COLLEGE OF ARTS AND SCIENCES 25 CHRISTINE KRESCHOLLEK to the amount of shear force we’re applying to them. It’s a new method we’ve developed … it’s quite sensitive and pretty reliable,” she says. But, the team also needs to create a way to tag and track the proteins without changing their size or destroying the cells with which they interact. “On our side, we develop model proteins of different sizes to test whether they move differently in function of flow. We are also trying to develop a way to tag the glypican-1 protein selectively without modifying its size dramatically,” Thévenin says. “We can label the protein with an antibody, but the antibody is gigantic—about three times the size of glypican-1. Conventionally, we could attach GFP, green fluorescent protein, onto it, but it changes the size, affecting its response to flow. So, we’re trying to develop a way to tag the protein on the surface of living cells, without being toxic to cells; without modifying the size of the protein dramatically; and only labeling that precise protein.” The goal, Thévenin says, is to have two or three differently sized protein models and then determine if size correlates to how they move and respond to flow. Honerkamp-Smith’s lab then utilizes a confocal fluorescence microscope to observe how the proteins move across the surface of the cells. “We’re able to collect three-dimensional images of where all these fluorescent proteins are, and we have a microfluidic flow channel, where neural growth. When these cells sense healthy blood flow, they produce nitric oxide, which, in turn, increases circulation and helps the blood more efficiently deliver nutrients and oxygen. When they don’t feel enough flow, the endothelial cells trigger immune responses including inflammation that, if chronic, can lead to heart disease, obesity, cancers, and a host of other health issues—which is why aerobic exercise is so important. A better understanding of the principles of the fluid mechanics behind cell signaling mechanisms, then, could someday lead to therapies that mitigate the development of cardiovascular diseases and other conditions. The project, in its second year, is exploring the flow-mediated transport of glypican-1, a protein that exists on the exterior and interior of the cell surface and initiates nitric oxide production in response to flow. Discovering the trigger mechanism, however, has been a challenge. “There’s something of a paradox in the field of flow sensing,” Honerkamp-Smith says. “Most of the time, we think about cells sensing mechanical force using individual proteins that change their shape. For instance, you might have a protein that’s folded up, and then, if a force pulls on it and partially unfolds it, that’s the signal. Or you might have an ion channel that’s closed, then a force pulls on it and opens it and some ions can go through, and that’s your signal. But, in the case of blood flow sensing, nobody really knows what molecule is doing the sensing. The shear force that blood flow applies to blood vessels is too weak to cause this kind of motion in individual proteins.” But, she says, “what we propose is that even if forces are very weak, they can move membrane proteins along the surface of the cell. Even a very weak force can push on each protein, like wind on a little sailboat, and that way, you could have proteins move over. When the cell notices all the proteins are to one side, it might say, ‘Ah! There’s a flow outside, pushing in that direction.’ Our ultimate goal is to really determine whether that’s what endothelial cells do to sense blood flow.” A Need to Measure Speed One of the first steps was to develop a method to measure protein speed, she says. “We’ve been exploring, in a model system, the movement of proteins under flow. We have a couple of papers out; one is a proof of principle demonstrating that we can make an artificial membrane approximately the size of a single cell— just a flat membrane on a glass surface. Then, we attach proteins and apply flow to it, and we can measure how fast the proteins move according A composite confocal microscope image of a single living COS-7 cell inside a flow chamber (far left).
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