This is the fourth interview in the series Research Spotlight, in which I share conversations that I have with faculty regarding their research, their journey within their field, and their field in a broader context.
Kathy Siwicki is the Howard A. Schneiderman Professor of Biology and Neurobiology. Her research focuses on understanding the neurobiology of courtship behavior and memory in the fruit fly Drosophila melanogaster.
This article is the first part of a two part interview with Professor Siwicki.
AIDAN REDDY: What research projects are you working on right now?
KATHY SIWICKI: The research that we’re doing is entirely with students. Students that work with me in my lab are doing experiments that investigate fruit fly behaviors with an aim towards ultimately understanding how brain systems and brain circuits are responsible for producing those behaviors. The behaviors we’ve been focusing on in recent years are mostly social behaviors, meaning male-female interactions, which we call courtship behavior, or male-male interactions, which are often aggressive. Many students have developed their own questions related to those behaviors and most of them are addressing some aspect of the effects of experience on the ways that the flies will behave. For example, the effect of prior courtship experience, on male-female interactions in the process of courtship. How does prior experience affect the way a male interacts with a female? How does prior experience with males affect the way a female responds to a male? We’re also looking at the fighting behaviors. If two males are together in an environment with a limited resource, they’ll fight, in a sense, to establish dominance over that resource. We’re looking at the outcomes of those fights (who’s the winner and who’s the loser) and how those males subsequently interact with females in courtship assays. Those are some examples of the kinds of questions students have addressed in recent years.
REDDY: A paper you co-authored as a postdoc was recently cited by the Nobel Prize Committee as a key publication in awarding the Prize in Physiology or Medicine. What was the paper about, and was there anything else that went along with that citation?
SIWICKI: I did that project as a postdoc – that’s when I began working with fruit flies. Prior to that, my training had been in neurobiology (which is what I teach here at Swarthmore). In the mid-1980’s, when I was finishing my PhD, I and many other neuroscientists of my generation were really impressed with the potential of using fruit flies, the little Drosophila melanogaster that almost everyone looks at at some point in an intro biology course. There were major technical breakthroughs around that time that allowed us to manipulate the fly genome, so we suddenly had this amazingly powerful system for studying these questions of how brain circuits are wired together to produce animal behavior. The particular behavior that we were studying in this project at Brandeis was circadian behavior. Circadian means “about a day.” It’s a word that refers to the daily rhythms of rest and activity that fruit flies and all other animals exhibit. For us, it’s sleep-wake cycles. All animals have some kind of sleep-wake cycle, certain times of day where they’re active and certain times of day when they’re resting. In addition, plants and fungi also have daily rhythms in their physiological properties. These rhythms are controlled by internal biological clocks. They’re not simply responses to the light-dark cycle (the fact that it’s sunny in the day and dark at night). When you take these organisms and put them in constant conditions (no light-dark cues), the rhythms continue to be exhibited. They continue to have daily peaks in activity and rest. Those rhythms persist with a period of about twenty four hours. That’s a circadian rhythm. A big question that biologists who studied these rhythms wanted to answer for many many decades, was, “How do these clocks work? What are the cells and molecules that actually ‘keep time’? Where’s the little clock inside of our bodies, inside of the fruit flies’ bodies, that controls these daily oscillations?”
