Mapping Cellular Traffic

Similar to the organs that keep our bodies running, our cells contain specialized subunits called organelles that perform the fundamental functions that drive life. Mitochondria, for example, turn glucose into chemical energy to fuel these activities. The lysosome breaks down cellular components that are no longer needed and recycles the building blocks to generate new materials.

But how do mitochondria move to the sites that need fuel? How do unneeded cellular components get to the lysosome? And when a cell divides, how do the organelles themselves distribute correctly to the new cells? Through the intricate, tightly controlled system superhighways, side streets, specialized transporters that is the cellular supply chain.

Within this transport system, the Cianfrocco lab is particularly focused on the delivery trucks: motor proteins called kinesin, dynein and myosin. Cruising along the cell’s transportation routes, they carry essential cargo toward the center of the cell (in the case of dynein) or export cargo to destinations around the cell (kinesin and myosin).

“If you think about a neuron that extends the full length of your leg, motor proteins carry sensory information from the outer perimeter back to the central nervous system,” explains Cianfrocco, who is also an assistant professor of biological chemistry at the U-M Medical School.

Without the delivery trucks, proteins and other molecules would have to rely on diffusion to move from one end of the cell to another. And while diffusion is technically possible (after all, the cytoplasm that surrounds everything within the cell is fluid), the process would take long enough to bring cellular functions to a halt.

Cianfrocco compares it to a tennis ball in JELL-O. The tennis ball is not likely to move on its own, but something could drag the ball fairly easily. For cellular transport, that something is motor proteins.

“We can see this in the way the rabies virus hijacks this system to infect a host,” he explains. “The virus hitches a ride on motor proteins to get to the spine or brain, before any symptoms set in. So the time between infection and the onset of symptoms depends on how far the infection site is from the spine or the brain, or how far the virus has to travel. If the virus was left to diffuse on its own, it would take thousands of years to see any symptoms.”

Recently, Cianfrocco and his colleagues have turned their attention to deciphering how these delivery trucks stop — because, he notes, “you can’t load the truck if it never stops moving.”

Employing cryo-electron microscopy (cryo-EM), a process that uses electron beams to reveal the 3D structures of molecules flash-frozen in vitreous ice, his lab recently discovered how one protein acts as a parking boot on kinesins. The aptly named kinesin-binding protein latches on to one of the kinesin’s two feet. Once the protein attaches, the kinesin can no longer contact or move along its path.

“Because they are involved in virtually every cellular operation, kinesins must be carefully controlled,” Cianfrocco says. 

This kinesin-binding protein, for example, is just one of the methods cells use to inhibit kinesin movement. Yet when this protein is mutated, it alone can cause a rare but serious autosomal disorder called Goldberg-Shprintzen syndrome.

Figuring out how these molecular delivery trucks pause for loading is just the beginning of what Cianfrocco hopes to map out when it comes to intracellular transport. He’s also working out how that truck secures its cargo and starts moving again.

“In my lab we spend a lot of time watching these proteins walk and trying to figure out: How does that happen? How does the cargo tell the motor protein to move it?” Cianfrocco says.

To answer that, he has turned to myosin and a bit of baker’s yeast.

Ever since she made a fortuitous discovery as a postdoctoral researcher, Lois Weisman has been fascinated by cellular transportation.

Studying yeast, she noticed that when a new cell was budding on the mother cell, the lysosome started to dance around before projecting a small portion of itself into the budding daughter cell.

“This was before people had a good way of looking at cells with live imaging. It was just serendipitous that I was able to find this,” recalls Weisman, a faculty member at the LSI and professor of cell and developmental biology at the Medical School. “And it raised all of these questions about how this occurs: How do you get that kind of movement in a cell?”

In her own lab, Weisman continues to explore the twisted paths that those questions have opened — including the path traversed by the myosin type V motor protein.

In mammals, type V myosins deliver cargo involved in processes ranging from skin pigmentation to brain and gut function. Minor mutations in one of these proteins cause Griscelli syndrome, which results in neurological defects and unusually light hair and skin color.

Weisman’s lab investigates myosin V’s counterpart in yeast to disentangle the processes that allow this motor protein to load and deliver its vital cargo.

The lab has published several papers explaining how myosin releases its cargo at the right destination. Now, through a collaboration with the Cianfrocco lab, the researchers are studying the other end of that journey: how the cargo gets loaded onto myosin in the first place.

Splitting her time between the Weisman and Cianfrocco labs, graduate student Lily Hahn is using live-cell imaging and cryo-EM to understand how an adapter protein hitches cargo onto myosin for delivery.

