Casein kinase 1a mediates a two-step subunit remodeling mechanism to regulate the FRQ-FRH circadian clock complex

Domain: biology
Published: 2026-01-08
Editorial outcome: major_revision

Abstract
The circadian clock of Neurospora operates through a negative feedback loop in which FREQUENCY (FRQ), along with FRQ-interacting RNA helicase (FRH) and casein kinase 1a (CK1a), inhibits its transcriptional activator, WHITE COLLAR COMPLEX (WCC), via phosphorylation. CK1a, anchored to FRQ, hyperphosphorylates FRQ at its intrinsically disordered regions in a slow, temperature-independent manner, forming a module suited for molecular timekeeping. However, the molecular processes triggered by FRQ’s hyperphosphorylation have remained unclear. Here we show that FRH, the folded binding partner of disordered FRQ, decodes FRQ’s time-dependent phosphorylation state by triggering a two-step remodeling of the FRQ-FRH complex: initially, two FRH molecules bind a FRQ dimer, keeping it inactive by blocking its interaction with WCC. Slow phosphorylation eventually triggers the dissociation of one FRH, thereby activating the complex by exposing a WCC-binding site. Due to the slow and stochastic nature of phosphorylation, the release of the second FRH occurs with a delay, promoting nuclear export and subsequent degradation of FRQ. This ensures precise activation and inactivation of FRQ and positions FRH as a hub for decoding temporal phosphorylation information.

Introduction
Circadian clocks are fascinating molecular devices capable of accurately measuring time and orchestrating the physiology and behavior of organisms in sync with the 24-h environmental day-night cycle. The circadian clock of the filamentous fungus Neurospora crassa is based on a delayed transcriptional-translational negative feedback loop: The heterodimeric circadian transcription factor White Collar Complex (WCC), composed of a White Collar-1 (WC-1) and WC-2 subunit, activates transcription of the circadian clock gene frequency (frq)1–3. The FRQ protein, 85% of which consists of intrinsically disordered regions (IDRs)2,4,5, dimerizes via a coiled-coil in its N-terminal region6. FRQ-Interacting RNA-helicase, FRH, binds to the C-terminal third of FRQ and protects FRQ from premature degradation7. FRH is an orthologue of the conserved MTR-4 helicase7, which has essential functions in various aspects of RNA metabolism8. Helicase activity of FRH is not required for its moonlighting function in the circadian clock5. Casein kinase 1a (CK1a) binds to the central part of FRQ via the spatially separated FRQ-CK1-interaction Domains 1 and 2, FCD1 and FCD29,10. The FRQ-FRH-CK1a complex (FFC) interacts weakly and dynamically with WCC and facilitates its inactivation through phosphorylation by CK1a11. In the course of a circadian period, FRQ is progressively hyperphosphorylated leading to its inactivation and degradation12–14. More than 100 phosphorylation sites in FRQ have been identified15,16, the vast majority located in its IDRs15,17,18. Hyperphosphorylation is associated with circadian timekeeping and triggers a poorly characterized conformational change in the FFC10,19. FCD-bound CK1a is sufficient to phosphorylate FRQ on a circadian timescale and in a temperature-insensitive manner, and thus the FFC constitutes a molecular module suited to measure time18,20–22. Complete compensation of the clock at high temperature requires additionally phosphorylation of FRQ by casein kinase 223.

Despite extensive knowledge of the components of the core clock and their interactions, it is still unclear how time is measured at the molecular level, which is the essential function of a circadian clock. This also applies to the circadian clocks of metazoans, vertebrates, and invertebrates.

For example, it is not yet known how FRH protects FRQ from degradation, or whether this is its sole function. Furthermore, it is not known when and how the FFC interacts with WCC. Most significantly, we do not understand what kind of conformational change could be triggered by the partially redundant multisite phosphorylation of IDRs in FRQ and how this allows the FFC to perform its functions at the right time.

Answering these questions in Neurospora is a technical challenge and in many cases, not possible. For example, homologues of CK1a and FRH in yeast and mammals have essential functions outside the clock24–28 and Neurospora knock-out strains are not available. FRQ and WC-1, without their binding partners, FRH and WC-25,29,30, respectively, are not expressed at a sufficient level to pursue complex biochemical analyses. Furthermore, Neurospora grows as a syncytium and forms dense hyphal networks with many nuclei that are rapidly transported along the hyphae31, making live-cell microscopy challenging. In addition, the WCC is a light receptor that resets the circadian clock in response to blue light stimuli32,33. Therefore, analyzing the subcellular distribution and dynamics of clock proteins by imaging in living cells is technically challenging.

To overcome these challenges, we opted to heterologously express fluorescently tagged Neurospora clock proteins in U2OStx cells, enabling us to analyze their interactions and subcellular dynamics using a live cell imaging system. This straightforward approach enabled us to explore processes that are challenging or impossible to study in a cell-free in vitro system or in Neurospora. Based on these observations, we were then able to design specific experiments for validation in Neurospora that we might not have originally considered.

The approach yielded unexpected insights into the timekeeping mechanisms of the core clock. Our data reveal that the phosphorylation state of FRQ’s IDRs is decoded through its interaction with its folded partner, FRH. FRH blocks the nuclear export signal (NES) of FRQ and the binding site for WCC, initially allowing accumulation of inactive inhibitor complexes that are activated with a delay by slow phosphorylation of FRQ. Phosphorylation eventually triggers the sequential stepwise release of FRH molecules. FFC subunit remodeling regulates FRQ’s delayed interaction with WCC, as well as its nuclear export and degradation.

Results
FRQ contains three nuclear localization signals
To analyze the subcellular localization of FRQ, we constructed plasmids expressing full-length and truncated FRQ versions with either an N-terminal mKate2 (mK2) or a C-terminal mNeonGreen (mNG) moiety under control of a tetracycline-inducible CMV promoter (Fig. 1A and Supplementary Fig. 1A). The proteins were heterologously expressed in U2OStx cells via transient transfection, and the fluorescently tagged proteins were analyzed using an Incucyte live-cell imaging system. Overexpressed mK2-FRQ and FRQ-mNG accumulated in nuclear foci (Fig. 1B left panels, 1C and Supplementary Fig. 1B). FRQ6B2 denotes a DHF to AAA substitution of amino acid residues 774–776 in FRQ, which is part of a larger FRH binding site, FFD, and abolishes binding of FRH29,34, and FRQ9 is a protein truncated after aa 66235, entirely lacking the FRH binding site (Fig. 1A). mK2-FRQ6B2 formed nuclear foci like full-length mK2-FRQ and mK2-FRQ9 formed nuclear foci, which were slightly smaller (Fig. 1B). The data suggest that FRQ has the potential to self-interact and form thermodynamically stable foci when overexpressed.

FRQ contains three polybasic sequences that could function as nuclear localization signal, NLS (Fig. 1A and Supplementary Fig. 1C). We expressed mK2-tagged fragments of FRQ, each containing one of the three polybasic sequences, mK2-FRQNLS1 (aa 1–412), mK2-FRQNLS2 (aa 557–638), and mK2-FRQC-term (aa 630–989) (Fig. 1A). The proteins localized to the nucleus, although a substantial portion of mK2-FRQC-term was detected in the cytosol. In contrast, mK2-FRQC-term-NLS (aa 720–989), a C-terminal fragment lacking the polybasic sequence, accumulated in the cytosol (Fig. 1A, D). These data suggest that FRQ contains three NLSs, henceforth referred to as NLS1, NLS2, and NLS3.

Impact of NLSs on FRQ function in Neurospora
We then created Neurospora strains expressing FRQ versions with mutated NLSs (Supplementary Fig. 1C) under control of the native frq promoter, alongside a control strain expressing wild-type (WT). These FRQ-mutant strains also expressed a frq-lucP reporter to enable analysis of circadian rhythms in vivo through bioluminescence recordings. The strains, grown in 96-well plates, were exposed to 12 h light, 12 h dark, and 12 h light and then transferred to the dark. The control strain, expressing WT FRQ, displayed a robust bioluminescence rhythm in constant darkness (Fig. 2A). Strains expressing FRQmNLS1 (NLS1: RRKKR to RQKKQ) displayed a WT period which slowly damped (Fig. 2B), suggesting that FRQmNLS1 must be a dimer in contrast to FRQΔNLS1, which is a monomer and has lost all clock functions36. This finding suggests that the arrhythmic phenotype of FRQΔNLS1 is likely due to the disrupted FRQ dimerization.

Strains expressing FRQmNLS2 (NLS2: RRKKRK to RGQERK) displayed a long period rhythm and strains expressing FRQmNLS3 (NLS3: RRKRR to RGQGR) displayed an early circadian phase and a short period rhythm damping slightly after several days in the dark (Fig. 2C, D). A triple mutant, FRQmNLS1/2/3, displayed a long period rhythm that damped rapidly in constant darkness (Fig. 2E). The data indicate that the NLSs are crucial for robust circadian oscillations, although some residual clock functions are retained in their absence. Subcellular fractionation revealed that all FRQ-NLS mutants, including FRQmNLS1/2/3, displayed nuclear-cytoplasmic distribution similar to WT FRQ (Supplementary Fig. 1D). In Neurospora, FRQ shuttles between the nucleus and the cytosol37. Although it is enriched in the nucleus4,38, the majority of FRQ resides in the cytosol. This distribution is in part a consequence of the low nuclear-to-cytoplasmic volume ratio in filamentous fungi. However, since FRQ9 was confined to the nucleus (Supplementary Fig. 1D), the C-terminal third of FRQ likely harbors a NES that governs FRQ’s subcellular distribution. Because FRQmNLS1/2/3 is exclusively cytosolic in U2OStx cells, our data suggest that FRQmNLS1/2/3 must employ an additional mechanism to enter the nucleus in Neurospora, potentially through interaction with FRH.

FRH binding governs FRQ self-interaction and localization
To analyze the subcellular localization of FRQmNLS1/2/3 alone and the influence of FRH, we generated plasmids encoding mK2-FRQmNLS1/2/3 and FRH C-terminally tagged with mNG, and expressed them separately or together in U2OStx cells. When expressed alone, FRH-mNG was evenly distributed in the nucleus and mK2-FRQmNLS1/2/3 accumulated in cytosolic foci, indicating the absence of additional NLSs (Supplementary Fig. 2A). Upon co-expression, the phenotype depended on the ratio of the transfected plasmids (Fig. 3A, B). At a DNA ratio of 16.5 ng FRH-mNG to 50 ng mK2-FRQmNLS1/2/3 (hereafter referred to as sub-saturating FRH levels), FRH-mNG co-localized with mK2-FRQmNLS1/2/3 in cytosolic foci (Fig. 3A). At saturating FRH levels (50 ng: 50 ng), however, both proteins were evenly distributed in the nucleus (Fig. 3B). These results indicate that the proteins interact at both expression ratios. Importantly, saturating amounts of FRH-mNG impaired mK2-FRQmNLS1/2/3 foci formation and promoted nuclear translocation of the NLS-deficient protein. Since each FRQ monomer has a single FRH binding site, our data strongly suggest that cytosolic species of FRH-mNG represent heterotrimeric complexes of one FRH-mNG molecule bound to mK2-FRQmNLS1/2/3 dimers. At saturating FRH levels, binding of two FRH-mNG molecules interferes with foci formation and drives nuclear import of the heterotetrameric complex. The data further suggest that mK2-FRQmNLS1/2/3 must contain a NES that competes with FRH-mediated import.

mK2-FRQ, which possesses three NLSs forms nuclear foci on its own (see Fig. 1B). When co-expressed with sub-saturating FRH-mNG levels, both proteins accumulated in nuclear foci, whereas saturating FRH-mNG levels disrupted foci formation (Fig. 3C, D).

We then expressed mK2-FRQ6B2 together with saturating FRH-mNG. mK2-FRQ6B2 accumulated in nuclear foci, which did not contain FRH-mNG; the latter was homogenously dispersed in the nucleus (Fig. 3E). This confirms that the 6B2 mutation disrupts FRQ-FRH binding29.

