List of Symbols α half-opening angle of conical indenter δ indentation of soft sample d deflection of AFM cantilever d0 zero deflection of the free AFM cantilever off the surface d1 lower
Trang 186 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 will eventually separate the two daughter cells and may 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
d0 zero deflection of the free AFM cantilever (off the surface)
d1 lower limit of deflection in range of analysis
d2 upper limit of deflection in range of analysis
E elastic or Young’s modulus of sample
F loading force of AFM cantilever tip
F1 lower limit of loading force in range of analysis
F2 upper limit of loading force in range of analysis
Fcone loading force predicted from the Hertz model for a conical indenter
Fparaboloid loading force predicted from the Hertz model for a parabolic indenter
FWLC force needed to extend a polymer molecule within the framework
of the worm-like chain model
kb Boltzmann’s constant
kc force constant of AFM cantilever
L contour length of polymer molecule
Trang 24 Measuring Elastic Properties of Living Cells 87
lp persistence length
rcone radius of contact area between conical tip and sample
rparaboloid 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
z1 lower limit of sample base height in range of analysis
z2 upper limit of sample base height in range of analysis
z0 sample base height at point of contact between tip and sample
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Trang 6CHAPTER 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
Trang 792 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
Trang 85 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 (100μm) piezo moving the Petri dish and
(B) short-range (15μm) 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
7μm/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◦C in CO2
(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 100μm is not enough to separate these cells from each other.
Trang 994 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 better define the contact area between sensor and cells, a sphere of 60μm in diameter
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 60μm 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.
Trang 105 Cell Adhesion Measured by Force Spectroscopy 95
Fig 3 Schematics (B) and images of a sephacryl (A) and a glass (C) sphere (diameter 60μm) 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 SiO2or a Si3N4surface 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 27μm/s However, for low velocities the influence