Circadian regulation of the light input pathway in Neurospora crassa - PubMed (original) (raw)
Circadian regulation of the light input pathway in Neurospora crassa
M Merrow et al. EMBO J. 2001.
Abstract
FREQUENCY (FRQ) is a critical element of the circadian system of Neurospora. The white collar genes are important both for light reception and circadian function. We show that the responsiveness of the light input pathway is circadianly regulated. This circadian modulation extends to light-inducible components and functions that are not rhythmic themselves in constant conditions. FRQ interacts genetically and physically with WHITE COLLAR-1, and physically with WHITE COLLAR-2. These findings begin to address how components of the circadian system interact with basic cellular functions, in this case with sensory transduction.
Figures
Fig. 1. Circadian time course of frq and wc-1 RNA and protein levels. frq+ was grown at 25°C in constant darkness. Samples were harvested at 2 h intervals and frq RNA (A), FRQ protein (B), wc-1 RNA (C) and WC-1 protein (D) levels were measured. Specific RNA signals were normalized to rRNA (RNA/ribo. rel. units) and specific protein signals were normalized to amido black-stained blots (rel. protein concentration). In rhythmic time series (A, B and C), the highest value was set equal to 1; in (C), the average signal was set equal to 0.5. See Materials and methods for curve fits. The initial light to dark transfer of the cells corresponds to dusk and is designated circadian time (CT) 12. See Materials and methods for determination of CT. Gray areas indicate subjective night (i.e. the half of the full circadian cycle that corresponds to darkness in a light:dark cycle); open areas are subjective day.
Fig. 2. Regulation of frq and wc-1. Mycelial pads from frq+, Δ_wc-1_, _frq_10 and frq_9 were incubated in darkness for 28 h and then RNA and protein extracts were prepared. (A) frq RNA is reduced in Δ_wc-1. frq RNA was detected by northern blotting (upper panel), quantified and normalized to rRNA levels (lower panel). The columns in the graphs represent averages of four experiments (standard deviations are given at the top of each bar). The controls _frq_10 and frq_9 show no frq or elevated RNA, respectively, as expected. (B) FRQ protein is reduced in Δ_wc-1. FRQ protein was detected by western blotting. To control for equal loading of the gel, a portion of the blot was stained with amido black (lower panel). The controls _frq_10 and frq+LL (constant light) show no FRQ protein or elevated levels, respectively. (C) wc-1 RNA is reduced in _frq_10 and _frq_9. RNA was analyzed as described above. (D) WC-1 protein levels are reduced in _frq_10 as shown by western blotting. The amido black-stained membrane indicated equal loading (lower panel).
Fig. 3. A bifurcated light input pathway: FRQ is required for conidiation but not for carotenogenesis. (A) FRQ rescues light-regulated conidiation in _frq_10. Race tubes were inoculated with frq+ (bd) or _frq_10 _qa-2p_-frq (_bd frq_10 his3::his3qa-2p-frqhis), which expresses a His-tagged version of FRQ under the control of the inducible qa-2 promoter. The race tubes were incubated in 12 h light:12 h dark (LD 12:12) cycles with or without quinic acid (QA). Approximately 4.5 cycles are shown. (B) QA-induced expression of FRQ is unaffected by light. _frq_10 _qa-2p_-frq was harvested in either the light or dark portion of the third day of an LD 12:12 cycle. At time 0, FRQ expression was induced by QA. Mycelia were harvested after 6 and 12 h and FRQ was analyzed by western blotting (left panel). Signals were quantified by densitometry. Relative expression levels (rel. FRQ conc., right panel) are the average of triplicate samples, normalized for loading based on amido black-stained membranes. (C) Induction of carotenoids by light does not require FRQ. Carotenoids were induced in frq+ (thick line), frq_10 (thin line) and Δ_wc-1 with 4 µE/m2/s of light for 5 h. Samples were extracted with hexane and absorption spectra were determined. The frq+ extract was diluted 2-fold relative to the other samples. (D) The fluence threshold of carotenogenesis is independent of FRQ. Carotenoids were induced in frq+ (filled circles) and _frq_10 (open circles) over the indicated range of fluences. Absorption of carotenoids was determined at 445 nm. Values >1.0 were measured as dilutions for accuracy. The black arrow indicates the fluence rate at which half-maximal light induction of carotenoids occurs for both strains. For comparison, the gray arrow indicates the fluence threshold for light-dependent conidial band formation in frq+ (Merrow et al., 1999; Roenneberg and Merrow, 2001).
