Nano imaging
Computer image show venus fly-trap movement of nanosensor molecule (Wolf Frommer)
DB: This is Earth and Sky. For the first time ever, scientists have measured the levels of important chemicals in brain cells, in real time, at the level of a single cell.
JB: The neurotransmitter glutamate is thought to be the major chemical that increases nerve cell activity in the human brain. Too much glutamate is thought to contribute to conditions such as Alzheimer’s and Parkinson’s disease. And the details of just how these chemicals move through the brain have been a bit fuzzy.
DB: We spoke with plant biologist Wolf Frommer of the Carnegie Institute. He headed a team that developed what are called nanosensors – in this case protein molecules from jellyfish that are genetically fused with different colored fluorescent tags at their ends. The sensor protein specifically binds to glutamate.
Wolf Frommer: And when they do this, they undergo a venus flytrap movement…and when it closes, it makes a movement.
JB: And that movement creates a change in the color of the fluorescent light that gives the scientist a picture of glutamate levels at the brain cell.
Wolf Frommer: And that’s a huge advance which is relevant for many fields, and directly the glutamate sensors are beautiful tools to study phenomena of glutamate toxicity in the brain.
DB: Specifically relating to Alzheimer’s and Parkinson’s disease. That’s our show. We have more about nanosensors at earthsky.org. With thanks to the National Science Foundation, we’re Block and Byrd for Earth and Sky.
Wolf Frommer is a plant biologist with the Carnegie Institution at located on the campus of Stanford University. Although his work is with plant cells, the processes he studies are very much related to those found in the human body. Earth and Sky’s Jorge Salazar talked with Dr. Frommer about imaging brain cells with nanotechnology.
ES: Thanks for talking with us today. Can you give me a little background into the work you’re doing with how plants use sugars, and eventually, how this all translates to something as seemingly unrelated as Alzheimer’s research?
Frommer: We’ve been interested in the question of how sugars, which are made in leaves of plants, are transported to the seeds or to the roots, which by themselves are dependent on the supply of which sugars from the leaves. The same is actually true in a similar sense for nitrogen, which is transported in the form of amino acids, and we have used tricks to get a hand on the proteins, which are responsible for exchanging these compounds across the plasma membrane, which is basically encapsulating every cell.
If you have a multicellular organism, you need to transport these compounds from cell to cell and get them into the veins of the plant, and transport them to whatever location where they are needed. And, of course, photosynthesis energy is used to assimilate CO2 from the air, and the nitrogen that’s coming from the soil into sugars like sucrose and amino acids. So we use a trick, by using yeast cells, which could not grow because they didn’t have the transporters, we introduced the plant transporters, and actually we introduced many different genes, and the genetic code is universal – we can introduce any gene from any organism, for example, into yeast, and if the proteins made correctly, it will be more or less the same protein like it would be in the plant. This would allow the yeast cell to grow again if it takes up sucrose by these plant transporters into yeast.
So that’s how we originally identified these proteins. But, we could characterize them into very much detail, but still we don’t really, perfectly understand how the plant transport processes work. We don’t know where the sugar is released. We don’t know exactly where the loading is happening, because most of the time we cannot see the sugars or the amino acids where it is, where it is released, what the concentration is inside individual cells, or even within compartments of individual cells.
We needed a technology which allows us to look from the outside, into the cells, and at what time we have, which concentration of a sugar or an amino acid. That’s a little bit of a background – and that led to the development of these genetically encoded nanosensors for sugars and amino acids.
ES: How do these nanosensors work?
Frommer: The analogy that you can use is, if you take two tuning forks, which are normally used to tune musical instruments, which are identical in length and give the same tone, then you can take one of those tuning forks and excite it by hitting it on the rim of the table, and bring it close to the other one without touching it. And what you will notice is a classical physics classroom experiment, that you will see that the second music fork, although it’s not touching the first one, will start to vibrate as well. And that is a phenomenon that is called resonant energy transfer. So there’s an energy of resonance, which is transferred between the one tuning fork and the other one. Essentially, you can do this also with light and excite one molecule, which, when it’s excited with a special color of light, it will respond to this by producing a special color of light, and you can have the second one. And if they are close enough to each other, without touching each either, they will also transmit resonance energy. So the second one will also start to vibrate, or in other words, to send out light. And in this case, we do not use two identical fluorescent proteins, but two which are whatever we use in this case – I’ll explain in a minute – two which are slightly different in their light properties, in their spectral properties. So, if we excite the Cyan, or the blue version of it, we get special wavelengths of light coming out of it. And if we bring the second one, which in our case is the yellow one, close to it, we’ll get yellow light coming from that one. So we can measure, in essence, the distance between the two fluorescent probes. And by looking, exciting one of them, looking at how much light is coming from the yellow, and how much is coming from the blue. And the trick is, now you can link these to a third compound, a so–called recognition element, which, for example, recognizes glucose, or in the most recent study that we have done for glutamate, an important neurotransmitter. And we have selected very carefully from the proteins that are available in nature, some proteins from bacteria which specifically recognized these compounds. And when they do this, they undergo a venus flytrap movement, so they close around the glucose or the other one closes around the glutamate, specifically, and when it closes, it makes a movement. And now you already see how this is in principle working. If we attach the two fluorescent compounds to different parts of the protein which is moving, then we move the two closer together or further apart, depending on the presence of the analyte, the glucose or glutamate. And we can measure the light, and the light color, in essence, will be different depending on the amount of sugar which is present. And the flourophors, which we are using, are proteins which are from jellyfish, and they have been modified to have these different colors. So essentially, we are putting together three different proteins – one is the cyan version, the so–called green fluorescent protein, then followed by the glucose, or in this case the glutamate binding protein from bacteria. And this then fused on the other side by a yellow version of the green fluorescent protein from the jellyfish. This fusion is not done on the protein level, but the proteins are encoded by DNA, so we make these constructs at the DNA level, and if we have fused the genes, then you will have the machinery from the organism reading this and assembling one protein from this, which contains at the end these two fluorescent proteins. And since they are genes, and since the genetic code is universal, we can introduce them into any organism, and the same protein will be made, no matter whether we bring this gene construct into a bacterium, into a fungal cell, a plant cell, or an animal cell.
Our thanks to:
Wolf B. Frommer
Carnegie Institution
Department of Plant Biology
Stanford, CA