The C-terminally truncated protein mK2-FRQ9 also accumulated in nuclear foci regardless of FRH-mNG levels (Fig. 3F). Finally, when we co-expressed mK2-FRQC-term, which contains NLS3, with saturating FRH-mNG (Fig. 3G), both proteins localized evenly throughout the nucleus, as expected. However, comparison with mK2-FRQC-term expressed alone (Fig. 1D) revealed more pronounced nuclear localization in the presence of FRH-mNG, suggesting that FRH enhances nuclear accumulation of this fragment. Quantifications are summarized in Supplementary Table 1.

Together, these findings indicate that tight anchoring of FRH to its binding domain (FFD) in the 6B2 region of FRQ prevents FRQ accumulation in nuclear foci, a process that requires additional FRQ regions outside the FRH anchoring site. In the absence of site-specific tight anchoring, FRH does not interact with FRQ. Anchoring thus increases the local FRH concentration, which may facilitate weak interactions with additional FRQ regions that would otherwise remain undetectable. Such multivalent FRQ-FRH interactions have recently been demonstrated in peptide array studies34.

FRQ interacts with the WCC in absence of FRH
In Neurospora, WC-1 is rapidly degraded without its stabilizing partner WC-2 and, hence, cannot be easily analyzed30. We tagged WC-1 with mNG and WC-2 with mK2 to study their expression and localization in U2OStx cells. WC-1-mNG was well expressed and localized predominantly in the cytosol, while mK2-WC-2 localized to the nucleus and formed foci (Supplementary Fig. 2B). When expressed together, WC-1-mNG was depleted from the cytosol and co-localized with mK2-WC2 in nuclear foci (Supplementary Fig. 2C), indicating interaction of the two WCC subunits.

In Neurospora, the FFC complex interacts weakly and dynamically with the WCC7,11 and inactivates WCC through phosphorylation by CK1a11. To investigate whether FRH is necessary for this interaction, we co-expressed in U2OStx cells WC-1-mNG with mK2-FRQ in absence of WC-2 and FRH. WC-1-mNG, which localized mainly to the cytosol when expressed by itself (Supplementary Fig. 2B), co-localized with mK2-FRQ in nuclear foci (Fig. 4A), demonstrating that WC-1 by itself interacted with FRQ (in ~90% of cells). Upon co-expression with mK2-FRQ6B2, which has a mutation in the FRH binding site, WC-1-mNG was also recruited into the nucleus, but a substantial fraction of WC-1-mNG remained cytosolic (Fig. 4B). The data suggest that WC-1-mNG interacted with mK2-FRQ6B2 but potentially with reduced affinity. In contrast, WC-1-mNG did not co-localize with the C-terminally truncated mK2-FRQ9, which formed nuclear foci (Fig. 4C).

Next, we expressed mK2-WC-2 together with FRQ-mNG (Supplementary Fig. 2D, left). Both proteins formed nuclear foci, which overlapped, suggesting that WC-2 on its own also interacts with FRQ.

To study the interaction of FRQ with the WCC, we co-expressed mK2-FRQ with WC-1-mNG and untagged WC-2 (Fig. 4G) as well as FRQ-mNG with untagged WC-1 and mK2-WC-2 (Supplementary Fig. 2D, right). As expected, mK2-FRQ as well as FRQ-mNG formed nuclear foci. WC-1-mNG was enriched in all mK2-FRQ foci (Fig. 4D) and all FRQ-mNG foci contained mK2-WC-2 (Supplementary Fig. 2D), suggesting interaction of FRQ with WCC in absence of FRH. Some WCC foci remained separate, likely due to the saturation of the FRQ foci with WCC, causing excess WCC to accumulate in distinct foci.

When WC-1-mNG and untagged WC-2 were co-expressed with mK2-FRQ6B2, WC-1-mNG was enriched in the mK2-FRQ6B2 foci (Fig. 4E), indicating that the 6B2 region is not required for the interaction of FRQ with WCC. A few WC-1-mNG (WCC) foci remained distinct, suggesting excess of WCC over mK2-FRQ6B2. In contrast, when WC-1-mNG and untagged WC-2 were co-expressed with mK2-FRQ9, WC-1-mNG was not enriched in mK2-FRQ9 foci (Fig. 4F).

To determine whether these expression patterns are cell-type specific, we also expressed the tagged clock proteins in HEK293T cells (Supplementary Fig. 3A, B). The tagged proteins displayed the same localization and colocalization patterns in HEK293T cells as in U2OStx cells.

Recent deletion studies suggested that the N-terminal half of FRQ is required for its interaction with WCC in Neurospora39. However, as these deletions are predicted to disrupt FRQ dimerization (e.g., FRQΔ149–193) and CK1a recruitment (e.g., FRQΔ482–510), they may impact WCC interaction indirectly.

The overexpression and co-localization in U2OStx cells indicate that the C-terminal third of FRQ is necessary with both WC-1 and WC-2, even in the absence of FRH. Because the 6B2 mutation in FRQ may weaken but does not eliminate WCC binding, it suggests that the WCC binding region overlaps with, but is distinct from, the FRH binding site. This raises the question of how FRH modulates this interaction.

FRH blocks WCC binding to FRQ
To directly analyze whether WCC interacts with the C-terminal portion of FRQ, WC-1-mNG was co-expressed with mK2-FRQC-term (Fig. 5A, left panels). WC-1-mNG; which is mainly cytoplasmic on its own, was recruited into the nucleus and uniformly dispersed just like mK2-FRQC-term. These findings indicate that the C-terminal third of FRQ is sufficient for interaction with WC-1. Thus, WCC and FRH both bind to the C-terminal third of FRQ. When WC-1-mNG and mK2-FRQC-term were coexpressed with untagged FRH, mK2-FRQC-term displayed nuclear localization, while WC-1-mNG remained predominantly in the cytoplasm (Fig. 5A, right), just like in the absence of mK2-FRQC-term (see Supplementary Fig. 2B). Similarly, when WC-1-mNG and mK2-FRQ were co-expressed, WC-1-mNG was recruited from the cytosol into the nucleus and colocalized with mK2-FRQ in nuclear foci (Fig. 5B, left). In presence of FRH, mK2-FRQ was homogeneously dispersed in the nucleus, indicating its interaction with saturating amounts of FRH, while WC-1-mNG remained predominantly cytoplasmic (Fig. 5B, right).

To further analyze the impact of FRH on the interaction of FRQ with the WCC, we co-expressed WC-1-mNG and untagged WC-2 together with mK2-FRQ and either low amounts of untagged FRH, allowing co-localization of FRQ and FRH in nuclear foci, or with high amounts of FRH, interfering with foci formation (see Supplementary Fig. 4A, B). At low levels of FRH (Supplementary Fig. 4A, left), mK2-FRQ accumulated in nuclear foci. WC-1-mNG co-localized with these foci, suggesting WCC interacted with FRQ. At high levels of FRH (Supplementary Fig. 4A, right), mK2-FRQ was homogeneously dispersed in the nucleus, indicating that saturating amounts of FRH interfered with the self-interaction of mK2-FRQ. WC-1-mNG accumulated in nuclear foci, indicating that it assembled with untagged WC-2. The WC-1-mNG (WCC) foci, however, did not contain mK2-FRQ (Supplementary Fig. 4A, right). The data suggest that FRQ saturated with FRH does not interact with WCC.

For additional analysis, we expressed mK2-WC-2 and untagged WC-1 together with untagged FRQ and either high or low amounts of FRH-mNG. At low amounts, FRH-mNG was enriched in mK2-WC-2 (WCC) foci (Supplementary Fig. 4B, left), indicating that unlabeled FRQ in complex with sub-saturating amounts of FRH-mNG interacted with WCC. When expressed at a high level, FRH-mNG was dispersed in the nucleus, while mK2-WC-2 (WCC) foci persisted (Supplementary Fig. 4B, right).

The data strongly suggest that FRH is not only unnecessary for the interaction of FRQ with WC-1 and WC-2, but it may even interfere with it. WCC and FRH bind to overlapping but distinct sites within the C-terminal third of FRQ. The strong anchoring of FRH inhibits WCC binding when FRH is present in high, presumably saturating amounts, but permits WCC-FRQ interaction at lower, sub-saturating FRH concentrations. This finding was unexpected, as it had been suggested that FRH is necessary for the interaction between WCC and FFC26. This suggestion was based on data showing convincingly that WCC did not interact with FRQ complexes containing a mutant FRH with an R806H substitution.

To investigate further, we expressed WC-1-mNG and mK2-FRQ with either unlabeled FRH or FRHR806H. Both FRH and FRHR806H competed with the nuclear recruitment of WC-1-mNG by mK2-FRQ (Supplementary Fig. 5). However, FRHR806H was a stronger competitor than FRH, supporting the notion that the R806H substitution may act as a dominant-negative mutation, leading to tighter binding to FRQ, potentially explaining why FRQ did not interact with WCC in the frhR806H Neurospora strain26. However, further analysis is required to uncover the mechanism underlying the R806H mutation.

Phosphorylation of FRQ by CK1 induces its dissociation from FRH and nuclear export
Having shown that FRH blocks binding of WCC to FRQ, we asked whether and how the interaction of FRQ with WCC is regulated. In Neurospora, FRQ is gradually hyperphosphorylated by CK1a, which inactivates FRQ and favors its degradation12–14. To analyze the impact of CK1a, we co-expressed in U2OStx cells untagged CK1a with FRH-mNG and either mK2-FRQ, mK2-FRQ6B2 or mK2-FRQ9. The three FRQ proteins were hyperphosphorylated in a CK1a-dependent fashion (Fig. 6A). This indicates that CK1a phosphorylated mK2-FRQ in U2OStx cells while endogenous kinases did not support similar hyperphosphorylation of the overexpressed FRQ proteins.

Next, we examined the CK1a-mediated effect on mK2-FRQ localization by widefield microscopy 6 h after doxycycline (DOX) induction (Fig. 6B). In absence of CK1a, mK2-FRQ localized exclusively in nuclear foci (see also Fig. 1C), while mK2-FRQ formed nuclear and cytosolic foci when CK1a was co-expressed, indicating that the kinase promoted nuclear export of mK2-FRQ.

Subsequently, we examined the dynamics of CK1a-mediated effects on mK2-FRQ and FRH by Incucyte time-lapse live-cell imaging (Fig. 6C–E). Initially, mK2-FRQ and FRH-mNG co-localized in the nucleus. mK2-FRQ did not form foci, indicating that it interacted with saturation levels of FRH (Fig. 6C). After several hours, CK1a induced the formation of large assemblies of mK2-FRQ in the cytosol, while FRH-mNG remained confined to the nucleus (Fig. 6C and Supplementary Fig. 6A). Interestingly, mK2-FRQ also formed focal assemblies in the nuclei (Fig. 6B, C), suggesting that FRH-mNG, despite being present in excess, could no longer saturate phosphorylated mK2-FRQ. In absence of CK1a, mK2-FRQ alone or together with FRH-mNG remained nuclear at all times and no cell with cytoplasmic FRQ foci was observed (Supplementary Fig. 6B). When CRM-1-dependent nuclear export was inhibited with leptomycin B, mK2-FRQ remained in the nucleus in all cells despite the presence of CK1a (Fig. 6C lower panels, Supplementary Fig. 6A). These data indicate that phosphorylation by CK1a caused dissociation of mK2-FRQ from FRH-mNG and supported its export from the nucleus. mK2-FRQ6B2, which cannot interact with FRH-mNG, was also exported from the nucleus in an LMB-sensitive manner when co-expressed with FRH-mNG and CK1a, but with substantially faster kinetics than mK2-FRQ (Fig. 6D and Supplementary Fig. 6C), indicating that FRH bound to wt FRQ delayed its nuclear export. The data suggest that phosphorylation inactivated the positively charged NLSs, potentially through neutralization by negatively charged phosphosites or -clusters generated by CK1a. In contrast, mK2-FRQ9 was not exported from the nucleus when co-expressed with CK1a and FRH-mNG (Fig. 6E and Supplementary Fig. 6D), suggesting that the C-terminal third of FRQ contains a NES.

