Use the force

The ability of cells to bond to each other involves many processes and can reveal much about biological function. Here we learn why - in the world of cell adhesion studies - force spectroscopy is just the ticket    

Cell–cell adhesion is a complex process involved in the tethering of cells to make tissues, inter cellular communication, cell migration as well as the development and metastasis of cancer. Proteins and other specific molecules regulate these adhesive interactions between cells and there are a wide range of these receptor-ligand complexes.

Intracellular adhesion is not limited to just cells from the same organism, indeed viruses use binding molecules to attach themselves to host cells before they invade the cell and replicate. For instance, the human immunodeficiency virus (HIV) targets the CD4 receptors on the surface of T-cells using the gp120 protein. By discovering more about the different stages of the attachment process, researchers may be able to design drugs that prevent viruses from infecting cells. Because cell membranes are covered by a large number of different ligands and receptors, the contribution of each of the individual molecules to any adhesion event needs to be investigated before an accurate picture of the mechanism can be built up.

Sometimes, a cell may be imaged using the AFM prior to performing force spectroscopy experiments, (Figure 1). Alternatively, the optical microscope is used to position the cantilever over the cell to generate data containing information on both specific and non-specific contributions to adhesion and in-situ experiments can be conducted to distinguish which molecules are involved.

The technique allows the real-time study of molecular interactions on the nano scale without the need for chemical labeling and is sensitive enough to characterise biomolecular interactions such as the unfolding forces of single proteins or forces of a single chemical bond.

Force measurements became possible on the early AFM systems thanks to the work of researchers such as Colton and Gaub who defined the mathematical models and published many early results. However, until the launch of the CellHesion module for the NanoWizard and NanoWizard II BioAFMs (JPK Instruments AG, Berlin, Germany), research into this area had been hampered by the lack of a commercially available instrument that could be used to study cell-cell adhesion. This is because force spectroscopy experiments on the adhesion of two cells can often require an effective pulling range of up to 80μm due to the extrusion of membrane tethers during cell separation. A routine AFM does not have this ability.

Recently, JPK have extended the possibilities with the launch of the CellHesion 200 system. This combines a 100 mm force-distance scanner (based on AFM technology) with an inverted microscope to enable users to measure samples with both techniques simultaneously. Users can identify the location of a cell using the optical microscope before investigating interaction forces between the sample and a functionalised probe tip.

The inverted microscope enables users to study their samples using advanced and established fluorescence techniques such as TIRF, FRET, FCS, FRAP, FLIM, Ca2+ response, and laser scanning microscopy as well as optical contrast enhancement methods like phase contrast and DIC. All these techniques require that the sample is kept immobilised while imaging is in progress, making the cantilever tip-scanning approach essential if simultaneous imaging with both techniques is to be achieved.

Biological materials need to be studied in their natural state, i.e. in liquid or solution, if denaturing is to be avoided. The need to pre-dry samples before their analysis has become a thing of the past, as the modern AFM provides the experimenter with the ability to study samples while still in their native liquid environments. This avoids the process of having to try to convince oneself that an image is “real” and not just an artifact caused by the drying process.

Working in liquid demands that no sensitive parts can be positioned below the sample level, making a sample scanning approach unsuitable. All parts that come into contact with the sample are easy to exchange, replace and rigorously clean. The AFM is completely sealed and different fluid cells are available to ensure accurate environmental control.

Innovative instrumentation design is at the heart of the success of this technology. There is no room for uncertainty during single molecule AFM experiments and simultaneous single molecule fluorescence. This is all vital for the life sciences researcher wishing to push the boundaries of understanding at the nanoscale.

The central principle behind acquiring force spectroscopy data using an atomic force microscope is based on bringing a cantilever-bound cell into contact with a second cell or a surface coated with the ligand or receptor of interest and monitoring cantilever deflection during separation.

There are a number of important steps needed to acquire force spectroscopy data and these are:

• Cantilever functionalisation
The cantilever must be functionalised with a molecule to which the cell of interest will bind to more strongly than it will to the surface of interest.

• Cantilever calibration
The cantilever deflection needs to be calibrated using inbuilt routines in the JPK SPM software so that the force measurements can be accurately calculated in Newtons.

• Introduction of cells into the temperature controlled sample chamber
Those cells that are to be bound to the cantilever then need to be introduced into the module before an appropriate cell can be selected using the inverted light microscope using either phase contrast or fluorescence microscopy.

• Cell attachment to cantilever
The functionalised cantilever can then be placed over the top of the selected cell and gently lowered onto the top of the cell. After a few seconds contact time the cantilever can be retracted with the cell attached. This whole process can be monitored using the light microscope.

