Depth matters: cells grown on nano-porous anodic alumina respond to pore depth.

Recent experiments have shown unambiguously that living cells respond to the nano-topography of surfaces they grow on-specifically, the fate of stem cells grown on nano-porous titania or alumina have been shown to be decided by the pore size. However, most experiments have focused on pore size or pitch. Here we show that in addition to pore size and pitch, the depth of the pores has a profound effect on cell morphology and the arrangement of the actin cytoskeleton.

Early contacts between T lymphocytes and activating surfaces.

Cells continually probe their environment to adapt their behaviour. A current challenge is to determine how they analyse nearby surfaces and how they process information to take decisions. We addressed this problem by monitoring human T lymphocyte attachment to surfaces coated with activating anti-CD3 or control anti-HLA antibodies. Interference reflection microscopy allowed us to monitor cell-to-surface apposition with a few nanometre vertical resolution during the first minutes following contact. We found that (i) when a cell fell on a surface, contact extension was preceded by a lag of several tens of seconds. (ii) During this lag, vertical membrane undulations seemed to generate transient contacts with underlying surfaces. (iii) After the lag period, the contact area started increasing linearly with a rate of about 1.5 µm(2) s(-1) on activating surfaces and about 0.2 µm(2) s(-1) on control surfaces. (iv) Concomitantly with lateral surface extension, the apparent distance between cell membranes and surfaces steadily decreased. These results are consistent with the hypothesis that the cell decision to spread rapidly on activating surfaces resulted from the integration of information yielded by transient contacts with these surfaces generated by membrane undulations during a period of about 1 min.

Is there a predictable relationship between surface physical-chemical properties and cell behaviour at the interface?

There is much interest in predicting and controlling the outcome of interaction between artificial surfaces and living cells. However, although there is an impressive amount of information on the behaviour of many cell populations deposited on a variety of surfaces, there is presently no available theory to explain or even summarize these data. Indeed, it is not even obvious that such a theory may exist. The aim of the present review is to emphasize the problems encountered when one attempts to build such a theory. Three sequential steps of cell surface interactions are considered: 1) protein adsorption is a preliminary step liable to involve irreversible interaction between the surface and several hundreds of molecular species occurring in blood or plasma. 2) the second step is the formation of adhesive bonds. Several theoretical frameworks were suggested to account for this step, including DLVO theory, physical chemistry of surfaces, and formation of specific ligand-receptor bonds. It is concluded that present evidence supports the latter approach, although this involves serious difficulties. 3) The last step is the triggering of a specific cell program such as apoptosis, proliferation, migration, differentiation or activation. Recent evidence suggests that in addition to the nature and amount of stimulated surface receptors, additional cues such as substratum mechanical or topographical properties may significantly affect cell behaviour.

Cell fitting to adhesive surfaces: A prerequisite to firm attachment and subsequent events.

Cell adhesion usually involves extensive shape reorganization. This process is important because i) it is required for efficient cross-linking of interacting surfaces by adhesion receptors the length of which does not exceed several tens of nanometers and ii) it influences subsequent cell differentiation and activation. This review focuses on the initial phase of cell deformation, preceding the extensive reorganization process known as spreading. This first phase includes local flattening at the micrometer scale and membrane alignment at the nanometer level, resulting in fitting of the cell to an adhesive surface. Three main points are considered. First, experimental methods available to study cell apposition to a surface are described, with an emphasis on interference reflection microscopy. Second, selected experimental evidence is presented to show that there is a quantitative relationship between "adhesiveness" and "contact extension", and some theoretical models aimed at relating these parameters are briefly sketched. Third, experimental data on the kinetics of initial contact extension are described and possible mechanisms for driving this extension are discussed, including nonspecific forces, receptor-mediated interactions, active cell movements or passive membrane fluctuations. It is concluded that both passive physical phenomena and random active cell movements are possible candidates for the initial triggering of contact extension.

Diffusion of microspheres in shear flow near a wall: use to measure binding rates between attached molecules.