To narrow down the NES, we divided the C-terminal third of FRQ into three parts, deleting amino acid residues 631–756, 757–888, and 889–989 in mK2-FRQ (Supplementary Fig. 7A), respectively, and expressed each modified protein along with CK1a in U2OStx cells (Supplementary Fig. 7B). After DOX induction, we observed cytosolic accumulation of mK2-FRQΔ631–756 and mK2-FRQΔ889–989, suggesting that CK1a-mediated phosphorylation favored cytoplasmic localization by inactivating the NLSs and/or activating the NES. In contrast, mK2-FRQΔ757–888 remained nuclear, demonstrating its NES had been compromised by the deletion. Since this deleted segment also includes the FRH binding region, it seems likely that FRH can mask the NES, thereby regulating FRQ’s nuclear export in a phosphorylation-dependent manner.

Our data indicate that in the absence of phosphorylation, the three NLSs in full-length FRQ dominate functionally over the NES, resulting in strong nuclear enrichment of FRQ. When FRQ is hyperphosphorylated by CK1a, it is exported to the cytosol, likely due to NLS neutralization. FRH slows this phosphorylation-driven export of FRQ by providing additional nuclear import capacity via its NLS and by masking FRQ’s NES.

Native FFC dissociates upon phosphorylation of FRQ by CK1a
In Neurospora, all detectable FRQ is present in a complex with FRH, which protects FRQ from degradation. FRQ6B2 or FRQ9, which cannot interact with FRH, are rapidly degraded. Hence, they accumulate only at low levels, despite being synthesized in excessive amounts due to nonfunctional negative feedback29,40.

Since free FRQ does not accumulate at significant levels in Neurospora, it is challenging to investigate whether gradual hyperphosphorylation triggers its dissociation from FRH. Proteasome inhibitors are not very effective and lead to pleiotropic effects when administered at high doses and for longer time periods41. To circumvent degradation of free FRQ, we prepared native Neurospora extract from cells expressing 2×FLAG-tagged FRH and hyperphosphorylated FRQ in vitro by adding recombinant CK1a and ATP (Fig. 7A). An untreated extract was used for comparison. Subsequently, 2×FLAG-FRH was subjected to immunoprecipitation. In the control reaction without CK1a, hypophosphorylated FRQ co-immunoprecipitated with 2×FLAG-FRH (Fig. 7B left). In contrast, upon incubation with CK1a and ATP, hyperphosphorylated FRQ no longer co-immunoprecipitated with 2×FLAG-FRH (Fig. 7B right).

Size exclusion chromatography of untreated native Neurospora cell extract revealed that FRH co-eluted with FRQ in a high molecular mass fraction, indicating their interaction (Supplementary Fig. 8A, B). Given FRH’s abundance relative to FRQ, most FRH molecules eluted in a lower molecular mass fraction, consistent with prior findings36. Conversely, when the cell extract was treated with CK1a and ATP, FRH no longer co-eluted with FRQ in the high molecular mass fraction (Supplementary Fig. 8A, B).

These findings strongly suggest that the phosphorylation of native FRQ by CK1a triggers its dissociation from FRH.

Stoichiometry of FRQ-FRH complexes
FRQ is a dimer, and in Neurospora most FRQ dimers appear to bind a single FRH molecule5, although monomeric FRQ variants are capable of binding FRH36. Our data from U2OStx cells suggest that FRQ dimers (FRQ2) can assemble into heterotetramers with two FRH molecules (FRQ2FRH2). These tetramers can be converted into heterotrimers (FRQ2FRH1) by displacement of one FRH through CK1a-dependent phosphorylation of FRQ. Hence, in Neurospora, where CK1a is present, tetramers, trimers, and even FRQ dimers may accumulate in distinct proportions, depending on the circadian phase.

To directly assess the subunit composition of FRQ-FRH complexes in the absence of CK1a, we expressed the relevant proteins in HEK293T cells. FLAG-FRH, mK2-FRH, and mK2-FRQ were co-expressed, and native extracts were subjected to FLAG immunoprecipitation. mK2-FRH efficiently co-precipitated with FLAG-FRH (Fig. 8A, right panels). In contrast, in control IPs lacking either FLAG-FRH or mK2-FRQ, mK2-FRH remained in the supernatant (Fig. 8A, left and middle panels). These results demonstrate that dimeric FRQ can recruit two FRH molecules. Notably, a substantial fraction of mK2-FRH underwent nonspecific proteolytic clipping (see Fig. 8A left panel), generating an FRH fragment (FRH*) that co-migrated with FLAG-FRH and displayed similar behavior to full-length mK2-FRQ.

To examine the subunit composition of FRQ-FRH complexes in Neurospora, we generated a strain expressing GFP-tagged FRH in addition to endogenous FRH. GFP pulldown revealed co-precipitation of very low levels of endogenous FRH (Fig. 8B). However, this was expected since FRH expression levels are roughly 10-fold higher than average FRQ levels7,36 and FRH-GFP is expressed at levels similar to endogenous FRH. Under these conditions, only ~5% of endogenous FRH and ~5% of FRH-GFP molecules could be maximally bound to FRQ. Therefore, even in the best-case scenario—where every FRQ dimer binds two FRH molecules—a maximum of only ~2.5% of endogenous FRH could be detected in a GFP pulldown. Given that FRH is progressively released through FRQ phosphorylation, the actual fraction of tetrameric complexes is likely substantially lower.

To verify that the low co-IP of endogenous FRH with FRH-GFP was genuine, we included two controls (Fig. 8C). First, a GFP pulldown from a strain lacking GFP-tagged FRH produced no endogenous FRH signal. Second, in a strain lacking FRQ (frq10), GFP-tagged FRH did not co-immunoprecipitate with endogenous FRH, confirming that the co-IP was mediated by FRQ.

Together, these findings strongly support that FRQ dimers can bind two FRH molecules in Neurospora, which are released stepwise upon CK1a phosphorylation.

Assuming that FRQ were produced at a constant rate, and the release of individual FRH molecules occurs independently, the steady-state ratio of FRQ2FRH2 to FRQ2FRH1 would be 1:2. Under physiological conditions, however, negative feedback suppresses FRQ synthesis, resulting in progressive conversion of FRQ2FRH2 into FRQ2FRH1. Consequently, FRQ2FRH1 would become the predominant FRQ species5, while unbound FRQ2 is degraded and does not accumulate.

Methods
Plasmids
The N-terminal mKATE-2 (mK2) tag, derived from pmKate2-N and the C-terminal mNeonGreen (mNG) tag, derived from pMaCTag-0743, were inserted into pcDNA™4/TO downstream of an inhibitory TetR-responsive cytomegalovirus (CMV-TetO) promoter by cloning with overlapping regions using appropriate primers44. In short, a linear vector and an insert DNA fragment carrying a 30 bp sequence overlap were transfected and recombined in vivo in E. coli. Accordingly, the cDNAs of the Neurospora crassa genes, frh, ck1a, frq and fragments of frq were inserted into pcDNA™4/TO, pcDNA™4/TO-mK2 and pcDNA™4/TO-mNG as indicated. The 6B2 mutation as well as the premature stop codon of the FRQ9 mutant were introduced in the pcDNA™4/TO-mK2-frq by site directed mutagenesis using the QuikChange II kit (Agilent).

FRQ-NLS mutants for expression in Neurospora were introduced by site directed mutagenesis into pBM60-frq, resulting in pBM60-frqmNLS1, frqmNLS2, -frqmNLS3 and -frqmNLS1/2/3 (QuikChange II kit, Agilent). For FRH-GFP, pMF30931 (expressing β-tub-gfp under the control of the ccg-1 promoter) was used as source vector for cloning and the tub ORF replaced by frh.

Primers and Oligos used for cloning are listed in Supplementary Data 1.

Western blots
Western blotting was performed as previously described45. Chemiluminescence signals were detected using X-ray films, which were then developed in Konica Minolta SRX-101A Medical Film Processor. Ponnçeau staining of Western blots was used as loading control (Supplementary Data).

U2OST-REx and HEK293T cells and culture conditions
U2OST-REx (U2OStx) and HEK293T (ATCC) cells were maintained in a 5% CO2 incubator at 37°C in Dulbecco’s modified Eagle’s medium (DMEM, Thermo Fisher Scientific) supplemented with 10% fetal bovine serum (Thermo Fisher Scientific), 1% penicillin-streptomycin (Thermo Fisher Scientific), and Hygromycin B (50 μg/mL, Invivogen) Leptomycin B (Merck; CAT# L2913-.5UG) was added at a final concentration of 20 nM one hour after DOX induction.

Protein expression
U2OStx cells were seeded in 96-well plates 24 h prior to transfection (Corning, CAT# 3598). A fixed amount of plasmid DNA (150 or 200 ng as indicated), pcDNA™4/TO plasmids with the genes of interest balanced with empty vector, was transfected per well using Xfect™ Transfection Reagent (Takara Bio) according to the manufacturer’s instructions. Protein expression was induced 24 h post transfection by addition of 10 ng/mL DOX. pcDNA4/TO-FRQ-mNG was co-transfected with pcDNA4/TO-FRQ at a ratio of 25 ng: 50 ng to avoid overexposure of mNG fluorescence.

HEK293T cells (60–80% confluence) were transfected using an in-house PEI reagent (adapted from Longo et al., 46). For transfection, 15 µg plasmid DNA was mixed with PEI at a 4:1 ratio in 2 mL DMEM. After 24 h, cells were scraped (Sarstedt, Cat.# 83.3950) and lysed in cell lysis buffer (25 mM HEPES pH 7.4, 140 mM NaCl, 2 mM EDTA, 0.5% Triton X-100, 1 mM NaF) supplemented with PMSF (1 mM), leupeptin (5 µg/mL), and pepstatin A (5 µg/mL). Protein extracts were sonicated for 5 min in an ultrasonic bath (Merck) and then collected after centrifugation. Protein concentration was measured using a NanoDrop (PeqLab).

Protein extraction for Western blot
Cells were seeded in 12-well plates 24 h before transfection. At either 4 or 14 h after DOX induction, cells were harvested directly in 2× protein sample buffer and boiled at 95°C for 7 min before Western blot analysis.

IncuCyte live-cell microscopy
After induction of transfected U2OStx cells with DOX, 96-well plates were placed in either the Incucyte ZOOM® or Incucyte® SX1 system (Sartorius) at 37°C. Images were acquired at 20× magnification at 1-h intervals for 48 h and then analyzed using the Incucyte® 2023A GUI (Sartorius) and Incucyte® 2016B GUI (Sartorius) software.

Widefield microscopy
U2OStx cells were transfected and induced with DOX. Six hours post-induction, cells were fixed with 4% PFA for 15 min at room temperature, followed by multiple washes with PBS. Coverslips were mounted using ProLong™ Glass Antifade Mountant with NucBlue™ Stain (Cat. #P36981) and sealed with nail polish. Imaging was performed on a Ni-E widefield microscope (Nikon Imaging Center, Heidelberg) using the DAPI (EX390/18, DM416, EM460/60) and Texas Red (EX562/40, DM 593, EM624/40) filter sets.

Quantification and statistical analysis
IncuCyte analysis: In each experiment, 20–30 cells were evaluated. Cells with fluorescence levels that were either too low or excessively high were excluded from the analysis. The cytoplasmic and nuclear distributions of mNG- and mK2-tagged proteins and their co-localization were examined as specified in each respective experiment. Interaction of FRQ and WC-1 or FRQ and WCC (Fig. 4) was determined by co-localization of WC-1-mNG in mK2-FRQ nuclear foci. ImageJ was used to analyze fluorescence intensity in ten cells. Regions of interest (ROIs) of 3×3 pixels were selected within nuclear foci formed by mK2-FRQ (“FRQ foci”) and within nuclear regions lacking foci (“outside foci”). The mean gray value of the green channel (WC-1-mNG) was measured for each ROI. The resulting values were plotted using BioRender (Created in BioRender.com).

Nuclear/cytoplasmic distribution of WC-1-mNG
The nuclear/cytoplasmic ratio of WC-1-mNG in cells expressing mK2-FRQC-term +/- FRH was determined using thresholding in ImageJ, as described by the Keith R. Porter Imaging Facility (University of Maryland, https://kpif.umbc.edu/image-processing-resources/imagej-fiji/determining-fluorescence-intensity-and-area). Briefly, to label the nucleus, the thresholding function was applied to the red channel (mK2-FRQC-term). This selection was then applied to the green channel (WC-1-mNG), where the threshold function was used to define the entire cell, thus allowing differentiation between the nucleus and the cytosol. The nuclear/cytoplasmic ratio was calculated by comparing the mean gray values of the nuclear and cytoplasmic regions.

