Graduate student Eric Waddell
As scientists, we sometimes do not challenge our long held assumptions, which leads to a skewed understanding of how something functions. We can represent this phenomenon as a sheet of paper. If we always view the paper from the side, all we see is a one dimensional plane. As a result, we might assume that the paper is nothing more than a thin line. However, if we look at the paper from another perspective, then we can understand that it is in fact a two dimensional plane. An understanding of the previous work within a field is necessary, but we should not allow it to blind us to unexplored avenues for a “solved” problem. Larrondo et al. took such an alternative approach when studying the Neurospora crassa circadian clock and discovered something quite interesting.
I’d like to start by providing you some background into the Neurospora clock, which is necessary before we get into the meat of the paper. The fungal clock model relies heavily on a negative feedback loop of FRQ production and degradation to determine the period length, as you can see in Figure 1. In the model, White Collar 1 and 2 form the White Collar Complex (WCC), which actively drives transcription of the frq gene. This in turn increases FRQ protein production. As FRQ levels rise, it dimerizes and forms the FFC (FRQ/FRH complex) with FRH. FFC then binds to WCC effectively stopping transcription of frq. As the FFC/WCC association continues it binds to kinases, CK-1 and CK-2, which begin to phosphorylate both FRQ’s and WCC. Hyperphosphorylation of FRQ then leads to FFC and WCC dissociation. FRQ is then directed to the degradation pathway by FWD-1, while WCC and FRH are recycled within the cell.
Figure 1 Simple Fungal Circadian Clock Model
What Larrondo et al. accomplished through luciferase reporting has rewritten the period determination mechanism of the fungal clock. The use of the luciferase reporting system in Neurospora crassa race tubes allows the researchers to measure frq and FRQ production in real-time. Larrondo et al. focus on knockouts of FWD-1, which is responsible for transferring FRQ to the proteasome for turnover. By stopping FRQ turnover, they can observe the changes in period using two methods of luciferase reporting. The first method attaches the frq promoter to the luciferase gene, allowing the researchers to explicitly look at transcription. The second method fused the luciferase protein to the c-terminus of the FRQ protein, allowing for direct measurement of protein translation. Luciferase experiments were performed in race tubes, where banding fungal colonies grow horizontally through the tube based on the period of the circadian clock. The researchers then looked at production of FRQ-luc in the initial colony and across the entire tube. They show previously unseen circadian oscillations in both the initial colony and the entire tube when FWD-1 is knocked out. This evidence illustrates that the period length continues even when FRQ turnover is halted, completely contradicting the current model of the fungal clock! To confirm that this is not a singular phenomenon they knock out COP9, another portion of the degradation machinery, to show the same circadian oscillation.
Once FRQ degradation was ruled out as the determinate of period length, the researchers needed to look for how the clock controls period length. They used the transcriptional luciferase reporter to look at the oscillation of frq when examining varying alleles. They looked at of frq, frq1, frq7, frqS900A, frqS548A, frqS538A, S540A, and frq without a c-terminus, frq5xS>D, and frq5xS>A all within the Wt and fwd-1 knockout lines. All of the point mutations from S to A are found within the PEST1 and PEST2 sites that are the phosphorylation targeting sites of CK-1 and CK-2. The alleles of frq and show drastically varying changes in period length in fwd-1 knockout compared to Wt. They conclude that period length variability is determined by the ability to phosphorylate FRQ and not by FRQ turnover. To confirm this the researchers inhibited kinase activity and saw a lengthened period in both Wt and fwd-1 knockout. Finally, Larrondo et al. studied the half-life of FRQ, which was previously thought to be a causative measure of period length. Their findings show that half-life predictions based on western blot assays did not hold true and that period length was relatively unaffected by the loss of FRQ turnover.
Figure 2 A model describing distinct roles for clock protein phosphorylation and degradation through time. Focusing on the new prediction that phosophorylation of FRQ drives the clock period.
All the evidence presented culminates in a new model of period determination for the Neurospora crassa clock. It highlights the newly proposed period determination mechanism, which is driven by clock-signaling phosphorylation (CSP) and termination-signaling phosphorylation (TSP), as seen in Figure 2. In this model, the researchers propose the idea that as FRQ is phosphorylated during the sequestering of WCC, there is a point within the cycle that hyperphosphorylation of FRQ transitions the protein out of the CSP and into the TSP. It is here that FRQ will then associate with FWD-1 and the signalosome and be targeted for degradation in the proteasome.
Christopher L. Baker, Jennifer J. Loros, Jay C. Dunlap.The circadian clock of Neurospora crassa. FEMS Microbiology Reviews Jan 2012, 36 (1) 95-110; DOI:10.1111/j.1574-6976.2011.00288.x
Larrondo, Luis F., Consuelo Olivares-Yañez, Christopher L. Baker, Jennifer J. Loros, and Jay C. Dunlap. “Decoupling circadian clock protein turnover from circadian period determination.” Science 347, no. 6221 (2015): 1257277.