The rate and distance-dependence of association between surface-attached molecules may be determined by monitoring the motion of receptor-bearing spheres along ligand-coated surfaces in a flow chamber (Pierres et al., Proc. Natl. Acad. Sci. U.S.A. 95:9256-9261, 1998). Particle arrests reveal bond formation, and the particle-to-surface distance may be estimated from the ratio between the velocity and the wall shear rate. However, several problems are raised. First, data interpretation requires extensive computer simulations. Second, the relevance of standard results from fluid mechanics to micrometer-size particles separated from surfaces by nanometer distances is not fully demonstrated. Third, the wall shear rate must be known with high accuracy. Here we present a simple derivation of an algorithm permitting one to simulate the motion of spheres near a plane in shear flow. We check that theoretical predictions are consistent with the experimental dependence of motion on medium viscosity or particle size, and the requirement for equilibrium particle height distribution to follow Boltzman's law. The determination of the statistical relationship between particle velocity and acceleration allows one to derive the wall shear rate with 1-s(-1) accuracy and the Hamaker constant of interaction between the particle and the wall with a sensitivity better than 10(-21) J. It is demonstrated that the correlation between particle height and mean velocity during a time interval Deltat is maximal when Deltat is about 0.1-0.2 s for a particle of 1.4-microm radius. When the particle-to-surface distance ranges between 10 and 40 nm, the particle height distribution may be obtained with a standard deviation ranging between 8 and 25 nm, provided the average velocity during a 160-ms period of time is determined with 10% accuracy. It is concluded that the flow chamber allows one to detect the formation of individual bonds with a minimal lifetime of 40 ms in presence of a disruptive force of approximately 5 pN and to assess the distance dependence within the tens of nanometer range.

Selective blocking of voltage-gated K+ channels improves experimental autoimmune encephalomyelitis and inhibits T cell activation.

Kaliotoxin (KTX), a blocker of voltage-gated potassium channels (Kv), is highly selective for Kv1.1 and Kv1.3. First, Kv1.3 is expressed by T lymphocytes. Blockers of Kv1.3 inhibit T lymphocyte activation. Second, Kv1.1 is found in paranodal regions of axons in the central nervous system. Kv blockers improve the impaired neuronal conduction of demyelinated axons in vitro and potentiate the synaptic transmission. Therefore, we investigated the therapeutic properties of KTX via its immunosuppressive and symptomatic neurological effects, using experimental autoimmune encephalomyelitis (EAE), an animal model for multiple sclerosis. The T line cells used to induce adoptive EAE were myelin basic protein (MBP)-specific, constitutively contained mRNA for Kv1.3. and expressed Kv1.3. These channels were shown to be blocked by KTX. Activation is a crucial step for MBP T cells to become encephalitogenic. The addition of KTX during Ag-T cell activation led to a great reduction in the MBP T cell proliferative response, in the production of IL-2 and TNF, and in Ca(2+) influx. Furthermore, the addition of KTX during T cell activation in vitro led a decreased encephalitogenicity of MBP T cells. Moreover, KTX injected into Lewis rats impaired T cell function such as the delayed-type hypersensitivity. Lastly, the administration of this blocker of neuronal and lymphocyte channels to Lewis rats improved the symptoms of EAE. We conclude that KTX is a potent immunosuppressive agent with beneficial effects on the neurological symptoms of EAE.

Effect of dietary lipid (soybean lecithin and triacylglycerol) on hepatic F-actin microfilaments in cyclosporine A-treated rats: image analysis by confocal laser scanning microscopy.

We studied and quantified the effect of cyclosporine A on hepatic F-actin on bile canalicular and basolateral membranes in rats fed either soybean lecithin, triacylglycerol-enriched diet, or low-fat diet by means of confocal laser scanning microscopy imaging. The phalloidin-FITC staining of F-actin was quite normal in the lecithin-cyclosporine A group but decreased significantly in the other cyclosporine A-treated groups (by 40% and 25% of control in triacylglycerol-cyclosporine A and cyclosporine A groups, respectively). The alteration of F-actin by cyclosporine A, related to cholestasis evidenced by a decrease in bile salt secretion, was prevented by dietary soybean lecithin and amplified by dietary soybean triacylglycerol.

Dynamic study of cell mechanical and structural responses to rapid changes of calcium level.