The work that was just cited for the Nobel Prize was the work that took place over about a decade in two labs at Brandeis, where I was working, and also in another lab at Rockefeller University. There were two lead investigators at Brandeis, Jeffrey Hall and Michael Rosbash, who were collaborators and worked together. I was in Hall’s lab, but we worked very closely with the people in Rosbash’s lab. The lab at Rockefeller that was led by Michael Young, and he’s the third person who’s being honored with this Nobel Prize. So, over that decade from the mid 1980’s to the mid 1990’s, those of us who were working in those three labs were trying to figure out how the fly clock worked. We started with three mutations that we were using as the keys to unlock the puzzle. The three mutations altered and disrupted the clock in different ways. There was the one mutation, called the short-period mutation, that made the fly’s clock run too fast. These were the early bird flies. They’d wake up early in the morning, earlier than a normal fly. Another mutation was the long-period mutation, where the flies were the night owls. They would have a longer-than-24-hour period in the endogenous clock, and, as a result of that, they would tend to stay up late at night. We all know early birds and night owls. The third mutation resulted in flies with no rhythm at all. They were arrhythmic, random in terms of when they were active and when they were resting in terms of time of day. There was no daily pattern in their activity-rest cycles. It turned out that all three of these mutations were in the very same gene! That was the first breakthrough. It actually happened at Caltech back in the 1960’s and published in 1971 by Konopka and Benzer. They named it the period gene. It was a remarkable discovery – that three mutations in the same gene had very different effects on the fly’s clock.
Come the 1980’s, there are new emerging methods for actually figuring out, “What’s the exact sequence of the DNA for this period gene? What the amino acid sequence of the protein that this gene encodes? How does that protein work in the fruit fly’s brain?” (We assumed it was in the brain, but it didn’t have to be.) How does the protein that’s encoded by this gene work to give the fly its endogenous daily patterns, endogenous daily rhythms in activity and rest? That kind of question had never been answered before — not just for circadian rhythms, but for really any other complex behavioral phenomenon. If you have a gene that interferes with or disrupts a particular kind of behavior, what protein does that gene encode and how does that gene actually have the effect that you’re observing in the whole animal? There are a lot of steps between a gene and an animal’s behavior. It was all a huge black box in the 1980’s. So what we did was basically decipher how the period gene was crucially involved in the fly’s endogenous biological clock. Our first breakthrough, really, was one that I made, and that was the discovery that the protein encoded by the gene itself goes through a daily rhythm in its levels. The levels of this PERIOD protein increase during the night, and then lights come on and levels of the protein start to go down and decrease during the day. When the lights go off at the end of the day, the protein levels start to go up again. These changes in the levels of PERIOD protein are not just responses to the light and dark. You turn the lights out completely, put the flies in constant darkness, and the PERIOD protein will continue to go through this twenty-four hour cycle. That was the discovery. Just asking, “Where and when do we find this protein being expressed in the fruit fly?” yielded this information, that it’s in very restricted types of brain cells, and, in those cells, it’s going up and down throughout the day.
Another one of my colleagues at Brandeis at the time was looking at the period gene’s RNA. In intro bio, we learn…
REDDY: I took AP Bio.
SIWICKI: So what happens between DNA and protein?
SIWICKI: DNA gets transcribed into RNA, RNA gets transcribed into protein. So, as soon as we discovered that the PERIOD protein levels were going up and down, the obvious question was, “What about the RNA? Are RNA levels also oscillating? Are the levels of protein going up and down in response to the levels of RNA going up and down?” (The protein is produced by translating that RNA.) So, another postdoc in the lab, Paul Hardin, decided to look at the period RNA levels specifically in the heads of fruit flies. He was looking at fruit fly heads to see if there was a daily rhythm in the levels of period RNA. Sure enough, Hardin found a rhythm in the levels of RNA, and I found this daily rhythm in the levels of the protein — both the products of this period gene. We considered the fact that there were two different rhythms in the levels of RNA and protein, and also that there was a six to eight hour delay between the time that the RNA levels would go up and the protein levels would go up (RNA levels would go up during the day, protein levels didn’t start to go up until the beginning of the night). As soon the protein levels would get to a relatively high level, that’s when the RNA levels would start to come down. That fact, that piece of data, suggested that maybe the protein was turning off the expression of the RNA, of its own gene. Negative feedback — PERIOD protein goes up, turns off the gene so that the period RNA is not transcribed anymore. As the RNA levels decline, the protein levels follow and decline. As soon as the protein levels get down below a certain level, the RNA comes back on again. That gave us this model – that the period gene is actually controlling its own expression through this negative feedback loop.
