Phosphorylation, disorder, and phase separation govern the behavior of Frequency in the fungal circadian clock

  1. Department of Chemistry & Chemical Biology, Cornell University, Ithaca, NY,14853
  2. Department of Biochemistry and Molecular Biology, SUNY Upstate Medical University, Syracuse, NY, 13210;
  3. Department of Molecular and Systems Biology, Geisel School of Medicine at Dartmouth, Hanover, NH 03755

Peer review process

Not revised: This Reviewed Preprint includes the authors’ original preprint (without revision), an eLife assessment, public reviews, and a provisional response from the authors.

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Editors

  • Reviewing Editor
    Volker Dötsch
    Goethe University Frankfurt, Frankfurt am Main, Germany
  • Senior Editor
    Volker Dötsch
    Goethe University Frankfurt, Frankfurt am Main, Germany

Reviewer #1 (Public Review):

Summary:
"Phosphorylation, disorder, and phase separation govern the behavior of Frequency in the fungal circadian clock" is a convincing manuscript that delves into the structural and biochemical aspects of FRQ and the FFC under both LLPS and non-LLPS conditions. Circadian clocks serve as adaptations to the daily rhythms of sunlight, providing a reliable internal representation of local time.

All circadian clocks are composed of positive and negative components. The FFC contributes negative feedback to the Neurospora circadian oscillator. It consists of FRQ, CK1, and FRH. The FFC facilitates close interaction between CK1 and the WCC, with CK1-mediated phosphorylation disrupting WCC:c-box interactions necessary for restarting the circadian cycle.

Despite the significance of FRQ and the FFC, challenges associated with purifying and stabilizing FRQ have hindered in vitro studies. Here, researchers successfully developed a protocol for purifying recombinant FRQ expressed in E. coli.

Armed with full-length FRQ, they utilized spin-labeled FRQ, CK1, and FRH to gain structural insights into FRQ and the FFC using ESR. These studies revealed a somewhat ordered core and a disordered periphery in FRQ, consistent with prior investigations using limited proteolysis assays. Additionally, p-FRQ exhibited greater conformational flexibility than np-FRQ, and CK1 and FRH were found in close proximity within the FFC. The study further demonstrated that under LLPS conditions in vitro, FRQ undergoes phase separation, encapsulating FRH and CK1 within LLPS droplets, ultimately diminishing CK1 activity within the FFC. Intriguingly, higher temperatures enhanced LLPS formation, suggesting a potential role of LLPS in the fungal clock's temperature compensation mechanism.

Biological significance was supported by live imaging of Neurospora, revealing FRQ foci at the periphery of nuclei consistent with LLPS. The amino acid sequence of FRQ conferred LLPS properties, and a comparison of clock repressor protein sequences in other eukaryotes indicated that LLPS formation might be a conserved process within the negative arms of these circadian clocks.

In summary, this manuscript represents a valuable advancement with solid evidence in the understanding of a circadian clock system that has proven challenging to characterize structurally due to obstacles linked to FRQ purification and stability. The implications of LLPS formation in the negative arm of other eukaryotic clocks and its role in temperature compensation are highly intriguing.

Strengths:
The strengths of the manuscript include the scientific rigor of the experiments, the importance of the topic to the field of chronobiology, and new mechanistic insights obtained.

Weaknesses:
This reviewer had questions regarding some of the conclusions reached.

Reviewer #2 (Public Review):

Summary:
This study presents data from a broad range of methods (biochemical, EPR, SAXS, microscopy, etc.) on the large disordered protein FRQ relevant to circadian clocks and its interaction partners FRH and CK1, providing novel and fundamental insight into oligomerization state, local dynamics, and overall structure as a function of phosphorylation and association. Liquid-liquid phase separation is observed. These findings have bearings on the mechanistic understanding of circadian clocks, and on functional aspects of disordered proteins in general.

Strengths:
This is a thorough work that is well presented. The data are of overall high quality given the difficulty of working with an intrinsically disordered protein, and the conclusions are sufficiently circumspect and qualitative to not overinterpret the mostly low-resolution data.

Weaknesses:
None

Reviewer #3 (Public Review):

Summary:
The manuscript from Tariq and Maurici et al. presents important biochemical and biophysical data linking protein phosphorylation to phase separation behavior in the repressive arm of the Neurospora circadian clock. This is an important topic that contributes to what is likely a conceptual shift in the field. While I find the connection to the in vivo physiology of the clock to be still unclear, this can be a topic handled in future studies.