Cell shape control is complex since it may involve multiple cytoskeletal components and metabolic pathways. Here we present a kinetic study of the mechanical and structural responses of cells from the monocytic THP-1 line to a rapid increase of cytosolic calcium level. Cells were exposed to ionomycin in a medium of varying calcium concentration and they were probed at regular intervals for (1) cortical rigidity as determined with micropipette aspiration, and (2) content and distribution of polymerized actin, myosin or ABP-280, as determined with flow cytometry and/or confocal microscopy. An increase of free intracellular calcium level induced: (1) a biphasic deformability change with marked stiffening within a second, and significant softening a minute later; (2) a biphasic change of actin polymerization with initial decrease (within less than a second) and rapid recovery (within a few seconds); (3) a topographical redistribution of microfilaments with an oscillatory behavior of the cortical fraction, while no substantial redistribution of myosin or ABP-280 was detected. It is suggested that a regulation of cell rigidity might be achieved without any structural change by suitable modulation of the lifetime of bridges formed between microfilaments by actin binding proteins.

Integrin (alpha) and beta subunit contribution to the kinetic properties of (alpha)2beta1 collagen receptors on human keratinocytes analyzed under hydrodynamic conditions.

The adhesion of keratinocytes to type I collagen or laminin 5 was studied in a laminar flow chamber. These experiments provided an insight into the binding kinetics of integrins in their natural environment and the effects of monoclonal antibodies specific for (alpha) and beta chains. Cells driven by a force too low to alter the natural lifetime of a single bond displayed multiple arrests. Studying the frequency and duration of these arrests yielded fairly direct information on the rate of bond formation (on-rate) and dissociation (off-rate). Off-rate values obtained on collagen or laminin 5 (0.06 seconds-1) were tenfold lower than values determined on selectins. Bond stability was strongly regulated by anti-beta1 chain antibodies since the off-rate was decreased sixfold by activating antibody TS2/16 and increased fivefold by inhibitory antibodies Lia1/2 or P4C10, whereas neutral antibody K20 had no effect on this parameter. Binding frequencies were not significantly changed by all these antibodies. In contrast, both binding frequency and off-rate were altered by antibodies specific for the (alpha)2 chain, suggesting that these antibodies interfered with ligand recognition and also with the ligand-beta1 chain interactions responsible for bond stabilization. The latter hypothesis was supported by the finding that the partial alteration of (alpha)2 chain function by inhibiting antibodies was corrected by anti-beta1 chain antibody TS2/16. These results could not be ascribed to allosteric changes of the functional region of beta1 integrin subunits regulated by TS2/16 since there was no competition between the binding of TS2/16 and anti-(alpha)2 chain antibodies. Interpreted within the framework of current concepts of integrin-ligand binding topology, these data suggest that ligand-alpha chain interactions may be qualitatively important in ligand recognition and also influence the formation of the ligand-beta1 subunit bonding involved in stabilization of the ligand-integrin complex by regulating its dissociation rate.

[Adhesion molecules and cancer]. / Les molécules d'adhésion en cancérologie.

INTRODUCTION: This review was aimed at summarizing recent advances in the understanding of cell adhesion in order to discuss the possible relevance of new knowledge to the exploration of cancer patients and elaboration of therapeutic strategies. CURRENT KNOWLEDGE AND KEY POINTS: During the last 10 years, many adhesion molecules were identified, thus allowing to determine their tissue distribution and functional regulation. The concept of adhesiveness was refined. It is now well known that adhesive rate (i.e., the minimal contact time required for bond formation) and binding strength (i.e., the minimal force required to detach bound cells) are distinct parameters. They may be regulated independently, and influence the cell behavior in different ways. It is now possible to achieve accurate control of tumor cell adhesiveness, either by inhibiting an adhesive mechanism (through monoclonal antibodies, competitive ligands, or inhibition of receptor expression with antisense strategy or gene knock-out) or by promoting a binding mechanism (with receptor transfection or pro-inflammatory stimulation). FUTURE PROSPECTS AND PROJECTS: Recent progress opens new possibilities for diagnosis and treatment. First, the interpretation of experimental data may be improved. Cell adhesive behavior is not entirely accounted for by the density of membrane adhesion receptors. Indeed, adhesion is influenced by receptor connection to the cytoskeleton and structure of the cell coat. An adhesion receptor may be anti-metastatic through an increase in tumor cohesion and cell differentiation, or pro-metastatic, through facilitation of cell migration towards a target tissue. New therapeutic strategies may include anti-adhesive procedure aimed at preventing metastasis formation. The potential importance of a better control of inflammatory processes is also emphasized in view of the influence of these processes on the expression of adhesion molecules.