Neurospora crassa culture conditions
Conidial suspensions in 1 M sorbitol were prepared from strains grown (5–7 days) on standard solid growth medium (2.2% agar, 0.3% glucose, 0.17% L-arginine, 1× Vogel’s medium, and 0.1% biotin). Standard growth medium for liquid cultures contained 2% glucose, 0.17% L-arginine, and 1× Vogel’s medium. Liquid cultures were inoculated with conidia and grown in constant light at 25°C for 2 to 3 days if not indicated otherwise.

To express hypophosphorylated FRQ, 48 h light-grown liquid cultures were transferred to the dark for 10 h (essentially leading to the degradation of “old” FRQ) and then exposed to light for 3 h to induce a burst of FRQ expression before mycelia were harvested and native protein extract was prepared. Strains used in this study are listed in Table 1.

Native protein extraction. To extract native protein from Neurospora, mycelial tissue was ground in liquid nitrogen using a precooled mortar and pestle. The resulting powder was suspended in an extraction buffer containing 50 mM Hepes-KOH (pH 7.4), 137 mM NaCl, 10% (v/v) glycerol, and 5 mM EDTA, with 1 mM phenylmethylsulfonyl fluoride (PMSF), leupeptin (5 μg/ml), and pepstatin A (5 μg/ml). Protein concentration was measured using a NanoDrop (PeqLab). Subcellular fractionation was performed as described ref. 37. 100 mg of total extract and cytosol and 50 mg of nuclei (determined by Bradford assay) were loaded on SDS PAGE.

Luciferase reporter assay. The luciferase assay was performed as described previously47 with slight modifications: 96-well plates containing growth medium including 75 µM D-lucferin were inoculated with 3×104 conidia per well and incubated in constant darkness at 25°C for 3 days. The plate was then transferred to an incubator at 25°C with an EnSpire Multilabel Reader (PerkinElmer). Bioluminescence was measured in 30 min intervals. The following light/dark regime was used: 2 h darkness, 12 h light, 12 h darkness, 12 h light, constant darkness. Light intensity used was 30 µE. Data was normalized to average bioluminescence levels during light exposure.

Signal processing and damped sinewave analysis of luciferase data. Signal data were processed and analyzed using a custom Python script with AI assistance of Google Gemini, which can be provided upon request. The analysis focused on the time series data starting at t = 40.0 h (= shortly after cultures were released into constant darkness, i.e., into free-run). Raw signal data were first smoothed to reduce noise by applying a 1D uniform filter with a window size of ±4 h. This smoothed signal was then detrended by subtracting a linear regression line to remove any long-term drift or baseline shift. This process isolated the oscillatory component of the signal for subsequent fitting. The damping coefficient (γ) was determined by fitting the detrended data to a damped sinewave model using a least-squares optimization method (scipy.optimize.curve fit). The model used is given by the equation:

f(t) = A * e^-γt * cos(ωt + ϕ) + C

where A is the initial amplitude, γ is the damping coefficient, ω is the angular frequency, ϕ is the phase shift, and C is the vertical offset. Initial guesses for the parameters, particularly for the angular frequency (ω), were informed by a Fast Fourier Transform of the detrended data to ensure a robust fit. The damping coefficient (γ) was calculated as an output parameter of this fitting process. The period (τ) was calculated using the fundamental relationship:

τ = 2π / ω:

In vitro phosphorylation. For in vitro phosphorylation of FRQ, Neurospora whole cell lysate was incubated overnight at 4°C with 2 µg purified recombinant CK1a18 per mg of lysate in a buffer containing a final concentration of 50 mM Hepes/KOH pH 7.4, 120 mM NaCl, 11.3 mM MgCl2, 12.5 mM ATP, 1× PhosStop (Roche), 1 mM PMSF, 1 µg/ml leupeptin, 1 µg/ml pepstatin.

Co-immunoprecipitation. FLAG Co-IP was performed as described36. Briefly, 20 µl of M2 FLAG Sepharose beads (Sigma) were washed twice in PBS. 4 mg of whole cell lysate from a strain expressing N-terminally 2×FLAG-tagged FRH was then added to the beads and filled to 500 µl with PBS supplied with protease inhibitors (Roche) and incubated for 3 h at 4°C. Supernatant was removed, beads washed twice, and bound protein eluted by boiling at 95°C for 5 min in 2× Laemmli. 400 µg of the input and supernatant were loaded together with amounts of IP corresponding 1× (for FLAG/FRH decoration) and 9× (for FRQ decoration) equivalents of the input. Western blots were probed with antibodies against FRQ and FLAG (FRH).

GFP pulldown was performed with GFP-Trap agarose beads (Chromotek). 40 µl of bead slurry was equilibrated in PBS supplemented with protease (PI) and phosphatase (P-stop) inhibitors. For each reaction, 8 mg of protein extract in 200 µl extraction buffer was adjusted to a final volume of 600 µl with PBS + PI + P-stop and incubated with equilibrated beads at 4°C for 6 h on a rotating wheel. Supernatant was removed, beads washed twice, and bound protein eluted by boiling at 95°C for 5 min in 100 µl 2× Laemmli. For SDS–PAGE analysis, equivalents of 400 µg Total extracts (T) and supernatant (S) were loaded. For immunoprecipitation (IP), 1×, 4×, and 8× equivalents of 400 µg were analyzed.

