 《European biophysics journal : EBJ》1999,28(4):312-316
Scanning force microscopy was used for the determination of the elastic properties of living cells in their culture conditions.
The studies were carried out on human epithelial cells. Two similar lines of normal cells (Hu609 and HCV29) and three cancerous
ones (Hu456, T24, BC3726) were measured using the scanning force microscope in order to collect the force versus indentation
curves. The BC3726 line originates from the HCV29 cell line which was transformed by the v-ras oncogene. To evaluate their elastic properties, Young's modulus values were determined. The present study has shown that
normal cells have a Young's modulus of about one order of magnitude higher than cancerous ones. Such a change might be attributed
to a difference in the organisation of cell cytoskeletons and requires further studies.
Received: 30 April 1998 / Revised version: 17 December 1998 / Accepted: 21 January 1999 相似文献
5.
Tamayo J 《Journal of structural biology》2003,141(3):189-197
In the first part of this work, human chromosomes were characterized by atomic force microscopy (AFM) in air and in aqueous solution. The analysis of the images suggests that the last level of organization consists of a radial arrangement of chromatin loops which are anchored to a fiber which is folded giving a pattern of bands which differs in volume. Here the pattern of bands observed by AFM is compared to the cytogenetic map at the 850-band level. Thus thicker and thinner bands are identified as G and R bands, respectively. Finally a model is proposed which links genome sequence, cytogenetics, and chromosome structure. 相似文献
6.
Atomic force microscopy (AFM) has been used to probe the interaction between porcine stomach mucin and a mucoadhesive polymer, pectin, with different chemical characteristics. Images were produced detailing the structures of mucin, pectin and the mixtures of pectin and mucin, in either 0.1 N hydrochloric acid or deionized water. The AFM images of the pectin–mucin mixture in acidic medium showed no association between pectin and mucin. The large aggregates observed after mixing pectin and mucin in deionized water revealed the association between pectin and mucin, probably by the H-bonding. Increasing of pectin in the mixture with mucin resulted in a shift of zeta potential of the mixture to a higher negative value. The electrostatic repulsion with the same charges of pectin and mucin may cause an uncoiling of polymer chains, which facilitated chain entanglement and bond formation. The particle size of the mixtures of pectin and mucin depended on the proportion of either pectin or mucin in the mixture. The results suggested that the mucoadhesion of pectin could be due to the adsorption mechanism on the mucin molecules or electrostatic repulsion between pectin and mucin. 相似文献
7.
Kanno T Yamada T Iwabuki H Tanaka H Kuroda S Tanizawa K Kawai T 《Analytical biochemistry》2002,309(2):196-199
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. 相似文献
8.
9.
Desmin filaments form the intermediate filament system of muscle cells where they play important role in maintaining mechanical integrity and elasticity. Although the importance of desmin elasticity and assembly-disassembly dynamics in cellular mechanics is being increasingly recognized, the molecular basis of neither desmin's elasticity nor its disassembly pathway is well understood. In the present work, we explored the topographical structure of purified and reconstituted desmin filaments by using scanning force microscopy. With the addition of divalent cation chelators ethyleneglycoltetraacetic acid or ethylenediaminetetraacetic acid, the filaments disassembled on a time scale of hours to days into stable, thin fibrillar components with variable (up to micrometer) length, smooth surface and uniform thickness, which are identified as protofibrils. Desmin protofibrils appear as elastic structures with a persistence length of 51.5 nm, and their Young's modulus (10.6 MPa) far exceeds that of the mature filament (3.7 MPa). Protofibrillar bundling within the desmin filament results in high longitudinal tensile strength at a large bending flexibility. The stability of protofibrils suggests that desmin may disassemble along a pathway quite distinct from its assembly. 相似文献
10.
11.
We have performed a very extensive investigation of chromatin folding in different buffers over a wide range of ionic conditions similar to those found in eukaryotic cells. Our results show that in the presence of physiological concentrations of monovalent cations and/or low concentrations of divalent cations, small chicken erythrocyte chromatin fragments and chromatin from HeLa cells observed by transmission electron microscopy (TEM) show a compact folding, forming circular bodies of approximately 35 nm in diameter that were found previously in our laboratory in studies performed under very limited conditions. Since TEM images are obtained with dehydrated samples, we have performed atomic force microscopy (AFM) experiments to analyze chromatin structure in the presence of solutions containing different cation concentrations. The highly compact circular structures (in which individual nucleosomes are not visible as separated units) produced by small chromatin fragments in interphase ionic conditions observed by AFM are equivalent to the structures observed by TEM with chromatin samples prepared under the same ionic conditions. We have also carried out experiments of sedimentation and trypsin digestion of chromatin fragments; the results obtained confirm our AFM observations. Our results suggest that the compaction of bulk interphase chromatin in solution at room temperature is considerably higher than that generally considered in current literature. The dense chromatin folding observed in this study is consistent with the requirement of compact chromatin structures as starting elements for the building of metaphase chromosomes, but poses a difficult physical problem for gene expression during interphase. 相似文献
12.
