Microrheology of cells with magnetic force modulation atomic force microscopy.

We propose a magnetic force modulation method to measure the stiffness and viscosity of living cells using a modified AFM apparatus. An oscillating magnetic field makes a magnetic cantilever oscillate in contact with the sample, producing a small AC indentation. By comparing the amplitude of the free cantilever motion (A0) with the motion of the cantilever in contact with the sample (A1), we determine the sample stiffness and viscosity. To test the method, the frequency-dependent stiffness of 3T3 fibroblasts was determined as a power law k(s)(f) = α + ß(f/f¯)(γ) (α = 7.6 × 10(-4) N m(-1), ß = 1.0 × 10(-4) N m(-1), f¯ = 1 Hz, γ = 0.6), where the coefficient γ = 0.6 is in good agreement with rheological data of actin solutions with concentrations similar to those in cells. The method also allows estimation of the internal friction of the cells. In particular we found an average damping coefficient of 75.1 µN s m(-1) for indentation depths ranging between 1.0 µm and 2.0 µm.

Comparison of the viscoelastic properties of cells from different kidney cancer phenotypes measured with atomic force microscopy.

The viscoelastic properties of human kidney cell lines from different tumor types (carcinoma (A-498) and adenocarcinoma (ACHN)) are compared to a non-tumorigenic cell line (RC-124). Our methodology is based on the mapping of viscoelastic properties (elasticity modulus E and apparent viscosity η) over the surface of tens of individual cells with atomic force microscopy (AFM). The viscoelastic properties are averaged over datasets as large as 15000 data points per cell line. We also propose a model to estimate the apparent viscosity of soft materials using the hysteresis observed in conventional AFM deflection-displacement curves, without any modification to the standard AFM apparatus. The comparison of the three cell lines show that the non-tumorigenic cells are less deformable and more viscous than cancerous cells, and that cancer cell lines have distinctive viscoelastic properties. In particular, we obtained that E(RC-124) > E(A-498) > E(ACHN) and η(RC-124) > Î·(A-498) > Î·(ACHN).

Direct, high-resolution measurement of furrow stiffening during division of adherent cells.

It is unclear whether cell division is driven by cortical relaxation outside the equatorial region or cortical contractility within the developing furrow alone. To approach this question, a technique is required that can monitor spatially-resolved changes in cortical stiffness with good time resolution. We employed atomic force microscopy (AFM), in force-mapping mode, to track dynamic changes in the stiffness of the cortex of adherent cultured cells along a single scan-line during M phase, from metaphase to cytokinesis. Video microscopy, which we used to correlate the AFM data with mitotic events identified by light microscopy, indicated that the AFM force-mapping technique does not perturb dividing cells. Here we show that cortical stiffening occurs over the equatorial region about 160 seconds before any furrow appears, and that this stiffening markedly increases as the furrow starts. By contrast, polar relaxation of cells does not seem to be an obligatory event for cell division to occur.

Double power-law viscoelastic relaxation of living cells encodes motility trends.

Living cells are constantly exchanging momentum with their surroundings. So far, there is no consensus regarding how cells respond to such external stimuli, although it reveals much about their internal structures, motility as well as the emergence of disorders. Here, we report that twelve cell lines, ranging from healthy fibroblasts to cancer cells, hold a ubiquitous double power-law viscoelastic relaxation compatible with the fractional Kelvin-Voigt viscoelastic model. Atomic Force Microscopy measurements in time domain were employed to determine the mechanical parameters, namely, the fast and slow relaxation exponents, the crossover timescale between power law regimes, and the cell stiffness. These cell-dependent quantities show strong correlation with their collective migration and invasiveness properties. Beyond that, the crossover timescale sets the fastest timescale for cells to perform their biological functions.

From molecules to cells: imaging soft samples with the atomic force microscope.

Since its invention a few years ago, the atomic force microscope has become one of the most widely used near-field microscopes. Surfaces of hard sample are imaged routinely with atomic resolution. Soft samples, however, remain challenging. An overview is presented on the application of atomic force microscopy to organic samples ranging from thin ordered films at molecular resolution to living cells. Fundamental mechanisms of the image formation are discussed, and novel imaging modes are introduced that exploit different aspects of the tip-sample interaction for local measurements of the micromechanical properties of the sample. As examples, images of Langmuir-Blodgett films, which map the local viscoelasticity as well as the friction coefficient, are presented.

Direct observation of enzyme activity with the atomic force microscope.

