A circadian rhythm is a cyclic process that repeats every 24 hours. Whether or not you realize it, your behaviors are largely dictated by a circadian rhythm. The light/dark cycle, for example, determines when you’re awake or asleep (generally, that is). It’s not just your conscious decisions that follow this pattern either – all of your cells follow a 24-hour circadian rhythm as well. In fact, most organisms (even bacteria) exhibit a rhythmic pattern. Many behaviors operate on a rhythmic basis, independent of light and dark cycles too. This is proven in experiments where the organism is kept under complete dark or light conditions. In a way, this is how the organism is able to “predict” the light and dark cycles it has previously been entrained to. Conversely, some physiological systems are severely impacted under such conditions. Interestingly, there are anticipatory effects both in behavior and on a molecular level. The hormone GLP-1 for example, will be secreted in anticipation to a regular feeding time. Under constant light conditions however, this pattern of secretion is completely abolished.
Cells have oscillating machinery, operating on a 24-hour (this exact time varies depending on the organism) basis. This allows the organism to respond appropriately, both biochemically and in behavior. The term circadian clock, here, refers to the biochemical processes that operate in this oscillating pattern over a 24-hour period.
Learning From A Fruit Fly
Synchronicity is important – the cell’s actions are largely a response to external stimuli. If a cell falls out of sync with the light/dark cycle, it’ll exhibit the wrong behavior. A classic example of this is a plant cell preparing for photosynthesis while it’s dark.
D. melanogaster, the fruit fly, is often used in research as a simple model for developing the basis of our understanding of the biochemical complexity in humans. All of the cells in D. melanogaster follow a circadian pattern, synchronized to the light/dark cycle – a property humans do not share (Tataroglu, 2014). Human cells, while having their own circadian processes, are largely governed by the “master clock” located in the suprachiasmatic nucleus (SCN) – an area of the hypothalamus, situated right above the optic nerves. This makes D. melanogaster an easy subject of study.
The genes period (per) and timeless (tim) were identified to be the crucial regulators of D. melanogaster cellular circadian rhythms. The protein products from these genes oscillate, under a normal 12-hour light/dark cycle, going down in the morning and up at night (Tataroglu, 2014). These proteins work in conjunction with the transcription factors CLOCK (CLK) and CYCLE (CYC). CLK and CYC promote the transcription of PER and TIM proteins (Tataroglu, 2014) and together, these 4 regulate the circadian clock in D. melanogaster cells.
In D. melanogaster cells, per and tim oscillate together and are lowest around dawn and peak around dusk (Taylor, 2008). After their peak, these gene’s (and thus, protein’s) levels decrease throughout the night, reaching their minimum around 8 AM. CLK and CYC work together, forming a heterodimer to act as a transcription factor. Transcription factors are proteins that modulate gene expression. They scan along DNA until they reach their binding site – a specific sequence of DNA they recognize, promoting or repressing gene expression. This is one level of regulation, ensuring a gene in a given cell is expressed appropriately. Controlling gene transcription controls the amount of mRNA produced, which in turn controls the amount of protein produced. Here, the CLK/CYC heterodimer bind to the specific sequence CACGTG, found in the gene control region of many genes. This binding site is known as an E-box, and when the CLK/CYC heterodimer binds, they activate the transcription of the downstream gene. And so, the CLK/CYC heterodimer can upregulate many genes. Of these are the per and tim genes (vri and Pdp1e are other circadian genes). As previously mentioned, per and tim mRNA will build up, generating peak levels of their protein around dusk.
When PER and TIM protein levels are low in the morning. As such, CLK/CYC heterodimers will be very active, binding to the E-boxes of the per and tim genes. The CLK/CYC heterodimers will increase gene expression, subsequently creating more protein product over the course of the day until levels peak. But why do they peak? Why stops protein levels from increasing indefinitely? When PER and TIM levels are high enough (around dusk), they’ll begin to form heterodimers. This is a timely, ~12-hour process as a number of post-translational modifications are necessary for the formation of this heterodimer, as well as modification to the heterodimer itself. As a heterodimer, the PER/TIM complex enters the nucleus of the cell and inhibits CLK/CYC activity, thereby inhibiting their own production (Taylor, 2008). Now, as with anything biology related, there are alternative pathways and mechanisms that are possible or have been proposed – we’ll see an example of this shortly.
But First, More on Post-translational Modifications
While all of the post-translational modifications necessary for the PER/TIM heterodimer to enter the nucleus aren’t known, we do know that phosphorylation plays a key role (Hara, 2011). Phosphate carries a negative electrical charge. So, when a protein is “phosphorylated”, its structure is altered to accommodate this change in charge. The phosphorylation of PER acts as a tag, signaling for its degradation (Grima, 2002). When TIM is phosphorylated however, there seems to be an opposite affect – entry into the nucleus is accelerated (Cyran, 2005). Moreover, TIM seems to suppress the breakdown of phosphorylated PER.
