Cell adhesion force microscopy

The monohydroxy bile acid, taurolithocholate (TLC), causes cholestasis in vivo and in isolated perfused livers. It is also cholestatic in vitro and, in this study using isolated rat hepatocyte couplets, causes a reduction of the accumulation of (fluorescent) bile acid in the canalicular vacuoles (cVA) of this polarized cell preparation. The hepatoprotective bile acid, tauroursodeoxycholate (TUDCA), partially protects against the action of TLC when added at the same time. It also partially reverses the cholestatic effect if added after the cells have been exposed to TLC. A second hepatoprotective compound, S-adenosyl-L-methionine (SAMe) also not only partially protects against the action of TLC when added at the same time, but it too is able to partially reverse the cholestatic effect. Neither hepatoprotective agent is fully effective alone, but their effects are additive. In combination, a full restoration of cVA is observed in moderate cholestasis, but not in severe cholestasis. We discuss briefly some possible mechanisms involved in the additive mode of action of both hepatoprotective compounds. In summary, we show for the first time that SAMe and TUDCA can exert an additive effect in the amelioration of TLC-induced cholestasis in isolated rat hepatocyte couplets. This finding may be of possible clinical relevance.     

Simultaneous height and adhesion imaging of antibody-antigen interactions by atomic force microscopy

Specific molecular recognition events, detected by atomic force microscopy (AFM), so far lack the detailed topographical information that is usually observed in AFM. We have modified our AFM such that, in combination with a recently developed method to measure antibody-antigen recognition on the single molecular level (Hinterdorfer, P., W. Baumgartner, H. J. Gruber, K. Schilcher, and H. Schindler, Proc. Natl. Acad. Sci. USA 93:3477-3481 (1996)), it allows imaging of a submonolayer of intercellular adhesion molecule-1 (ICAM-1) in adhesion mode. We demonstrate that for the first time the resolution of the topographical image in adhesion mode is only limited by tip convolution and thus comparable to tapping mode images. This is demonstrated by imaging of individual ICAM-1 antigens in both the tapping mode and the adhesion mode. The contrast in the adhesion image that was measured simultaneously with the topography is caused by recognition between individual antibody-antigen pairs. By comparing the high-resolution height image with the adhesion image, it is possible to show that specific molecular recognition is highly correlated with topography. The stability of the improved microscope enabled imaging with forces as low as 100 pN and ultrafast scan speed of 22 force curves per second. The analysis of force curves showed that reproducible unbinding events on subsequent scan lines could be measured.     

Probing single biomolecules with atomic force microscopy

An ovary implanted into the spleen of an ovariectomized rat develops into a luteinized tumor, growing in response to gonadotrophins. Previously, it was shown that in vivo Buserelin, a gonadotrophin-releasing hormone (GnRH) analog, inhibited tumor growth. To determine if GnRH had a direct effect on tumor cells, the presence of GnRH receptors as well as the endocrine effects of buserelin were studied on tumoral tissue. GnRH receptors were present in luteoma in similar concentrations and dissociation constant (Kd) to control estrous ovaries. In vivo treatment with buserelin did not modify luteoma GnRH receptors. In organ incubations, luteoma secreted significantly higher estradiol and lower progesterone than estrous ovaries; addition of buserelin did not modify steroid secretion. The same difference in basal steroid secretion between luteoma cells and luteal cells superovulated prepubertal ovaries was observed in cell cultures. Although luteinizing-hormone (LH)-stimulated progesterone in both kinds of cells, buserelin significantly inhibited LH-stimulated progesterone only in luteoma cells. These results describe clear differences in basal steroid secretion between tumoral and normal tissue. Furthermore, they show that luteoma possess GnRH receptors similar to those in normal ovarian tissue, and that GnRH analogs have endocrine effects on these cells. Therefore, a direct effect of buserelin on luteoma cells can be postulated.     

Imaging biomolecule arrays by atomic force microscopy

We describe here a method for constructing ordered molecular arrays and for detecting binding of biomolecules to these arrays using atomic force microscopy (AFM). These arrays simplify the discrimination of surface-bound biomolecules through the spatial control of ligand presentation. First, photolithography is used to spatially direct the synthesis of a matrix of biological ligands. A high-affinity binding partner is then applied to the matrix, which binds at locations defined by the ligand array. AFM is then used to detect the presence and organization of the high-affinity binding partner. Streptavidin-biotin arrays of 100 x 100 microns and 8 x 8 microns elements were fabricated by this method. Contact and noncontact AFM images reveal a dense lawn of streptavidin specific to the regions of biotin derivatization. These protein regions are characterized by a height profile of approximately 40 A over the base substrate with a 350-nm edge corresponding to the diffraction zone of the photolithography. High resolution scans reveal a granular topography dominated by 300 A diameter features. The ligand-bound protein can then be etched from the substrate using the AFM tip, leaving an 8 A shelf that probably corresponds to the underlying biotin layer.     

