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Structural biologist Naoko Mizuno likes decision making. She studies it — down to its molecular underpinnings. How do proteins on cell surfaces connect cells to their surroundings and help the cell react? Why do axons, which act as a steady backbone, decide to branch? How do small moments culminate into things we notice?

“I find it very interesting to connect what we are seeing in a neuron or on a molecular level to how people behave or move,” Mizuno said.

A headshot of a woman
Photo courtesy of Naoko Mizuno.

Head of the Laboratory of Structural Cell Biology at the National Heart, Lung and Blood Institute, she’s also co-organizing the third Molecular Neurobiology Workshop, a conference to build connections — not only between molecules and cells — but also between researchers.

When scientists with different backgrounds and perspectives come together, “then you see, ‘Ah, that’s how it connects,’” she said. “You suddenly get a different level of understanding.”

Mizuno started her research career at the University of Tokyo’s Komaba Campus, nestled in a stylish town south of the city and famous for its cherry blossoms. There, she studied microtubules, which determine cell shape, organize cellular components and establish routes for transport proteins.

After finishing her Ph.D. at the University of Texas Southwestern Medical Center, she completed a postdoctoral fellowship at the National Institute of Arthritis and Musculoskeletal and Skin Diseases. In 2012, Mizuno moved to the Max Planck Institute of Biochemistry in Germany, where she founded her lab.

Eventually, her team landed on a finding that stirred new questions in her work: a mechanism that caused axons to split into new neural pathways.

For the first time, the researchers identified and visualized how a gregarious protein, SSNA1, accumulated at forks along axons and caused the microtubules defining an axon’s shape to branch. The findings offered a key insight into how microtubules help guide brain development and form the intricate networks that define human behavior.

While microtubules are key players in many cellular processes, such as mitosis and polarization, they play another essential role in neuronal cells. Bound together like parallel fibers in a rope, microtubules stabilize axons and help connect their branches to neighboring cells.

Though researchers knew that certain signaling events triggered axon branching, they had yet to explain the mechanics of how one axon splits into two. The search began when Mizuno and her team first observed SSNA1 accumulating at axon branch sites in primary neurons, suggesting that it might be involved in the remodeling of cytoskeleton microtubules into new branches.

A figure showing neurons and their branch sites
Mizuno found that SSNA1, a protein now known to be indispensable to neuronal development, accumulated at axon branch sites, highlighted in the boxes above with SSNA1 shown in red. Image: “Direct induction of microtubule branching by microtubule nucleation factor SSNA1” by Basnet, N., Nedozralova, H., Crevenna, A.H. et al.

To test their hypothesis, researchers combined multi-spectrum analyses, including cryo-electron microscopy (cryo-EM) and super-resolution imaging using DNA-PAINT. While DNA-PAINT showed the influence of SSNA1 on the microtubule network, cryo-EM enabled scientists to visualize SSNA1-induced microtubule branches on a molecular level.

Unexpectedly, the structural analysis revealed that SSNA1 played a direct role in microtubule branching, finally unraveling the molecular mechanism behind the team’s initial observations. Going back to neurons, they demonstrated its impact on axon branching and neuronal development.

The findings set Mizuno on a quest for other, higher-level guidance factors, a natural derivation of her work’s organizing principle: to explain, with precision, the driving forces behind biology.

Recently, her team used cryo-electron tomography to visualize how organelles and cytoskeletal structures remodel and reorganize themselves around newly formed axon branches in mouse neurons.

Remarkably, they found that axon branches also function as cellular synthesis hubs to support branching and outgrowth activities far away from the cell body, where a neuron’s nucleus is. It’s the first roadmap of protein influence at axon junctions and, eventually, on the formation of neural networks.

An image of branching microtubules
Mizuno’s team showed that, rather than grafting daughter microtubules onto existing ones, SSNA1 prompted existing ones to sprout entirely new branches. The image above, taken using cryo-electron microscopy, shows branched microtubules. Image: “Direct induction of microtubule branching by microtubule nucleation factor SSNA1” by Basnet, N., Nedozralova, H., Crevenna, A.H. et al.

“Now is the time that our mind’s limitations can be released by using combinations of different technologies, different expertise, with different disciplines,” Mizuno said.

At the Neurobiology Workshop, hosted by the European Molecular Biology Organization and co-sponsored by the Institute for Protein Innovation, top scientists from around the world will gather to share insights on protein function in biological systems. It’s an opportunity to openly discuss nascent ideas against the backdrop of a beachside resort on the Greek island of Crete.

In its third installment, the workshop uniquely attracts neurobiologists, structural biologists and biophysicists, cultivating warm and productive relationships. Mizuno attended the workshop’s inaugural 2018 meeting, also held in Crete. She was drawn in by the workshop’s primary organizer, Elena Seiradake, a professor at the University of Oxford.

“I felt immediately comfortable,” Mizuno said. “I think people have an active effort to be inclusive. And then that propagates to the sort of spine of the conference.”

For some, the inclusivity spawns long-term collaborations. After attending the conference in 2018, Mizuno partnered with Osaka University professor Junichi Takagi, whom she met there, to study the structure and function of a key cell surface receptor, integrin alpha-5 beta-1 (α5β1).

Leveraging Mizuno’s interest in cell shaping mechanisms and Takagi’s expertise in cell surface signaling, the pair uncovered the protein’s structure and published their findings in Science Advances. They found a new mechanism by which fibronectin, a glycoprotein and ligand of α5β1 found in the extracellular matrix, activates α5β1, improving the scientific understanding of integrins and their impact on disease.

That collaborative spirit is in tune with the path Mizuno said her career has taken. Starting with small questions, she turned her attention over time to larger ones that required multiple perspectives to answer.

“We have a certain level of depth that we like to reach,” she said. “I think that’s a very fulfilling experience to have the same expectation, but different targets that could mingle and synergize together.”


Writer: Halle Marchese, halle.marchese@proteininnovation.org
Source: Naoko Mizuno, naoko.mizuno@nih.gov


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