YSM Issue 97.1

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FOCUS Biochemistry How Firefighters Fight the Fire While much remains unknown about how neutrophils produce extracellular glycoRNA, biologists know that neutrophils aren’t recruited until they are needed. When tissue is injured or inflamed, it must somehow signal for neutrophils to come and bind to the tissue. Lu likens this process of neutrophil recruitment to firefighters rushing to a fire. Upon injury, damaged cells release proinflammatory proteins called cytokines that signal nearby endothelial cells to express selectins, like P-selectin, which are normally tightly controlled. This “fire” prompts the neutrophils, acting as firefighters, to attend to the site of injury. “Selectins are like glue that try to capture circulating white blood cells—mostly neutrophils because of their abundance—which starts the neutrophil infiltration process,” Wu said. P-selectin– neutrophil–glycoRNA binding is central to this glue-like interaction, but this is likely just part of the picture. Wu and Lu performed an experiment called RNA sequencing on glycoRNA isolated using click chemistry from three kinds of neutrophils in the mouse bloodstream and several human cell lines. Analyzing the mapping of these RNAs to the mouse or human genomes indicated specific rules that could govern RNA glycosylation. Lu describes these rules as a “licensing step.” Although the existence of these rules is not currently known, Lu speculates that they may involve special RNA sequences, structures, or modifications. Wu expressed that it may not be sufficient for an RNA to be glycosylated for recognition by the P-selectin; P-selectin’s specificity may include the RNA as well. These discoveries are only the first steps in characterizing the specificity of the P-selectin-neutrophil-glycoRNA interaction, but the methodology used in this study will likely inform future explorations of extracellular protein-glycoRNA interactions across the body. PHOTOGRAPHY BY EMILY POAG Ningning Zhang (left) and Wenwen Tang (right) discuss an image shown on the computer screen. How Did GlycoRNAs Get Outside of the Cell? The authors proposed two potential models to explain the mechanism by which glycoRNAs moved from the cytoplasm inside the cell to the outer surface of neutrophils. In the cell-to-cell model, cellular RNAs are released from one cell and are captured on an adjacent cell’s surface. On the other hand, in the cell-autonomous model, the production and transport of cellular RNAs to the cell surface occur in the same cell. To differentiate between these models, a co-culture experiment was conducted. One group of neutrophils was labeled with the Ac4ManNAz sugar and a fluorescent green dye, while another group was only labeled with a fluorescent red dye. These cells were then mixed and incubated together. Afterward, the researchers found only strong fluorescent signals in the green cells but not the red cells, confirming the second cellautonomous model of the production and transportation of glycoRNAs across the cell membrane. Once they had confirmed the cellular origin of glycoRNAs, Lu and Wu began to consider the pathways by which glycoRNAs could leave the cells. One such pathway was inspired by the C. elegans worm. In this model organism, the Sidt1 gene was found to encode RNA transporters that facilitated the uptake of digested RNA in the gut into the cells of the worm across cellular membranes. Therefore, the Yale scientists reasoned that the Sidt genes expressed in neutrophils could be facilitating the transport of RNAs across the cell membrane. To test this hypothesis, they ABOUT THE AUTHORS disrupted the expression of both Sidt1 and Sidt2 in cells in a knockdown experiment. As a result, the presence of Ac4ManNAz-labeled glycoRNAs was abolished, highlighting the crucial role of Sidt RNA transporters in the presence of glycoRNAs in cells. Importantly, the Sidt-knockdown cells also exhibited a significant reduction in in vivo recruitment to inflammatory sites, underscoring the essential role of Sidt genes in the functionality of neutrophils. The Future of the RNA World This novel collaboration between a neutrophil biologist and an RNA biologist is only the beginning of a growing field focused on glycoRNA interactions outside the cell. Lu and Wu are both eager to continue their collaboration and begin answering the many questions opened by this paper. As this field is still in its infancy, Lu suggests that it will take some work to even begin elucidating the initial mechanistic questions, such as exploring the molecular pathways involved in making extracellular glycosylated RNAs and figuring out the environments in which they are selectively produced or glycosylated. “We can’t work on all of these questions, so we have to be careful about picking the lower-hanging fruits first to work on. Then, I expect many people will start to work on it,” Lu said. After that, Wu and Lu hope other labs explore more disease-specific questions, like studying the disease conditions in which these RNAs are dysregulated and whether there are any therapeutic or diagnostic roles for extracellular RNAs. ■ KENNY CHENG RISHA CHAKRABORTY KENNY CHENG is a first-year student majoring in Molecular, Cellular, and Developmental Biology in Pauli Murray College. Outside of YSM, Kenny carries out research on ornate, large, extremophilic (OLE) RNAs in the Breaker lab and is an editorial associate for the Yale School of Medicine and the Yale Medicine Magazine. RISHA CHAKRABORTY is a third-year Neuroscience and Chemistry major in Saybrook College. In addition to writing for YSM, Risha plays trumpet for the Yale Precision Marching Band and La Orquesta Tertulia, volunteers at YNHH, and researches Parkinson’s Disease at the Chandra lab in the Yale School of Medicine. She enjoys cracking jokes, having “philosophical” discussions with her friends, and having boba with her PLees at the Asian American Cultural Center. THE AUTHORS WOULD LIKE TO THANK Dr. Jun Lu and Dr. Dan Wu for their time and enthusiasm for their research. FURTHER READING: Zhang, N., Tang, W., Torres, L., Wang, X., Ajaj, Y., Zhu, L., Luan, Y., Zhou, H., Wang, Y., Zhang, D., Kurbatov, V., Khan, S. A., Kumar, P., Hidalgo, A., Wu, D., & Lu, J. (2024). Cell surface RNAs control neutrophil recruitment. Cell, 187(4): 846-860. https://doi.org/10.1016/j.cell.2023.12.033 24 Yale Scientific Magazine March 2024

