Sick Papes Interviews Jonathan Tang
Sick Papes sat down with Jonathan Tang to discuss his recent paper “A Nanobody-Based System Using Fluorescent Proteins as Scaffolds for Cell-Specific Gene Manipulation.” Sick Papes also did this interview the old fashioned way, like an idiot, by recording and transcribing rather than emailing and relaxing. Hence the big delay. But like rediscovering a bottle of once lost liquor in your dirty clothes pile, you should be excited: Jonathan took advantage of antibodies from a camel to make GFP – the head honcho fluorescent protein for cellular visualization – friggin’ functional, as the key scaffolding ingredient in a threesome of transcriptional hedonism. He calls these nanobodies “transcription devices” and if you F with GFP, you better start getting creative.
Jonathan Tang et al. 2013. A Nanobody-Based System Using Fluorescent Proteins as Scaffolds for Cell-Specific Gene Manipulation. Cell 154, 928–939
SP: I’d like to start this interview with a quote from the movie Nacho Libre. Have you ever seen Nacho Libre?
SP: Okay well it’s a movie where Jack Black plays a Mexican wrestler and at one very important point in the movie he says, “Under the clothes we find the man and beneath the man we find… his nucleus.” What type of man are you and what’s up with your nucleus?
JT: Okay. I guess on the surface I’m pretty shy person. Underneath I am someone who wants to change the world. And my nucleus? I hope it’s functioning well, with little UV induced damages.
SP: You’ve invented a technology for retrofitting transgenic organisms called “transcription devices dependent on GFP.” Can you explain GFP and “transcription devices” to our readership?
JT: GFP stands for green fluorescent protein, it was discovered in a jellyfish 40-50 years ago and emits green fluorescent light. GFP has been put to use as a tool in molecular biology since 1994, allowing researchers to tag proteins and visualize cellular processes. This has been a powerful, Nobel prize winning tool for researchers. Regarding “transcription devices,” that’s a fancy name I gave to an engineered, hybrid transcription factor. The idea is to make the device activate transcription, but only when its two independent protein parts are tethered to GFP. If GFP is present in the cell, the device be fully formed and functional to initiate transcription in a downstream gene of interest.
SP: Most SP readers have a 3rd grade education. How would you explain this technology to a 3rd grader?
JT: I don’t know if a 3rd grader would understand genes, but here it goes: the transcription devices are present throughout an organism but remain inactive except in cells with GFP. In these cells, devices tether themselves to GFP and initiate events inside cells to turn on other genes.
SP: How does this interaction actually work?
JT: It’s based on protein-protein interactions. The devices are binding proteins derived from camel antibodies that bind to GFP with high affinity. The reason why we use camel antibodies is because the antigen recognition domain is contained in a single peptide segment. Conventional antibodies are hard to express in cells as they are made of two proteins joined by a breakable disulfide bond. I found that there were pairs of the these camel antibodies that recognized different parts of GFP and could co-occupy GFP. This allows one to simultaneously tether different protein domains on to GFP. In this case, I tethered a DNA binding domain with one camel antibody and a transcription activation domain with another. GFP is then the scaffold for forming an active transcription factor which can then initiate transcription of any gene of interest downstream of its genome binding site.
SP: It seems like you kind of owe the camel a lot. Pretend I’m a camel. What would you like to say to me?
JT: I guess for the sake of the next Nature paper, tell me why you evolved single chain antigen binding domains. Please tell me that. If you do, I’ll pay you back with a lot of water.
SP: Like middle-author type water?
JT: Like a gallon of water. Other than that, thank you very much you solved my problem.
SP: At any point in your project, did you make a scale model of GFP in your kitchen to try and identify where the transcription devices would bind?
JT: Not quite like that, but I did do a look at a lot of crystal structures and computational models of GFP structure, along with the structures of the GFP binding nanobodies. I just kept trying to fit them together. Turns out it was a waste of my time, because in the end I had to empirically pair-wise test all 6 nanobodies.
SP: How happy were you when you found a pair that worked ?
JT: I was pretty happy actually. I was in the lab at like 5 o’clock in the morning (editors note, JT is a confirmed night owl) and there was nobody around. I was just so happy, but there was no one to tell. Then my adrenaline went up and I walked around the building looking for people to tell but there was still no one. So I went back to work.
SP: That was a breakthrough moment in the project?
JT: Yes. There was no way to know if the system would work at all. I only had the six nanobodies which were generously given to us.
SP: It’s amazing how a couple of hours of positivity can energize us to wade through a swamp of disappointment that can last years.
JT: That’s true. Prior to that moment there was a lot of disappointment.
SP: Did you try other systems?
JT: Yes, I tried to make a Cre recombinase into a GFP-dependent into Cre recombinase. It was a naïve idea. But those experiments were helpful, as they did told me that the nanobodies could direct GFP to distinct compartments in the cell. That was the first clue the reagents I had could tether GFP.
SP: How do you think about, or visualize, these high affinity interactions between the nanobody devices and GFP? Like a key in a lock? Bugs on a windshield?
JT: Yes, I think of them as locks and keys, they simply come together. Without the high affinity, I don’t think it would work very well.
SP: One of my favorite things about this paper is that the technology that’s developed is not only an important proof of concept, but is immediately of practical use. What is the current utility of your GFP nano-bodies? Where do you see this technology going forward?
JT: One of the exciting applications is for retrofitting transgenic GFP animals. Many GFP lines have been engineered to express GFP in genetically defined populations of cells. For example, the GENSAT (http://www.gensat.org/index.html) project has over 1,500 mouse lines for labeling neuron populations in the brain and retina. Now the question is can we use these nanobody devices to perturb function in GFP expressing cells. One idea would be to turn on the light-sensitive ion channel channelrhodopsin in GFP expressing brain cells to make these neurons fire action potentials with light. As we show in the paper, with this technology you can probe the downstream brain circuitry from the cells that were previously only visualizable. In the long term, this paper suggests that GFP itself is a great transgene. Because it can be used for anything. You can imagine building many types of synthetic systems that use GFP to turn on or off different cellular processes, by mating GFP animals to other animals carrying nanobody devices or by introducing these devices with viruses.
SP: Have you thought about targeting your transcriptional devices to other proteins too?
JT: You mean using other proteins for turning on the system? That’s something that we’re looking into doing.
SP: One more question for you, on behalf of the molecular aficionado readership: In your paper, you note that there are pros and cons to your devices vs. traditional recombinases for manipulating genomes. Can you explain those pros and cons?
JT: The current system uses three components to form the hybrid transcription factor. While this is not as efficient as functional single molecules like Cre, it does have the ability to achieve more specificity through intersectional expression of the individual components. The other problem we experienced was having too much GFP, which sequesters individual nano-devices without the paired binding necessary for transcriptional activation. So the nano-devices have to be tested with each transgenic line. Unlike with Cre, which tends to enact permanent changes in a cell and its progeny, this system can also be reversible, dependent on the continued presence of GFP.
Thanks Jonathan you’re the man!