Applied Atomic Force Microscopy Research Articles

Imaging CFTR: A Tail to Tail Dimer with a Central Pore

The cystic fibrosis transmembrane conductance regulator (CFTR) is a protein that belongs to the superfamily of ATP binding cassette (ABC) transporters. Mutations in the CFTR gene cause cystic fibrosis, an autosomal recessive disorder. The function of CFTR is versatile. It can serve as a regulatory protein, as a membrane transporter and as an ion channel. Dimerization of CFTR is necessary for full ion channel function although structural details of CFTR in native membrane are yet unknown. In order to identify CFTR in native plasma membrane we applied atomic force microscopy (AFM) to inside-out oriented membrane patches of CFTR-expressingXenopus laevis oocytes after cAMP stimulation. First, oocytes were injected with CFTR-cRNA and, three days later, voltage-clamped verifying successful CFTR expression and incorporation into the plasma membrane. Then, plasma membrane patches were isolated, placed inside-out on appropriate substrate and incubated with gold-labelled antibodies against the C-terminus of CFTR. Finally, the intracellular surface of the plasma membrane was scanned by AFM. In close vicinity to the immunogold labels we detected ring-like structures with bipartite symmetry. The substructure of the ring, formed by the extramembrane protein domains of CFTR, is consistent with the model of a CFTR dimer. Derived from AFM molecular mass analysis of the intramolecular domains we conclude that two CFTR molecules line up in parallel, tail by tail, forming a pore in its center. This molecular arrangement could represent the CFTR chloride channel configuration, operative in native plasma membrane.

Atomic force microscopy study of chromosome surface structure changed by protein extraction

We applied atomic force microscopy (AFM) to investigate the surface structure of barley chromosome in combination with a chemical treatment method. As a result, we have obtained high-resolution topographic images of granular structures with a diameter of ca. 50 nm on the surface of critical-point dried metaphase chromosomes. Treatment with 2 M NaCl significantly modified the chromosome surface structure: surface roughness was increased and chromosome thickness was decreased. The NaCl treatment extracted two major proteins with molecular weights of 4000 and 20,000 Da. These proteins might be belonging to non-histone protein families that do not contain any aromatic amino acid. The results demonstrate the advantage of the combined method of high-resolution AFM imaging and chemical treatments for understanding nano-scale surface structures of the chromosome.

An Attempt to Observe Surface Morphology of NaKC 4 H 4 O 6 ·4H 2 O by Atomic Force Microscope

We applied atomic force microscopy (AFM) for studying the surface topography of NaKC 4 H 4 O 6 ·4H 2 O (Rochelle Salt). In particular, temperature variation of the surface images was studied. Below the upper Curie point, a regular undulation of the crystal surface, which reflects monoclinic twin boundaries, was observed for the c -plate surface.

An Attempt to Observe Surface Morphology of NaKC 4 H 4 O 6 ·4H 2 O by Atomic Force Microscope

We applied atomic force microscopy (AFM) for studying the surface topography of NaKC 4 H 4 O 6 ·4H 2 O (Rochelle Salt). In particular, temperature variation of the surface images was studied. Below the upper Curie point, a regular undulation of the crystal surface, which reflects monoclinic twin boundaries, was observed for the c -plate surface.

The structure of intramolecular triplex DNA: atomic force microscopy study

We applied atomic force microscopy (AFM) for direct imaging of intramolecular triplexes (H-DNA) formed by mirror-repeated purine-pyrimidine repeats and stabilized by negative DNA supercoiling. H-DNA appears in atomic force microscopy images as a clear protrusion with a different thickness than DNA duplex. Consistent with the existing models, H-DNA formation results in a kink in the double helix path. The kink forms an acute angle so that the flanking DNA regions are brought in close proximity. The mobility of flanking DNA arms is limited compared with that for cruciforms and three-way junctions. Structural properties of H-DNA may be important for promoter-enhancer interactions and other DNA transactions.

