Push and Pull on the Smallest Scale: Whitman Fellow “Democratizes” the Study of Cellular Forces
The inside of our cells is constantly in motion. DNA helices are winding and unwinding. Motor proteins drag payloads and yank cellular components to and fro. Ion channels are flinging open and slamming shut. Cilia flap in sync, helping us cough and circulating our brain fluid.
These processes, and the thousands more like them that keep our bodies running, are fundamental to our understanding of the cell. Yet, with limited exceptions, we have no idea how much force these proteins generate or respond to.
Measuring these forces “should be a routine thing,” said Yuan Ren, a postdoctoral scientist in the Julien Berro lab at Yale University and a Whitman Fellow this summer at the Marine Biological Laboratory (MBL). Unfortunately, existing force measurement mechanisms are slow and difficult to use accurately. Ren is working on a project to solve this that he hopes will “democratize force measurement in cells.”
What once took years now takes weeks
The reason that these cellular forces are so mysterious is their microscopic scale. Forces in cells are so small that they are measured in piconewtons, or about one trillionth the force that an apple exerts when pulled by gravity. Being so miniscule, the best way to measure them is with miniscule devices. Devices made of proteins serve this role well, since they can be introduced into the cell using CRISPR, and can be designed to send a signal that indicates the strength of the force being measured.
Currently, the best force measurement approach we have is based on a process called Förster resonance energy transfer (FRET), which acts as a proxy to turn small changes in distance (typically less than five nanometers) into changes in fluorescence intensity or lifetime. The change in distance is correlated to the magnitude of force by using a miniscule protein spring. Unfortunately, the molecular structure used in FRET is so large that inserting one of these devices into a protein can alter its shape and functions, skewing results. Worse still, the process of performing the technique takes multiple years; you could spend an entire graduate degree measuring the force at just one place on one protein.
FRET was an important first step in force measurement -- after all, Ren said, “prior to that tool there was nothing” -- but the challenges of using it has made force measurement in cells difficult, time-consuming, and prohibitively expensive. Ren’s project aims to change that using a different measurement technique altogether: the unwinding of coiled coils.
Coiled coils are one of the most common shapes, or motifs, in proteins. They consist of two helices wrapped around each other like the strings that form a rope. These strands are bound to each other tightly, but every coiled coil has a force at which they will unwind.
What Ren has done is taken coiled coils that unwind at a known force, then hidden a short peptide in the linker of the coils that is only exposed once the coils have unwound. When a coil unwinds, it triggers a variety of responses, indicating that that coiled coil’s known force has been reached. The readout is no longer restricted to FRET. He then can add one of these coiled coils with a known unwinding force to a protein, see if its force unwinds it, then repeat this process using an array of coils. Eventually, he can narrow down the force he is measuring to a very precise range.
This technique is precise, and more importantly, fast -- the entire process of inserting the coiled coil and measuring the protein’s force takes only a week. Even with the time it takes to repeat the measurement with different coiled coils, Ren can determine a given force’s strength in as little as one or two months.
Uncoiling coiled coils
For this technique to work, Ren needs a large variety of coiled coils. To make them, he engineers different coiled coils by changing their amino acid makeup. In doing so, he alters the strength of the bond between the two coils, making them easier or harder to pull apart.
The next challenge, then, is measuring how much force it takes to crack open this newly made coiled coil. This seems borderline impossible: What device could possibly manipulate such a tiny protein?
Jie Yang in Yongli Zhang’s lab at Yale University is in charge of this step of the process, and the way he does it is nothing short of incredible. First, he attaches a bead to each end of the new coiled coil. Then, he blasts each bead with an extremely focused laser that is so powerful that it actually exerts a force on the bead, trapping it. Then, slowly and carefully, he moves the lasers apart, pulling the beads with them. At some point, the coiled coil snaps open. Because the lasers exert a known force themselves, Yang can calculate the force required to pull the coiled coil apart, down to the nearest piconewton.
Armed with a vast array of precisely-measured coiled coils and a technique that can be performed quickly and cheaply, Ren hopes to change the way that we understand the forces in cells. In particular, he is fascinated by mechanosensitive channels, membrane proteins that open and close in response to external forces. These channels are vitally important: they govern everything from our body awareness to our sense of hearing. “I can talk and you can hear me because of mechanosensitive channels,” Ren said.
Understanding these processes on a microscopic level may seem trivial, but faulty mechanosensitive channels are behind everything from tinnitus to deafness to seizures. Studying them could be key to unlocking cures. After all, Ren said, “before we measure something, we don't know if it's there.” There are also certainly more mechanosensitive channels to be discovered, which this technique could help illuminate.
Ren and colleagues at Yale have already measured 12 forces acting on five different proteins in yeast cells, mammalian cells and worm axons, but measuring every one of the myriad cellular forces is too big a job for one lab. Ren, as well as his advisor Berro, want to establish this technique in the scientific community in the hopes of opening up new avenues for future research. That is the main reason Ren decided to come to MBL for the summer.
“The collaborative research atmosphere here is the best,” he said, and it gives him a chance to introduce his technique to other scientists. “For people that develop tools, you do want those tools to be used,” he said, so “you want to meet potential users.” He also values the chance to work with scientists from other disciplines, especially MBL-CZI Imaging Scientist Abhishek Kumar, whose microscopes have been invaluable to his work. Together they have shown that force can be visualized through bioluminescence in single cells.
“The measurement of forces is a really basic question that will eventually have a lot of practical uses,” Ren said. “But many people may not think it's important.” He appreciates that “MBL really values basic research,” giving ideas like Ren’s a chance to flourish.