Size exclusion chromatography. A Superose 6 Increase G 10/300 GL column was used in an Äkta Pure system (GE Life Sciences/Cytiva). The column was equilibrated with gel filtration buffer (25 mM HEPES/KOH (pH 7.4), 140 mM NaCl, 1 mM EDTA pH 8.0, 1% glycerol, 0.05% Triton X-100). 10 mg native Neurospora protein extracts were loaded onto a 200 µL loop and chromatography was performed at 4°C with a flow rate of 0.5 mL/min. Fractions of 490 µL were collected, and protein was precipitated by adding 122.5 µL of 50% TCA (w/v). The samples were incubated on ice for 10 min and then centrifuged (4°C, 14,000×g, 10 min). Pellets were washed with 1 mL acetone, air-dried for 3 min at room temperature, and resuspended in 90 µL of 2× Laemmli buffer. Samples were boiled at 95°C for 5 min. 45 µL was loaded on SDS-PAGE, followed by Western blotting for analysis.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "Schunke et al. developed a heterologous cell-based system (U2OS cells) to study Neurospora clock proteins using live-cell\nmicroscopy. They expressed FRQ, WCC, FRH, and CK1a, analyzing FRQ localization, FRQ-WCC/FRH interactions, and\nCK1a-mediated FRQ phosphorylation. Their findings suggest CK1a-dependent phosphorylation remodels the FRQ-FRH\ncomplex in a two-step process. While this system offers novel insights into clock regulation, the conclusions require\nvalidation in Neurospora, and the omission of key literature weakens the contextual framework. Addressing these points\nwould strengthen the study’s impact.\nMajor points:\n1. Figure 1, “mK2-FRQ6B2 formed nuclear foci like full-length mK2-FRQ and mK2-FRQ9 formed nuclear foci”. This is not\nreal in Neurospora, in which FRQ is mainly localized in the cytosol, despite its essential role in the nucleus (PMID: 9482720,\nPMID: 21300798). Although the authors also created these relevant strains in Neurospora, they do not examine their\nlocalization, nor cited prior localization studies. One explanation is that CK1a is not co-expressed with FRQ in the cells, as\nindicated by the data in Figure 6. However, it remains puzzling to discuss the localization of FRQ in U2OS cells when it\ndoes not align with the in vivo published data in Neurospora.\n2. DAPI staining is essential to confirm nuclear localization. This is needed at least in Figure 1 or Figure S1, if not in other\nfigures.\n3. Figures 1 and 2, the authors found that three NLS are required for FRQ localization. Previous work identified NLS1 as\ncritical for FRQ nuclear localization and clock functon(PMID: 9482720). The authors report that only combined NLS1-3\nmutations disrupt FRQ nuclear localization and reduce rhythm amplitude. Since they generated Neurospora NLS mutants\n(Figure 2), they should assess FRQ localization in these strains (NLS1, NLS2, NLS3, and NLS1-3).\n4. Figure 6A and Figure S5A, “After several hours, CK1a induced the formation of morphous assemblies of mK2-FRQ 286 in\nthe cytosol, while FRH-mNG remained confined to the nucleus (Fig. 6B, S5A)”. The claim that CK1a expression shifts FRQ\nto the cytosol is unsupported; Figure S5A shows FRQ remains nuclear. This contradicts expectations and requires\nclarification.\n5. Figure S6B, the subcellular distribution of this mutant mK2-FRQΔ631-756 (nuclear vs. cytosolic) is unclear and should be\nexplicitly addressed.\n6. “Phosphorylation of FRQ by CK1 induces its dissociation from FRH and nuclear export”. This conclusion also contradicts\nprevious work (PMID: 9482720, PMID: 21300798). Previous studies indicate that FRQ phosphorylation does not\nsignificantly influence its cellular localization but does affect its interaction with WCC. In this study, CK1a expression plays\nan important role in FRQ localization. Could they discuss the differences?\n7. Figure 8, “Slow multisite phosphorylation of FRQ by CK1a triggers with a delay remodeling of an initially inactive nuclear\nhetero-tetrameric complex with FRQ2FRH2 stoichiometry (and bound CK1a) into an active trimeric FRQ2FRH1 complex\nthat interacts with and inhibits WCC”. Is the ratio between FRQ and FRH derived from the overexpression of proteins in\nU2OS cells or from existing literature? If it is based on overexpression in U2OS cells, it may not be accurate for Neurospora.\nWithout evidence from Neurospora or supporting literature, the model should either omit precise ratios or cite relevant\nstudies.\n8. The authors miss many relevant literatures. A part of them is listed below.\nReferences (PMID: 38526948, PMID: 24316221) are required when the authors mentioned IDR of FRQ in “The FRQ protein,\n85% of which consists of intrinsically disordered regions (IDRs)2, dimerizes via a coiled-coil in its N-terminal region 3” or\nother relevant part.\nThe references (i.e. PMID: 31554810, PMID: 34182774) are required for “FCD-bound CK1a is sufficient to phosphorylate\nFRQ on a circadian timescale and in a temperature-insensitive manner, and thus the FFC constitutes a molecular module\nsuited to measure time 16,18.”\nReferences are needed here,“For example, CK1a and FRH have essential functions outside the clock and cannot be\ndeleted or inactivated. FRQ and WC-1 without their binding partners, FRH and WC-2, respectively, are not expressed at a\nsufficient level to pursue complex biochemical analyses.”\nReferences (PMID: 12098706, PMID: 12098705) are required here, “In addition, the WCC is a light receptor that resets the\ncircadian clock in response to blue light stimuli.”\nReferences are required for “In Neurospora, WC-1 is rapidly degraded without its stabilizing partner WC-2 and, hence,\ncannot be easily analyzed.”\nMinor points：\n1. Figure 3C-E, The FRH:FRQ ratio is noted in the legend but not the main text.\n2. For Figures S1 and S5, Combine multi-panel figures onto single pages for clarity.\n3. “It has been reported that specific mutations in the C-terminal third of FRQ affect its interaction with WCC but not with\nFRH”.“third”or “part”？", "reviewer_2": "This manuscript addresses the question of the functional roles of FRQ phosphorylation by CK1a and FRH binding, two\naspects of FRQ function that are clearly important for circadian rhythms in the fungal model system Neurospora crassa, but\nwhich lack a firmly established mechanistic role. There have been challenges studying CK1a and FRH in Neurospora\nbecause they are both essential genes. The authors established a heterologous expression system for the fungal proteins in\nhuman U2OS osteosarcoma cells, enabling them to look at how FRH binding and CK1a phosphorylation affect nuclear\nlocalization and interaction with white collar (WC) proteins, which serve as the central transcriptional regulator of the\nNeurospora circadian clock. They conclude that the well-documented slow hyperphosphorylation of FRQ by CK1a remodels\nthe FRQ-FRH-CK1a (FFC) complex, and make the important discovery that FRH competes with the white collar complex\n(WCC) and regulates nuclear localization and protein turnover, providing a new model that helps to explain the biochemical\nbasis for the delay in FFC regulation of the WCC in the clock mechanism. Their evidence for this new model is compelling,\neven if it mostly originates from the heterologous system. Mutations based off the U2OS data are tested in Neurospora\ncycling assays and some in vitro biochemistry provides strong support for the central claim of this work. Overall, this is an\nexciting piece of work that offers a new explanation for the delay in transcriptional regulation in a circadian clock, leading to\nmany additional studies that can follow up in more detail on the specific sites and mechanisms. However, looking at the\nfigures alone, it is often hard to synthesize the important points, significantly detracting from the impact of the work. There are\nsome comments and suggestions below for improvements that might help convey this information better to readers.\n1. It might be helpful to denote graphically which constructs in Figure 1 can interact with FRH. Also, the microscopy images\ncould be smaller and perhaps be accompanied by a graphical summary of the data to assist in interpretation at a glance.\n2. It would be nice to get damping rates for the data in Figure 2.\n3. The use of red-green coloring throughout the figures should be changed in consideration of color-blind viewers.\n4. It might help to have cartoons of the constructs and interactions alongside the microscopy images in Figure 3, 4, and 6,\nsimilar to Figure 5.\n5. The data in Fig. S7 is compelling and complementary to the co-IP in Figure 7. I suggest including it in the main figure.\n6. It would be nice to have data of FRH NLS mutant(s) to support the claim made in Figure 3e and lines 177-180.", "reviewer_3": "Summary:\nThis paper explores the molecular events governing FRQ localisation, participation in complexes and stability using\ncombined mutational and microscopy-based approaches. Owing to the intractability of N. crassa to live cell microscopy the\nauthors elect to establish a heterologous overexpression model (using U2OS cells) to explore the interplay between\nFRH, FRQ and phosphorylation (of FRQ) by casein kinase 1. They characterise a series of FRQ mutants in terms of their\nnuclear/cytoplasmic distribution and relate this to downstream effects on the circadian clock. They later use the same\nmutants to study both the distribution and colocalization of FRQ with various binding partners.\nThe findings regarding FRH’s regulatory influence on FRQ localization and interactions are of substantial interest to the\nwider circadian biology community. However, several methodological and interpretive blind-spots should be addressed\nbefore the manuscript is suitable for publication.\nStrengths:\nThis manuscript provides a compelling and novel system for dissecting the spatial regulation and molecular interactions of\nNeurospora crassa clock proteins using a heterologous human cell model. Using this platform, the authors identify sites for\nregulation of nuclear import and export and demonstrates the role of FRH in modulating localisation. They link the nuclear\nmis-localisation of different NLS mutants to circadian phenotypes in N. crassa. In general, the paper is clearly written and\nwell presented, and it does strongly suggest a novel role for FRH in regulating the interaction of WCC and FRQ.\nI am also interested if the authors can rationalise the different NLS mutation phenotypes (Figure2), i.e NLS2 and NLS3 have\nopposing effects on circadian period, which is an interesting and counter-intuitive (at least to me) observation. Further to this,\ndoes introducing the 6B2 mutation to native FRQ abolish rhythmicity, particularly when combined with the triple NLS mutant.\nSimilarly, would a FRQ-NES mutant have a similar circadian phenotype.\nWeaknesses:\n1. In general, quantification and appropriate statistical tests should be provided for all microscopy data and western blots.\n2. Studying typically low-abundance proteins in the context of over-expression has inherent problems, it is plausible that\nsome of the interactions which are reported here are, in part, a function of their being present at a significantly higher copy\nnumber than would be expected in N. crassa, though this is caveated by the authors. In particular, the formation of nuclear\naggregates is common consequence of over-expressing transcriptional factors, e.g., Buchberger et al., PLoS One, 2013.\nGiven the size of the foci (several microns) it is plausible that FRQ and FRH may be in nuclear aggregates/inclusions or\ninteracting with other large macromolecular assemblies, RNA, other membrane less organelles or nuclear bodies in a\nfashion that does not reflect their endogenous function and sub-nuclear localisation. It would be revealing to perform some\nsimple IP or pull-down experiments with affinity-tagged constructs.\nFurthermore, over-expression artefacts are possibly exacerbated by the combined choice of host (U2OS) and the highly\nprocessive CMV-tet promoter. Since the promoter is tet-responsive it should be possible to titrate the expression level with of\nat least one tagged protein with doxycycline. Furthermore, where plasmids have been co-transfected one cannot assume\nequal partitioning of transcriptional and translational machinery, i.e. the observed response when combining different\nmutants is not just a feature of the biochemical activities of the expressed proteins. Moreover, although fine at the population\nlevel it is not reasonable to assume that each dually fluorescent cell has taken up an equimolar ratio of plasmids to that in\nthe transfection mixture unless validated e.g. by calibrating the relative fluorescence intensities.\nSince the authors are using a heterologous system, I would have greater confidence in the conclusions of this paper if some\nmicroscopy could be recapitulated in at least one other cell-line. This would demonstrate that what is observed here is due\nto the intrinsic properties of the specific proteins and not cell-line specific.\n2. The authors make claims about the overlapping, or not, of WC1 and FRQ foci in Figure 4H-I, respectively. Firstly I do not\nagree that this is particularly apparent in the figures themselves, nonetheless one would expect some degree of overlap\neven if they were independently and randomly distributed. Frq-9 signal also appears somewhat more diffuse than Frq-6B2\nand so one may make the interpretation it is simply interacting with WC at a lower stoichiometry. Proper quantification of the\ndegree of colocalization should be provided using established methods.\nThe method of quantification for figure 5A is unusual and rather opaque. It appears that cells are classified as exhibiting\neither nuclear or cytoplasmic localisation of WC-1-mNG, based on the relative fluorescent intensities from each\ncompartment. This approach fails to capture the spectrum of localization, from predominantly nuclear to predominantly\ncytoplasmic. Nonetheless, since observations are not independent (as one goes up the other goes down), neither are they\ncontinuous, therefore it invalidates the choice of statistical test. I would recommend a simple nuclear/cytoplasm ratio be\nreported instead, this would preserve underlying biological variation and better facilitate comparison between +FRH/-FRH\nconditions.\n3. The authors make multiple allusions to the ability of FRQ to homodimerize and form the basis of various intermediate\ncomplexes. Though reduced foci formation by smaller fragments is consistent with a role of the entire molecule forming\nintermolecular multivalent interactions it doesn’t necessarily prove that foci formation is caused by a biologically relevant\nassembly process (phase-separation or otherwise), nor does it necessarily mean they are functionally important for clock\noutput. Some FRAP experiments on the various FRQ foci would go some way to demonstrating this.