“These are very foundational cellular events that we might take for granted,” Hahn says. “But when cells divide, whether they are cancer cells or budding yeast, they have to make sure the right amount of each organelle gets transported to the new cells. And to understand how that happens, or how it goes wrong in disease, we need to understand the fundamental mechanisms.”

While teasing apart the genetic drivers of these mechanisms, Weisman and her colleagues stumbled upon another serendipitous discovery: a pathway that they are studying affects not only how cargo moves within cells, but also how cells themselves move.

“When I see something new, I just think, ‘I wonder how it works,’ and I want to study it,” Weisman says.

In this case, “it” is an enzyme that makes a critical, but very low-abundance, signal inside cells — so low that Weisman’s lab is still one of the only research groups in the world that can measure it at all. The enzyme and its signal have recently attracted greater attention as a potential drug target to treat neurodegenerative diseases, cancers and even COVID-19.

The enzyme, called PIKfyve, helps keep the cellular recycling system in balance at the lysosome. But researchers in Weisman’s lab recently discovered that PIKfyve also plays an unexpected role in the pathway that transports sensors to the cell surface. In particular, it recycles sensors called integrins, which help control cell migration.

This latest study also opened a new route to understanding PIKfyve’s role in neurodegeneration. Minor mutations in this sensor recycling pathway have been tied to various neurological diseases. Weisman’s lab is now investigating whether the enzyme’s role close to the cell surface could be necessary for proper communication between nerve cells. 

“We know that completely blocking PIKfyve in animal models is lethal,” Weisman says. “But if we can tease apart its various roles, we could perhaps begin to design therapeutics that target specific pathways that PIKfyve regulates.”

While Weisman and Cianfrocco are deciphering how proteins move within cells, and even how some of these movements allow the entire cell tomigrate, their colleague Melanie Ohi is focused on what happens when these proteins run into their confining barrier: the plasma membrane.

“How do cells deal with the barrier of a membrane? Because it is an actual barrier, and the cell needs to haveways to alter or overcome it,” says Ohi, a faculty member at the LSI and a professor of cell and developmental biology at the Medical School. “They need to form channels that allow things to pass into and out of the cell, but they also need to be able to rearrange the architecture of that membrane.”

Recently, her lab revealed the 3D structure of a key engineer involved in that architectural rearrangement: the caveolin-1 protein (or Cav1 for short).

The cell membrane is not a rigid wall just holding in the contents of the cell. This dynamic surface constantly remodels to support normal cell functions, growth and even cellular migration. One of the major ways cells alter this membrane is through caveolae (“little caves”) that fold the membrane in on itself. These tiny indentations offer one entryway into the cell, but they can also be released when the cell surface is under tension, allowing the membrane to expand without snapping apart.

The Cav1 protein attaches to the inside of the cell membrane, laying the foundation for the caves. The protein was discovered more than 30 years ago, and research has shown that mutations in this critical cave-builder lead to cardiovascular and muscular disorders in humans. But without a clear picture of what the protein looks like, scientists have not been able to precisely map how it works, or where its disease-causing vulnerabilities lie.

“People are really interested in this protein because it’s one of the major ways that cells are regulating signaling at the plasma membrane, and there are many mutations that create disease,” says Ohi, whose lab has been studying Cav1 since 2015. “But it’s just a really challenging protein to work with.”

Earlier this year, Ohi and her colleagues had a major breakthrough, with the help of some Escherichia coli. Caveolin proteins are naturally found only in multi-celled animals (including humans). But the Ohi lab used genetically modified E. coli bacteria as Cav1 factories.

With a healthy supply of purified proteins from E. coli, the researchers turned to cryo-EM to uncover the 3D structure of the full protein. The recently published structure differs markedly from some of the prevailing models and theories about Cav1’s shape, and thus its mode of function and malfunction, Ohi says.

“It is kind of a classic example of why we do structure analysis,” she explains. “Now that we have a full structure, we can begin to make informed mutations to study the functions in disease. It just opens a whole new avenue for understanding how this protein works.”

The Ohi lab employs bacteria as more than just protein manufacturers. The lab also studies how these organisms build their own machines to transport infectious agents across the cell membrane into their hosts.

One of these bacterial species, Helicobacter pylori, is a leading cause of stomach ulcers and the strongest known risk factor for stomach cancer. It spreads infection by forming protein machines that inject a harmful protein directly into the host’s gastric cells.

“And once it gets inside, this protein messes up all the transportation systems in the cell,” Ohi says.

Her lab is investigating the structures of these machines across several bacterial species, in hopes of identifying weaknesses that could be exploited to halt the delivery of infectious material.

“Whether you’re looking across cells or within the cell itself, so much depends on these complicated, overlapping transport systems,” Ohi says. “The more we can understand about the fundamental biology that drives cellular transport, the better we can predict and capitalize on its roles in health and disease.”