Visualizing life on biomembranes by atomic force microscopy

Since its invention in 1986, the atomic force microscope (AFM) has become one of the most widely used near-field microscopes. Surfaces of hard samples are imaged routinely with atomic resolution. Soft biological samples, however, are still challenging. In this brief review, the AFM technique is introduced to the experimental biologist. We discuss recent data on imaging molecular structures of biomembranes, and give detailed information on the application of the AFM with two representative examples. One is imaging plasma membrane turnover of transformed renal epithelial cells during migration in vivo, and the other is visualizing macromolecular pore complexes of the nuclear envelope of aldosterone-sensitive kidney cells.     

Molecular dynamic study for nanopatterning using atomic force microscopy

The atomic force microscopy (AFM)-based nanolithographic technique is currently used to directly machine material surfaces and fabricate nanocomponents for microelectromechanical systems (MEMS) devices. In the current study, three-dimensional molecular dynamic (MD) simulations with potentials based on the Morse function were performed to evaluate the effect of crystallographic factors and process variables on the nanodeformation characteristics of the nanolithography processing of monocrystalline copper. Moreover, the effects of process variables (tool shape, cutting speed, and ploughing depth) on the nanostructural pattern are investigated. The simulation results revealed that the crystal orientation and ploughing direction had a significant influence on varying the forces (cutting force, thrust force, and width-direction force); the nature of the nanodeformation ahead of the tool; and the surface quality of the machined material.     

Filipin-induced lesions in planar phospholipid bilayers imaged by atomic force microscopy

Filipin is a macrolide polyene with antifungal activity belonging to the same family of antibiotics as amphotericin B and nystatin. Despite the spectroscopy and electron microscopy studies of its interaction with natural membranes and membrane model systems, several aspects of its biochemical action, such as the role of membrane sterols, remain to be completely understood. We have used atomic force microscopy (AFM) to study the effect of filipin on dipalmitoylphosphatidylethanolamine bilayers in the presence and absence of cholesterol. The bilayers were prepared by Langmuir-Blodgett deposition over mica and imaged under water. It was shown that filipin-induced lesions could only be found in membranes with cholesterol. In close agreement with electron microscopy results, we have reported the presence of densely packed circular protrusions in the membrane with a mean diameter of 19 nm (corrected for convolution with AFM tip) and 0.4 nm height. Larger circular protrusions (90 nm diameter and 2.5 nm height) and doughnut-shaped lesions were also detected. These results demonstrate that filipin-induced lesions in membranes previously observed by electron microscopy are not biased by artifacts resulting from sample preparation. Filipin aggregates in aqueous solution could also be imaged for the first time. These polydisperse spherical structures were observed in samples with and without cholesterol.     

Fibrous long spacing collagen ultrastructure elucidated by atomic force microscopy

Fibrous long spacing collagen (FLS) fibrils are collagen fibrils in which the periodicity is clearly greater than the 67-nm periodicity of native collagen. FLS fibrils were formed in vitro by the addition of alpha1-acid glycoprotein to an acidified solution of monomeric collagen and were imaged with atomic force microscopy. The fibrils formed were typically approximately 150 nm in diameter and had a distinct banding pattern with a 250-nm periodicity. At higher resolution, the mature FLS fibrils showed ultrastructure, both on the bands and in the interband region, which appears as protofibrils aligned along the main fibril axis. The alignment of protofibrils produced grooves along the main fibril, which were 2 nm deep and 20 nm in width. Examination of the tips of FLS fibrils suggests that they grow via the merging of protofibrils to the tip, followed by the entanglement and, ultimately, the tight packing of protofibrils. A comparison is made with native collagen in terms of structure and mechanism of assembly.     

Direct visualization of collagen-bound proteoglycans by tapping-mode atomic force microscopy

In 1984, the first flow cytometry data file format was proposed as Flow Cytometry Standard 1.0 (FCS1.0). FCS 1.0 provided a uniform file format allowing data acquired on one computer to be correctly read and interpreted on other computers running a variety of operating systems. That standard was modified in 1990 and adopted by the Society of Analytical Cytology as FCS 2.0. Here, we report on an update of the FCS 2.0 standard which we propose to designate FCS 3.0. We have retained the basic four segment structure of earlier versions (HEADER, TEXT, DATA and ANALYSIS) in order to maintain analysis software compatibility, where possible. The changes described in this proposal include a method to collect files larger than 100 megabytes (not possible in earlier versions of the standard), the inclusion of international characters in the TEXT portions of the file, a method of verifying data integrity using a 16-bit cyclic redundancy check, and increased keyword support for cluster analysis and time acquisition. This report summarizes the work of the ISAC Data File Standards Committee. The complete and detailed FCS 3.0 standard is available through the ISAC office [Sherwood Group, 60 Revere Drive, Ste 500, Northbrook, IL 60062, phone: (847) 480-9080 ext. 231, fax: (847) 480-9282, E-mail: isac@sherwood-group.com] or through the internet at the ISAC WWW site, http://nucleus.immunol.washington.edu/ISAC.ht ml.     