Studying receptor-mediated cell adhesion at the single molecule level.

Cell adhesion is essentially mediated by specific interactions between membrane receptors and ligands. It is now apparent that the mere knowledge of the on- and off-rate of association of soluble forms of these receptors and ligands is not sufficient to yield accurate prediction of cell adhesive behavior. During the last few years, a variety of complementary techniques relying on the use of hydrodynamic flow, atomic force microscopy, surface forces apparatus or soft vesicles yielded accurate information on i) the dependence of the lifetime of individual bonds on applied forces and ii) the distance dependence of the association rate of bound receptors and ligands. The purpose of this review is, first to recall the physical significance of these parameters, and second to describe newly obtained results. It is emphasized that molecular size and flexibility may be a major determinant of the efficiency of receptor mediated adhesion, and this cannot be studied by conventional methods dealing with soluble molecules.

Thrombin-activated human endothelial cells support monocyte adhesion in vitro following expression of intercellular adhesion molecule-1 (ICAM-1; CD54) and vascular cell adhesion molecule-1 (VCAM-1; CD106).

Thrombin, a central molecule in coagulation, is also involved in inflammation. Notably, thrombin induces endothelial neutrophil adhesion, P- and E-selectin expression, and chemokine production. We show here that thrombin induces expression of intercellular adhesion molecule-1 (ICAM-1; CD54) and vascular cell adhesion molecule-1 (VCAM-1; CD106) on human umbilical vein endothelial cells (HUVECs) associated with increased adhesion of monocytes. Thrombin increased mRNA steady-state levels and expression of ICAM-1 over 24 hours. Thrombin-induced VCAM-1 expression exhibited unusual kinetics, reaching maximum levels after 6 to 12 hours, but decreasing to near baseline after 24 hours. Thrombin activity on HUVECs was mediated through interaction with its specific receptor, because ICAM-1 and VCAM-1 expression were similarly induced by the 14-amino acid thrombin receptor-activating peptide. Thrombin-induced ICAM-1 and VCAM-1 expression was significantly inhibited by hirudin, but not by interleukin-1 receptor antagonist or anti-tumor necrosis factor alpha monoclonal antibody (MoAb). Thrombin-activated HUVECs significantly increased greater numbers of adhering THP-1 macrophagic cells, peripheral blood mononuclear cells, or purified monocytes than unstimulated HUVECs. This adhesion was inhibited by anti-CD18 and anti-CD49d MoAb, demonstrating that thrombin-induced ICAM-1 and VCAM-1 were functional. These results show that, in addition to selectins, thrombin directly induces a cytokine-independent expression of adhesion molecules of the Ig superfamily on HUVECs that may support firm leukocyte attachment during inflammation.

Experimental study of the interaction range and association rate of surface-attached cadherin 11.

We describe a method allowing quantitative determination of the interaction range and association rate of individual surface-attached molecules. Spherical beads (1.4 micro(m) radius) were coated with recombinant outer domains of the newly described classical type II cadherin 11, a cell adhesion molecule. Beads were driven along cadherin-coated surfaces with a hydrodynamic force of approximately 1 pN, i.e., much less than the mechanical strength of many ligand-receptor bonds. Spheres displayed periods of slow motion interspersed with arrests of various duration. Particle position was monitored with 50 Hz frequency and 0.025 micro(m) accuracy. Nearly 1 million positions were recorded and processed. Comparison between experimental and computer-simulated trajectories suggested that velocity fluctuations might be related quantitatively to Brownian motion perpendicular to the surface. The expected amplitude of this motion was of order of 100 nm. Theoretical analysis of the relationship between sphere acceleration and velocity allowed simultaneous determination of the wall shear rate and van der Waals attraction between spheres and surface. The Hamaker constant was estimated at 2.9 x 10(-23) J. The frequency of bond formation was then determined as a function of sphere velocity. Experimental data were consistent with the view that the rate of association between a pair of adhesion molecules was approximately 1.2 x 10(-3) s-1 and the interaction range was approximately 10 nm. It is concluded that the presented methodology allows sensitive measurement of sphere-to-surface interactions (with approximately 10 fN sensitivity) as well as the effective range and rate of bond formation between individual adhesion molecules.

Thyrotropin chronically regulates the pool of thyroperoxidase and its intracellular distribution: a quantitative confocal microscopic study.

