How Our Genes Reassemble Themselves to Get Work Done

Joan Pulupa sometimes thinks of her work as akin to watching a soccer match. Photos from the match can only reveal a tiny fraction of what has actually taken place on the field. To truly understand what happened — how the players moved up and down the pitch, how they worked together to score a goal — a video replay is far superior.

Inside cells, genes and proteins behave like those football players: gathering, dispersing, then gathering again in new formations. As these shifts occur, our cells are simultaneously performing the various tasks needed to sustain life. In her work, Pulupa studies live cells under the microscope to uncover the relationship between this gene assembly and cellular work.

During her doctoral research at Rockefeller University under the mentorship of Sanford M. Simon, Pulupa used these techniques to better understand the workings of the nuclear pore, a complex that regulates what enters and exits a cell’s nucleus. As part of her current postdoctoral research at Columbia University’s Zuckerman Institute, Pulupa is working in Stavros Lomvardas’ lab to employ similar techniques aimed at detangling the relationship between gene activity and our sense of smell.

Pulupa is now in her second year as a Junior Fellow with the Simons Society of Fellows. In 2015, while at Rockefeller University, she received the Gilliam Fellowship for Advanced Studies from the Howard Hughes Medical Institute. We recently discussed her work; our conversation has been edited for clarity.

What technologies do you use in your research?

One of the main tools that I use is live cell fluorescence microscopy, an imaging technology that allows us to see and analyze the actions of moving tissues under the microscope. By attaching fluorescent ‘tags’ to our protein of interest or adding differently colored tags to distinguish the activity of a DNA sequence from, say, that of a protein, we can track our desired protein(s) or molecules as they move around and do work inside the cell.

Fluorescence microscopy of living samples was developed in the early 1990s, made possible by the discovery that fluorescent proteins could be expressed in various cell types and organisms, thereby using them as fluorescent reporters or tags to label proteins. For me, this technique has been absolutely critical toward my goal of observing cellular activity in as close to a native environment as possible.

What inspired your interest in this area of research?

I like to use the analogy of a soccer match. A game is 90 minutes long, and maybe only three goals are scored the entire time. If you take photos of the match at random moments, they may not tell you much. You’d need to watch videos of what happens — especially at key times, such as when the players moved with the ball toward the goalkeeper — to see the actions and reactions in that crucial part of the match.

Our genes are like those players; they are active participants in how the body’s cells — from heart cells to immune cells to neurons — provide nutrients, information and energy to our tissues. The only way to truly understand these processes is to watch the action of our genes, proteins and cells in real time, and thankfully, today’s technology enables me to do just that.

How did you deploy live cell imaging during your doctorate at Rockefeller University?

At Rockefeller, I studied the nuclear pore complex. It acts as a gatekeeper to prevent unwanted molecules from getting inside the nucleus, while allowing so-called nuclear ‘cargo’ to enter unhindered. A group of proteins on the nuclear pore’s surface regulate this activity, but we didn’t really have a way to visualize how they do so.

As part of my doctoral research, we developed specialized microscopy to solve this problem, which is still, to my knowledge, the only such microscope in existence. The technology, called polarized-total internal reflection fluorescence microscopy, or pol-TIRFM, uses polarized light to interact with proteins at the exact moment they are regulating nuclear cargo, enabling us to visualize the dynamics of these proteins. When we deliberately altered the activity of these proteins, their behavior subsequently changed. This was the first direct visual evidence from living cells that the nuclear pore changes shape relative to conditions inside the cell. We published this work in eLife in 2020.

How has your focus changed since arriving at Columbia University?

If you think of my doctorate as being focused on the nucleus’ ‘gatekeeper,’ my postdoctoral research is looking at how genes operate within the nucleus. Specifically, I’m studying olfactory neurons, which govern a mouse’s sense of smell. These neurons are very unusual; although they each contain the genetic material to make 2,000 different receptors, mature neurons each make one and only one type of receptor. To distinguish between odors, different neurons must ‘choose’ to make different receptors.

How does all but one receptor gene know to turn itself off? How do genes and the proteins they encode work to make this happen? These are the questions I’m most excited to answer, and I’ve developed both visualization and tagging infrastructure to support my work. Preliminary results have shown that there’s an abundance of protein activity around the active odor receptor gene, more than would be expected normally. This gives us a clue that the active olfactory receptor gene is unique, and its environment allows it to be expressed when all other olfactory receptor genes are repressed, but we’re still not sure how. This is what I’m now working to uncover.

One good thing about working with mice is that their sense of smell is very similar to ours. This means that we can be confident that the work we are doing in mice has some relevance to our experience as humans.

Finally, what are your thoughts about the Simons Junior Fellowship?

It’s helped me create a network of different scientists around the city. I’ve adopted Carol Mason, a neuroscientist at Columbia and Senior Fellow with the Simons Society of Fellows, as a mentor. She has been very helpful to me, especially as I navigate the relatively new-to-me world of neuroscience after my time in cell imaging and cell biology. With that support, I see how my work fits within the larger field of neuroscience, and that’s been wonderful.