Scientists observe key subcellular process through an IVM microscope
“There is nothing new under the sun,” wrote the 19th
century satirist Ambrose Bierce, repeating the famous lament from the Old Testament Book of Ecclesiastes. He then winked to his readers, “But there are a lot of old things that we don’t know.” Bierce raises a valid point: How does one discover the old things under the sun that exist on the nanoscale and about which our understanding remains rudimentary? This challenge resides at the heart of biomedical research. For some, the way forward is to grow cells in culture, study them under carefully controlled conditions, and plug their data into the time-tested scientific paradigms. For others, the solution is to push the technological envelope, and let the biology fall where it may.
Dr. Roberto Weigert, chief of NIDCR’s Intracellular Membrane Trafficking Unit, has chosen to take the latter route. His envelope-pushing technology is called intravital microscopy, or IVM, an umbrella term for microscopic techniques that directly track the biochemical activity within the tissues and cells of living animals. Weigert and his colleagues have constructed their own IVM microscope that, like that old General Electric jingle, continues to “bring good things to life” in mouse studies. The most recent good thing is an extremely high-resolution view of a critical biological process of mice (and men) called regulated exocytosis. Never heard of it? Regulated exocytosis generates the neurotransmitters that just allowed you to read this sentence, the insulin helping your cells metabolize your last meal, and the amylases in your saliva that will help you break down your next meal. But, as Weigert and colleagues recently discovered, there’s more to regulated exocytosis than meets a Western blot. They used their microscope in live rodents to capture a somewhat different picture of the process in acinar cells of the submandibular salivary glands. Weigert discusses this different picture, its implications for understanding regulated exocytosis, and the value of high-powered imaging strategies in biology. The group’s paper is found in the August 16 issue of the Proceedings of the National Academy of Sciences.
Let’s start with the obvious first question: What is regulated exocytosis?
The prefix “exo” means out of, and “cyto” refers to the cell. So, it is the regulated secretion of molecules out of the cell. Regulated exocytosis is a common occurrence in neurons, endocrine, and exocrine cells. The latter being the specialized cells that secrete enzymes, electrolytes, and other biological factors on an as-needed basis to help the body respond to the world around it. Secretory cells are found throughout the body, including acinar cells in the salivary glands, which serve as the investigative model and basis of the current paper.
How does the regulation work?
It is a multi-step process that starts in an organelle called the Golgi apparatus, a sort of specialized post office in the cytoplasm that packages proteins after their synthesis in the ribosomes. In the case of certain specialized molecules — hormones, digestive enzymes, neurotransmitters — they are bundled into protein granules. Think of the granules as the cellular equivalent of bubble wrap. These bubble-like granules proceed to the plasma membrane with their cargo and park themselves there for a while. When the appropriate signal comes, the granules fuse to the plasma membrane and ultimately release their molecular contents into the extracellular space to carry out their specialized functions.
One of the points made in the introduction of your paper is worth repeating here. Science has assembled over the years a broad conceptual framework to define regulated exocytosis. But within this framework, multiple, often contradictory, models can exist that detail exactly how the process might work.
That’s right. The point is most of these models are based on work in cell culture and other inherently artificial experimental environments. You are a writer. If I wanted to learn how you write, I’d watch you click away at the keyboard in your office. I wouldn’t want to parachute you onto a desert island with a small bag of provisions and say, “Okay, now write.”
I’d be stuck in survival, not writing, mode.
Exactly. Until recently, technological necessity — and thus tradition — dictated that we parachute cells into the best-available experimental systems. These systems have been invaluable over the years to get us into the biological ballpark, so to speak. But now we are bumping up against their inherent limitations as artificial environments. We need to turn our investigative lens in a new direction to work out the specifics. We need to catch cells sitting at the keyboard in real time and observe them as they enter in the code for regulated exocytosis.
Acini in the mouse submandibular salivary gland.
And that new direction is intravital microscopy, or IVM?
Yes, this is the direction that we have taken. This permits us to watch in real time our biological process of interest in a more physiological context. It also means we don’t have to worry about creating a meaningful experimental system. The system already is there and physiologically relevant. I really do hope that other scientists who wonder how their work in cell culture might translate into physiologic terms will give IVM a try. We have a series of collaborations with researchers who are interested in utilizing this technique. Usually they spend a few weeks in my lab to get training on IVM in order to answer their specific question.
I was surprised to learn that IVM, conceptually at least, isn’t new. It dates back to the 19th century, and the high-resolution observations of German pathologist Julius Cohnheim. He observed the movement of immune cells in frogs.
You’re absolutely right. Two things have changed over the last few decades. One, many of the recent technical advances in visualizing tissues and molecules, for example, green fluorescent probes, have been folded into IVM studies. That has catalyzed a technological bonanza: High specificity in tagging molecules, extremely high-resolution visualization, and studies in intact, living systems. Two, strategies have been developed to minimize motion artifacts.
What do you mean?
