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The crucial role of the biopolymer "Von Willebrand factor" (VWF) in blood platelet binding is tightly regulated by the shear forces to which the protein is exposed in the blood flow. Under high-shear conditions, VWFs ability to immobilize blood platelets is strongly increased due to a change in conformation which at sufficient concentration is accompanied by the formation of ultra large VWF bundles (ULVWF). However, little is known about the dynamic and mechanical properties of such bundles. Combining a surface acoustic wave (SAW) based microfluidic reactor with an atomic force microscope (AFM) we were able to study the relaxation of stretched VWF bundles formed by hydrodynamic stress. We found that the dynamical response of the network is well characterized by stretched exponentials, indicating that the relaxation process proceeds through hopping events between a multitude of minima. This finding is in accordance with current ideas of VWF self-association. The longest relaxation time does not show a clear dependence on the length of the bundle, and is dominated by the internal conformations and effective friction within the bundle.

This study demonstrates an innovative application of atomic force microscopy (AFM). The combination of high-resolution AFM technology and tape stripping is presented as a tool for the structure analysis of human stratum corneum (SC) at a nanometer scale. Topographic images with a vertical resolution of about 10 nm of the SC are presented. Topographical and structural differences between aged and young skin can be observed. Aged skin SC is characterized by an increased single-cell surface area, prominent intercellular gaps and enhanced cell surface roughness. The use of AFM in combination with other already established methods, e.g. tape stripping in the field of dermatological research will give new insights to the structure, function and morphodynamics of SC.

Atomic force microscopy is a powerful technique used to investigate the surface of living cells under physiological conditions. The resolution of the instrument is mainly limited by the softness of living cells and the interactions with the scanning tip (cantilever). Atomic force microscopy, in combination with myosin-functionalized cantilevers, was used in the detection of ATP concentrations in solution and on living cells. Functionally active tips were used to scan the surface of cells in culture and to show that the CFTR+ cell line (S9) had a basal surface ATP concentration that could be detected with atomic force microscopy (n = 10). ATP-dependent signals were not detectable in cells scanned with noncoated or heat-inactivated enzyme-coated tips (n = 9). Enzymatically active tips may serve as a model for future development of atomic force microscopy biosensors that can simultaneously detect topographical and biologically important compounds at the surface microenvironment of living cells.

Proteins are usually identified by their molecular weights, and atomic force microscopy (AFM) produces images of single molecules in three dimensions. We have used AFM to measure the molecular volumes of a number of proteins and to determine any correlation with their known molecular weights. We used native proteins (the TATA-binding protein Tbp, a fusion protein of glutathione-S-transferase and the renal potassium channel protein ROMK1, the immunoglobulins IgG and IgM, and the vasodilator-stimulated phosphoprotein VASP) and also denatured proteins (the red blood cell proteins actin, Band 3 and spectrin separated by SDS-gel electrophoresis and isolated from nitrocellulose). Proteins studied had molecular weights between 38 and 900 kDa and were imaged attached to a mica substrate. We found that molecular weight increased with an increasing molecular volume (correlation coefficient = 0.994). Thus, the molecular volumes measured with AFM compare well with the calculated volumes of the individual proteins. The degree of resolution achieved (lateral 5 nm, vertical 0.2 nm) depended upon the firm attachment of the proteins to the mica. This was aided by coating the mica with suitable detergent and by imaging using the AFM tapping mode which minimizes any lateral force applied to the protein. We conclude that single (native and denatured) proteins can be imaged by AFM in three dimensions and identified by their specific molecular volumes. This new approach permits detection of the number of monomers of a homomultimeric protein and study of single proteins under physiological conditions at the molecular level.