That was the basic breakthrough in understanding how fruit fly circadian clocks work, and, in subsequent years, Michael Young’s lab at Rockefeller identified several other genes that interact with the PERIOD protein and the period gene. There’s more than one gene that’s involved in this loop, but the fundamental biology of how it works is this negative feedback loop. After we figured out that there was a negative feedback loop, then Young’s lab, and also the Hall and Rosbash group, discovered several additional genes that interact with period in regulating the specific timing of when one goes up and the other goes down, that kind of thing. So, the overall system involves really four core genes, and maybe another four or five that are involved in modulating (regulating) different features that are involved in the turning on and off of those four core genes, but the fundamental concept is this negative feedback loop — that the protein produced by this gene is turning off the expression its own gene, and thereby you get this nice daily oscillation in the protein — that, in turn, influences lots of other downstream processes that result in the fruit fly having a daily activity-rest pattern that continues in the absence of any other clues.
Cool, right? We figured out how the fly clock works, but why is that finding important enough to get a Nobel Prize? Really, who would think fruit fly circadian rhythms were going to be that fundamentally important in biology? I think there are of couple of reasons that it’s being recognized at this level. One is that our own human genomes are very similar to fruit fly genomes. We didn’t necessarily know this at the time, 30 years ago. We know now how similar our own DNA is to fruit fly DNA, and the fact is that mammalian, including human, circadian clocks function with the very same genes and proteins that the fruit fly clocks do. We didn’t know it at the time, but we were actually figuring out how human circadian clocks work. That was kind of lucky. It has medical implications in all of the issues that are associated with abnormal biological rhythms, mostly things like psychiatric disorders, but there are all sorts of issues. When people have abnormal circadian rhythms and they don’t sleep well, they’re more prone to depression and other types of affective disorders. Things like late-night shift work, as health care and factory workers do, can cause social issues. There are implications for human health and well-being that come from understanding how our circadian rhythms and biological clocks work. The medical relevance of the work is also something that we didn’t necessarily anticipate at the time that we did the studies, but has turned out to be a bigger deal that we might have imagined.
I think it’s also the fact that this sort of thing had never been done before. To take a gene (which is a very abstract concept, really) that was identified initially by the mutations that screwed up a particular complex behavior, like the regulation of the timing of an animal’s activity. It’s a pretty complicated to imagine how the brain does that, just like it’s pretty complicated to imagine how a particular gene that increase a person’s susceptibility to cancer or schizophrenia has that effect on a person. There are many, many genes that have effects on our behavior, and there’s a lot of interest in understanding how they do it. What’s the product of this gene, and how does it get expressed in what parts of the brain to influence the function of particular brain circuits that result in schizophrenic symptoms, or result in liver cancer, or anything else? So, making the connections between a gene and a complex organismal phenotype, especially a neurological behavioral phenotype, hadn’t ever been done before, so there was no roadmap for how to do it. We just had to kind of figure it out one step at a time. You answer whatever question you can, one step at a time, and you try to put the pieces together to come up with some model that you can then continue to test with one experiment after another. I think that’s another reason that this is being recognized, because it’s now a classic example of a specific gene that affects a specific behavior and, by golly, now we actually understand how it is that this gene and the regulation of its expression in particular cells in the fly brain results in the fly having either normal or abnormal circadian rhythms.
It was pioneering at the time. Now, there are tons and tons of genetic linkages for all kinds of conditions that still need to be deciphered at that mechanistic level. The link between the genotype and the phenotype is something that biologists want to understand. This was an early example of an effort to fill in that black box.
REDDY: That seems like something I maybe remember reading about in my high school biology class, or at least about circadian rhythms in general. It just goes to show how, in a few decades, something on the forefront can become something that you learn at the high school level class.
SIWICKI: That’s a pretty humbling experience, when you can look back on your career and see how that perspective has changed.
Next, stay tuned for how Professor Siwicki developed her interest in biology, and her take on the importance of scientific literacy in the United States.
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