\nRegarding figure 6, the blot lacks quantification and loading control, without this one cannot claim that endogenous CK1 or\nother kinases are not phosphorylating FRQ. To aid the comparison and draw a parallel with the blot, it would be helpful to\nhave some live cell microscopy without CK1a overexpression.\n4. Though the model the authors propose (Figure 8) is certainly plausible, it is not clear to me what is the direct evidence for\nthe existence of the specific intermediates in the data presented (i.e gradual dissociation of FRH), which is surprising given\nthe suggested slow phosphorylation kinetics. Additional experiments (ie. Native-PAGE/crosslinking etc.) would be required\nto interrogate the relative stoichiometries of the complex participants.\nMinor point: The Y-axes of Figure 2 are not consistent (‘.’ and ‘,’). It would be helpful to add the amplitude and damping rates\nif they intend to comment on these in the text."}, "author_response": "RESPONSE to REVIEWER COMMENTS\nTo all Reviewers:\nDear Reviewers,\nin our revised manuscript, we added a new paragraph at the end of the Results section to\ndirectly analyze the subunit composition of the FRQ-FRH complex in both HEK293T cells and\nNeurospora (Fig. 8).\nTo this end, we performed a pulldown of FLAG-tagged FRH from HEK293T cells co-expressing\nmK2-FRH with or without FRQ. In the presence of FRQ, we observed co-immunoprecipitation\nof mK2-FRH with FLAG-FRH, demonstrating that a FRQ dimer can simultaneously bind two FRH\nmolecules.\nWe also generated a Neurospora strain expressing, in addition to endogenous FRH, a GFP-\ntagged FRH copy. A GFP pulldown revealed co-precipitation of endogenous FRH.\nIMPORTANTLY, interpretation of this result requires consideration of the relative expression\nlevels:\nFRH is expressed at roughly 10-fold higher levels than FRQ, and FRH-GFP is expressed at levels\ncomparable to endogenous FRH. Thus, when immunoprecipitating FRH-GFP, at most ~5% of\nthe FRH-GFP molecules could be bound to FRQ. In the maximal scenario where every FRQ\ndimer binds two FRH molecules, half of the FRQ molecules in the pulldown would carry two\nFRH-GFP, and the other half would carry one FRH-GFP plus one endogenous FRH.\nConsequently, only ~2.5% of endogenous FRH would be detectable in the assay. Moreover,\nbecause the heterotetrameric FRQ₂FRH₂ complex is continuously converted into the trimeric\nFRQ₂FRH₁ complex, and negative feedback eventually suppresses FRQ synthesis, only a weak\nendogenous FRH signal is expected under physiological conditions.\nTo confirm that the co-IP signal is genuine, we performed two controls. First, a GFP pulldown\nfrom a strain lacking GFP-tagged FRH produced no endogenous FRH signal. Second, in an frq¹⁰\nstrain (FRQ deletion), GFP-tagged FRH did not co-precipitate endogenous FRH, indicating that\nco-IP is mediated by FRQ dimers. Together, these findings strongly support that FRQ can bind\ntwo FRH molecules in Neurospora.\nHurley et al. (2013; PMID: 24316221), using a strain expressing FLAG-tagged and V5-tagged\nFRH, reported FRQ bound to a single FRH as the predominant species. However, the sensitivity\nof their assay likely precluded detection of less abundant FRQ dimers containing both a V5-\nand a FLAG-tagged FRH.\nReviewer #1 (Remarks to the Author):\nSchunke et al. developed a heterologous cell-based system (U2OS cells) to study Neurospora\nclock proteins using live-cell microscopy. They expressed FRQ, WCC, FRH, and CK1a,\nanalyzing FRQ localization, FRQ-WCC/FRH interactions, and CK1a-mediated FRQ\nphosphorylation. Their findings suggest CK1a-dependent phosphorylation remodels the FRQ-\nFRH complex in a two-step process. While this system offers novel insights into clock\nregulation, the conclusions require validation in Neurospora, and the omission of key\nliterature weakens the contextual framework. Addressing these points would strengthen the\nstudy’s impact.\nMajor points:\n1. Figure 1, “mK2-FRQ6B2 formed nuclear foci like full-length mK2-FRQ and mK2-FRQ9\nformed nuclear foci”. This is not real in Neurospora, in which FRQ is mainly localized in the\ncytosol, despite its essential role in the nucleus (PMID: 9482720, PMID: 21300798).\nAnswer:\nIt has been shown that in Neurospora FRQ is concentrated in the nucleus, where it is active\nin negative feedback (see Fig’s below).\nHowever, the nuclei in filamentous fungi like Neurospora occupy only about 5-10 % of the\ncytosolic volume (I roughly calculated approximately 7% of the volume). Hence, the large\nfraction of cytosolic FRQ is in part due to the low nuclear/cytosolic volume ratio. In addition,\nour data suggest that the shuttling equilibrium of the FRQ2FRH1 heterotrimer, which is the\nmajor FRQ species in Neurospora (Hurley et al., 2013; PMID: 24316221) contributes to the\ncytosolic fraction.\n…. Although the authors also created these relevant strains in Neurospora, they do not\nexamine their localization, nor cited prior localization studies. One explanation is that CK1a is\nnot co-expressed with FRQ in the cells, as indicated by the data in Figure 6. However, it\nremains puzzling to discuss the localization of FRQ in U2OS cells when it does not align with\nthe in vivo published data in Neurospora.\nAnswer:\nAs suggested, we examined the localization of FRQ in Neurospora. All FRQ-NLS mutants,\nincluding FRQNLSmut1/2/3, display similar subcellular distribution like wt FRQ (Fig. S1D), as FRH\nbinding alone is sufficient to direct FRQ to the nucleus. In U2OStx cells, FRQNLSmut1/2/3 remains\ncytosolic in the absence of FRH but becomes nuclear in its presence (Fig. 3A,B).\nOur subcellular localization data from U2OS cells are consistent with published observations\nfrom Neurospora (see answer to query #3).\n2. DAPI staining is essential to confirm nuclear localization. This is needed at least in Figure 1\nor Figure S1, if not in other figures.\nAnswer:\nUnfortunately, the Incucyte system does not have the capability to detect DAPI. Therefore,\nnuclei were identified in all experiments using phase-contrast imaging. See example:\nFurthermore, by increasing the brightness/contrast of the images, low levels of proteins\nbecome detectable outside the foci. Unlike the foci, this fraction is uniformly distributed\nthroughout the nucleus, providing a reliable internal nuclear marker as shown below for Fig.\n3C:\nIn addition, free FRH is consistently localized to the nucleus and can be used to identify\nnuclei in the Incucyte instead of DAPI. Similarly, WC-2 is always nuclear.\nYet, as recommended by the reviewer, we include representative wide field microscopic\nimages in which nuclei are visualized with DAPI staining (Fig. 1C and Fig. 6B).\n3. Figures 1 and 2, the authors found that three NLS are required for FRQ localization.\nPrevious work identified NLS1 as critical for FRQ nuclear localization and clock function\n(PMID: 9482720). The authors report that only combined NLS1-3 mutations disrupt FRQ\nnuclear localization and reduce rhythm amplitude. Since they generated Neurospora NLS\nmutants (Figure 2), they should assess FRQ localization in these strains (NLS1, NLS2, NLS3,\nand NLS1-3).\nAnswer:\nAs suggested, we show the localization of FRQ mutants in Neurospora (see response to 1.).\nThe absence of nuclear localization of FRQ-ΔNLS (now NLS1) reported in PMID: 9482720\ncould not be reproduced. In our hands, FRQ lacking NLS1 remains nuclear in Neurospora.\nThis fact has been recognized for a long time. (Görl, 2002, PhD thesis, p. 80). Notably, FRQ9,\nwhich lacks its NES, is almost exclusively nuclear (see below & Fig. S1D).\nC = cytosol, N = nucleus\n4. Figure 6A and Figure S5A, “After several hours, CK1a induced the formation of amorphous\nassemblies of mK2-FRQ in the cytosol, while FRH-mNG remained confined to the nucleus\n(Fig. 6B, S5A)”. The claim that CK1a expression shifts FRQ to the cytosol is unsupported;\nFigure S5A shows FRQ remains nuclear. This contradicts expectations and requires\nclarification.\nAnswer:\nThe reviewer is right, there are also FRQ foci in the nucleus, in addition to cytosolic FRQ. Our\nstatement did not exclude this. Yet, we changed the sentence:\nAfter several hours, CK1a induced the formation of amorphous assemblies of mK2-FRQ in\nthe cytosol and in the nucleus, while FRH-mNG remained confined to the nucleus (Fig. 6B,\nS5A). This observation is fully consistent with our model and indeed matches our\nexpectations. According to our model, FRQ is exclusively cytosolic only when all three NLSs\nare inactivated and both FRH molecules are released (FRH possesses its own NLS and can\nshuttle FRQ back into the nucleus). Because phosphorylation is rather stochastic, numerous\nintermediates exist in which, even after some time of CK1a phosphorylation, one or more\nNLSs remain active and/or one FRH remains bound. These intermediates distribute between\nthe cytosol and nucleus, depending on their specific shuttling equilibrium. Figure S5A shows\nthe individual channels for FRQ and FRH, demonstrating that CK1a-phosphorylated FRQ is\npresent in both compartments, cytosol and nucleus.\nWe show in the revised version (Fig S5B) that without CK1a, FRQ as well as FRQ+FRH are\nconsistently nuclear, demonstrating that CK1a is required for its export.\nWe also provide a widefield microscopic image with DAPI (Fig. 6B) showing CK1a-dependent\ncytoplasmic localization of mK2-FRQ.\nTogether, the data suggest that the wild-type FRQ remaining in the nucleus in the presence\nof CK1a still has FRH bound.\nNotably, FRQ does not form nuclear foci when saturated with FRH, but does so at\nsubsaturating FRH levels (see Fig. 3C, D). In Fig. 6C (saturating FRH conditions), FRQ fails to\nform foci after 3 h of incubation with CK1a. However, after prolonged incubation with CK1a\n(e.g. 12 h), FRQ forms foci in the nucleus (and cytoplasm) despite excess FRH\n(homogeneously dispersed) in the nuclei, indicating that at least one FRH molecule has\ndissociated by the action of CK1a.\n5. Figure S6B, the subcellular distribution of this mutant mK2-FRQΔ631-756 (nuclear vs.\ncytosolic) is unclear and should be explicitly addressed.\nAnswer:\nWe have replaced the panel for mK2-FRQΔ631-756 with one showing clearer cytoplasmic\nlocalization. Below are three additional examples for the CK1a-induced cytosolic localization\nof mK2-FRQΔ631-756:\n6. “Phosphorylation of FRQ by CK1 induces its dissociation from FRH and nuclear export”.\nThis conclusion also contradicts previous work (PMID: 9482720, PMID: 21300798). Previous\nstudies indicate that FRQ phosphorylation does not significantly influence its cellular\nlocalization but does affect its interaction with WCC. In this study, CK1a expression plays an\nimportant role in FRQ localization. Could they discuss the differences?\nAnswer:\nThis is precisely why we expressed the clock proteins heterologously in U2OStx cells, as\nsome FRQ species are rapidly turned over and do not accumulate to significant levels in\nNeurospora.\nOur data suggest that the FRQ dimer remains cytosolic only when both FRH molecules are\ndissociated by CK1a and sufficient NLSs (the FRQ dimer has 6 NLSs) are inactivated. In\nNeurospora, FRQ dimers lacking FRH are unstable, rapidly degraded, and therefore do not\naccumulate in the cytosol at substantial levels, making reliable biochemical analysis\nimpossible. (e.g. below an experiment from our lab showing that FRQ does not accumulate\nin a Neurospora strain expressing the mutant FRHΔ100-150 (Hurley et al., 2013;\nPMID: 24316221) that cannot interact with FRQ.)\nIn Neurospora, most cytosolic FRQ species likely contain a single FRH molecule bound per\nFRQ dimer (Hurley et al., 2013; PMID: 24316221).\nAccording to our model, unmodified FRQ dimers with two FRH molecules reside in the\nnucleus, while partially phosphorylated FRQ species with one FRH bound shuttle between\nthe cytosol and nucleus. Given the high cytosol-to-nucleus volume ratio in filamentous fungi\n(~5%), these substoichiometric complexes are mainly cytoplasmic and may represent the\npredominant FRQ species detected in Neurospora. This explains why the majority of\ndetectable FRQ in Neurospora is cytosolic and likely contains substoichiometric amounts of\nFRH (PMID: 24316221), despite each FRQ molecule having its own FRH binding site.\n7. Figure 8, “Slow multisite phosphorylation of FRQ by CK1a triggers with a delay remodeling\nof an initially inactive nuclear hetero-tetrameric complex with FRQ2FRH2 stoichiometry (and\nbound CK1a) into an active trimeric FRQ2FRH1 complex that interacts with and inhibits\nWCC”. Is the ratio between FRQ and FRH derived from the overexpression of proteins in\nU2OS cells or from existing literature? If it is based on overexpression in U2OS cells, it may\nnot be accurate for Neurospora. Without evidence from Neurospora or supporting\nliterature, the model should either omit precise ratios or cite relevant studies.\nAnswer:\nIn our revised manuscript, we added a paragraph and an updated version of Fig. 8, directly\nshowing that FRQ dimers can bind two FRH molecules in HEK293T cells and in Neurospora\n(see responses to all reviewers above).\n8. The authors miss many relevant literatures. A part of them is listed below.\nReferences (PMID: 38526948, PMID: 24316221) are required when the authors mentioned\nIDR of FRQ in “The FRQ protein, 85% of which consists of intrinsically disordered regions\n(IDRs)2, dimerizes via a coiled-coil in its N-terminal region 3” or other relevant part.\nAnswer:\nWe have cited Hurley et al and Tariq et al., PMID: 24316221, PMID: 38526948,\nThe references (i.e. PMID: 31554810, PMID: 34182774) are required for “FCD-bound CK1a is\nsufficient to phosphorylate FRQ on a circadian timescale and in a temperature-insensitive\nmanner, and thus the FFC constitutes a molecular module suited to measure time 16,18.”\nAnswer:\nPMID: 31554810, PMID: 34182774 are included in the revised version.\nReferences are needed here, “For example, CK1a and FRH have essential functions outside\nthe clock and cannot be deleted or inactivated.”\nAnswer:\nWe have modified the sentence:\nFor example, homologues of CK1a and FRH in yeast and mammals have essential functions\noutside the clock and Neurospora knockouts are not available (Shi et al 2010,\nPMID: 19948888; Knippschild et al 2014, PMID: 24904820; Liang et al 1996, PMID: 8756671;\nPfaffenwimmer et al, PMID: 24968893; Jackson et al 2010, PMID: 20512111).