The atomic force microscope (AFM) was used to continuously follow height changes of individual protein molecules exposed to physiological stimuli. A AFM tip was coated with ROMK1 (a cloned renal epithelial potassium channel known to be highly pH sensitive) and lowered onto atomically flat mica surface until the protein was sandwiched between AFM tip and mica. Because the AFM tip was an integral part of a highly flexible cantilever, any structural alterations of the sandwiched molecule were transmitted to the cantilever. This resulted in a distortion of the cantilever that was monitored by means of a laser beam. With this system it was possible to resolve vertical height changes in the ROMK1 protein of >/=0.2 nm (approximately 5% of the molecule's height) with a time resolution of >/=1 msec. When bathed in electrolyte solution that contained the catalytic subunit of protein kinase A and 0.1 mM ATP (conditions that activate the native ion channel), we found stochastically occurring height fluctuations in the ROMK1 molecule. These changes in height were pH-dependent, being greatest at pH 7.6, and lowering the pH (either by titration or by the application of CO2) reduced their magnitude. The data show that overall changes in shape of proteins occur stochastically and increase in size and frequency when the proteins are active. This AFM "molecular-sandwich" technique, called MOST, measures structural activity of proteins in real time and could prove useful for studies on the relationship between structure and function of proteins at the molecular level.

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 +/- 0.02 nm. Multiple proteins protruded from the lipid bilayer into the cytosolic space with molecule heights between 1 and 20 nm. 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-gamma-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.

We combined the patch-clamp technique with atomic force microscopy (AFM) to visualize plasma membrane proteins protruding from the extracellular surface of cultured kidney cells (MDCK cells). To achieve molecular resolution, patches were mechanically isolated from whole MDCK cells by applying the patch-clamp technique. The excised inside-out patches were transferred on freshly cleaved mica and imaged with the AFM in air and under physiological conditions (i. e. in fluid). Thus, the resolution could be increased considerably (lateral and vertical resolutions 5 and 0.1 nm, respectively) as compared to experiments on intact cells, where plasma membrane proteins were hardly detectable. The apical plasma membrane surface of the MDCK cells showed multiple protrusions which could be identified as membrane proteins through the use of pronase. These proteins had a density of about 90 per micron(2), with heights between 1 and 9 nm, and lateral dimensions of 20-60 nm. Their frequency distribution showed a peak value of 3 nm for the protein height. A simplified assumption - modelling plasma membrane proteins as spherical structures protruding from the lipid bilayer - allowed an estimation of the possible molecular weights of these proteins. They range from 50 kDa to 710 kDa with a peak value of 125 kDa. We conclude that AFM can be used to study the molecular structures of membranes which were isolated with the patch-clamp technique. Individual membrane proteins and protein clusters, and their arrangement and distribution in a native plasma membrane can be visualized under physiological conditions, which is a first step for their identification.

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 almost routinely with atomic resolution. Soft biological surfaces, 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 three representative examples. One is imaging plasma membrane turnover of transformed renal epithelial cells during migration in vivo, another is visualizing a cloned and isolated potassium channel usually located in kidney, and a third is imaging macromolecular pore complexes of the nuclear envelope of aldosterone-sensitive kidney cells and of Xenopus laevis oocytes. The review ends with the conclusion that nuclear pores can serve as birthday candles on a Guglhupf.

The dynamics at the plasma membrane resulting from secretory vesicle docking and fusion and compensatory endocytosis has been difficult to observe in living cells primarily due to limited resolution at the light microscopic level. Using the atomic force microscope, we have been able to image and record changes in plasma membrane structure at ultrahigh resolution after stimulation of secretion from isolated pancreatic acinar cells. "Pits" measuring 500-2000 nm and containing 3-20 depressions measuring 100-180 nm in diameter were observed only at the apical region of acinar cells. The time course of an increase and decrease in "depression" size correlated with an increase and decrease of amylase secretion from live acinar cells. Depression dynamics and amylase release were found to be regulated in part by actin. No structural changes were identified at the basolateral region of these cells. Our results suggest depressions to be the fusion pores identified earlier in mast cells by freeze-fracture electron microscopy and by electrophysiological measurements. The atomic force microscope has enabled us to observe plasma membrane dynamics of the exocytic process in living cells in real time.