The height fluctuations on top of the protein lysozyme adsorbed on mica were measured locally with an atomic force microscope operated in tapping mode in liquid. Height fluctuations of an apparent size of 1 nanometer that lasted for about 50 milliseconds were observed over lysozyme molecules when a substrate (oligoglycoside) was present. In the presence of the inhibitor chitobiose, these height fluctuations decreased to the level without the oligoglycoside. The most straightforward interpretation of these results is that the height fluctuations correspond to the conformational changes of lysozyme during hydrolysis. It is also possible, however, that the height fluctuations are, at least in part, the result of a different height or elasticity of the transient complex of lysozyme plus the substrate.

Influence of microenvironment topography and stiffness on the mechanics and motility of normal and cancer renal cells.

The tumor microenvironment highly influences cancer cell modes and dynamics, above all during invasive and metastatic processes. When aiming at studying cancer cell behavior in vitro, the use of conventional cell culture systems, such as Petri dishes, fails in recapitulating the mechanical and topographical properties of the natural extracellular matrix (ECM). Here the versatility of stiffness-tunable hydrogels and the efficacy of the replica molding technique with silicone polymers are exploited, aiming at studying cancer and normal cell behavior with platforms able to capture the heterogeneities of the natural in vivo context. We compared the mechanical properties of normal and cancer renal cells on different stiffness value gels (with Young's moduli of 3, 17 and 31 kPa) by using atomic force microscopy (AFM) and investigated cell indentation phenomena on compliant gels with confocal microscopy. Moreover, we studied cell mechanics, morphology and migration on isotropic linear structures, spaced at 1.5 µm, aiming at mimicking the aligned fiber bundles typically observed at tumor borders. By using this approach, we could highlight differences in the way healthy and cancer renal cells react to changes in their microenvironment. Our results may potentially pave the way to unravel the complex processes involved in cancer progression, especially in tissue invasion and migration during metastasis formation.

Estimation of tamoxifen's efficacy for preventing the formation and growth of breast tumors.

BACKGROUND: Several randomized clinical trials have tested the hypothesis that tamoxifen is effective in preventing breast cancer. The largest such trial, the National Surgical Adjuvant Breast and Bowel Project's Breast Cancer Prevention Trial (BCPT), reported a 49% reduction in risk of invasive breast cancer for the tamoxifen group. However, it is unclear whether the effect of tamoxifen in this trial was mainly due to prevention of newly forming tumors or to treatment of occult disease. METHODS: We used various tumor growth models (i.e., exponential and Gompertzian [growth limited by tumor size]) and a computer simulation to approximate the percentage of detected tumors that were initiated after study entry. Maximum likelihood techniques were then used to estimate separately the efficacy of tamoxifen in treating occult disease and in preventing the formation and growth of new tumors. RESULTS: Under the assumptions of most of the growth models, the trial was sufficiently long for substantial numbers of new tumors to form, grow, and be detected during the trial. With the Gompertzian model and all available incidence data from the BCPT, it was estimated that 60% (95% confidence interval [CI] = 40%-80%) fewer new tumors were detected in the tamoxifen group than in the placebo group. Likewise, 35% (95% CI = 6%-63%) fewer occult tumors were detected in the tamoxifen group. With this model, the estimated incidence rate of invasive breast cancer among women in the placebo group of the BCPT was 7.7 (95% CI = 6.6-8.9) per 1000 women per year. Similar results were obtained with three exponential tumor growth models. CONCLUSIONS: These results support the concept that tamoxifen reduced cancer incidence in the BCPT through both treatment of occult disease and prevention of new tumor formation and growth. However, data from prevention trials may never be sufficient to completely distinguish prevention of new tumor formation from treatment of occult disease.

Direct observation of different surface structures on high-resolution images of native halorhodopsin.

Halorhodopsin (HR) was investigated with atomic force microscopic techniques (AFM) in aqueous solution. Two-dimensional (2D) crystals of HR were obtained by purifying an HR membrane fraction with the same buoyant density as the purple membrane (HR-PM) from the overexpressing strain Halobacterium salinarum D2. The membrane patches of HR were immobilized on mica. Images with a resolution up to 14 A were recorded. Crystals showed an orthogonal structure and the orientation of the molecules showed p42(1)2 symmetry; thus, alternate tetramers are inverted in the membrane. The crystal surface was found to display different structures depending on the imaging force used, indicating that some parts of the HR molecule are more rigid but others more compressible. From samples with single tetramers missing in the crystalline patches dimensions of the unit cell could be determined. Helix-connecting loops in single molecules of halorhodopsin were assigned. The images indicate that the large extracellular BC loop covers the whole molecule and is very flexible.

Chromosome abnormalities in malignant melanoma: clinical significance of nonrandom chromosome abnormalities in 206 cases.

