Taekjip Ha and his postdoc Xuefeng Wang have devised a simple but powerful strategy to measure the tiny forces exerted through single molecules in the cell. The University of Illinois scientists dreamed up a cellular tug-of-war between receptors anchored in the cell membrane and corresponding ligands that they tethered to a surface. To determine the forces, they simply monitored whether the tethers held and the cells adhered.

Cells actively sense and transmit forces, and we know that the stiffness of the environment can determine whether cancer cells become metastatic or whether embryonic stem cells differentiate into other cell types. Force also plays a critical role in processes such as cell adhesion, migration and immune function. Ultimately, events at the cellular scale depend on forces exerted by single molecules.

Ha's work evolved out of an earlier collaboration with Martin Schwartz at the University of Virginia to measure molecular forces using a tension-sensitive fluorescence resonance energy transfer (FRET) sensor. His group calibrated forces corresponding to FRET signals, and the Schwartz group took the biological measurements. “I got kind of unhappy that now the cell biologists are having all the fun,” says Ha. He proposed to Wang that he measure FRET at the cell surface, which would be easier than making measurements inside the cell. After a month of applying FRET sensors, Wang came up with the idea of using a tether that could rupture at a known force.

The method works by testing a series of tethers that can tolerate defined tensions before snapping. A receptor must bind its ligand with a force greater than the tolerance of the tether for the cell to snap the tether and detach from the surface. Ha points out that in most single-molecule methods relevant to cell biology, people apply the force, whereas in their system, the cell applies the force. “We are just measuring cell behavior, which is much easier to measure than single molecules; and by definition, what we are doing is physiologically relevant,” he says.

In creating the tethers, Wang and Ha were inspired by the work of Hermann Gaub and colleagues, which showed that pulling apart two strands of a short DNA fragment from opposite ends requires higher forces than does pulling apart strands from the same end (the former configuration in effect shears the DNA, whereas the latter unzips it). Wang and Ha also designed DNA fragments with seven intermediate attachment points corresponding to a stepwise series of tension tolerances.

The DNA tethers do have some caveats. They eventually fall apart from thermal fluctuations, a factor that limits studies to 5 or 6 hours, and they are only able to measure forces between 10 and 60 picoNewtons, but the researchers are working on new kinds of tethers to push those limits.

Using their tethering method, Wang and Ha found that integrin pulls its ligand in the extracellular matrix with far greater force than is estimated in the literature and that the Notch receptor pulls its ligand relatively weakly. Ha was most surprised by the fact that the same threshold is observed across a number of cell types. “In my talks, I often use the expression 'a quantum of force', and it's as if there is a quantized force value in mechanical signaling,” he says. Ligand density does not appear to affect binding thresholds above a level needed for adhesion.

The group is working on measuring forces in a number of contexts, from endocytosis to immune cell activation, and on testing ligands present in different micropatterns. Ha is also interested in studying multiple protein interactions at once and testing cells in clusters. He points out that fluorescently labeled tethers can be imaged to determine which remain intact and which break, and that different ligands can be labeled with unique colors. It should be possible to see how different cells within a cluster sense and respond to tension, as an approximation of what happens in tissues.