\n“FRQ and WC-1 without their binding partners, FRH and WC-2, respectively, are very\nunstable and thus not expressed at a sufficient level to pursue complex biochemical\nanalyses.”\nAnswer:\nAs suggested, we have included references:\nFRQ and WC-1 without their binding partners, FRH and WC-2, respectively, are very unstable\nand thus not expressed at a sufficient level to pursue complex biochemical analyses (Guo\n2010; PMID: 20159972; Hurley 2013; PMID: 24316221, Schafmeier 2008; PMID: 19141472)\nReferences (PMID: 12098706, PMID: 12098705) are required here, “In addition, the WCC is a\nlight receptor that resets the circadian clock in response to blue light stimuli.”\nAnswer\nReference PMID: 12098706, PMID: 12098705 are included in the revised version.\nReferences are required for “In Neurospora, WC-1 is rapidly degraded without its stabilizing\npartner WC-2 and, hence, cannot be easily analyzed.”\nAnswer\nReference PMID: 19141472 is included in the revised version.\nMinor points：\n1. Figure 3C-E, The FRH:FRQ ratio is noted in the legend but not the main text.\nAnswer:\nThe high and low FRH to FRQ ratios are noted in the text\n2. For Figures S1 and S5, Combine multi-panel figures onto single pages for clarity.\nAnswer:\nDone. Note that we have altered Fig. S1.\n3. “It has been reported that specific mutations in the C-terminal third of FRQ affect its\ninteraction with WCC but not with FRH”.“third”or “part”？\nAnswer:\nBoth is right, C-terminal third is more precise. We use “third” in the revised version.\nReviewer #2 (Remarks to the Author):\nThis manuscript addresses the question of the functional roles of FRQ phosphorylation by\nCK1a and FRH binding, two aspects of FRQ function that are clearly important for circadian\nrhythms in the fungal model system Neurospora crassa, but which lack a firmly established\nmechanistic role. There have been challenges studying CK1a and FRH in Neurospora because\nthey are both essential genes. The authors established a heterologous expression system for\nthe fungal proteins in human U2OS osteosarcoma cells, enabling them to look at how FRH\nbinding and CK1a phosphorylation affect nuclear localization and interaction with white\ncollar (WC) proteins, which serve as the central transcriptional regulator of the Neurospora\ncircadian clock. They conclude that the well-documented slow hyperphosphorylation of FRQ\nby CK1a remodels the FRQ-FRH-CK1a (FFC) complex, and make the important discovery that\nFRH competes with the white collar complex (WCC) and regulates nuclear localization and\nprotein turnover, providing a new model that helps to explain the biochemical basis for the\ndelay in FFC regulation of the WCC in the clock mechanism. Their evidence for this new\nmodel is compelling, even if it mostly originates from the heterologous system. Mutations\nbased off the U2OS data are tested in Neurospora cycling assays and some in vitro\nbiochemistry provides strong support for the central claim of this work. Overall, this is an\nexciting piece of work that offers a new explanation for the delay in transcriptional\nregulation in a circadian clock, leading to many additional studies that can follow up in more\ndetail on the specific sites and mechanisms. However, looking at the figures alone, it is often\nhard to synthesize the important points, significantly detracting from the impact of the work.\nThere are some comments and suggestions below for improvements that might help convey\nthis information better to readers.\n1. It might be helpful to denote graphically which constructs in Figure 1 can interact with\nFRH. Also, the microscopy images could be smaller and perhaps be accompanied by a\ngraphical summary of the data to assist in interpretation at a glance.\nAnswer:\nAll constructs containing the FRQ-FRH binding domain (FFD) including the 6B2 region should\ninteract with FRH. We have indicated this in Figure 1\n2. It would be nice to get damping rates for the data in Figure 2.\nAnswer:\nAs suggested, we have calculated damping rates (), which are indicated in Figure 2.\n3. The use of red-green coloring throughout the figures should be changed in consideration\nof color-blind viewers.\nAnswer:\nWe have changed the coloring.\n4. It might help to have cartoons of the constructs and interactions alongside the microscopy\nimages in Figure 3, 4, and 6, similar to Figure 5.\nAnswer:\nAs suggested by the reviewer, we added cartoons to Figure 3. However, this approach did\nnot work for Figure 4, S5 and 6. We attempted to reduce the size of the microscopic images\nand add cartoons, but this made the figure overly busy, cluttered, and confusing. Instead, we\nhave indicated the combination of proteins expressed, e.g.: FRQ + WC-1, … etc.\n5. The data in Fig. S7 is compelling and complementary to the co-IP in Figure 7. I suggest\nincluding it in the main figure.\nAnswer:\nWe thank the reviewer for the suggestion. In the light that we have already so many main\nfigure panels, we rather leave the gelfiltration analysis in the supplemental figures.\n6. It would be nice to have data of FRH NLS mutant(s) to support the claim made in Figure 3e\nand lines 177-180.\nAnswer:\nWhile additional insights are always valuable, such experiments would require considerable\ntime and resources, in particular as it is already well established that FRH is nuclear (see,\ne.g., Cheng et al. 2005 PMID: 15625191, and several other studies).\nWe demonstrate that FRH facilitates the translocation of FRQNLSmut1/2/3 from the cytosol to\nthe nucleus (Fig. 3A,B). Therefore, we do not consider it a high priority, within the scope of\nthis work, to experimentally identify and mutate the NLS of FRH.\nYet, following the reviewer’s comments, we re-examined the NLS predictions. FRH shows\nthree sub-threshold peaks that are absent in the cytosolic MTR4 homologs from yeast and\nhumans. An above-threshold signal is predicted for yeast Ski2; however, since Ski2 is\ncytosolic, this likely does not represent a true NLS. Consequently, experimentally identifying\nthe NLS(s) of FRH may prove to be labor-intensive. Given the uncertainty and potential for\nconfusion, we chose to delete the NLS prediction for FRH.\nReviewer #3 (Remarks to the Author):\nSummary:\nThis paper explores the molecular events governing FRQ localisation, participation in\ncomplexes and stability using combined mutational and microscopy-based approaches.\nOwing to the intractability of N. crassa to live cell microscopy the authors elect to establish a\nheterologous overexpression model (using U2OS cells) to explore the interplay between\nFRH, FRQ and phosphorylation (of FRQ) by casein kinase 1. They characterise a series of FRQ\nmutants in terms of their nuclear/cytoplasmic distribution and relate this to downstream\neffects on the circadian clock. They later use the same mutants to study both the distribution\nand colocalization of FRQ with various binding partners.\nThe findings regarding FRH’s regulatory influence on FRQ localization and interactions are of\nsubstantial interest to the wider circadian biology community. However, several\nmethodological and interpretive blind-spots should be addressed before the manuscript is\nsuitable for publication.\nStrengths:\nThis manuscript provides a compelling and novel system for dissecting the spatial regulation\nand molecular interactions of Neurospora crassa clock proteins using a heterologous human\ncell model. Using this platform, the authors identify sites for regulation of nuclear import\nand export and demonstrates the role of FRH in modulating localisation. They link the\nnuclear mis-localisation of different NLS mutants to circadian phenotypes in N. crassa. In\ngeneral, the paper is clearly written and well presented, and it does strongly suggest a novel\nrole for FRH in regulating the interaction of WCC and FRQ.\nI am also interested if the authors can rationalise the different NLS mutation phenotypes\n(Figure2), i.e NLS2 and NLS3 have opposing effects on circadian period, which is an\ninteresting and counter-intuitive (at least to me) observation.\nAnswer:\nThe reviewer is correct; this result does appear a bit counterintuitive. Unfortunately, we do\nnot yet understand why the effects on period are opposite. One possible explanation is that\nindividual NLS sequences are inactivated with different kinetics and/or differ in their “import\nstrength”. Hence, when only two NLSs are present in FRQ, period length may be determined\nby their combined import „strength“ relative to the export “strength” of the NES.\nFurther to this, does introducing the 6B2 mutation to native FRQ abolish rhythmicity,\nparticularly when combined with the triple NLS mutant. Similarly, would a FRQ-NES mutant\nhave a similar circadian phenotype.\nAnswer:\nThe 6B2 mutation is arrhythmic; therefore, all combinations involving 6B2 will also be\narrhythmic.\nWeaknesses:\n1. In general, quantification and appropriate statistical tests should be provided for all\nmicroscopy data and western blots.\nAnswer:\nWestern blots show qualitative data:\nFig 6A shows CK1a-dependent hyperphosphorylation. n =3\nFig 7B shows that CK1a treatment disrupts the Co-IP of FRQ with FRH. n =3\nMicroscopic images:\nFig 1: The vast majority (if not all) cells expressing the indicated FRQ variants exhibit the\nsubcellular localization shown in the corresponding figure panels.\nFig 3\nSame answer, the vast majority (if not all) cells expressing the indicated FRQ variants exhibit\nthe subcellular localization shown in the corresponding figure panels. Specifically, we have\nevaluated 20 cells per condition.\nFig 4\nWe have evaluated 20 cells per condition.\nA-E: interaction of FRQ with WC-1:\nFig. A,B: 20/20 cells exhibit the subcellular localization shown in the corresponding figure\npanels.\nFig. 4C: 19/20 cells exhibit the subcellular localization shown in the figure panel.\nFig. 4D is quantified in Fig 5A.\nE: 18/20 cells exhibit the subcellular localization shown in the figure panel.\nF-I: interaction of FRQ with WCC:\nFig. F-G: 20/20 cells exhibit the subcellular localization shown in the figure panels.\nI: 19/20 cells exhibit the subcellular localization shown in the figure panel. i.e. no co-\nlocalization of WCC with FRQ9.\n2. Studying typically low-abundance proteins in the context of over-expression has inherent\nproblems, it is plausible that some of the interactions which are reported here are, in part, a\nfunction of their being present at a significantly higher copy number than would be expected\nin N. crassa, though this is caveated by the authors.\nIn particular, the formation of nuclear aggregates is common consequence of over-\nexpressing transcriptional factors, e.g., Buchberger et al., PLoS One, 2013. Given the size of\nthe foci (several microns) it is plausible that FRQ and FRH may be in nuclear\naggregates/inclusions or interacting with other large macromolecular assemblies, RNA, other\nmembrane less organelles or nuclear bodies in a fashion that does not reflect their\nendogenous function and sub-nuclear localisation. It would be revealing to perform some\nsimple IP or pull-down experiments with affinity-tagged constructs.\nAnswer:\nAll clock protein interactions in Neurospora were previously identified through pulldown\nassays of wild-type and mutant proteins conducted by us and others. These interactions are\nvalidated/reproduced in our co-localization assays, with specificity confirmed by the absence\nof co-localization in the respective mutants. Moreover, the co-immunoprecipitation of FRH\nwith FRQ in HEK293T cells (Fig. 8A) provides additional evidence that the co-localizing\nproteins physically interact.\nFurthermore, over-expression artefacts are possibly exacerbated by the combined choice of\nhost (U2OS) and the highly processive CMV-tet promoter. Since the promoter is tet-\nresponsive it should be possible to titrate the expression level with of at least one tagged\nprotein with doxycycline.\nAnswer:\nWe have attempted this, but it was unfortunately not possible to titrate expression levels\nwith DOX, as it behaves like an all-or-nothing system. We can only speculate as to the\nreason. One possibility is DOX metabolism, which may require substantially higher\nconcentrations to sustain expression over time than are needed to initially induce\ntranscription. Consequently, at low DOX concentrations, genes may be partially induced at\nfirst, but as DOX levels fall below a threshold due to metabolism, protein levels do not\naccumulate to a detectable level. Hence, induction at levels sufficient to sustain detectable\nprotein expression is likely to exhibit switch-like behavior.\nFurthermore, where plasmids have been co-transfected one cannot assume equal\npartitioning of transcriptional and translational machinery, i.e. the observed response when\ncombining different mutants is not just a feature of the biochemical activities of the\nexpressed proteins. Moreover, although fine at the population level it is not reasonable to\nassume that each dually fluorescent cell has taken up an equimolar ratio of plasmids to that\nin the transfection mixture unless validated e.g. by calibrating the relative fluorescence\nintensities.\nAnswer:\nSuitable ratios of co-transfected plasmids were determined empirically. In U2OStx cells,\nindividual cells express varying amounts of the proteins. We monitored the cells over time\nand analyzed only those that expressed both tagged proteins at medium levels (i.e.,\nexcluding cells with extremely low or extremely high expression of one or both proteins).\nThe reviewer is of course correct. Detectable interaction depends on ratios, but also on\nconcentrations and binding affinities. For example, when analyzing the interaction of WC-1\nwith FRQ, we always see colocalization of WC-1 in FRQ nuclear foci but observe in several\ncases WC-1 remaining in the cytosol, likely due to WC-1 being in excess over FRQ or\nexpression levels not permitting saturation binding to nuclear FRQ. Our primary focus,\nhowever, is on whether interaction occurs - e.g., colocalization of WC-1 with nuclear FRQ\nfoci - or whether there is no interaction, such as no colocalization of WC-1 with FRQ9 foci.\nSince the authors are using a heterologous system, I would have greater confidence in the\nconclusions of this paper if some microscopy could be recapitulated in at least one other\ncell-line. This would demonstrate that what is observed here is due to the intrinsic\nproperties of the specific proteins and not cell-line specific.\nAnswer:\nAs suggested, we examined protein expression in HEK293T cells. The results (Fig. S2D,E)\nindicate that the proteins display behavior similar to that observed in U2OS cells.