Strengths: The ability to prepare purified versions of unphosphorylated FRQ and P-FRQ phosphorylated by CK-1 is a major advance that allowed the authors to characterize the role of phosphorylation in structural changes in FRQ and its impact on phase separation in vitro.

Weaknesses: The major question that remains unanswered from my perspective is whether phase separation plays a key role in the feedback loop that sustains oscillation (for example by creating a nonlinear dependence on overall FRQ phosphorylation) or whether it has a distinct physiological role that is not required for sustained oscillation.

Author Response

Reviewer #3 (Public Review):

Summary:

The manuscript from Tariq and Maurici et al. presents important biochemical and biophysical data linking protein phosphorylation to phase separation behavior in the repressive arm of the Neurospora circadian clock. This is an important topic that contributes to what is likely a conceptual shift in the field. While I find the connection to the in vivo physiology of the clock to be still unclear, this can be a topic handled in future studies.

Strengths: The ability to prepare purified versions of unphosphorylated FRQ and P-FRQ phosphorylated by CK-1 is a major advance that allowed the authors to characterize the role of phosphorylation in structural changes in FRQ and its impact on phase separation in vitro.

Weaknesses: The major question that remains unanswered from my perspective is whether phase separation plays a key role in the feedback loop that sustains oscillation (for example by creating a nonlinear dependence on overall FRQ phosphorylation) or whether it has a distinct physiological role that is not required for sustained oscillation.

The reviewer raises the key question regarding data suggesting LLPS and phase separated regions in circadian systems. To date condensates have been seen in cyanobacteria (Cohen et al, 2014, Pattanayak et al, 2020) where there are foci containing KaiA/C during the night, in Drosophila (Xiao et al, 2021) where PER and dCLK colocalize in nuclear foci near the periphery during the repressive phase, and in Neurospora (Bartholomai et al, 2022) where the RNA binding protein PRD-2 sequesters frq and ck1a transcripts in perinuclear phase separated regions. Because the proteins responsible for the phase separation in cyanobacteria and Drosophila are not known, it is not possible to seamlessly disrupt the separation to test its biological significance (Yuan et al, 2022), so only in Neurospora has it been possible to associate loss of phase separation with clock effects. There, loss of PRD-2, or mutation of its RNA-binding domains, results in a ~3 hr period lengthening as well as loss of perinuclear localization of frq transcripts. A very recent manuscript (Xie et al., 2024) calls into question both the importance and very existence of LLPS of clock proteins at least as regards to mammalian cells, noting that it may be an artefact of overexpression in some places where it is seen, and that at normal levels of expression there is no evidence for elevated levels at the nuclear periphery. Artefacts resulting from overexpression plainly cannot be a problem for our study nor for Xiao et al. 2021 as in both cases the relevant clock protein, FRQ or PER, was labeled at the endogenous locus and expressed under its native promoter. Also, it may be worth noting that although we called attention to enrichment of FRQ[NeonGreen] at the nuclear periphery, there remained abundant FRQ within the core of the nucleus in our live-cell imaging.

Cohen SE, et al.: Dynamic localization of the cyanobacterial circadian clock proteins. Curr Biol 2014, 24:1836–1844, https://doi.org/10.1016/j.cub.2014.07.036.

Pattanayak GK, et al.: Daily cycles of reversible protein condensation in cyanobacteria. Cell Rep 2020, 32:108032, https://doi.org/10.1016/j.celrep.2020.108032.

Xiao Y, Yuan Y, Jimenez M, Soni N, Yadlapalli S: Clock proteins regulate spatiotemporal organization of clock genes to control circadian rhythms. Proc Natl Acad Sci U S A 2021, 118, https://doi.org/10.1073/pnas.2019756118.

Bartholomai BM, Gladfelter AS, Loros JJ, Dunlap JC. 2022 PRD-2 mediates clock-regulated perinuclear localization of clock gene RNAs within the circadian cycle of Neurospora. Proc Natl Acad Sci U S A. 119(31):e2203078119. doi: 10.1073/pnas.2203078119.

Yuan et al., Curr Biol 78: 102129, 2022. https://doi.org/10.1016/j.ceb.2022.102129

Pancheng Xie, Xiaowen Xie, Congrong Ye, Kevin M. Dean, Isara Laothamatas , S K Tahajjul Taufique, Joseph Takahashi, Shin Yamazaki, Ying Xu, and Yi Liu (2024). Mammalian circadian clock proteins form dynamic interacting microbodies distinct from phase separation. Proc. Nat. Acad. Sci. USA. In press.

  1. Howard Hughes Medical Institute
  2. Wellcome Trust
  3. Max-Planck-Gesellschaft
  4. Knut and Alice Wallenberg Foundation