We report the cytogenetic abnormalities from a series of 206 primary malignant melanoma specimens referred to a single institution. A total of 169 out of 206 unique cases had chromosome breakpoints. A previously described statistical method was used to detect nonrandom distribution of chromosome breakpoints at the level of chromosome regions. Nonrandom occurrence of chromosome breakpoints (indicating that the observed number of breaks significantly exceeded the expected number of breaks) was detected in 28 regions, suggesting a hierarchy of genetic abnormalities in melanoma. Clinical variables and tumor characteristics were analyzed for associations with the presence of any nonrandom chromosome breakpoints; with individual, nonrandomly involved chromosome regions; and with paired, nonrandomly involved chromosome regions. No nonrandomly involved chromosome regions or pairs of regions appeared to significantly affect survival. These results identify recurring, nonrandom chromosome abnormalities in malignant melanoma. These results suggest that recurring, nonrandom chromosome alterations play a key role in the etiology and/or progression of malignant melanoma and identify targets within the genome for molecular genetic studies.

Can the light-addressable potentiometric sensor (LAPS) detect extracellular potentials of cardiac myocytes?

The light-addressable potentiometric sensor (LAPS) measures localized photo-induced currents from a silicon wafer, which are dependent on the local surface potential and on the intensity of the light pointer. In this study the ability of the LAPS to record extracellular potentials of adherent cells was investigated. Time dependent LAPS photocurrent signals that correlated in time with contractions were recorded from beating cardiac myocytes cultured on LAPS surfaces. Signals could be recorded both when the LAPS was biased to working points where the photocurrent was maximally sensitive to potential changes and when it was biased to working points where the photocurrent was insensitive to changes in surface potential. Therefore, signals could not be predominantly created by changes in extracellular potential and might be related to mechanical contractions. One possible explanation might be, that the cell-induced modulation of photocurrents arose as a result of cell shape changes. Such alterations in cell shape might have focused and defocused the light pointer and, thus, modulated its intensity. To further test this hypothesis, height changes of beating cardiac myocytes were measured with an atomic force microscope (AFM). They were found to match well with signals derived from LAPS measurements. Therefore, it can be concluded, that LAPS signals were mainly determined by the periodic changes in shape of beating heart cells, and this interference precludes the measurements of extracellular electrophysiological potentials from these cells.

EGF-stimulated lamellipod extension in adenocarcinoma cells.

The extension of lamellipodia has been triggered by the application of epidermal growth factor (EGF). We have used an atomic force microscope (AFM) to investigate this lamellipodial extension. During extension we could detect an increase in height from about 500 nm for the stable lamellipodium to typical values of 600-800 nm for the extending lamellipodium. The AFM was also used to determine the mechanical properties of the lamellipodium where we found a decrease of the elastic modulus by a factor of 1.4 at the same location within the same cell. Both findings are consistent with the cortical expansion hypothesis, suggesting that severing of actin filaments, leading to a swelling of the cytoskeleton, generates the protrusive force during lamellipodial extension.

Waiting times to appearance and dominance of advantageous mutants: estimation based on the likelihood.

The germinal center reaction (GCR) of vertebrate immunity provides a remarkable example of evolutionary succession, in which an advantageous phenotype arises as a spontaneous mutation from the parental type and eventually displaces the parental type altogether. In the case of the immune response to the hapten (4-hydroxy-3-nitrophenyl)acetyl (NP), as with several other designed immunogens, the process is dominated by a single key mutation, which greatly simplifies the modeling of and analysis of data. We developed a two-stage model of this process in which the primary stage represents the appearance and establishment of the mutant population as a stochastic process while the second stage represents the growth and dominance of the clone as a deterministic process, conditional on its time of establishment from stage one. We applied this model to the analysis of population samples from several germinal center (GC) reactions and used maximum-likelihood methods to estimate the waiting times to arrival and to dominance of the mutant clone. We determined the sampling properties of the maximum-likelihood estimates using Monte Carlo methods and compared them to their asymptotic distributions. The methods we present here are well-suited for use in the analysis of other systems, such as tumor growth and the experimental evolution of bacteria.

Drug-induced changes of cytoskeletal structure and mechanics in fibroblasts: an atomic force microscopy study.

The effect of various drugs affecting the integrity of different components of the cytoskeleton on the elasticity of two fibroblast cell lines was investigated by elasticity measurements with an atomic force microscope (AFM). Disaggregation of actin filaments always resulted in a distinct decrease in the cell's average elastic modulus indicating the crucial importance of the actin network for the mechanical stability of living cells. Disruption or chemical stabilization of microtubules did not affect cell elasticity. For the f-actin-disrupting drugs different mechanisms of drug action were observed. Cytochalasins B and D and Latrunculin A disassembled stress fibers. For Cytochalasin D this was accompanied by an aggregation of actin within the cytosol. Jasplakinolide disaggregated actin filaments but did not disassemble stress fibers. Fibrous structures found in AFM images and elasticity maps of fibroblasts could be identified as stress fibers by correlation of AFM data and fluorescence images.