WEATHERING Evolutionary Biology FEATURE THE STORM ART BY PATRICIA JOSEPH BY SAMANTHA LIU HOW TINY BUGS SURVIVE THE RAIN Over a still pond, a raindrop falls over a line of water striders. It engulfs one of the insects, launching it upward amid a water jet. Momentarily the water strider is airborne—then the jet collapses, whips back up. The bug is left submerged, twisting just beneath the glassy surface. The Gerridae insect family has long drawn scientists’ fascination with its unusual water-walking ability. But how these bugs fare on turbulent seas has just been uncovered in a new study by a team of physicists from Florida Polytechnic University. Led by assistant professor of mechanical engineering Daren Watson, researchers captured on stunning video how water striders survive simulated rain drops. After plunging subsurface with their water-repellent coat, they rise back upward atop a jetstream. When a second collision pulls the bugs underwater, they paddle along with swift strokes. As a result, these tiny striders can weather a violent rainstorm— an insight that surprisingly may extend into how microplastics persist in marine environments. “I think it’s my best work yet,” noted Watson. “We’ve answered a fundamental question, but there’s much to explore in studies to come.” Watson’s foray into aquatic insects began on his Florida campus, where he found himself curious about the bugs skimming across his local pools. He couldn’t understand how their millimeters-long bodies could survive a free-falling drop, much less a storm. “[As] opposed to us humans, these insects have nowhere to hide,” Watson noted. With a little guidance from the “Bug Closet” at University of Central Florida, and a lot of scouring from nearby ponds, Watson captured and reared a group of water striders in a mini aquarium. He placed twenty of these insects in a chamber with an elevated nozzle and a syringe A water strider balances atop a pool with its delicate legs. IMAGE COURTESY OF WIKIMEDIA COMMONS pump. To simulate rainfall, he directed water from the nozzle through a long, narrow channel, landing droplets one at a time onto the bugs. After some tinkering and tailoring, the results—rendered in crystalclear, 3200 frames-per-second video resolution—reveal a valiant battle by the Gerridae. While some scatter and leap away at an impending raindrop, a bug caught in the splash zone must bear its full brunt. “The force of the raindrop striking the water strider is significantly higher than the weight of the water strider,” Watson said (up to forty times higher, to be precise—imagine a small delivery truck crashing atop you). “But it does not cause the strider to die.” Hydrophobic, densely-packed hairs along the bug’s exoskeleton repel the water, forming a bubble, called the first crater, around its body. This crater generates a buoyant force that pulls the water strider back to surface, then shoots upward as a water jet—which a well-positioned bug can surf like a gentle tidal wave. But when this jet collapses back downward and forms a second crater, the collision this time comes faster and harsher. “You can think of it like you’re stretching a rubber band… [it’s] going to rapidly go back to its original position,” Watson said. As the bug sinks down again, the crater retracts so quickly that the swimming creatures struggle to follow. According to Watson’s calculations, if this crater’s acceleration exceeds a threshold value—5.7 times the acceleration due to gravity—it tears away from the insect body, leaving the water strider submerged beneath the cavity. Though some footage shows intrepid water striders pedaling their legs to come up for air, they do not always succeed. “They must be able to swim and survive below the waterline for a period of time,” Watson said. “And that also adds to their survival during rainfall.” He wonders if this is an evolutionary predisposition for creatures exposed to rain—why resurface if another drop will come plummeting down seconds later? But as a physicist, Watson is more proud of cracking the mathematics of the second crater’s growth and collapse, something never before reported. Noting water striders’ similarities in size and buoyancy to ocean microplastics, he looks forward to translating his findings toward studying these waterway pollutants. When [microplastics] get into our water bodies, how do they become submerged?” he said. “How are they going to be exposed to the fishes, the marine life within those water bodies? How does rainfall precipitate that exposure?” For now, Watson has moved on from sea critters and back toward his realm of inanimate objects, though his study has opened new doors for biologists and engineers alike. In the meantime, across the swamps of Florida, water striders continue to twist and swirl beneath our marine surfaces, braving the onslaught of rain. ■ www.yalescientific.org March 2024 Yale Scientific Magazine 25

