Atomic Force Microscopy in Cell Biology Episode 1 Part 6 potx

86 Manfred Radmacher extending lamellipodium is somewhat softer and less variable in time compared to the stable edge, which shows a well-pronounced and stable pattern of stress fibers running parallel to the edge. Another very exciting process is cell division, especially the formation of the cleavage furrow,which willeventually separatethe two daughtercells andmay involve mechanical changes in the mother cell. This process was first investigated by AFM by Dvorak and Nagao (Dvorak and Nagao, 1998), who took a single force curve at one position, which was very undefined, since dividing cells change shape and tend to move and rotate on the substrate. Therefore, it is difficult to stay at the same location of a cell. We used the AFM in line scan mode as described previously to measure the mechanics along a single scan line, which is positioned such that it will be crossing the newly formed cleavage furrow. Here we could unambiguously show that the furrow region stiffens before the furrow is visible in the topography (Matzke et al., 2000). V. Summary In this chapter I discussed the possibility of measuring elastic properties of living cells by AFM. One reason for using the AFM for this purpose is its ability to both measure locally the mechanics of a cell and to distinguish different regions of the cell. Since the AFM can be operated under physiological conditions cellular processes can be followed, for example, cytokinesis and the investigation of the migration of cells. List of Symbols α half-opening angle of conical indenter δ indentation of (soft) sample d deflection of AFM cantilever d 0 zero deflection of the free AFM cantilever (off the surface) d 1 lower limit of deflection in range of analysis d 2 upper limit of deflection in range of analysis E elastic or Young’s modulus of sample F loading force of AFM cantilever tip F 1 lower limit of loading force in range of analysis F 2 upper limit of loading force in range of analysis F cone loading force predicted from the Hertz model for a conical indenter F paraboloid loading force predicted from the Hertz model for a parabolic indenter F WLC force needed to extend a polymer molecule within the framework of the worm-like chain model k b Boltzmann’s constant k c force constant of AFM cantilever L contour length of polymer molecule 4. Measuring Elastic Properties of Living Cells 87 l p persistence length ν Poisson ratio r cone radius of contact area between conical tip and sample r paraboloid radius of contact area between parabolic tip and sample R radius of curvature for parabolic or spherical indenter T absolute temperature x extension of polymer molecule z sample base height z 1 lower limit of sample base height in range of analysis z 2 upper limit of sample base height in range of analysis z 0 sample base height at point of contact between tip and sample References A-Hassan, E., Heinz, W. F., Antonik, M. D., D’Costa, N. P., Nagaswaran, S., Schoenenberger, C-A., and Hoh, J. H. (1998). Relative micro-elastic mapping of living cells by atomic force microscopy. Biophys. J. 74(3), 1564 –1578. Arnoldi, M., Fritz, M., B¨auerlein, E., Radmacher, M., Sackmann, E., and Boulbitch, A. (2000). Bacterial turgor pressure can be measured by atomic force microscopy. Phys. Rev. E, vol. 62(1), 1034–1044. 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CHAPTER 5 Cell Adhesion Measured by Force Spectroscopy on Living Cells Martin Benoit Center for Nanoscience Ludwig-Maximilians-Universit ¨ at M ¨ unchen D-80799 M ¨ unchen, Germany I. Introduction II. Instrumentation III. Preparations of the Force Sensor for Measurements with Living Cells A. Cell-Surface Adhesion Force Measurements B. Adhesion Force Measurements between Cell Layers C. Cell–Cell Adhesion Force Measurements IV. Cell Culture A. HEC/RL Cell Culture on Coverslips B. JAR Cell Culture on Cantilever C. Dictyostelium Cell Culture V. Final Remarks References I. Introduction Cell-to-cell adhesion is essential for multicellular development and arrangement. Cells may carry several different adhesion molecules (Kreis and Vale, 1999), resulting in a high variability of the molecular repertoire of the cell surfaces. This variability is reflected in the broad pattern of adhesion-controlled cellular functions during development and adult life (Fritz et al., 1993; Springer, 1990; Vestweber and Blanks, 1999). To determine cell adhesion many techniques have been evolved, such as functionalized latex beads moved with optical tweezers (Choquet et al., 1997), microfluorescence assays or interferrometric techniques (Bruinsma et al., 2000), and centrifugation experiments, e.g., with cell spheroids (John et al., 1993; Suter et al., 1998). Viscoelastic properties of cells were measured by cell poking and even with spatial resolution by an atomic METHODS IN CELL BIOLOGY, VOL. 68 Copyright 2002, Elsevier Science (USA). All rights reserved. 