Dimensional and mechanical dynamics of active and stable edges in motile fibroblasts investigated by using atomic force microscopy.

The atomic force microscope (AFM) was employed to investigate the extension and retraction dynamics of protruding and stable edges of motile 3T3 fibroblasts in culture. Such dynamics closely paralleled the results of earlier studies employing video microscopy that indicated that the AFM force-mapping technique does not appreciably perturb these dynamics. Force scans permitted height determinations of active and stable edges. Whereas the profiles of active edges are flat with average heights of 0.4-0.8 micrometer, stable edges smoothly ascend to 2-3 micrometers within about 6 micrometers of the edge. In the region of the leading edge, the height fluctuates up to 50% (SD) of the mean value, much more than the stable edge; this fluctuation presumably reflects differences in underlying cytoskeletal activity. In addition, force mapping yields an estimate of the local Young's modulus or modulus of elasticity (E, the cortical stiffness). This stiffness will be related to "cortical tension," can be accurately calculated for the stable edges, and is approximately 12 kPa in this case. The thinness of the leading edge precludes accurate estimation of the E values, but within 4 micrometers of the margin it is considerably smaller than that for stable edges, which have an upper limit of 3-5 kPa. Although blebbing cannot absolutely be ruled out as a mechanism of extension, the data are consistent with an actin polymerization and/or myosin motor mechanism in which the average material properties of the extending margin would be nearly constant to the edge. Because the leading edge is softer than the stable edge, these data also are consistent with the notion that extension preferentially occurs in regions of lower cortical tension.

Imaging soft samples with the atomic force microscope: gelatin in water and propanol.

We have imaged mica coated with thin gelatin films in water, propanol, and mixtures of these two liquids by atomic force microscopy (AFM). The elastic modulus (Young's modulus) can be tuned from 20 kPa to more than 0.1 GPa depending on the ratio of propanol to water. The resolution is best in pure propanol, on the order of 20 nm, and becomes worse for the softer samples. The degradation in resolution can be understood by considering the elastic indentation of the gelatin caused by the AFM tip. This indentation becomes larger and thus the contact area becomes larger the softer the sample is. Therefore this study may be used to estimate the resolution to be expected with an AFM on other soft samples, such as cells. Nondestructive imaging was possible only by imaging at forces < 1 nN. This was difficult to achieve in contact mode because of drift in the zero load deflection of the cantilever, supposedly caused by temperature drift, but straightforward in tapping mode.

Granula motion and membrane spreading during activation of human platelets imaged by atomic force microscopy.

The redistribution of platelet constituents during activation is essential for their physiological function of maintaining hemostasis. We report here about real time investigations of the activation of native human platelets under physiological conditions from the initial formation of filopodia to the fully spread form by atomic force microscopy. We followed the trafficking of granules and their interaction with the plasma membrane within single cells. Our results show movement of certain granula towards the lamellipodia. Analysis of this rearrangement and the subsequent enlargement of the platelet surface reveals details of the membrane spreading process. Images of living cells are presented that show the distribution of cytoskeletal components and membrane-bound filaments at a resolution of better than 50 nm. The local minimum forces between the tip and the platelets were estimated to be smaller than 60 pN. A model for the elastic contributions of the glycocalix to the tip/membrane interaction was developed using the theory of grafted polymers.

Predicted and inferred waiting times for key mutations in the germinal centre reaction: evidence for stochasticity in selection.

The germinal centre reaction (GCR) is a fundamental component of the immune response to T-dependent antigens, during which the immunoglobulin (Ig) genes of B cells experience somatic hypermutation and selection. A maximum-likelihood method on DNA sequence data from 16 individual germinal centres was used to infer that the waiting time for position 33 key (high-affinity) mutations in the anti-(4-hydroxy-3-nitrophenyl) acetyl (NP) response is 8.3 days. This is in marked contrast to the prediction of a key mutant each generation (waiting time about 1/3 day) obtained from a simple model and parameters available in the literature. This disagreement is resolved in part by the finding that the targeted base occurs in a cold spot for hypermutation, raising the predicted waiting time to 2.3 days, although this value remains significantly lower than that inferred from the sequence data. It is proposed that the remaining disparity is attributable to some further stochastic process in the GCR: many early key mutations arise but fail to 'take root' within the GC, either due to emigration or failure of cognate T cell/B cell interaction. Furthermore, it is argued that the frequency with which position 33 mutations are found in secondary responses to NP indicates the presence of selection after the GCR.