26 ACUMEN • SPRING 2024 and then see if that’s correlated with the initiation of the signal transduction,” she says. “Everything that has to deal with lipid membranes is extremely difficult to study,” Thévenin says. “One, lipids are hydrophobic, and so the proteins that bind to them and interact with them are also hydrophobic. And two, you need to detect how one single protein, among all the others, moves with very, very tiny forces. It’s like observing the movement of a very small boat in an ocean from very far up.” Tiny Forces, Large Impact One of the most significant findings so far, Honerkamp-Smith says, is that “we can get dramatic rearrangement of the whole cell surface with such a tiny force applied to each individual protein. It’s hard to convey how small the forces are … piconewtons are the tiniest forces that people usually deal with, and these forces are 10 times smaller than that! So, we’re really looking at something extremely subtle that has a big effect on the outside of the cell. “I’m very pleased that we’ve seen protein gradients forming on the outside of living cells that were growing,” she says. “This is something I did expect to find, but it’s exciting to see it in reality. This isn’t published yet, so that’s a very new, in-progress result that we’re working on now.” Thévenin says the next step will be to look at glypican-1 in living cells and see whether it can be labeled, followed, flowed at different rates, and linked to a difference in cell response. “There is also part of the grant that is just looking at the lipids, without proteins,” he says. And, initial findings are already eye-opening. “As we’ve been doing experiments, we see that in addition to moving the proteins, just the lipids by themselves do surprising things in response to flow,” Honerkamp-Smith says. “There are shape changes we’ve observed that we’re still doing experiments on to try to figure out what is happening on a basic level.” “When you do an experiment, you open a can of worms,” Thévenin says. “You have more questions, more hypotheses to test and even more experiments to do—and that’s where the fun is.” ● we can apply a controlled amount of flow and also keep them at the right temperature and in their usual growth medium,” she says. “We’re also developing image analysis code to determine how fast proteins are moving in response to flows. “What we observed is what we were hoping to observe, which is that larger proteins go faster than smaller ones,” she says. “So, if you have a bigger sail, then you move faster under flow, and we can actually measure the forces that we’re applying to each individual protein. Those are really tiny forces. These are femtonewton-sized forces, which are incredibly small. We’re quite excited about that.” “I think the big challenge,” Honerkamp-Smith says, “is that in order to see the response, we have to do this with cells that are alive and happy. It’s not as hard to get beautiful images of cells if you’ve killed them and frozen them in place, but we need to do this with living, functioning cells.” In addition to the size restrictions Thévenin mentioned, the team also needs to find a location for the tag that won’t change the protein’s role. “We’re trying to get a very specific, very tiny fluorescent label onto these proteins, and then, we can apply flow, watch the protein move SCIENCE SOURCE A fluorescent light micrograph of cultured COS-7 cells.
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