• Target selection
The light microscope can now be used to select a target cell or region of a monolayer on which to collect force measurement data.

• Force curve acquisition
After setting the desired measurement parameters, the cantilever-bound cell is brought into contact with the targeted area (or cell) and cantilever deflection and separation distance monitored. By plotting the applied force calculated for the cantilever deflection against the separation distance, the software can calculate the force required to break the bonds that cause the cells to stick together.

Cell-cell adhesion operating principle

A single living cell is chemically bound to the cantilever sensor (e.g. fibronectin coating).	The cell is brought with a defined force into contact with the binding target (e.g. another single cell, molecular layer, implant surface) on a substrate slide, coverslip or Petri dish).
After a user-defined reaction time, the cell on the cantilever is seperated from the substrate cell by retracting the cantilever in vertical direction.	The cell at first resists causing the cantilever to bend. This is measured by a detector. Eventually, the cells will seperate and force required to break this adhesion may be calculated. Because, in physical terms, the cantilever is a spring, the actual adhesive forces and energies can be derived from the measured bending events that contribute to the adhesion. Typically, such experiments are repeated many times to build statistically viable data.

An example of applying force spectroscopy measurement is the study of Melanoma binding to endothelial cells. The progression of malignant melanoma tumor cells can be tracked with changes in cell surface markers. Untransformed melanocytes in membranes have a low proliferative capacity as their cell cycle is rigidly controlled by their interaction with surrounding keratinocytes. On transformation to melanoma cells stop displaying the E-cadherin surface markers that promote contact with the keratinocytes and instead display N-cadherin which promotes cell-cell interactions with fibroblasts and endothelial cells. This changes the way the cells interact with their environment causing them to invade the dermis and metastasize, significantly worsening a patient’s prognosis.

Understanding the various steps involved in the transformation process and the role that different markers play in cell signaling and adhesion could enable researchers to develop drugs that halt tumor progression.

Puech and coworkers  have used CellHesion force spectroscopy protocols to

Figure 1:AFM image of rat kidney cells

study the changing adhesion profiles of cells as they transform into cancer cells. The team injected WM115 melanoma cells into a JPK BioCell before selecting and attaching a cell to a ConA-coated cantilever.

A series of force distance curves were acquired at 37°C to determine the strength of the interactions between the cells and a fibronectin-coated surface. The force curves indicate a number of discrete unbinding events after the initial rupture of bonds.

After the control curves were collected, an RGD peptide was added to block any integrin-mediated binding. These curves show only an initial unbinding event, indicating that initial unbinding consists predominantly of non-specific interactions between the cell and the fibronectin glass surface. The downstream events can therefore be ascribed to specific integrin-fibronectin interactions.

Force-distance curves of WM115 interaction with fibronectin coated surface. The grey curve represents the approach curve, the blue curve corresponds to a typical retract curve and the green after blocking with RGD peptide	Force-distance curves of WM115 cell binding to HUVEC monolayers, blue curves correspond to the control retract curve and the green to the retract curves after the addition of EG

Traditional cell binding assays studying the adhesion of melanoma cells have used human umbilical vein endothelial cell (HUVEC) and Peuch et al. investigated whether previous studies could be replicated using force spectroscopy, rather than cellular binding experiments that can take 24 hours to complete. The adhesion between cantilever-bound WM115 cells and a surface-bound HUVEC monolayer was studied using the CellHesion module. Curves were collected at a pulling distance of 60μm after being in contact with the surface for either 5 or 20 seconds.

The experiments were then repeated after the addition of the chelating agent EGTA (ethylene glycol tetraacetic acid) that blocks Ca2+ dependent cadherin-mediated binding. While the initial unbinding event occurs in both sets of force curves, many of the subsequent interactions observed in the control curves are not observed after the addition of EGTA. As the treatment with the EGTA does not block all of the discrete unbinding events it suggests that cadherin is not the only specific receptor on the melanoma cell surface that interacts with the HUVEC cells.

The process of monitoring cell-cell interactions allows researchers to distinguish

Atomic force (left) and corresponding fluorence (right) images of melanoma cells with FITC-phalloidin labeled actin

between the various interactions that occur between cells and substrates. These interactions play a key role in not only tethering cells, also in intercellular communication, tissue formation, cell migration and the development and metastasis of tumors. The complexity of the cell adhesion process and subsequent signaling means that only a sensitive technique like force spectroscopy can really distinguish between the various cell-cell interactions that enable us to live. The extended pulling range of CellHesion 200 and its integration with an inverted optical microscope opens the door to a new field of research that will enable researchers to gain deeper insights into the development and progression of both healthy and cancerous cells and potentially lead to the discovery or validation of new drug targets.