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PubMed2000

Pushing, pulling, dragging, and vibrating renal epithelia by using atomic force microscopy.

Renal physiologists focus on events that take place on and around the surfaces of cells. Various techniques have been developed that follow transport functions at the molecular level, but until recently none of these techniques has been capable of making the behavior of molecular structures visible under physiological conditions. This apparent gap may be filled in the future by the application of atomic force microscopy. This technique produces an image not by optical means, but by "feeling" its way across a surface. Atomic force microscopy can, however, be modified in a number of ways, which means that besides producing a high-resolution image, it is possible to obtain several types of data on the interactions between the ultrastructural components of cell membranes (such as proteins) and other biologically active molecules (such as ATP). In this review we describe the recent use of the atomic force microscope in renal physiology, ranging from experiments in intact cells to those in isolated renal transport protein molecules, include examples of these extended applications of the technique, and point to uses that the microscope has recently found in other areas of biology that should prove fruitful in renal physiology in the near future.

Animals↗

PubMed2004

Exploring the electronic and mechanical properties of protein using conducting atomic force microscopy.

In interfacing man-made electronic components with specifically folded biomacromolecules, the perturbative effects of junction structure on any signal generated should be considered. We report herein on the electron-transfer characteristics of the blue copper metalloprotein, azurin, as characterized at a refined level by conducting atomic force microscopy (C-AFM). Specifically, the modulation of current-voltage (I-V) behavior with compressional force has been examined. In the absence of assignable resonant electron tunneling within the confined bias region, from -1 to 1 V, the I-V behavior was analyzed with a modified Simmons formula. To interpret the variation of tunneling barrier height and barrier length obtained by fitting with the modified Simmons formula, an atom packing density model associated with protein mechanical deformation was proposed and simulated by molecular dynamics. The barrier heights determined at the minimum forces necessary for stable electrical contact correlate reasonably well with those estimated from bulk biophysical (electroanalytical and photochemical) experiments previously reported. At higher forces, the tunnel barrier decreases to fall within the range observed with saturated organic systems. Molecular dynamics simulations revealed changes in secondary structure and atomic density of the protein with respect to compression. At low compression, where transport measurements are made, secondary structure is retained, and atomic packing density is observed to increase linearly with force. These predictions, and those made at higher compression, are consistent with both experimentally observed modulations of tunneling barrier height with applied force and the applicability of the atom packing density model of electron tunneling in proteins to molecular-level analyses.

Azurin↗

PubMed2006

Highly compact folding of chromatin induced by cellular cation concentrations. Evidence from atomic force microscopy studies in aqueous solution.

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.

Animals↗

PubMed2004

Direct atomic force microscopy observations of monovalent ion induced binding of DNA to mica.

Multivalent ions in solution are known to mediate attraction between two like-charged molecules. Such attraction has proved useful in atomic force microscopy (AFM) where DNA may be immobilized to a mica surface facilitating direct imaging in liquid. Theories of DNA immobilization suggest that either 'salt bridging' or fluctuation in the positions of counter ions about both the mica surface and DNA backbone secure DNA to the mica substrate. Whilst both theoretical and experimental evidence suggest that immobilization is possible in the presence of divalent ions, very few studies identify that such immobilization is possible with monovalent ions. Here we present direct AFM evidence of DNA immobilized to mica in the presence of only monovalent ions. Our data depict E. coli plasmid pBR322 adsorbed onto the negatively charged mica both after short (10 min) and long (24 h) incubation periods. These data suggest the need to re-explore current theories of like-charge attraction to include the possibility of monovalent interactions. We suggest that this DNA immobilization strategy may offer the potential to image natural processes with limited immobilization forces and hence enable maximum conformational freedom of the immobilized biomolecule.

Aluminum Silicates↗

PubMed2000

H-NS mediated compaction of DNA visualised by atomic force microscopy.