Force Measurement and Inhibitor Binding Assay of Monomer and Engineered Dimer of Bovine Carbonic Anhydrase B

We applied atomic force microscopy (AFM) to study the intramolecular mechanics of the globular protein molecule, bovine carbonic anhydrase B. The immobilized protein on an amino-functionalized silicon wafer was pulled from its N- and C-termini after being covalently cross-linked to the AFM tip, and the relationship between the tensile force applied on the protein and its extension was recorded. The native enzyme (having 261 residues with two Cys added at its ends, and in a theoretical stretching length of 96 nm) was extended only to 13 ± 2 nm under physiological conditions before disruption of the covalent cross-linking system. Contrary to the above observation, an engineered dimer was extended to about 110 nm even in the absence of the denaturant. The difference was ascribed to the presence or presumed absence of a “knot” structure at the C-terminal end of the two forms, respectively. When a specific inhibitor was added to the experimental solution, native monomers (sp activity = 88% of the wild type enzyme) were extended to 28 ± 4 nm, whereas dimers (sp activity = 46%) were extended to about 56 ± 3 nm, suggesting that both monomeric units in the dimer could bind inhibitor molecules, which was further corroborated by a titration experiment using a fluorescent inhibitor. Thus, one of the monomeric units in the engineered dimer was concluded to be enzymatically inactive but capable of binding inhibitors.

Plasma membrane protein clusters appear in CFTR-expressing Xenopus laevis oocytes after cAMP stimulation.

Membrane trafficking of the cystic fibrosis transmembrane conductance regulator (CFTR) is supposed to be an important mechanism controlled by the intracellular messenger cAMP. This has been shown with fluorescence techniques, electron microscopy and membrane capacitance measurements. In order to visualize protein insertion we applied atomic force microscopy (AFM) to inside-out oriented plasma membrane patches of CFTR-expressing Xenopus laevis oocytes before and after cAMP-stimulation. In a first step, oocytes injected with CFTR-cRNA were voltage-clamped, verifying successful CFTR expression. Water-injected oocytes served as controls. Then, plasma membrane patches were excised, placed (inside out) on glass and scanned by AFM. Before cAMP-stimulation plasma membranes of both water-injected and CFTR-expressing oocytes contained about 200 proteins per micron 2. Molecular protein masses were estimated from molecular volumes measured by AFM. Before cAMP-stimulation, protein distribution showed a peak value of 11 nm protein height corresponding to 475 kDa. During cAMP-stimulation with 1 mM isobutylmethylxanthine (IBMX) plasma membrane protein density increased in water-injected oocytes to 700 proteins per micron 2 while the peak value shifted to 7 nm protein height corresponding to 95 kDa. In contrast, CFTR-expressing oocytes showed after cAMP-stimulation about 400 proteins per micron 2 while protein distribution exhibited two peak values, one peak at 10 nm protein height corresponding to 275 kDa and another one at 14 nm corresponding to 750 kDa. They could represent heteromeric protein clusters associated with CFTR. In conclusion, we visualized plasma membrane protein insertion upon cAMP-stimulation and quantified protein distribution with AFM at molecular level. We propose that CFTR causes clustering of plasma membrane proteins.

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.

Atomic Force Microscopic Observation of Three-Dimensional Morphological Changes of Neurons When Stimulated by a Neurotransmitter

As the first step in the study of morphological changes in neurons associated with their functional changes, we applied atomic force microscopy (AFM) for the observation of fine three-dimensional morphological changes in rat cerebellar granule cells stimulated by an agonist of glutamate receptors, N-methyl-D-aspartate (NMDA). The AFM revealed that NMDA changed the cross-sections of cell bodies from a trapezoid-like form to a triangle-like form within a minute. The fine hill-like structures on the top surfaces of the cell bodies became wider during the same period. These results were suggested to be induced by the depolymerization of filamentous actin triggered by the entry of Ca2+ via cation channels complexed with the activated NMDA receptors.

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.

THE ATOMIC FORCE MICROSCOPE DETECTS ATP-SENSITIVE PROTEIN CLUSTERS IN THE PLASMA MEMBRANE OF TRANSFORMED MDCK CELLS

Plasma membrane proteins are supposed to form clusters that allow ‘functional cross-talk’ between individual molecules within nanometre distance. However, such hypothetical protein clusters have not yet been shown directly in native plasma membranes. Therefore, we developed a technique to get access to the inner face of the plasma membrane of cultured transformed kidney (MDCK) cells. The authors applied atomic force microscopy (AFM) to visualize clusters of native proteins protruding from the cytoplasmic membrane surface. We used the K+channel blocker iberiotoxin (IBTX), a positively charged toxin molecule, that binds with high affinity to plasma membrane potassium channels and to atomically flat mica. Thus, apical plasma membranes could be ‘glued’ with IBTX to the mica surface with the cytosolic side of the membrane accessible to the scanning AFM tip. The topography of these native inside-out membrane patches was imaged with AFM in electrolyte solution mimicking the cytosol. The plasma membrane could be clearly identified as a lipid bilayer with the characteristic height of 4.9±0.02nm. Multiple proteins protruded from the lipid bilayer into the cytosolic space with molecule heights between 1 and 20nm. Large protrusions were most likely protein clusters. Addition of the proteolytic enzyme pronase to the bath solution led to the disappearance of the proteins within minutes. The metabolic substrate ATP induced a shape-change of the protein clusters and smaller subunits became visible. ADP or the non-hydrolysable ATP analogue, ATP-γ-S, could not exert similar effects. It is concluded that plasma membrane proteins (and/or membrane associated proteins) form ‘functional clusters’ in their native environment. The ‘physiological’ arrangement of the protein molecules within a cluster requires ATP.

