Although the protein tau, which is associated with Alzheimer’s and other neurodegenerative diseases, has been heavily studied for decades, its role in maintaining cell function is poorly understood.

Normally, tau binds to tube-like structures called microtubules that support cells to help stabilize them. But in diseases like Alzheimer’s, tau dissociates or no longer associates properly with the microtubules, causing them to destabilize and fall apart, losing their ability to maintain cell shape and function. The protein accumulates in the cytoplasm as fibrillar aggregates which are associated with cell death. 

Now, a team of researchers at the University of Pennsylvania is using a single molecule technique to better understand tau and how it interacts with the protein tubulin, which chains together, or polymerizes, to form the microtubules. The technique incorporates fluorophores, or molecules which emit photons when excited by visible light, to figure out properties of tau that would otherwise have gone unseen.

Their paper, published in the Proceedings of the National Academy of Sciences, sheds new light on tau’s interactions with tubulin and how it helps stabilize the microtubules that keep cells in shape.

According to the results, tau binds to multiple tubulin subunits, increasing the local tubulin concentration, which then facilitates polymerization into microtubules. The findings suggest that this interaction may be important to controlling microtubule dynamics.

“Tau’s interaction with soluble tubulin has really been overlooked,” said Elizabeth Rhoades, an associate professor of chemistry at Penn who contributed to the findings. “Understanding how tau binds to and interacts with tubulin is important not only for understanding its native role and how it actually stabilizes microtubules but may turn out to be really critical for understanding what happens in disease when you start to destabilize this interaction and the microtubules begin to fall apart.”

The research was started while Rhoades was at Yale University, though the students initially working on the project have since moved on to other things. Rhoades continued the research when she came to the Department of Chemistry in the School of Arts & Sciences at Penn, where she collaborated with postdoc Ana Melo, who was the main researcher on the project, and other students.

According to Rhoades, tau belongs to a class called intrinsically disordered proteins. Unlike most proteins, it doesn’t form a stable, compact structure and so the way it interacts with binding partners is different from what might be expected.

To probe tau, the researchers used a technique called single molecule Förster resonance energy transfer, or FRET, which allows them to measure distances within a molecule.

First, the researchers attached two fluorophores to tau. Then they excited the first fluorophore, the donor, so that it would emit photons or transfer energy to the second fluorophore, the acceptor. This would cause the acceptor to emit photons in a distance dependent matter.

“If the two probes are close together, the interaction is very strong and you get very efficient transfer of energy,” Rhoades said. “When they’re further apart, the interaction is much weaker and therefore you get a less efficient transfer of energy. So you can quantify the number of photons you get from each fluorophore and use that as a means of calculating the distance between the two fluorophores.”

Unlike other techniques, which use high concentrations of tau, single molecule FRET allows the researchers to investigate the first step before polymerization.

“This technique allows us to explore interactions we’d never be able to see using high concentrations,” said Melo.

The researchers found that tau forms something called a fuzzy complex, meaning it maintains its disordered extended state and combines to multiple subunits of tubulin through short stretches of amino acids within the protein.

“In this case, disorder matters for the function of tau,” Melo said. “The most interesting thing we’ve seen is that the individual repeats within the microtubule binding region that directly interface with tubulin expand to accommodate tubulin binding, despite a lack of extension in the overall dimensions of this region. These results suggest that tau retains its intrinsically disordered features that provide enough flexibility to bind multiple tubulin dimers.”

Melo explained that this research is important because, while there has been significant focus on tau aggregation, there are still many open questions about its biological function. It has been proposed that neurodegenerative disease arises both from aggregation and the loss of this function.

“If you better understand the biological function of tau, you can have a big picture of how everything works,” said Melo. “You can start to study how mutations affect interaction with tubulin and all of the mechanisms of polymerization. Once you understand all these steps you can better describe the role of tau in these pathologies.”

Rhoades said that the next step in the research is to collaborate with Penn’s Perelman School of Medicine to use a technique called hydrogen-deuterium exchange mass spectrometry. This will allow them to get a more detailed picture of which regions of tau bind to tubulin and whether there are any differences between the tubulin and microtubule bound forms of the protein. The latter may provide insight into how tau regulates its interactions with the two binding partners.

Another long-term project that Melo is working on is looking for these interactions between tau and tubulin in cells.

“We’ve shown this interaction in vitro, and now we need proof in vivo,” Melo said. “This would open a whole new area of research.”