The Escherichia coli H-NS protein is a nucleoid-associated protein involved in gene regulation and DNA compaction. To get more insight into the mechanism of DNA compaction we applied atomic force microscopy (AFM) to study the structure of H-NS-DNA complexes. On circular DNA molecules two different levels of H-NS induced condensation were observed. H-NS induced lateral condensation of large regions of the plasmid. In addition, large globular structures were identified that incorporated a considerable amount of DNA. The formation of these globular structures appeared not to be dependent on any specific sequence. On the basis of the AFM images, a model for global condensation of the chromosomal DNA by H-NS is proposed.

Bacterial Proteins↗

PubMed1996

Extent of sperm chromatin hydration determined by atomic force microscopy.

Volume measurements were performed on intact bull and mouse sperm heads and amembranous sperm nuclei, both in the fully hydrated (fluid cell) and dehydrated (air-dried on glass coverslips) states by atomic force microscopy (AFM). Data were obtained by analyzing a small population of cells/nuclei, as well as by performing repeated measurements on single cells imaged following the addition of increasing concentrations of propanol. Results show that the volume of fully hydrated, intact sperm heads and amembranous sperm chromatin particles are at least twice the volume of their air-dried counterparts. Dehydration occurs rapidly in air, and the reduction in volume of chromatin induced by water loss appears to be completely reversible. These studies demonstrate that both mouse and bull sperm chromatin are extensively hydrated in the native state, and are not as compact as previous studies have suggested.

1-Propanol↗

PubMed2006

Orientation of a monoclonal antibody adsorbed at the solid/solution interface: a combined study using atomic force microscopy and neutron reflectivity.

Conformational orientations of a mouse monoclonal antibody to the beta unit of human chorionic gonadotrophin (anti-beta-hCG) at the hydrophilic silicon oxide/water interface were investigated using atomic force microscopy (AFM) and neutron reflectivity (NR). The surface structural characterization was conducted with the antibody concentration in solution ranging from 2 to 50 mg.L(-1) with the ionic strength kept at 20 mM and pH = 7.0. It was found that the antibody adopted a predominantly "flat-on" orientation, with the Fc and two Fab fragments lying flat on the surface. The AFM measurement revealed a thickness of 30-33 A of the layer formed in contact with 2 mg.L(-1) antibody in water, but, interestingly, the flat-on antibody molecules formed small nonuniform clusters equivalent to 2-15 antibody molecules. Parallel AFM scanning in air revealed even larger surface clusters, suggesting that surface drying induced further aggregation. The AFM study thus demonstrated that the interaction between protein and the hydrophilic surface is weak and indicated that surface aggregation can be driven by the attraction between neighboring protein molecules. NR measurements at the solid/water interface confirmed the flat-on layer orientation of adsorbed molecules over the entire concentration range studied. Thus, at 2 mg.L(-1), the adsorbed antibody layer was well represented by a uniform layer with a thickness of 40 A. This value is thicker than the 30-33 A observed from AFM, suggesting possible layer compression caused by the tip tapping. An increase in the antibody concentration to 10 mg.L(-1) led to increasing surface adsorption. The corresponding layer structure was well represented by a three-layer model consisting of an inner sublayer of 10 A, a middle sublayer of 30 A, and an outer sublayer of 25 A, with the protein volume fractions in each sublayer being 0.22, 0.42, and 0.10, respectively. The structural transition can be interpreted as a twisting and tilting of segments of the adsorbed molecules, driven by an electrostatic repulsion between them that increases with the surface packing density. Hindrance of antigen access to antibody binding sites, resulting from the change in surface packing, can account for the decrease in antigen binding capacity (AgBC) with increasing surface density of the antibody that is observed.

Animals↗

PubMed1998

Surface roughness of orthodontic wires via atomic force microscopy, laser specular reflectance, and profilometry.