We present a novel slit scanning confocal microscope with a CCD camera image sensor and a virtual slit aperture for descanning
that can be adjusted during post-processing. A very efficient data structure and mathematical criteria for aligning the virtual
aperture guarantee the ease of use. We further introduce a method to reduce the anisotropic lateral resolution of slit scanning
microscopes. System performance is evaluated against a spinning disk confocal microscope on identical specimens. The virtual
slit scanning microscope works as the spinning disk type and outperforms on thick specimens. 相似文献
13.
Effects of acetic acid treatment on plant chromosome structures analyzed by atomic force microscopy 总被引:3,自引:0,他引:3
Sugiyama S Yoshino T Kanahara H Shichiri M Fukushi D Ohtani T 《Analytical biochemistry》2004,324(1):39-44
Acetic acid treatment has been frequently used to remove cellular contaminants from plant chromosome samples for structural analyses by scanning electron microscopy and atomic force microscopy (AFM). We evaluated the effects of various concentrations of acetic acid treatments on barley chromosome structures by using AFM. The long-term 45% acetic acid treatment significantly damaged the chromosome structures, although the treatment effectively removed the cellular contaminants. On the other hand, the treatment with 15% acetic acid could not obtain sufficiently clean chromosome samples and the chromosome surface structures could not be observed. In contrast, we obtained clean chromosome preparation without severe damage by using an intermediate concentration (30%) of acetic acid treatment. In the centromeric region, we could observe fiber structures with a width of 100 nm, which were composed of ca. 50-nm granules and aligned to the axes of chromosomes. Thus, AFM analysis of chromosomes appropriately treated with acetic acid will provide important insights into the organization of higher-order structures of plant chromosomes. 相似文献
14.
M. Girasole G. Pompeo A. Congiu-Castellano A. Serafino G. Boumis 《生物化学与生物物理学报:生物膜》2007,1768(5):1268-1276
A novel approach to the study of RBCs based on the collection of three-dimensional high-resolution AFM images and on the measure of the surface roughness of their plasma membrane is presented. The dependence of the roughness from several parameters of the imaging was investigated and a general rule for a trustful analysis and comparison has been suggested. The roughness of RBCs is a morphology-related parameter which has been shown to be characteristic of the single cells composing a sample, but independent of the overall geometric shape (discocyte or spherocyte) of the erythrocytes, thus providing extra-information with respect to a conventional morphology study. The use of the average roughness value as a label of a whole sample was tested on different kinds of samples. Analyzed data revealed that the quantitative roughness value does not change after treatment of RBCs with various commonly used fixation and staining methods while a drastic decrease occurs when studying cells with membrane-skeletal alteration both naturally occurring or artificially induced by chemical treatments. The present method provides a quantitative and powerful tool for a novel approach to the study of erythrocytes structure through an ultrastructural morphological analysis with the potential to give information, in a non-invasive way, on the RBCs function. 相似文献
15.
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 相似文献
16.
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. 相似文献
17.
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. 相似文献
18.
The molecular details of adhesion mechanics in phospholipid bilayers have been studied using atomic force microscopy (AFM). Under tension fused bilayers of dipalmitoylphosphatidylcholine (DPPC) yield to give non-distance dependent and discrete force plateaux of 45.4, 81.6 and 113±3.5 pN. This behaviour may persist over distances as great as 400 nm and suggests the stable formation of a cylindrical tube which bridges the bilayers on the two surfaces. The stability of this connective structure may have implications for the formation of pili and hence for the initial stage of bacterial conjugation. Dimyristoylphosphatidylcholine (DMPC) bilayers also exhibit force plateaux but with a much less pronounced quantization. Bilayers composed of egg PC, sterylamine and cholesterol stressed in a similar way show complex behaviour which can in part be explained using the models demonstrated in the pure lipids. 相似文献
19.
Deng X Zhang L Zhang Y Yan Y Xu Z Dong S Fu S 《Biochemical and biophysical research communications》2006,346(4):1228-1233
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. 相似文献
20.
Structure, force, and energy of a double-stranded DNA oligonucleotide under tensile loads 总被引:2,自引:0,他引:2
The end-to-end stretching of a duplex DNA oligonucleotide has been studied using potential of mean force (PMF) calculations
based on molecular dynamics (MD) simulations and atomic force microscopy (AFM) experiments. Near quantitative agreement between
the calculations and experiments was obtained for both the extension length and forces associated with strand separation.
The PMF calculations show that the oligonucleotide extends without a significant energetic barrier from a length shorter than
A-DNA to a length 2.4 times the contour length of B-DNA at which the barrier to strand separation is encountered. Calculated
forces associated with the barrier are 0.09±0.03 nN, based on assumptions concerning tip and thermal-activated barrier crossing
contributions to the forces. Direct AFM measurements show the oligonucleotide strands separating at 2.6±0.8 contour lengths
with a force of 0.13±0.05 nN. Analysis of the energies from the MD simulations during extension reveals compensation between
increases in the DNA-self energy and decreases in the DNA-solvent interaction energy, allowing for the barrierless extension
of DNA beyond the canonical B form. The barrier to strand separation occurs when unfavorable DNA interstrand repulsion cannot
be compensated for by favorable DNA-solvent interactions. The present combination of single molecule theoretical and experimental
approaches produces a comprehensive picture of the free energy surface of biological macromolecular structural transitions.
Received: 2 June 1998 / Revised version: 25 January 1999 / Accepted: 11 February 1999 相似文献