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.

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.

Volume dynamics in migrating epithelial cells measured with atomic force microscopy.

Migration of transformed renal epithelial cells (transformed Madin-Darby canine kidney cells, MDCK-F cells) relies on the activity of a Ca(2+)-sensitive K+ channel (IK channel) that is more active at the rear end of these cells. We have postulated that intermittent IK channel activity induces local cell shrinkage at the rear end of migrating MDCK-F cells and thereby supports the cytoskeletal mechanisms of migration. However, due to the complex morphology of MDCK-F cells we have not yet been able to measure volume changes directly. The aim of the present study was to devise a new technique employing atomic force microscopy (AFM) to measure the volume of MDCK-F cells in their physiological environment and to demonstrate its dependence on IK channel activity. The spatial (x, y' and z) co-ordinates of each pixel of the three-dimensional image of MDCK-F cells allow calculation of the volume of the column "underneath" a given pixel. Thus, total cell volume is the sum of all pixel-defined columns. The mean volume of 17 MDCK-F cells was 2500+/-300 fl. Blockade of the IK channel with the specific inhibitor charybdotoxin (CTX) increased cell volume by 17+/-4%; activation of IK by elevating the intracellular [Ca2+] with the Ca2+ ionophore ionomycin decreased cell volume by 19+/-3%. Subtraction images (experimental minus control) reveal that swelling and shrinkage occur predominantly at the rear end of MDCK-F cells. In summary, our experiments show that AFM allows the measurement not only of total cell volume of living cells in their physiological environment but also the tracing of local effects induced by the polarized distribution of K+ channel activity.

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.

Investigating the cytoskeleton of chicken cardiocytes with the atomic force microscope.

We have investigated living chicken cardiocytes with an atomic force microscope (AFM). Cytoskeletal structures like stress fibers can easily be imaged with the AFM. Here we have also measured the cell's elastic properties. By taking force curves as a function of lateral position (force mapping) we could compare the elastic properties at different locations of the same cell. In the lamellipodal region investigated here in detail, the elastic moduli range from around 10 up to 200 kPa on top of stress fibers. By degradation with cytochalasin B we can estimate to what extent the elastic properties of this type of cell are determined by the actin network.

AFM imaging and elasticity measurements on living rat liver macrophages.

The authors investigated the morphology and the elastic properties of living cultured rat liver macrophages (Kupffer cells) with an atomic force microscope (AFM). Continuous imaging and elasticity mapping of individual cells in physiological buffer was carried out for several hours without damaging the cells as judged by their persistent undisturbed morphology. Dynamic events such as protrusive activity were observed in time course. The importance of the cytoskeleton for the mechanical properties of the cell has been investigated by measuring the cell's elasticity as a function of position. Chemical disassembly of the actin network by applying 10 microgram/ml cytochalasin B decreased the cell's average elastic modulus seven-fold within less than 40 minutes. Treating the cells with 0.1 micrograms/ml latrunculin A resulted in a two-fold decrease in the elastic modulus merely in the perinuclear region after 40 minutes, whereas other parts of the cell were not affected.

Recognition of damage in polarizing transmission-grating facets.

An optical setup was built for microscopic damage inspection on transmission-grating facets composed of a gold-wire structure. Contrast improvement was achieved by exploiting the polarizing properties of these gratings in the near-infrared region. Spatial filtering yields an additional contrast enhancement and reduces unwanted signals caused by the periodic support structure. An image-processing algorithm is developed that evaluates the number and the size of the faults in a grating facet with high accuracy from only one digital image.