The surface roughness of orthodontic archwires is an essential factor that determines the effectiveness of arch-guided tooth movement. Using the non-destructive techniques of atomic force microscopy (AFM) and of laser specular reflectance, the surface roughness of 11 nickel-titanium orthodontic wires, a stainless steel and a beta-titanium wire was measured. The results were compared with those obtained using surface profilometry. The smoothest wire, stainless steel, had an optical roughness of 0.10 micron, compared with 0.09 micron from AFM and 0.06 from profilometry. The surface roughness for the beta-titanium wire measured by all three methods was approximately 0.21 micron, while that of the NiTi wires ranged from 0.10 to 1.30 microns. As the surface roughness not only affects the effectiveness of sliding mechanics, but also the corrosion behaviour and the aesthetics of orthodontic components, the manufacturers of orthodontic wires should make an effort to improve the surface quality of their products.

Corrosion↗

PubMed2007

Shape-engineered fibroblasts: cell elasticity and actin cytoskeletal features characterized by fluorescence and atomic force microscopy.

The regulation of cell shape, which determines cell behaviors including adhesion, spreading, migration, and proliferation in an engineered artificial extracellular milieu, is an important task in tissue engineering and in development of functional biomaterials. To deepen the understandings of shape-dependent cell mechanics, the cell elasticity and structural features of the actin cytoskeleton (CSK) were characterized for shape-engineered fibroblasts; round and spindle-shaped cells cultured on photolithographically microprocessed surfaces, employing the cellular microindentation tests and fluorescence observation of actin CSK by the combination of atomic force microscopy (AFM) and fluorescence microscopy (FM). The relationships among cell elasticity, the structural features of actin CSK, and engineered cell shape were analyzed and compared with those of control cells that had been cultured on nonprocessed surfaces (termed naturally extended cells). Results showed that the spindle-shaped cells with sparse or no apical stress fibers (ASFs) exhibited similar stiffness to that of the naturally extended cells with dense ASFs. The elasticity of spindle-shaped cells was affected only slightly by the stress fiber (SF) density, which is in marked contrast to the significant correlation shown between cell elasticity and SF density in naturally extended cells. This result implies that the elasticity of regionally restricted adhesion-surface-induced shape-engineered cells, particularly of highly elongated cells, is affected predominantly by cell shape rather than by structural features of SFs.

3T3 Cells↗

PubMed2000

Bacterial turgor pressure can be measured by atomic force microscopy.

We report a study of the deformability of a bacterial wall with an atomic force microscope (AFM). A theoretical expression is derived for the force exerted by the wall on the cantilever as a function of the depths of indentation generated by the AFM tip. Evidence is provided that this reaction force is a measure for the turgor pressure of the bacterium. The method was applied to magnetotactic bacteria of the species Magnetospirillum gryphiswaldense. Force curves were generated on the substrate and on the bacteria while scanning laterally. With the mechanical properties so gained we obtained the spring constant of the bacterium as a whole. Making use of our theoretical results we determined the turgor pressure to be in the range of 85 to 150 kPa.

Cell Wall↗

PubMed2001

Atomic Force Microscopy and Scanning Electron Microscopy Reveal Genome-dependent Ultrastructure of Seed Surface.

Both scanning electron microscopy (SEM) and contact mode imaging via atomic force microscopy (AFM) have been utilized to elucidate the ultrastructure of mung bean seed surfaces. The results indicate: 1) that AFM is useful in the examination of seed surface ultrastructure ex-vaccuo without the need for additional complex preparative procedures; and 2) that both the cotyledon and seed coat of different strains of mung beans bear specific ultrastructural details unique to each strain. To our knowledge, these are the first AFM images of seed surfaces.

Journal Article↗

PubMed1999

Dynamics of astrocyte adhesion as analyzed by a combination of atomic force microscopy and immuno-cytochemistry: the involvement of actin filaments and connexin 43 in the early stage of adhesion.