0091-679X/02 $35.00 91 92 Martin Benoit force microscope (AFM) in either force modulation mode or more recently by force volume techniques (Domke et al., 2000;Goldmann et al., 1998;Hoh and Schoenenberger, 1994; Radmacher et al., 1996; Zahalak et al., 1990). Adhesion between single cells, e.g., granulocytes and target cells, was measured in the past using mechanical methods, such as micropipette manipulations (Evans, 1985, 1995) or hydrodynamic stress (Chen and Springer, 1999; Curtis, 1970). With the development of piconewton instrumentation based on AFM technology (Binnig et al., 1986), the force resolution and the precision of positioning have allowed measurements at the single-molecule level (Gimzewski and Joachim, 1999; M¨uller et al., 1999; Oesterhelt et al., 2000). Forces for conformational transitions in polysaccharides (Marszalek et al., 1999; Rief, Oesterhelt et al., 1997) for the unfolding of proteins (Oberhauser et al., 1998; Rief, Gautel et al., 1997; Smith et al., 1999) and for stretching and unzipping of DNA (Rief et al., 1999; Strunz et al., 1999) were measured. Unbinding forces of individual ligand–receptor pairs were determined (Baumgartner et al., 2000; Florin et al., 1994; Hinterdorfer et al., 1996; M¨uller et al., 1998) and the basic features of the binding potentials were reconstructed (Grubm¨uller et al., 1995; Merkel et al., 1999). Recently, the first steps toward cell adhesion mea- surements with AFM technology were made (Domke et al., 2000; Razatos et al., 1998; Sagvolden et al., 1999). Several theories have been developed to describe the processes which are involved while separating cells by either modeling single independent contacts or picturing more elaborate mechanisms such as molecular clustering (Evans and Ritchie, 1997; Kuo et al., 1997; Ward et al., 1994; Ward and Hammer, 1993). In this section a new AFM-based experimental platform to investigate cell-to-cell interactions in vivo down to the molecular level will be described, immobilizing living cells to a force sensor. Epithelial cells (RL95-2 and HEC-2-A) from human endometrium as a substrate for an artificially rebuilt human trophoblast (JAR) are used to distinguish molecular adhesion processes involved not only in embryo–uterus interactions but also between individual cells of Dictyostelium discoideum to measure the adhesion force of single-contact site A proteins. To obtain reproducible results, the complexity of living cells demands recording, estimating, and pinpointing a large variety of parameters. Therefore the contact-force is controlled down to 30 pN during the contact between well-studied cell types in a defined cell culture environment. II. Instrumentation The cell adhesion force spectrometer with an integrated optical microscope is special- ized for force measurements on living cells. As a force sensor, a standard AFM cantilever is placed underneath a Perspex holder. The force signal is obtained from the deflection of the laser beam (Fig. 1) and plotted as force versus piezo position (e.g., Fig. 5). The spring constant of the cantilever in each experiment is determined using the thermal noise technique reported earlier (Florin et al., 1995). By using sensors with a low spring constant, less force is applied to a cell when touched. The force resolution lies between 20 5. Cell Adhesion Measured by Force Spectroscopy 93 Fig. 1 Schematic of the adhesion force spectrometer with a light microscope below the Petri dish. The sensor mounted on a Perspex holder is placed from above in the Petri dish with the detecting laser unit. Two versions of cell adhesion force spectrometers: (A) long-range (100 μm) piezo moving the Petri dish and (B) short-range (15 μm) piezo moving the force sensor. and 3 pN and is recorded together with the piezo position at a precision of 1 ˚ A in either 256 pts (12 bit) or 32,768 pts (16 bit) per trace. The frequency of data collection is 60 kHz and the noise can be reduced by either filtering or averaging. For position- ing, the sample is manually driven by an x-y stage mounted on a high-precision z piezo-actuator (100 μm) 1 with a strain gauge for long-range cell interactions (Thie et al., 1998) (Fig. 1A). To detect shorter range interactions the Perspex holder is moved by a high-precision z piezo actuator (15 μm) which is equipped with a strain gauge (Dettmann et al., 2000) (Fig. 1B). The z piezo velocity was typically set between 1 and 7 μm/s. Slower velocities often interfere with drift effects basically caused by cell movement, while at higher velocities hydrodynamics influence the measurement. The lateral sample displacement is disabled during most of the experiments reported here. The approach of the sensor to the surface stops automatically if a certain threshold force is reached. This force can be kept constant within a certain range by a feedback loop compensating movements of the cells or piezo drift, especially if contacts last several minutes. Mea- surements are performed in an appropriate medium for living cells in a cell culture dish. To achieve long-time measurements standard cell culture conditions at 37 ◦ CinCO 2 (5% v/v) can be applied. The cells are monitored using the light microscope during the entire experiment. 