The enzymes, double time (DBT), casein kinase 2 alpha (CKII), and shaggy (Sgg) are key regulators of PER and TIM, phosphorylating them, thereby affecting their ability to translocate to the nucleus. DBT and CKII phosphorylate PER, while Sgg phosphorylates TIM (Lin 2002; Cyran, 2005). Conversely, there are enzymes that remove phosphate groups. Here, protein phosphatase 2A (PP2A) and protein phosphatase 1 (PP1) do just that – it removes a phosphate group from PER. The actual coordination of these enzymes (and surely others yet to be discovered) isn’t fully known, but we do know they regulate entry of the PER/TIM heterodimer into the nucleus.
In the Nucleus
Once in the nucleus, PER/TIM inhibit CLK/CYC, preventing them from activating the transcription of per and tim genes. As I touched upon earlier, their have been challenges to this current mechanism. It has, for example, been demonstrated that PER can translocate into the nucleus in D. melanogaster cells in the absence of the tim gene, provided DBT is inhibited (Cyran, 2005). Maybe it’s the case where TIM helps to ferry PER into the nucleus, but it’s solely PER that inhibits CLK/CYC activity – we do not yet know.
Regardless, when the PER/TIM heterodimer enters the nucleus around dusk, CLK and CYC will be sequestered, preventing them from heterodimerizing. When this happens, the production of PER and TIM proteins will cease. The store of PER and TIM proteins that exist in the cytoplasm of the cell will start being degraded via ubiquitin proteasome pathway, mediated by a protein called Slmb (Grima, 2002). This is why levels of PER and TIM drop throughout the night – the rate of degradation surpasses that of protein production.
The PER and TIM proteins inside the nucleus take much longer to degrade, taking until the morning. And, at this point, CLK and CYC will be “free” to initiate this entire cycle over again.
Bringing It All Together
This seemingly abstract mechanism does, in fact, influence D. melanogaster behavior. The CLK/CYC heterodimer acts as a transcription factor for many other genes besides per and tim. You can probably gather then that this heterodimer upregulates the genes necessary for daytime behavior. This mechanism is highly conserved, and we’ll see much of it again when discussing circadian rhythm in mammals. There’s one last, crucial protein to consider before moving forward.
At the beginning of this article, I mentioned how D. melanogaster cells are able to synchronize with the light/dark cycle. How is this possible? Cryptochrome (CRY) is a protein that’s responsive to light. Light will activate this protein’s function, whose role is to hasten the breakdown of PER and TIM.
The physiological implications of this are considerable. If, for example, the organism is exposed to light at some inappropriate time (maybe as it begins to get dark, but before PER and TIM reach their peak), CRY becomes active, subsequently leading to the accelerated degradation of PER and TIM. This could, in effect, shift the molecular clock backwards. It’s a cellular attempt to adapt to whatever the light/dark cycle the environment presents – you can imagine how this disruption can impact normal physiological functioning.
This is a mechanism that’s carried over to us, as mammals, and the foundation for many circadian studies. I’ll introduce the mammalian system in the next post and, as you’ll see, it will share many similarities with D. melanogaster.
Cyran, S. A., Yiannoulos, G., Buchsbaum, A. M., Saez, L., Young, M. W., & Blau, J. (2005). The double-time protein kinase regulates the subcellular localization of the Drosophila clock protein period. The Journal of neuroscience : the official journal of the Society for Neuroscience, 25(22), 5430–5437. https://doi.org/10.1523/JNEUROSCI.0263-05.2005
Grima B, Lamouroux A, Chélot E, Papin C, Limbourg-Bouchon B, Rouyer F. The F-box protein slimb controls the levels of clock proteins period and timeless. Nature. 2002;420(6912):178‐182. doi:10.1038/nature01122
Hara, T., Koh, K., Combs, D. J., & Sehgal, A. (2011). Post-translational regulation and nuclear entry of TIMELESS and PERIOD are affected in new timeless mutant. The Journal of neuroscience : the official journal of the Society for Neuroscience, 31(27), 9982–9990. https://doi.org/10.1523/JNEUROSCI.0993-11.2011
Lin JM, Kilman VL, Keegan K, et al. A role for casein kinase 2alpha in the Drosophila circadian clock. Nature. 2002;420(6917):816‐820. doi:10.1038/nature01235
Tataroglu, O., & Emery, P. (2014). Studying circadian rhythms in Drosophila melanogaster. Methods (San Diego, Calif.), 68(1), 140–150. https://doi.org/10.1016/j.ymeth.2014.01.001
Taylor, P., & Hardin, P. E. (2008). Rhythmic E-box binding by CLK-CYC controls daily cycles in per and tim transcription and chromatin modifications. Molecular and cellular biology, 28(14), 4642–4652. https://doi.org/10.1128/MCB.01612-07