We observed the time-dependent morphological alteration of astrocytes during their adhesion by atomic force microscopy (AFM) and investigated the relationships between this morphological alteration and the localization of actin filaments and connexin 43 by immunocytochemistry. The fine processes observed as fine ridge-like structures by AFM were closely concerned with actin filaments by immunocytochemistry. During the adhesion of astrocytes, actin filaments appeared to be aligned regularly beyond the borders among different cells. Detectable connexin immunoreactivity was changed in the following regions: 1) the tips of fine cell processes and the cell margin when astrocytes started to adhere; 2) the border of cells when astrocytes tightly adhered; and 3) non-specific sites when astrocytes became a cluster. In the former two cases, the immunopositive spots for connexin were observed to colocalize with the tips of cell processes with actin filaments. These results strongly suggest that connexin associated with actin filaments at the tip of cell processes plays an important role in the early stage of the adhesion of astrocytes. These observations afford valuable clues for understanding the glial communication.

Actin Cytoskeleton↗

PubMed2005

Atomic force microscopy of collagen structure in bone and dentine revealed by osteoclastic resorption.

Mineralised tissues such as bone consist of two material phases: collagen protein fibrils, secreted by osteoblasts, form model structures for subsequent deposition of mineral, calcium hydroxyapatite. Collagen and mineral are removed in a three-dimensional manner by osteoclasts during bone turnover in skeletal growth or repair. Bone active drugs have recently been developed for skeletal diseases, and there is revived interest in changes in the structure of mineralised tissues seen in disease and upon treatment. The resolution of atomic force microscopy and use of unmodified samples has enabled us to image bone and dentine collagen exposed by the natural process of cellular dissolution of mineralised matrix. The morphology of bone and dentine has been analysed when fully mineralised and after osteoclast-mediated bone resorption, and compared with results from other microscopy techniques. Banded type I collagen, with 66.5+/-1.4 nm axial D-periodicity and 62.2+/-7.0 nm diameter, has been identified within resorption lacunae in bone and 69.4+/-4.3 nm axial D-periodicity and 140.6+/-12.4 nm diameter in dentine substrates formed by human and rabbit osteoclasts, respectively. This observation suggests a route by which the material and morphological properties of bone collagen can be analysed in situ, compared with collagen from non-skeletal sites, and contrasted in diseases of medical importance, such as osteoporosis, where skeletal tissue is mechanically weakened.

Acid Phosphatase↗

PubMed2000

Glial cells with differential neurite growth-modulating properties probed by atomic force microscopy.

Lateral (L) and medial (M) midbrain astrocytes differ in their ability to support neuritic growth (L, permissive; M, non-permissive) with properties of M glia depending on heparan sulfate (HS). Here we show by atomic force microscopy that the surfaces of formaldehyde-fixed astrocytes differ by conspicuous 250 nm protrusions in L and by a HS-dependent fibrillar network in M glia, thus, demonstrating correlations between cell surface morphology and functional properties.

Animals↗

PubMed2002

Atomic force microscopy investigation of fibroblasts infected with wild-type and mutant murine leukemia virus (MuLV).

NIH 3T3 cells were infected in culture with the oncogenic retrovirus, mouse leukemia virus (MuLV), and studied using atomic force microscopy (AFM). Cells fixed with glutaraldehyde alone, and those postfixed with osmium tetroxide, were imaged under ethanol according to procedures that largely preserved their structures. With glutaraldehyde fixation alone, the lipid bilayer was removed and maturing virions were seen emerging from the cytoskeletal matrix. With osmium tetroxide postfixation, the lipid bilayer was maintained and virions were observable still attached to the cell surfaces. The virions on the cell surfaces were imaged at high resolution and considerable detail of the arrangement of protein assemblies on their surfaces was evident. Infected cells were also labeled with primary antibodies against the virus env surface protein, followed by secondary antibodies conjugated with colloidal gold particles. Other 3T3 cells in culture were infected with MuLV containing a mutation in the gPr80(gag) gene. Those cells were observed by AFM not to produce normal MuLV on their surfaces, or at best, only at very low levels. The cell surfaces, however, became covered with tubelike structures that appear to result from a failure of the virions to properly undergo morphogenesis, and to fail in budding completely from the cell's surfaces.