1 Especially nerve cells tend to form strongly adhering membrane tethers (Dai and Sheetz, 1998) over distances of millimeters. Even 100 μm is not enough to separate these cells from each other. 94 Martin Benoit III. Preparations of the Force Sensor for Measurements with Living Cells To immobilize cells on the force sensor without harming them is most crucial for operating the cell adhesion force spectrometer (Fig. 1). Here a single cell or, alternatively, a whole monolayer of epithelial cells will be immobilized to the sensor (Figs. 2A and 2B). Since most cells express adhesion molecules on their surface, a very gentle method of immobilization is establishing a matching connection to these molecules. A. Cell-Surface Adhesion Force Measurements To determine which proteins to use for immobilizing the respective cells, as a first step, we characterized the adhesion forces by probing the cells with differently functionalized sensors. To distinguish the adhesion of the coating to be tested from the nonspecific interaction between surface and cell, a treatment had to be found to inactivate the sensor surface prior to applying the functionalizing molecules. 1. Immobilization of a Sphere to the Sensor To betterdefine the contact area between sensor and cells,a sphere of 60 μm indiameter from either sephacryl or glass is fixed at the end of a cantilever. The spheres are mounted to the cantilevers (DNP-S Digital Instruments, Santa Barbara, CA; or Microlever, Park Scientific Instruments, Sunnyvale, CA) in the following manner. A tiny spot of epoxy glue (UHU plus endfest 300, B¨uhl, Germany) is applied to the tip of a cantilever using a patch-clamp glass electrode. Then a single Sephacryl S-1000 sphere (Pharmacia, Freiburg, Germany) or a glass sphere (G 4649; Sigma, Deisenhofen/Germany), about 60 μm in diameter, which sticks electrostatically to a cannula (Terumo No. 20, Leuven/Belgium) is placed on the epoxy. To cure the epoxy, the microsphere-mounted cantilever is then heated at 90 ◦ C for 45 min. Another method is described in Holmberg et al. (1997). Before use, the cantilevers were sterilized in 70% ethanol for 2 h and washed thoroughly in distilled water. Sensor tips and spheres fixed to the force sensor (Fig. 3) with various coatings were tested on the cells of interest. Fig. 2 Schematics and light microscopic image of (A) a single-cell (Dictyostelium discoideum; the cells on the cover slide are out of focus) and (B) a layer of cells (osteoblasts) on a glass sphere immobilized on a force sensor. 5. Cell Adhesion Measured by Force Spectroscopy 95 Fig. 3 Schematics (B) and images of a sephacryl (A) and a glass (C) sphere (diameter 60 μm) glued to a force sensor. 2. Passivated Force Sensors The following protocol, derived from Johnsson et al. (1991), proved useful for prepar- ing sensors with a sufficiently low nonspecific interaction with cells. First the Si–OH layer of either a SiO 2 oraSi 3 N 4 surface is ammino-silanized with N  -(3-(trimethoxysilyl)-propyl)-diethylentriamin (Aldrich) at 80 ◦ C for 10 min to obtain an amino-functionalized surface. It is then washed in ethanol and completely crosslinked for 10 min in water at 80 ◦ C. A phosphate-buffered saline (pH 7.4) (PBS Sigma) solution of 10 mg/ml of carboxymethylamylose (Sigma) is activated with 20 mg/ml N-hydroxy- succinimide (NHS, Aldrich) and 20 mg/ml 1-ethyl-3-(3-dimethylaminopropyl)carbodi- imide (EDC, Sigma) for 2 min. The tip is then incubated with the NHS-activated amylose for 15 min, rinsed three times in PBS, incubated with 0.5 mg/ml ethanolamine (Sigma) in PBS for 1–2 h, and intensively rinsed in PBS. Other preparation techniques with PEG have also proved to sufficiently passivate surfaces for proteins and cells (Bruinsma et al., 2000; Willemsen et al., 1998). 3. Results From the “deadhesion force versus piezo position traces” (e.g., Fig. 4 or Fig. 5) adhesion can be characterized in an initial approach by measuring the maximum adhesion force. As shown later, other adhesion parameters will be derived from these traces. If a sphere is lowered onto a soft cell surface, the area of interaction increases with the indentation which leads to an enhancement of the adhesion signal. The adhesion strength is not only dependent on the indentation force (here 3 ± 1 nN) while the cells are brought and held in contact as mentioned earlier but also, as shown in Fig. 5, on the duration of the contact. This is probably due to the fact that the cell shape adapts to the sphere’s surface and more and more molecules can interact with this surface with time. The adhesion to the sepacryl spheres is enhanced by at least 50% compared to that to a glass sphere, in agreement with their structured and therefore larger surface. Changing the velocity of retraction leads to a fairly linear relation between separation speed and adhesion in the range between 2 and 27 μm/s. However, for low velocities the influence [...]