Animals breathe, their hearts beat, and their appendages twitch. The combined effect under very high magnification is like watching a 6.0 earthquake. Everything shakes and blurs out of focus. We have developed approaches that better stabilize the organ of interest and thereby minimize the motion artifacts. At that point, it was just a matter of generating more powerful optics.
In fact, your group is the first to crack the subcellular barrier. Would you explain that?
Sure. IVM has been used for many years to image structures all the way down to a single cell. But once we solved the movement issue, as I mentioned, we maximized the optics and looked for the first time inside the cell. Or, as you said, cracked the subcellular barrier. We now can track the trafficking of molecules within the cell essentially in real time. That’s the basis for the current paper. We used extremely high-resolution confocal IVM to image the submandibular salivary glands of live rodents.
Well, let’s go to the paper and its three novel findings. The first involves the control switch that initiates regulated exocytosis. What was the accepted textbook model?
The model was still a work in progress. The scientific literature presented two conflicting scenarios. In the first scenario, the release of secretory proteins is controlled primarily by what’s called the β-adrenergic receptor. It’s a protein that, like many receptors, has a kinked, telephone cord-like structure with domains inside of the cell, within the plasma membrane, and on the cell surface. The domain on the cell surface receives the appropriate extracellular signal, stimulating the vesicles to fuse to the plasma membrane and proceed with exocytosis. The second scenario stipulates that the β-adrenergic receptor has a partner. It’s called the muscarinic receptor, which plays a key role in stimulating the salivary gland to secrete water. The two receptors must intertwine on the cell surface and generate synergistically a signal to proceed with exocytosis.
What did you see?
We saw directly in the animal that the sole control switch is the β-adrenergic receptor. Why the contradiction? The answer may be that when you explant the glands or culture the cells under artificial conditions, the second receptor kicks in and functions there in a way that you don’t see in the animal. And, again, we tried to stimulate the muscarinic receptor.
The second discovery has to do with how the vesicles fuse with the plasma membrane. What was the standard textbook model?
The model laid out a process in which the granules first fuse with the membrane. Thereafter, the granules fuse together like links in a chain and are excreted in unison. Compound exocytosis it is called. The process has been described extensively ex vivo [out of the body] and in cell culture systems.
But you didn’t see that in the live animal?
No, it doesn’t occur at all. We looked for signs of compound exocytosis. We wanted to reproduce it. But we soon realized that it doesn’t occur. Look at this short clip. Focus on the granules in black. You’ll notice a white aura as they fuse individually with the plasma membrane. The arrows point them out:
Here’s the process slowed down in still frames:
We actually have an explanation for why this isn’t seen in cell culture and other experimental systems.
Every time that you explant a gland and try to isolate the cells, you introduce a mechanical or enzymatic disruption. You affect the cell’s cytoskeleton. And it’s the cytoskeleton that avoids and prevents these compound events. In fact, we found interesting steps that have been proposed previously — but never proven — on how the granule, once it gets out of the plasma membrane, collapses and release its contents.
And that takes us to the third novel discovery.
Exactly. After the granule fuses, there is an instant recruitment of actin, the cytoskeleton protein, and myosin, which is a mortar that basically forms a contracting scaffold. The contraction basically pushes the granule in a way that we don’t understand in biophysical terms. It can be just pulling the membrane or enlarging the opening on the other side. It is something that we are working on. Here’s another short clip to help you visualize the process. It shows actin coating the granule in a thickened white aura. Then you’ll see the granules collapse and disappear. Take a look:
So it is a very efficient process. It’s the difference between driving a Mercedes and a freight train?
Right, and this kind of phenomenon could not be seen in the ex vivo systems — and for a very good reason. The gland, per se, is an extremely complex structure. It has various cells and ducts that constantly secrete protein and fluid. All of this activity creates a dynamic pressure within the gland that, in turn, affects the dynamics of the membranes. The actin that surrounds the gland is important to prevent this pressure from disrupting the granules, their fusion to the plasma membrane, and the timing of their secretion. In the explanted glands, you interrupt the ducts, you don’t get pressure. So, you won’t see the phenomenon.
You’re using IVM in basic research studies. What about its potential applications diagnostically?
We are developing a microscope for diagnostics. So are several other research groups around the country and worldwide. Aside from the optics, there are practical advantages with IVM. For example, it’s possible to image the architecture of an organ at the cellular level without labeling anything with fluorescent tags. That eliminates the need for contrasting agents, which can have some toxicity, need to be metabolized, and represent a time-consuming step at the front end of the imaging process.
How would IVM be employed diagnostically in the oral cavity?
There are two major potential applications. One is imaging the salivary glands for signs of Sjögren’s syndrome. The other is to examine suspicious oral lesions for early molecular signs of a developing cancer. Our thinking is the oral cavity, with its diversity of hard and soft tissues, is an excellent place to pilot test the technology for diagnostic use. That’s what we’re doing. But we’re also interested in applying IVM to image the gastrointestinal tract, bladder, lungs, and eyes, to name a few tissues.
Thanks for talking about IVM.