3T3 Cells↗

PubMed2000

P67-phox-mediated NADPH oxidase assembly: imaging of cytochrome b558 liposomes by atomic force microscopy.

NADPH oxidase activity depends on the assembly of the cytosolic activating factors, p67-phox, p47-phox, p40-phox, and Rac with cytochrome b(558). The transition from an inactive to an active oxidase complex induces the transfer of electrons from NADPH to oxygen through cytochrome b(558). The assembly of oxidase complex was studied in vitro after reconstitution in a heterologous cell-free assay by using true noncontact mode atomic force microscopy. Cytochrome b(558) was purified from neutrophils and Epstein-Barr virus-immortalized B lymphocytes and incorporated into liposomes. The effect of protein glycosylation on liposome size and oxidase activity was investigated. The liposomes containing the native hemoprotein purified from neutrophils had a diameter of 146 nm, whereas after deglycosylation, the diameter was reduced to 68 nm, although oxidase activity was similar in both cases. Native cytochrome b(558) was used after purification in reconstitution experiments to investigate the topography of NADPH oxidase once it was assembled. For the first time, atomic force microscopy illustrated conformational changes of cytochrome b(558) during the transition from the inactive to the active state of oxidase; height measurements allow the determination of a size of 4 nm for the assembled complex. In the processes that were studied, p67-phox displayed a critical function; it was shown to be involved in both assembly and activation of oxidase complex while p47-phox proceeded as a positive effector and increased the affinity of p67-phox with cytochrome b(558), and p40-phox stabilizes the resting state. The results suggest that although an oligomeric structure of oxidase machinery has not been demonstrated, allosteric regulation mechanisms may be proposed.

Cell-Free System↗

PubMed2001

Interfacial water dielectric-permittivity-profile measurements using atomic force microscopy.

The arrangement of water molecules at charged aqueous interfaces is an important question in biology, electrochemistry, and geochemistry. Theoretical studies suggest that the molecules become arranged in several layers adjacent to a solid interface. Using atomic force microscopy we have measured the water dielectric-permittivity profile perpendicular to mica surfaces. The measured variable permittivity profile starting at epsilon approximately 4 at the interface and increasing to epsilon=80 about 10 nm from the surface suggests a reorientation of water molecule dipoles in the presence of the mica interfacial charge.

Aluminum Silicates↗

PubMedDate not supplied

Enzymatic degradation processes of lamellar crystals in thin films for poly[(R)-3-hydroxybutyric acid] and its copolymers revealed by real-time atomic force microscopy.

Enzymatic degradation processes of flat-on lamellar crystals in melt-crystallized thin films of poly[(R)-3-hydroxybutyric acid] (P(3HB)) and its copolymers were characterized by real-time atomic force microscopy (AFM) in a phosphate buffer solution containing PHB depolymerase from Ralstonia pickettii T1. Fiberlike crystals with regular intervals were generated along the crystallographic a axis at the end of lamellar crystals during the enzymatic degradation. The morphologies and sizes of the fiberlike crystals were markedly dependent on the compositions of comonomer units in the polyesters. Length, width, interval, and thickness of the fiberlike crystals after the enzymatic degradation for 2 h were measured by AFM, and the dimensions were related to the solid-state structures of P(3HB) and its copolymers. The width and thickness decreased at the tip of fiberlike crystals, indicating that the enzymatic degradation of crystals takes place not only along the a axis but also along the b and c axes. These results from AFM measurement were compared with the data on crystal size by wide-angle X-ray diffraction, and on lamellar thickness and long period by small-angle X-ray scattering. In addition, the enzymatic erosion rate of flat-on lamellar crystals along the a axis was measured from real-time AFM height images. A schematic glacier model for the enzymatic degradation of flat-on lamellar crystals of P(3HB) by PHB depolymerase has been proposed on the basis of the AFM observations.

Biophysical Phenomena↗