The regulation of thyroperoxidase (TPO) expression and of its intracellular distribution was studied in porcine thyroid cells cultured on porous bottom filters. Cells were cultured for 18 days in the absence or in the presence of thyrotropin (TSH) and with or without iodide. Microsomes were purified and analyzed by electrophoresis. TPO was detected by immunoblotting with polyclonal anti-porcine TPO antibodies and quantified by scanning the bands. The amount of TPO was increased 2-fold by TSH. High concentrations of iodide (1-50 microM, added daily) decreased the level of TPO. Confocal microscopy served to determine the intracellular localization of TPO and its quantitative distribution. Intracellular and surface-located TPO was detected by fluorescein-labeled antibodies on saponin-treated cells. Quantitative confocal microscopy showed that TSH increased the total amount of TPO 2-fold as for immunoblotting. The highest amount of TPO was found in the perinuclear area and between the nucleus and the Golgi apparatus. Only 4% of TPO was present on the apical surface and about 1% on the basolateral membrane; the remainder (about 95%) was inside the cells. TSH did not change these relative contents. TSH modified the intracellular distribution of the enzyme, increasing the TPO pool from the perinuclear area to apical membrane. This domain could be a site of storage of TPO. Adding a physiological concentration of iodide (0.5 microM, daily) did not influence the intracellular distribution of TPO. We concluded that chronic TSH stimulation 1) increased 2-fold the pool of TPO but did not change the relative proportion of TPO inside the cells and on the apical surface, and 2) modified the intracellular distribution of vesicular TPO, the major part of which was accumulated in the perinuclear and cytoplasmic area under the subapical domain of the polarized cells.

Use of a laminar flow chamber to study the rate of bond formation and dissociation between surface-bound adhesion molecules: effect of applied force and distance between surfaces.

It has recently been shown that much information on the behaviour of surface-bound adhesion molecules could be obtained by monitoring the motion of receptor-coated particles along ligand-derivatized surfaces in the presence of a hydrodynamic force of a few pN. This procedure is expected to allow direct monitoring of the formation and dissociation of individual bonds. We present experimental results on the interaction between streptavidin-coated spheres (1.4 microns diameter) and control or biotinylated mica surfaces in a laminar flow chamber. Moving spheres are found to display numerous arrests whose frequency is markedly increased (5-13-fold) in the presence of biotin groups. For a given shear rate, the binding frequency is strongly dependent on the sphere-surface separation. Indeed, this frequency displayed a 14-fold decrease when the velocity increased from 7 to 15 microns s-1 for a wall shear rate of 20 s-1. Furthermore, the lifetime of observed arrests was of the order of several seconds, i.e. 5-50-fold higher than previously determined on models such as selectin-ligand, CD2-CD48 or cadherin-cadherin. Finally, this lifetime did not decrease when the wall shear rate was increased from ca. 10 to 40 s-1.

Insulin stimulates haptotactic migration of human epidermal keratinocytes through activation of NF-kappa B transcription factor.

Insulin-mediated cell motility as well as the role of transcription factors in insulin-activated intracellular signal events have not been extensively studied. In this report we have examined whether insulin could mediate haptotactic migration of cultured human epidermal keratinocytes through activation of transcription factor NF-kappa B. Insulin caused a dose-dependent stimulation of keratinocyte migration that maximally reached 2-fold at 2 x 10(-7) M hormone. This phenomenon was independent of the nature of the extracellular matrix component (collagen I or laminin5/nicein) on which the cells migrated, indicating that a specific integrin-ligand complex is not required. A 10(-7) M insulin treatment of keratinocytes resulted in activation of a major kappa B DNA binding complex within 15 to 30 minutes, which was identified as the p65/p50 NF-kappa B heterodimer by electrophoretic mobility shift assays. The activation induced nuclear translocation of cytosolic pools of NF-kappa B factor. Pyrrolidine dithiocarbamate and N-acetyl-leucinyl-leucinyl-norleucinal H (two compounds that differentially inhibit I kappa B alpha degradation and, thus, NF-kappa B activation) reversed the insulin-stimulated keratinocyte haptotactic migration without affecting insulin receptor activation. These compounds inhibited the insulin-induced nuclear translocation of NF-kappa B as detected by confocal laser scanning microscopy. Taken together our experiments demonstrate that insulin stimulates haptotactic migration of human epidermal keratinocytes through activation of NF-kappa B transcription factor. They emphasize the ability of insulin to stimulate keratinocyte movement and provide a first clue to the mechanism of insulin-induced haptotactic signaling.