Fig. 4. Robust light induction of wc-1 RNA requires FRQ. frq+ and _frq_10 were incubated for 28 h in the dark and then exposed to 4 µE/m2/s of light. Samples were harvested over 2 h. (A) wc-1 and rRNAs were detected by northern blot analysis. (B) wc-1 RNA was quantified and normalized to the amount of rRNA. The maximal signal was set equal to 1. Data are the average of duplicate samples and representative of three experiments.
Fig. 5. Light responses are regulated by the circadian system. Time of day-specific light induction of wc-1 RNA (A), frq RNA (B) and al-1 RNA (C). Mycelial pads were incubated for 16 h (∼CT05, unfilled circles) and 27 h (∼CT17, filled circles) in the dark and then exposed to light (0.4 µE/m2/s). After the indicated time periods, RNA was prepared, analyzed by northern blotting and quantified based on rRNA values. The maximal signal was set equal to 1. (D) Light-induced carotenogenesis over the course of a circadian cycle. frq+ (filled squares) and _frq_10 (open squares) mycelia were transferred from light to dark. After the indicated time in darkness, samples were exposed to light for 5 h to induce carotenogenesis. Carotenoids were extracted with hexane and quantified by absorption at 445 nm. Circadian time of frq+ is shown on top; the gray background indicates subjective night.
Fig. 6. Interaction of FRQ with WC-2 and WC-1. (A) Co-precipitation of FRQ with WC-2 (right panel) and control cell extracts (left panel). For immunoprecipitations, cell extracts were prepared from age-matched mycelial pads grown in DD for the indicated amounts of time. Samples were immunoprecipitated with anti-WC-2 serum (right panel), separated by SDS–PAGE and probed with antibodies against FRQ on western blots. Control cell extracts include those from wc-2-234w, _frq_10 and a strain wild-type at frq and wc-2 loci (frq+) at DD12 (∼CT01) and DD20 (∼CT10). These were probed for FRQ and indicate the relative abundance and phosphorylation state of FRQ protein at these times. (B) Co-precipitation of FRQ with WC-1. frq+, frq_10, Δ_wc-1 and wc-2-234w were grown in constant light (LL). Extracts were prepared and subjected to immunoprecipitation with antibodies against WC-1. The precipitates were analyzed by SDS–PAGE and blots were probed with antibodies against FRQ. The cell extract of frq+ cultures represents 4% of the input in the co-precipitation experiment and is shown in the right lane as an estimate of the efficiency of the co-precipitation.
Fig. 7. The molecular components of the Neurospora circadian system and the light input pathway (LIP) are inseparable. (A) Our results are compatible with a bifurcation within the LIP, with one branch leading to light-dependent (or, in the absence of light, circadian) conidiation and the other to light-induced carotenogenesis. In terms of regulation by light, WC-1 is required for both branches, while FRQ is only essential for regulated conidiation. Both are important for circadian rhythmicity. In addition, the circadian system modulates the branch regulating carotenoid synthesis, possibly via FRQ. Within the LIP, the WCs and FRQ form a functional unit responsible for both light reception and circadian modulation. (B) Genetic interactions within the Neurospora LIP. Although not specified, a basal activation of wc-1 and frq is presumed, in addition to the inferred positive effect on wc-1 RNA levels by FRQ or WC-1 (see Figure 2). Also, WC-2 is involved in frq regulation (Crosthwaite et al., 1997), but this aspect was not addressed in this work.
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