\n2. The authors make claims about the overlapping, or not, of WC1 and FRQ foci in Figure 4H-\nI, respectively. Firstly I do not agree that this is particularly apparent in the figures\nthemselves, nonetheless one would expect some degree of overlap even if they were\nindependently and randomly distributed. Frq-9 signal also appears somewhat more diffuse\nthan Frq-6B2 and so one may make the interpretation it is simply interacting with WC at a\nlower stoichiometry.\nAnswer:\nThe C-terminally truncated FRQ9 protein(G663TER) does not interact with WC-1 in\nNeurospora. Truncations beyond aa663 are WCC binding deficient (e.g. Wang et al. 2023\nPMID 37220856), which is consistent with our observations, i.e. WC-1 never colocalized with\nFRQ9 foci (comp. Figs. 4A,C):\nProper quantification of the degree of colocalization should be provided using established\nmethods.\nThe method of quantification for figure 5A is unusual and rather opaque. It appears that cells\nare classified as exhibiting either nuclear or cytoplasmic localisation of WC-1-mNG, based on\nthe relative fluorescent intensities from each\ncompartment. This approach fails to capture the spectrum of localization, from predominantly\nnuclear to predominantly cytoplasmic.\nNonetheless, since observations are not independent (as one goes up the other goes down),\nneither are they continuous, therefore it invalidates the choice of statistical test. I would\nrecommend a simple nuclear/cytoplasm ratio be reported instead, this would preserve\nunderlying biological variation and better facilitate comparison between +FRH/-FRH\nconditions.\nAnswer:\nAs suggested, we have quantified in Fig. 5A for 30 cells the nuclear/cytoplasm intensity ratio\nof WC-1-mNG fluorescence in absence of FRH (WC-1-mNG more nuclear) versus the\npresence of FRH (WC-1-mNG more cytosolic).\n3. The authors make multiple allusions to the ability of FRQ to homodimerize and form the\nbasis of various intermediate complexes. Though reduced foci formation by smaller\nfragments is consistent with a role of the entire molecule forming intermolecular multivalent\ninteractions it doesn’t necessarily prove that foci formation is caused by a biologically\nrelevant assembly process (phase-separation or otherwise), nor does it necessarily mean\nthey are functionally important for clock output. Some FRAP experiments on the various FRQ\nfoci would go some way to demonstrating this.\nAnswer:\nThe molecular basis of foci formation and its physiological relevance are important\nquestions, but they are beyond the scope of this study and therefore not the focus of our\ninvestigations. In the present work, we use foci formation merely as a convenient tool to\nstudy colocalization (using the appropriate controls/mutants to avoid artifacts).\nNevertheless, we are currently exploring this topic and have already made some intriguing,\nunexpected and exciting preliminary observations.\nRegarding figure 6, the blot lacks quantification and loading control, without this one cannot\nclaim that endogenous CK1 or other kinases are not phosphorylating FRQ.\nTo aid the comparison and draw a parallel with the blot, it would be helpful to have some\nlive cell microscopy without CK1a overexpression.\nAnswer:\nIn U2OStx cells FRQ, as well as FRQ6B2 and FRQ9, reach over time a higher phosphorylation\nstate in cells co-expressing CK1a than in cells that do not express CK1a. This does not exclude\nthat endogenous kinases do not phosphorylate FRQ to some (minor) extent.\nWe changed the wording in the main text:\nThis indicates that CK1a phosphorylated mK2-FRQ in U2OStx cells while endogenous kinases\ndid not support similar hyperphosphorylation of the overexpressed FRQ proteins.\nHowever, potential phosphorylation by endogenous U2OS kinases does not impact FRQ\nexport to the cytosol.\nAs suggested, we therefore include (as Fig. S5B) a time course showing that FRQ without\nCK1a remains nuclear at all times:\n4. Though the model the authors propose (Figure 8) is certainly plausible, it is not clear to\nme what is the direct evidence for the existence of the specific intermediates in the data\npresented (i.e gradual dissociation of FRH), which is surprising given the suggested slow\nphosphorylation kinetics. Additional experiments (ie. Native-PAGE/crosslinking etc.) would\nbe required to interrogate the relative stoichiometries of the complex participants.\nAnswer:\nWe show pulldown from HEK293T cells and Neurospora, demonstrating that a FRQ dimer\ncan bind two FRH molecules (see answer to all reviewers above).\nConcerning the stepwise release, and assuming that the CK1a-dependent dissociation of the\ntwo FRH molecules from the FRQ dimer is not strictly coupled but occurs independently, we\nprovide here a theoretical calculation to illustrate the effect of slow phosphorylation on FRH\nrelease:\nAssume a FRQ dimer initially binds two FRH molecules. If slow phosphorylation by CK1\ntriggers FRH release at a rate of e.g. 0.25 h⁻¹ (t ≈ 2.8 h, median), the average waiting time\n1/2\n(mean) for dissociation of the first FRH - producing an FRQ₂FRH₁ species - equals t = 1/(2x\n0.25 h⁻¹) = 2 h.\nThe second FRH dissociates after an additional average waiting time of 1/(0.25 h⁻¹) = 4 h.\nConsequently, in this theoretical example, the average time delay before inactive FRQ₂FRH₂\nbecomes active via release of the first FRH is 2 h, and the resulting time window during\nwhich species with only one FRH bound (FRQ₂FRH₁) remain active is 4 h.\nIf FRQ were produced at a steady rate, the ratio of FRQ₂FRH₂ to FRQ₂FRH₁ would be about\n1:2. In a physiological context (Neurospora), where negative feedback operates to shut down\nFRQ synthesis, FRQ₂FRH₂ would be converted to FRQ₂FRH₁, which would become the\npredominant FRQ species (FRQ₂ is degraded and does not accumulate). The experimental\nobservation that FRQ₂FRH are detected in HEK293T cells and with the expected low\n2\nabundance in Neurospora (see new Fig. 8) is consistent with the stepwise release model.\nMinor point: The Y-axes of Figure 2 are not consistent (‘.’ and ‘,’). It would be helpful to add\nthe amplitude and damping rates if they intend to comment on these in the text.\nAnswer: Done.\nRESPONSE TO REVIEWER COMMENTS\nWe thank all reviewers for the continued engagement with our manuscript.\nReviewer #1 (Remarks to the Author):\nThe authors have addressed my concerns.\nWe thank the Reviewer\nReviewer #2 (Remarks to the Author):\nThe authors have done an acceptable job addressing my prior comments. The addition of the cartoons makes it easier to\nmove between the text and figures to process the information. There are still many questions remaining that will require\nfuture studies to dissect, but given that this type of biochemical study is not possible in the fungal system, I think there is\nvalue in proposing this new model. Although the conclusions here depend on overexpression in a heterologous system, I\nam\nI am satisfied with the biochemical data supporting the conclusions.\nI do have two minor comments that should be easy to address.\nThe new co-IP data in figure 8 should state in the figure what protein is being precipitated. This is done in figure 7 and it is\nstandard practice.\nDone.\nThe cartoon present in figure 8 has different coloring than the ones used throughout the other figures. It may make sense\nto unify the coloring here.\nColor has been changed\nReviewer #3 (Remarks to the Author):\nIn the revised manuscript the authors have made a comprehensive attempt to address several important points. However,\nsome methodological and interpretative concerns remain about the steps the authors have taken to mitigate the risk of\nconfirmation bias, because standard practices such as loading controls, quantification, statistical analyses are still not\nconsistently employed throughout.\nMore generally the authors are of course entitled to their opinions, and presumably find their interpretation very\nconvincing. Whilst I find their interpretations plausible, I do not personally find the supporting evidence even comes close\nto being unambiguous. If the authors are reluctant to do further experiments and analyses then I would encourage then to\ntone down the confidence with which they present their interpretation. Starting with the abstract, I suggest that they\nshould be clear to distinguish their interesting experimental observations from their informed speculations.\nWe have softened the wording in the abstract: … We present data suggesting that FRH …\nMore detailed responses are below:\nReviewer response:\nDespite the qualitative nature of the data, loading controls are still recommended to ensure equal loading/transfer and not\naddressed in the methods. Tubulin loading controls are shown for supplementary figure 1. It is not clear why they are not\nshown for other blots.\nWe show Ponceau S staining of the membranes as loading controls in the supplementary\ndata.\nReviewer response:\nUsing established metrics of co-localization would add rigor to this study and add substance to the claims made in the text.\nIf 20 cells were evaluated then why not show the quantification of the evaluation.\nWe have quantified the images as requested:\nFig 3: Co-localization of FRH-mNG with mK2-FRQ foci was quantified by evaluation of foci in\n20 cells for each FRQ variant. The quantitative results are presented in Supplementary Table\n1.\nFig. 4: We have measured the enrichment/density of WC-1-mNG (-/+ untagged WC-2) in\nnuclear foci of mK2-FRQ (4A, D), mK2-FRQ6B2 (4B, E) and mK2-FRQ9 (4C, F).\nTo accommodate the new quantification, Fig. 4 was reorganized. The co-localization of mK2-\nWC-2 with FRQ-mNG has been moved to Supplementary Figure 2D. The co-localization of\nWC-1-mNG with the mK2-FRQ C-terminal construct is shown and quantified in Figure 5A. The\nmain text was revised accordingly.\nReviewer response:\nAlthough evidence from Neurospora is supportive of the author’s general thesis, for the previously mentioned reasons, foci\nformation in of itself does not provide sufficient proof that localization occurs through the specific interactions mentioned.\nTo establish U2OS overexpression as a useful system for studying mechanisms of Neurospora protein complexes there is an\nobligation to demonstrate that foci are not artefactual. The IP presented in figure 8 is a step towards this but it is unclear\nwhy this was done only in HEK293 and not also in U2OS.\nThe IP was done in HEK293T cells because the transfection efficiency of U2OStx cells is low.\nIt is well established that FRQ interacts with FRH, whereas the mutant versions FRQ6B2 and\nFRQ9 do not. It is also firmly demonstrated that FRQ interacts with WCC, while the FRQ9\nmutant fails to do so.\nOur co-localization approach faithfully reproduces these known interactions and the impact\nof mutations, validating our approach. Therefore, we find no basis for the reviewer’s concern\nthat the observed co-localization could represent an artifact in U2OS cells. It is difficult to\nsee what kind of artifact the reviewer has in mind that could precisely reproduce the\nestablished interactions and mutant phenotypes and subcellular localization and still be\nregarded as artificial?\nMoreover, it is very well to say that cells are selected based on a medium level of expression, but it is not clear from the\ntext how this is determined.\nMedium expression levels correspond to those shown in the figure panels. We use time-\nlapse microscopy, which allows us to select cells that neither express at very low levels\n(predominantly at early time points) nor at excessively high levels (predominantly at late\ntime points). Evaluation of both extremes do not yield different results but are less suitable\nfor clear visual presentation in the figures.\nReviewer response:\nThe point I intended to make is that Figure H and Figure I appear similar. How this interaction occurs in Neurospora is\nsomewhat irrelevant. The onus is on the authors to demonstrate this system faithfully reflects endogenous interactions.\nWithout proper quantification of the degree of localization, the reader is in the position of having to take the authors\nexplanations of the images in good faith.\nAs requested, we have now quantified the data in Fig. 4 as answered above. Again, I want to\nstress that all FRQ or FRQ6B2 foci contained WC-1 while we did not find FRQ9 foci containing\nWC-1.\nReviewer response/Figure 8:\nThe figure legend and main text appear to suggest different experimental set-ups.\nMain text:\n“FLAG-FRH and mK2-FRH were co-expressed with, or without mK2-FRQ”\nIn any case, I found this figure and the accompanying text confusing. The way the blots are presented is not helpful. Some\nof the blots are overloaded, and it is not clear what the arrows are pointing to in the upper panel of 8a.\nWe apologize for the mix-up. In the revised version, we replaced the IP and adapted the text\nand figure legend accordingly. We also present a schematic illustrating the rationale of the CoIP\nand include two types of negative control FLAG-IPs:\n1. Omitting FLAG-FRH: No co-IP of mK2-FRH and mK2-FRQ.\n2. Omitting mK2-FRQ: No co-IP of mK2-FRH with FLAG-FRH.\nCritically, this experiment is not suitable to infer complex stoichiometry, SDS-PAGE is a bulk measurement and cannot\ndefinitively establish the presence of said complexes.\n8b/c similarly to 8a is not suitable to infer stoichiometry. The combined experiments only weakly support the suggested\nmodel.\nThe question we sought to address was whether a FRQ dimer can bind two molecules of\nFRH. To test this, we used two immunologically distinguishable versions of FRH. The rationale\nbehind the Co-IPs shown in Fig. 8 is that if a FRQ dimer can indeed bind two FRH molecules,\nit should be possible to co-precipitate one FRH variant via the specific tag of the second. This\ntype of approach is widely used.\nIn panel 8A (HEK cells), mK2-FRH is detected in an IP of FLAG-FRH (FLAG-IP). The co-IP is\ndependent on (mediated by) mK2-FRQ.\nIn panels 8B and 8C (Neurospora), endogenous untagged FRH is detected in an IP of GFP-FRH\n(GFP-pulldown). The co-IP is dependent on (mediated by) FRQ.\nTogether, these results establish that complexes with 2:2 (FRQ₂FRH₂) stoichiometry exist. See\nalso answer above.\n“To directly examine the subunit composition of FRQ-FRH complexes in the absence of CK1a,\nwe expressed the proteins in HEK293T cells”. This statement is not strictly true, HEK293T cells also possess endogenous\nCK1a.\nHEK cells do not express CK1a. They endogenously express CK1δ, which does not\nphosphorylate FRQ to a detectable extent and does not support dissociation of FRQ-FRH\ncomplexes."}]

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