Analysis by confocal laser scanning microscopy imaging of undilated bile canaliculi F-actin staining in the hepatocytes of human extrahepatic cholestatic liver.

Many studies have demonstrated the role of bile canalicular microfilaments in bile secretion and bile flow. It is now admitted that modification of bile canalicular network of microfilaments play a role in dysfunction of bile secretion observed in many cases of cholestasis. This work intends to study F-actin, a major component of microfilaments, in human hepatocytes in extrahepatic cholestasis. Normal and extrahepatic cholestatic liver were studied. F-actin was stained with fluorescent phallotoxin and quantified by using confocal laser scanning microscopy and an image analysis method. Mean specific fluorescence (MSF) of bile canaliculi was measured. Since dilated and bile plugged canaliculi were rarely observed in cholestatic liver sections, only undilated bile canaliculi were analysed. Bile canalicular MSF was significantly increased (p < 0.05) in cholestatic hepatocytes (1.3 to 1.7 fold higher than in controls). These data demonstrate a pericanalicular thickening of F-actin microfilaments in human extrahepatic cholestatis, similar to that described in literature in many cases of human intrahepatic and extrahepatic cholestasis cases as well as in experimentally induced cholestasis. However, further studies are needed to understand this increase in F-actin pericanalicular microfilaments in human extrahepatic cholestasis.

The dependence of the association rate of surface-attached adhesion molecules CD2 and CD48 on separation distance.

The kinetics of bond formation between spherical beads coated with CD48 and CD2-derivatized surfaces was studied with a flow chamber. For a given shear rate, the binding frequency was exquisitively sensitive to the particle velocity. Flow equations were used to derive the particle-to-surface distance from the velocity, thus yielding a relationship between this distance and the binding rate. Numerical values of the binding site densities allowed absolute determination of the rate of association between two individual molecules as a function of the distance between attachment points. In our model, this rate was about 0.03 s-1 at 10 nm separation, and it was inversely proportional to the cube of the distance.

Interest of image processing in cell biology and immunology.

Microscopy is a basic tool for cell biologists. Recent progress of electronics and computer science made powerful methodologies for digital processing of microscopic images easily available. These methods allowed impressive increase of the power of conventional microscopy. Dramatic image enhancement may be achieved by combination of filtering techniques, computer-based deblurring and contrast enhancement. Quantitative treatment of digitized images allows absolute determination of the density of different components of the observed sample, including antigens, intracellular calcium and pH. Morphometric studies are also greatly facilitated by image processing techniques. The capture of fast phenomena may be performed by transfer of small portion of microscopic images into computer memory as well as particular use of confocal microscopy. Finally, improved display of experimental data through coded colors or other procedures may enhance the amount of information that can be conveyed by visual examination of microscopical images. The purpose of the present review is to describe the basic principles of image processing and exemplify the power of this approach with a variety of illustrated applications to conventional, fluorescence or electron microscopy as well as confocal microscopy.

Determination of the lifetime and force dependence of interactions of single bonds between surface-attached CD2 and CD48 adhesion molecules.

We studied single molecular interactions between surface-attached rat CD2, a T-lymphocyte adhesion receptor, and CD48, a CD2 ligand found on antigen-presenting cells. Spherical particles were coated with decreasing densities of CD48-CD4 chimeric molecules then driven along CD2-derivatized glass surfaces under a low hydrodynamic shear rate. Particles exhibited multiple arrests of varying duration. By analyzing the dependence of arrest frequency and duration on the surface density of CD48 sites, it was concluded that (i) arrests were generated by single molecular bonds and (ii) the initial bond dissociation rate was about 7.8 s-1. The force exerted on bonds was increased from about 11 to 22 pN; the detachment rate exhibited a twofold increase. These results agree with and extend studies on the CD2-CD48 interaction by surface plasmon resonance technology, which yielded an affinity constant of approximately 10(4) M-1 and a dissociation rate of > or = 6 s-1. It is concluded that the flow chamber technology can be an useful complement to atomic force microscopy for studying interactions between isolated biomolecules, with a resolution of about 20 ms and sensitivity of a few piconewtons. Further, this technology might be extended to actual cells.