Direct measurement of hydrogen bonding in DNA nucleotide bases by atomic force microscopy

We have used self-assembled purines and pyrimidines on planar gold surfaces and on gold-coated atomic force microscope (AFM) tips to directly probe intermolecular hydrogen bonds. Electron spectroscopy for chemical analysis (ESCA) and thermal programmed desorption (TPD) measurements of the molecular layers suggested monolayer coverage and a desorption energy of about 25 kcal/mol. Experiments were performed under water, with all four DNA bases immobilized on AFM tips and flat surfaces. Directional hydrogen-bonding interaction between the tip molecules and the surface molecules could be measured only when opposite base-pair coatings were used. The directional interactions were inhibited by excess nucleotide base in solution. Nondirectional van der Waals forces were present in all other cases. Forces as low as two interacting base pairs have been measured. With coated AFM tips, surface chemistry-sensitive recognition atomic force microscopy can be performed.     

A convenient method of aligning large DNA molecules on bare mica surfaces for atomic force microscopy

Large DNA molecules remain difficult to be imaged by atomic force microscopy (AFM) because of the tendency of aggregation. A method is described to align long DNA fibers in a single direction on unmodified mica to facilitate AFM studies. The clear background, minimal overstretching, high reproducibility and convenience of this aligning procedure make it useful for physical mapping of genome regions and the studies of DNA-protein complexes.     

Structures and dynamic motion of laminin-1 as observed by atomic force microscopy

Laminins are a family of multifunctional extracellular matrix glycoproteins that play important roles in the development and maintenance of tissue organization via their interactions with cells and other extracellular matrix proteins. To understand the structural basis of laminins' functions, we examined the motion of laminin-1 (Ln-1) in physiological buffers using atomic force microscopy. While many Ln-1 molecules assumed the expected cruciform structure, unexpected dynamic movements of the Ln-1 arms were observed in aqueous environments. These dynamic movements of the Ln-1 arms may contribute to the diversity of laminin functions.     

Voltage and pH-induced channel closure of porin OmpF visualized by atomic force microscopy

Gram-negative bacteria are protected by an outer membrane in which trimeric channels, the porins, facilitate the passage of small solutes. The pores are formed by membrane-spanning antiparallel beta-strands, which are connected by short turns on the periplasmic side and long loops on the extracellular side. Voltage and pH-dependent conformational changes of these extracellular loops have now been visualized by atomic force microscopy of two-dimensional crystals of Escherichia coli porin OmpF. The observed conformational changes accompany the closure of the channel entrance, and suggest that this is a mechanism that the cells have evolved to protect themselves from drastic changes of the environment.     

Force-mediated kinetics of single P-selectin/ligand complexes observed by atomic force microscopy

Leukocytes roll along the endothelium of postcapillary venules in response to inflammatory signals. Rolling under the hydrodynamic drag forces of blood flow is mediated by the interaction between selectins and their ligands across the leukocyte and endothelial cell surfaces. Here we present force-spectroscopy experiments on single complexes of P-selectin and P-selectin glycoprotein ligand-1 by atomic force microscopy to determine the intrinsic molecular properties of this dynamic adhesion process. By modeling intermolecular and intramolecular forces as well as the adhesion probability in atomic force microscopy experiments we gain information on rupture forces, elasticity, and kinetics of the P-selectin/P-selectin glycoprotein ligand-1 interaction. The complexes are able to withstand forces up to 165 pN and show a chain-like elasticity with a molecular spring constant of 5.3 pN nm-1 and a persistence length of 0.35 nm. The dissociation constant (off-rate) varies over three orders of magnitude from 0.02 s-1 under zero force up to 15 s-1 under external applied forces. Rupture force and lifetime of the complexes are not constant, but directly depend on the applied force per unit time, which is a product of the intrinsic molecular elasticity and the external pulling velocity. The high strength of binding combined with force-dependent rate constants and high molecular elasticity are tailored to support physiological leukocyte rolling.     

Slow cellular dynamics in MDCK and R5 cells monitored by time-lapse atomic force microscopy

We have examined dynamic events that occur on a time scale of minutes in an epithelial monolayer of Madine-Darby Canine Kidney (MDCK) cells and in ras-transformed MDCK cells by atomic force microscopy (AFM). Cells were imaged under physiological conditions, and time-lapse movies representing approximately 60 s real time per frame were assembled. In normal MDCK cells, two types of protrusions in the apical plasma membrane exhibit dynamic behavior. First, smooth bulges formed transiently over the time scale of minutes to tens of minutes. Second, spike-like protrusions appear initially as bulges, extend well above the apical surface and, finally, seem to detach. R5, an oncogenic transformant derived from MDCK cells, grows very flat on glass. During AFM imaging, these cells sometimes round up and detach from the substrate. In light microscopic observations of parallel preparations, cells rarely detach, suggesting that this is an active response of these cells to irritation by the AFM tip. R5 cells often extend processes that are supported by actin stress fibers. During imaging with the AFM, these processes withdraw at a rate of 1-5 microns/min, similar to that observed by light microscopy. During the withdrawal, movement of the stress fibers can be clearly seen. In the flat periphery of these cells, the transport of intracellular particles along cytoskeletal elements was seen. In addition, we have observed two types of wave-like movements through the cell, which appear to be an organized rearrangement of cytoplasm. One type of wave moves radially out from center of the cell while the other moves circularly along the cell periphery.