Looking between cells

Before arriving at Janelia three years ago, Postdoctoral Scientist Antonio Fiore was designing and building optical instruments like microscopes and spectrometers.

Fiore, a physicist by training, came to the Pedram Lab to try something new.

“I focused on the physics rather than investing in the biological applications of the optics I was developing,” Fiore says. “I came to the Pedram Lab in search of a different kind of impact, joining a team that explores areas of biology that need new tools, while keeping a connection to light microscopy”.

So far, Fiore’s new direction is paying off.

Fiore and Janelia Group Leader Kayvon Pedram, along with a team of researchers, have developed Rhobo6, a light microscopy probe that gives scientists an unprecedented look at the extracellular matrix—the collection of organized molecular structures that fills the spaces between cells in our bodies.  

The extracellular matrix supports and gives structure to our cells and tissues: It provides a scaffold for cells to grow in, dictates the mechanical properties of tissues, and supplies pathways for cells to travel.

“If our bodies are a community of cells, we can think of the extracellular matrix as the infrastructure that the community builds,” Pedram says. “So, trying to understand tissue biology without considering the extracellular matrix is like trying to understand a city while ignoring all the buildings, roads, and trains. You’ll miss a lot.”

Looking between cells

Despite its importance, the extracellular matrix hasn’t been investigated as deeply as its intracellular counterparts. That’s because it is difficult to peer between cells and characterize extracellular structures without perturbing them.

To overcome these barriers, the team set out to develop an easier way for researchers to use light microscopy to study these extracellular structures.

They designed a probe, Rhobo6, that does not permeate cells but stays in the surrounding area. Within those extracellular spaces, the molecule reversibly binds to glycans, one of the most abundant biomolecules of the extracellular matrix. Upon binding, Rhobo6 increases its fluorescence. As a result of this reversible, fluorogenic binding, researchers can visualize extracellular matrix structure in live tissues and animals without interfering with native biological processes.

Rhobo6 could also be useful in studying diseases linked to changes in the extracellular matrix and in diagnostic imaging, according to the researchers. The team collaborated with Valerie Weaver’s lab at the University of California San Francisco to use Rhobo6 in surgical imaging of breast tumors in live animals, finding stark differences between the matrix surrounding primary tumors and nearby healthy tissue.   

A collaborative effort

The team says Janelia has been an ideal environment for developing and testing Rhobo6, which required a multidisciplinary effort.

For example, Group Leader Shaohe Wang was involved in the project from the beginning and was instrumental in setting up experiments to test the probe in the mouse salivary glands that his lab studies. Pratik Kumar, a Postdoctoral Scientist in the Lavis Lab, helped test the chemical stability of Rhobo6 over time. Members of the Rubin, Shroff, and Ahrens labs contributed to testing of Rhobo6 in fruit flies, roundworms, and zebrafish, showing that Rhobo6 is compatible with various commonly used model organisms.

These collaborations not only benefitted the project, but were also helpful to Pedram and Fiore, who were both relatively new to Janelia.

“Tony and I have many more friends and collaborators on campus than we would otherwise have had if it weren’t for this project,” Pedram says.

Guoqiang Yu, a Postdoctoral Scientist in the Pedram Lab and a co-author on the new study, has also scaled up the synthesis of Rhobo6, enabling the team to share the probe widely with the scientific community.

“I’m looking forward to seeing the gorgeous images of extracellular matrix that researchers are going to capture,” Fiore says. “And I’m even more excited about the new questions we and others will be able to answer with this tool.”