WEATHERING<br />

Evolutionary Biology<br />

FEATURE<br />

THE STORM<br />

ART BY<br />

PATRICIA<br />

JOSEPH<br />

BY SAMANTHA LIU<br />

HOW TINY BUGS SURVIVE THE RAIN<br />

Over a still pond, a raindrop falls over a line of water striders. It<br />

engulfs one of the insects, launching it upward amid a water<br />

jet. Momentarily the water strider is airborne—then the<br />

jet collapses, whips back up. The bug is left submerged, twisting just<br />

beneath the glassy surface.<br />

The Gerridae insect family has long drawn scientists’ fascination with<br />

its unusual water-walking ability. But how these bugs fare on turbulent<br />

seas has just been uncovered in a new study by a team of physicists from<br />

Florida Polytechnic University. Led by assistant professor of mechanical<br />

engineering Daren Watson, researchers captured on stunning video how<br />

water striders survive simulated rain drops. After plunging subsurface<br />

with their water-repellent coat, they rise back upward atop a jetstream.<br />

When a second collision pulls the bugs underwater, they paddle along<br />

with swift strokes.<br />

As a result, these tiny striders can weather a violent rainstorm—<br />

an insight that surprisingly may extend into how microplastics<br />

persist in marine environments.<br />

“I think it’s my best work yet,” noted Watson. “We’ve answered a<br />

fundamental question, but there’s much to explore in studies to come.”<br />

Watson’s foray into aquatic insects began on his Florida campus,<br />

where he found himself curious about the bugs skimming across his<br />

local pools. He couldn’t understand how their millimeters-long bodies<br />

could survive a free-falling drop, much less a storm. “[As] opposed to us<br />

humans, these insects have nowhere to hide,” Watson noted.<br />

With a little guidance from the “Bug Closet” at University of Central<br />

Florida, and a lot of scouring from nearby ponds, Watson captured and<br />

reared a group of water striders in a mini aquarium. He placed twenty<br />

of these insects in a chamber with an elevated nozzle and a syringe<br />

A water strider balances atop a pool with its delicate legs.<br />

IMAGE COURTESY OF WIKIMEDIA COMMONS<br />

pump. To simulate rainfall, he directed water from the nozzle through<br />

a long, narrow channel, landing droplets one at a time onto the bugs.<br />

After some tinkering and tailoring, the results—rendered in crystalclear,<br />

3200 frames-per-second video resolution—reveal a valiant battle<br />

by the Gerridae. While some scatter and leap away at an impending<br />

raindrop, a bug caught in the splash zone must bear its full brunt.<br />

“The force of the raindrop striking the water strider is significantly<br />

higher than the weight of the water strider,” Watson said (up to forty<br />

times higher, to be precise—imagine a small delivery truck crashing<br />

atop you). “But it does not cause the strider to die.”<br />

Hydrophobic, densely-packed hairs along the bug’s exoskeleton repel<br />

the water, forming a bubble, called the first crater, around its body. This<br />

crater generates a buoyant force that pulls the water strider back to<br />

surface, then shoots upward as a water jet—which a well-positioned bug<br />

can surf like a gentle tidal wave.<br />

But when this jet collapses back downward and forms a second crater,<br />

the collision this time comes faster and harsher. “You can think of it like<br />

you’re stretching a rubber band… [it’s] going to rapidly go back to its<br />

original position,” Watson said.<br />

As the bug sinks down again, the crater retracts so quickly that<br />

the swimming creatures struggle to follow. According to Watson’s<br />

calculations, if this crater’s acceleration exceeds a threshold value—5.7<br />

times the acceleration due to gravity—it tears away from the insect body,<br />

leaving the water strider submerged beneath the cavity.<br />

Though some footage shows intrepid water striders pedaling their legs<br />

to come up for air, they do not always succeed. “They must be able to<br />

swim and survive below the waterline for a period of time,” Watson said.<br />

“And that also adds to their survival during rainfall.”<br />

He wonders if this is an evolutionary predisposition for creatures<br />

exposed to rain—why resurface if another drop will come plummeting<br />

down seconds later? But as a physicist, Watson is more proud of cracking<br />

the mathematics of the second crater’s growth and collapse, something<br />

never before reported. Noting water striders’ similarities in size and<br />

buoyancy to ocean microplastics, he looks forward to translating his<br />

findings toward studying these waterway pollutants.<br />

When [microplastics] get into our water bodies, how do they become<br />

submerged?” he said. “How are they going to be exposed to the fishes,<br />

the marine life within those water bodies? How does rainfall precipitate<br />

that exposure?”<br />

For now, Watson has moved on from sea critters and back toward his<br />

realm of inanimate objects, though his study has opened new doors for<br />

biologists and engineers alike. In the meantime, across the swamps of<br />

Florida, water striders continue to twist and swirl beneath our marine<br />

surfaces, braving the onslaught of rain. ■<br />

www.yalescientific.org<br />

March 2024 Yale Scientific Magazine 25

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