Dr. Sanford Simon is a researcher at The Rockefeller University in New York City. Some of his lab's current lines of research include the cell biology of tumorigenesis, the assembly of membrane proteins, and mechanisms of endo- and exocytosis. Recently, Simon teamed up with fellow researchers Nolwenn Jouvenet and Paul Bieniasz to study how viruses bud from cells. They chose to focus on HIV's assembly mechanism. Thanks to the novel application of total internal reflection, they became the first scientists to see the virus forming and budding in real time. Their findings are published in the June 2008 issue of Nature. Scientist Live spoke with Dr. Simon about his research.
1. How did you come to work on this project and what prompted you to turn to a total internal reflection microscope?
Very often in science you have a global idea of where you want to go with a particular area of research and you start exploring different paths. Along the way, you find things that you realise haven't been addressed before or, if they have been addressed before, you can address them in ways that provide new insights. In other words, you go off an a tangent. In this particular case, going back to the 1970's, I had been studying how cells can move molecules across their membranes, a barrier that keeps the outside out and inside in, something essential to the viability of the cell. Anything that might compromise the membrane's integrity also compromises the cell's integrity.
My lab started looking at everything from how cells get proteins across membranes to what happens when cells have cargo to secrete (for example when a nerve secretes a neurotransmitter). We realised that we were very limited in our ability to see these events so what we did was borrow a technique from physics that allowed us to look at them on a very thin plane like a cell's outer membrane.
2. Had you studied HIV before? If not, why now?
I had attended a series of meetings where people were debating where HIV assembles and how it gets out of cells. There were two very different models. One model said that the molecules that form the virus particle assembled one at a time at the cell's surface, the other said that they assembled inside the cell at various compartments and then travelled to the membrane, where they were released as one large bolus. It occurred to me that we were looking at similar questions - how do molecules get out of cells. Moreover, I had studied the compartment in which some argued HIV assembled and I knew how to watch it being released from the cell. I had no experience with HIV and I was extremely fortunate to hook up with Paul Bieniasz and Nolwenn Jouvenet: Two virologists here at Rockefeller who were independently interested in the very same questions. Our strengths complemented each other beautifully in this project.
3. What were the key challenges in the study?
The problem was no one ever imaged a single HIV virus assembling before. Not only that but nobody had ever imaged a single virus of any sort assembling before, so, when we were watching these little spots appear, the biggest challenge was to figure out if we were watching virus particles assembling and not some artifact.
4. How does total internal reflection microscopy work and what are the size parameters of what it can see?
It is easier to follow a molecule if you make if fluorescent. However, the wavelength of light, about half a micron, limits our ability to resolve different molecules. We wanted to see molecules coming together in a space that was one tenth of that size. A total internal reflection microscope looks like a regular microscope, but it plays with an optical trick to allow you to selectively excite fluorophores in a plane that is only as thick as one-tenth the wavelength of light. When you shine a beam of light on a surface at a steep angle, most of the light reflects back. When I learned about this in school, they used to say everything is completely reflected internally. That is because no energy is propagated on the other side of the of the interface. However, you actually have a small field of light on the opposite side of the surface. This is a standing field: It does not propagate way. However, it is able, over a very short distance, to excite fluorophores We had been using this kind of microscope for a long time but not for this purpose.
5. What did this technique reveal about HIV?
Amongst other things, we did not know how long the process would take. It turns out the assembly time is in the order of six or seven minutes. We also discovered that we can separate out different stages in the assembly of HIV. We found that we could separate a step when all the basic proteins that form virion are initially made. At first they look like a diffuse haze in the cell. It is at this phase that the first components get recruited to the membrane that surrounds the cell, lining the inside surface of the membrane that surrounds the cell. Then, as they contact the membrane, they start to assemble, increasing in brightness appearing like stars in the sky. Next we could resolve a phase where the nascent virion is no longer exchanging units for the cell. It has reached its final form. Beyond that, there is an additional step when all of a sudden, the virion completely detaches from the membrane to the point that molecules even as small as a proton can not move between the cell and the virus.
6. What are the technique's possibilities in the future?
We can go in two different directions. We can now identify when each separate component of HIV assembles and come up with a complete description of the birth of the virus. The other direction is to apply this technique to other viruses and see what we can learn about them.
7. What is next for the Simon Lab?
All of the above. Primarily, we are trying to look at single events in biology There's often a real advantage to looking at individuals because there are things you can learn from individuals that you can't learn from looking at a whole population.