Visualization of supercoiled DNA with atomic force microscopy in situ.

Tertiary structure of supercoiled DNA is a significant factor in a number of genetic functions and is apparently affected by environmental conditions. We applied atomic force microscopy (AFM) for imaging the supercoiled DNA deposited at different ionic conditions. We have employed a technique for the sample preparation that permits high-resolution AFM imaging of DNA bound to the surface in buffer solutions without drying the sample (AFM in situ). The AFM data show that at low ionic strength, DNA molecules are loosely interwound supercoils with an irregular shape. Plectonemic superhelices are formed in high-concentration, near-physiological salt solutions. At such ionic conditions, superhelical loops are typically separated by regions of close helix-helix contacts. The data obtained show directly and unambiguously that overall geometry of supercoiled DNA depends dramatically on ionic conditions. This fact and the formation of close contacts between DNA helices are important features of supercoiled DNA related to its biological functions.

Atomic Force Microscope Observation of Ferroelectrics: Barium Titanate and Rochelle Salt

We applied atomic force microscopy (AFM) to study the surface morphology of ferroelectric BaTiO3 (barium titanate, BT) and NaKC4H4O6·4H2O (Rochelle salt, RS). At room temperature, an undulation of the crystal surface was observed around the 90° domain boundary in BT and around the monoclinic domain boundary in RS.

Immunolocalization of lamins and nuclear pore complex proteins by atomic force microscopy.

The nuclear envelope functions as a selective barrier separating the nuclear from the cytosolic compartment. Nuclear pore complexes (NPCs) mediate nuclear import and export of macromolecules and, therefore, are potential regulators of gene expression. In this study we applied atomic force microscopy (AFM) to visualize the three dimensional (3D) structure of individual NPCs in the absence and presence of two different antibodies, one directed against a pore protein (gp62) and another directed against Xenopus lamin LIII, a component of the nuclear lamina, a filament meshwork localized on the nucleoplasmic side of the nuclear envelope (NE) adjacent to and interacting with NPCs. Using 12-nm gold-labelled secondary antibodies and transmission electron microscopy we could clearly localize the primary single anti-gp62 antibody on NPCs and the primary single anti-LIII antibody between NPCs. Using AFM, the secondary antibodies against anti-gp62 could be detected as particles 7 nm in height on the nucleoplasmic face of NPCs. The secondary antibodies against anti-LIII could be clearly identified between NPCs. The secondary antibodies, attached to a 12-nm colloidal gold particle and visualized on glass, revealed similar shapes and heights as found on NEs. According to the 3D images, the volume of a single gold particle conjugated with secondary antibodies was 10203 nm3. This volume is equivalent to the volume of 38 IgG molecules associated with one individual gold particle. A similar volume of 11987 nm3 was calculated from a model assuming that the 150-kDa IgG molecules perfectly cover the spherical gold particle. We conclude that AFM can be used for identifying antibodies or other macromolecules associated with biomembranes.

A Study of the Surface Texture of Polycrystalline Phosphor Films Using Atomic Force Microscopy

ABSTRACTStimulable phosphor thin films are being investigated for use as optical data storage media. We have successfully applied atomic force microscopy (AFM) to the measurement of the surface texture of these films. Determination of the surface texture of the films is important for evaluating the effect of surface quality on optical scatter. In other thin film material systems it has been found that the surface “bumps” revealed by AFM correspond to grains in the film. This is not the case for the stimulable phosphor films used in our study. We have determined the grain size of our phosphor films by transmission electron microscopy (TEM) and x-ray diffraction (XRD). The grain size from TEM and XRD does not correlate with the size of the AFM surface “bumps.” For example, in two of the five films studied, the XRD derived grain size varies by a factor of two but the size of the surface “bumps” remains the same. We conclude that the texture of the film surface is not directly determined by the grain size of the phosphor material.