... RL/JAR Time (min) Continuous Discontinuous Continuous Discontinuous 1 10 15 20 30 40 87 55 24 24 11 13 0 0 0 0 0 0 15 4 67 38 19 6 5 0 0 0 5b 4 14 b a HEC b After never showed discontinuous deadhesion, while RL does one of these experiments, a cell was found loosened on the mono- layer laser reflex, typical features can be recognized As in Fig 5, the adhesion increases with increasing contact time in Fig 7... known to interact with JAR cells via fibronectin-binding proteins (certain integrins; Thie and Ramunddal, unpublished data), these experiments corroborate our assumption that this discontinuous adhesion is due to a specific interaction This conclusion is supported by previous work of other groups which had investigated integrin–cytoskeleton interactions in other cell types where integrin–ligand binding promotes... the invasive trophoblast, RL cells, and the receptive uterine epithelium From centrifugation experiments (John et al., 19 93) HEC cells are supposed to represent the nonreceptive uterine epithelium 1 Immobilizing a Monolayer of Cells to a Force Sensor Before seeding cells, the sensor is carefully rinsed in alcohol and water, precoated with polylysins, laminins, fibronectins, or other adhesive coatings,4... glycoprotein, can be singled out by genetic manipulations (Faix, 19 99) In D discoideum, csA participates in cell aggregation, the transition from the single -cell to the multicellular stage (Ponte et al., 19 98) Thus, csA is undetectable in growth-phase cells but is expressed upon starvation (Murray et al., 19 81) In developing cells of the aggregation stage, csA covers roughly 2% of the total cell surface... amylose chain become longer If the binding pocket is potentially inactivated by binding the amylose with NH2 groups too close to the pocket, a soluble binding partner lacking any NH2 groups could be added during activation to protect the binding site The ligand must then be washed out carefully before the experiment 3 5 Cell Adhesion Measured by Force Spectroscopy 99 strong for all cells at the initial... cross-talk between trophoblast and uterine epithelium leading to specific cell cell binding, i.e., a redistribution/upregulation/ activation of adhesion systems at the free cell pole (Albers et al., 19 95; Denker, 19 94; Thie et al., 19 95, 19 96) In this context, it is of interest that a discontinuous JAR–RL interaction is observed only after prolonged contact of both partners This could be due to the time... embryo–uterus interactions With this preparation, the mechanisms of the interaction between human trophoblasttype JAR cells (JAR) with the two human uterine epithelial cell lines RL95-2 (RL) and HEC -1- A (HEC) were investigated RL cells, in contrast to HEC cells, are supposed to respond to the contact with JAR cells in a specific way (John et al., 19 93; Thie et al., 19 97, 19 98) 2 Results JAR-coated force sensors... single cells by combining singlemolecule force spectroscopy with genetic manipulation for the measurement of deadhesion forces at the resolution of individual cell- adhesion molecules In general single cells behave different from cells in tissue But, to resolve single-molecule events instead of cooperative molecular effects, it is necessary to minimize contact area, contact time, and contact force Two... epoxy glue 5 Cell Adhesion Measured by Force Spectroscopy 10 1 Fig 7 Typical adhesive force curves for (A) HEC -1- A and (B) RL95-2 cells resulting when a JAR-coated sensor, (C) a bovine serum albumin (BSA)-coated sensor, or a (D) fibronectin (FN)-coated sensor is retracted after periods of 1 40 min time of contact at approximately 5 nN (Thie et al., 19 98) 10 2 Martin Benoit Table I Listing of JAR Experimentsa... surface area (Beug, Katz, and Gerish, 19 73) CsA molecules react with each other (homophilic interaction), forming noncovalent bonds linking the surfaces of adjacent cells (Kamboj et al., 19 88), which are anchored in the plasma membrane by a ceramide-based phospholipid (Stadler et al., 19 89) 1 Immobilizing a Single Living Cell to a Force Sensor As shown by Razatos et al (19 98) even bacteria can be fixed to . by atomic force microscopy. J. Cell Sci. 10 7, 11 05 11 14. Ingber, D. E. (19 93). Cellular tensegrity: defining new rules of biological design that govern the cytoskeleton. J. Cell Sci. 10 4, 61 3 62 7. Janmey,. Experiments a HEC/JAR RL/JAR Time (min) Continuous Discontinuous Continuous Discontinuous 1 87 0 15 4 0 10 55 0 67 0 15 24 0 38 0 20 24 0 19 5 b 30 11 0 6 4 40 13 0 5 14 b a HEC never showed discontinuous deadhesion,. Magnetospirillum Gryphiswaldense investigated by atomic force microscopy. Appl. Phys. A 66 , S 61 3 –S 61 7 . Ashkin, A., and Dziedzic, J. M. (19 89). Internal cell manipulation using infrared laser traps. Proc.