The molecular workings of a eukaryotic cell are so complex and sophisticated, you might think the cell had a mind of its own. “It’s amazing! How does the cell do all of this?” says J. Christopher Fromme, Molecular Biology and Genetics.
The cells, of course, don’t have minds. They make all of their decisions through the interactions of molecules. “Just using the structure of molecules, cells can make all of these decisions, often very simple, binary decisions that build up to more complicated phenomena,” Fromme says. “But it’s the structure of molecules that provides the fundamental basis for life.”
Understanding how the molecular structures of proteins enact decisions in the cell is at the heart of Fromme’s research. The particular decisions he studies are those made at the Golgi complex, often referred to as the Grand Central Station of eukaryotic cells. It’s here where nearly a third of the cell’s proteins are sorted—and therefore where abundant decisions are made. When a newly made protein arrives at the Golgi, for example, the complex needs to determine where it needs to go next and how. This involves a dizzying number of steps and regulatory checks.
“It seems super complicated—how is it possible that cells keep all of it straight? That’s fundamentally what we’re trying to figure out,” Fromme says. “We want to know how the cell knows to turn on and off these pathways.”
To Flip a Switch at the Golgi
Here’s an example of how the process works in broad strokes: when the cell needs to secrete a protein, the protein is first made at the endoplasmic reticulum (ER) and transferred in a vesicle to the Golgi, an organelle that, in textbook drawings, resembles stacked folds of ribbon. The vesicle carrying the protein fuses with the membrane of the Golgi, and the protein cargo passes through the folds.
On the other side, at the trans-Golgi, a lot of sorting happens. Specific vesicles are made depending on the destination of the proteins, and the protein is then transported in its vesicle to where it’s needed. These pathways all happen in reverse at the Golgi as well, when the cell takes proteins from its surface into its interior.
Most of these pathways are turned on and off by what Fromme refers to as molecular switches—a broad class of proteins called GTPases. If the GTPases are like light switches that make the traffic stop and go, the proteins that flip them on are called GEFs (guanine nucleotide exchange factors).
“The light switches, the GTPases, are interesting, but what’s more interesting to my lab is that there’s this other molecule deciding to turn that switch on,” Fromme says.
Since Fromme began his lab at Cornell nine years ago, he’s been pursuing questions about GEFs: How do they get to the right place? How do they know it’s okay to turn the switches on? Using yeast as a model, Fromme and his team have employed in-depth approaches to find surprising answers.
Crosstalk at the Golgi
One challenge to studying the GEFs biochemically is that the proteins are large and difficult to isolate in a test tube. But Fromme and his team have been able to isolate and purify several GEFs for the first time. “That’s part of where we’ve found our niche; we’re basically the first people to really do in-depth mechanistic studies on these proteins using biochemistry,” Fromme says.
Another difference in their approach is that they create synthetic membranes that resemble the Golgi’s membrane. This allows them to more accurately study the most crucial interactions, which often happen on the surface of the Golgi complex. “We reconstitute the reactions of the GEF proteins in a test tube, using the most physiologically accurate environment we can. Then we just start breaking things to see what happens,” Fromme says.
“It seems super complicated—how is it possible that cells keep all of it straight?”
When he was just beginning his lab, Fromme started using these methods on a GEF called Sec7, which functions at the last stage of the Golgi. There, it turns on the outgoing pathways that send proteins to the cell surface, endosomes, and lysosomes.
By chopping off pieces of Sec7, Fromme’s group determined the function of those pieces one by one. “What we found was that some of those domains inhibit Sec7 from doing its job inappropriately,” Fromme says. “So they bind to the active site and mask it, and when Sec7 is bound to the right membrane, it opens up. It’s a way to make sure the activity happens in the right place.” Called autoinhibition, this behavior is one answer to Fromme’s initial question of how the GEFs know when and where to flip their GTPase switches.
In later studies, Fromme’s lab found that some of the pieces of Sec7 are responsible for interacting with or talking to proteins involved in other GTPase pathways. “Once GTPase A is activated, it physically interacts with the GEF for GTPase B. So A says, hey, I’m activated, maybe you should be activated, too,” Fromme says. “Sec7 seems to be a signal integrator, a master regulator. It’s monitoring four different Golgi pathways and uses that information to decide to do its job, which is to activate this one pathway.”
Sec7 knows when to flip the GTPase switches because it’s listening to signals from other pathways. Fromme says one of the major contributions of his lab has been determining the extent to which this kind of communication, called cross talk, happens at the Golgi.
How the ARFs and Rabs Communicate
Since making breakthroughs with Sec7, Fromme’s lab has moved on to other GEFs, uncovering their roles and how they make their decisions. “Essentially the goal for my career is to figure out how all of the Golgi GEFs are regulated,” Fromme says.
With two grants from the National Institute of General Medical Sciences, Fromme is continuing to probe the GEFs for two classes of GTPases, the Arfs (one of which Sec7 activates) and the Rabs. They’ve answered longstanding questions about a key GEF that activates the Rab pathway and discovered the mechanisms by which the Rab and Arf pathways communicate with each other. They are also starting to provide more answers to the question of how the GEFs know where to go. One explanation is that they can sense slight alterations in the lipids found on different areas of the Golgi membrane.
The Challenge of Imaging the GEF Proteins
Another major effort in Fromme’s lab has been trying to image the atomic structure of the GEFs. “To really understand how these GEFs make decisions, we need to know their atomic structure,” Fromme says. “It’s clear the GEFs themselves have different states. When they’re flipping or not flipping the switch, they’re adopting different conformations. We need to see what those look like to design better experiments and to fully explain how they work.”
Efforts at imaging these proteins have proved challenging. X-ray crystallography hasn’t provided a full picture, as these large proteins have been very difficult to coax into crystals suitable for x-ray diffraction experiments.
A new technique—high-resolution, single-particle cryo-electron microscopy (cryo-EM)—however, is giving Fromme and his team, as well as his entire field, a new window to look through. “It’s hard to overstate how powerful this technology is,” Fromme says.
With support from a Guggenheim Fellowship, Fromme spent a sabbatical at the University of Cambridge and the Medical Research Council Laboratory of Molecular Biology in the United Kingdom to learn the new technique. On campus, his team will be able to use a newly acquired cryo-electron microscope at the Cornell Center for Materials Research. “All of our GEFs are awesome targets for cryo-EM,” he says. “We’ll essentially be taking this cellular logic all the way down to the atomic level to really understand the mechanisms.”
Fromme fell in love with this kind of work from the start. As an undergraduate at Cornell, he took a class from the professor who would become his lab adviser, Steven E. Ealick, Chemistry and Chemical Biology. “The class was all about using protein structures to explain their function. It sounds so simple, but that’s how everything works,” Fromme says. “Being exposed to that area of science was just transformative.”
Now, as faculty at Cornell, Fromme has come to appreciate the vast and varied expertise within the community—and also the common interests. “There are so many groups using different approaches to investigate the biology of membranes, and the structural biology community is great,” Fromme says. “I can’t imagine being as successful anywhere else.”