Figure 1.
Simplified representation of the Neurospora circadian clock.
Transcription factors WHITE COLLAR-1 (WC-1) and WHITE COLLAR-2 (WC-2) form a heterodimeric WHITE COLLAR COMPLEX (WCC). Early in the subjective night, the hypophosphorylated form of WCC (hypoWCC) activates the transcription of the frequency (frq) gene. Once hypoWCC activates transcription, it is degraded. The FREQUENCY protein (FRQ) accumulates, peaking around midday, and is progressively phosphorylated. Hyperphosphorylated FRQ is ubiquitinated and degraded by the proteosome. FRQ promotes phosphorylation of WCC by recruiting kinases, and phosphorylated WCC (hyperWCC) is inactive thus leading to decreased transcription of frq and consequently negative regulation of FRQ. Phosphorylated WCC is more stable than its hypophosphorylated form, thus the increase in FRQ level leads to a rise in overall WCC level.
Figure 2.
The Neurospora circadian clock model.
The symbol representations of compartments, species and reactions are shown in the right hand panel. Individual pathways are numbered starting with the transcription of the frq gene. frq = frequency, wc-1 = white collar-1, wc-2 = white collar-2, vvd = vivid, hypoFRQc = cytosolic hypophosphorylated FREQUENCY (FRQ) protein, hypoFRQn = nuclear hypophosphorylated FRQ, hyperFRQc = cytosolic hyperphosphorylated FRQ, hyperFRQn = nuclear hyperphosphorylated FRQ, WC-1c = cytosolic WHITE COLLAR-1 (WC-1) protein, WC-2c = cytosolic WHITE COLLAR-2 (WC-2) protein, hypoWCCc = cytosolic hypophosphorylated WHITE COLLAR COMPLEX (WCC), hypoWCCn = nuclear hypophosphorylated WCC, hyperWCCc = cytosolic hyperphosphorylated WCC, hyperWCCn = nuclear hyperphosphorylated WCC, aWCC = activated WCC, laWCC = light activated WCC, VVDc = cytosolic VIVID (VVD) protein, VVDn = nuclear VVD, WVC = WCC-VVD complex.
Figure 3.
(A) Experimental data showing the oscillation of frq mRNA and FRQ protein levels [41]. The period length is approximately 22 hours, and FRQ (red line) peaks 3–7 hours after frq mRNA (black line). (B) Simulated results showing the oscillation of frq mRNA and FRQ protein levels has a period of 21.6 hours, and FRQ peaks 4.4 hours after frq mRNA. Simulation begins 10 hours after a light to dark transfer, 10 data points per hour. (C) Experimental data showing wc-1 mRNA and WC-1 protein levels [42]. The level of wc-1 mRNA is nearly constant. WC-1 protein expression oscillates. (D) Simulated results. wc-1 mRNA (black line) level is constant and WC-1 oscillates. Simulation begins at the light to dark transfer, 10 data points per hour. (E) The level of WC-2 protein is 5–30 times higher than the average level of FRQ and WC-1 protein [43]. (F) In the model WC-2 protein is 10 times higher than the average level of FRQ and WC-1 proteins. Simulation begins at the light to dark transfer, 10 data points per hour are plotted.
Figure 4.
Distribution of parameters based on the value of their period and amplitude response coefficients.
The trend line indicates that period and amplitude response coefficients are negatively correlated. A = wc-1 translation, B = rate of basal transcription of wc-1, C = Michaelis constant of frq transcription, D = degradation of wc-1 mRNA, E = degradation of activated WCC.
Figure 5.
Reproduction of wc-2ER24 mutant and wc-1−, qa-WC-1 behaviour.
(A) Simulated results showing levels of frq RNA and light-activated WCCn before and after a light to dark transfer. 10 data points/h are plotted. When the light (red line) is turned off after 50 h, frq mRNA levels oscillate. (B) Simulated frq mRNA behaviour of the wc-2ER24 mutant and (C) the wc-1−, qa-WC-1 strain. In (B) and (C) frq mRNA oscillates in the dark but the oscillation dampens with time.
Figure 6.
Simulated results of light resetting, entrainment by light/dark (LD) cycles, and photoadaptation.
(A) The simulation consists of 60 h dark, 48 h constant light, 92 h constant dark. 10 data points per hour are plotted. The red line indicates light intensity. After the dark to light transfer, frq mRNA and FRQ protein increase and the oscillation is lost. Rhythmic expression begins without delay on return to darkness. (B) The simulation began with continuous dark for 60 hours, followed by three cycles of 12 h light: 12 h dark, before transfer to constant dark. The simulated clock can be entrained to 24 h with 12 h: 12 h light: dark cycles. (C) Experimental data shows the molecular behaviour of photoadaptation [25]. Light intensity (red line) of 5 mol·m−2·s−1 for 4 hours and 130 mol·m−2·s−1 for 5 h. vvd mRNA level (black line) is rapidly and transiently induced to high levels immediately after exposure to light. (D) The behaviour of vvd mRNA photoadaptation is reproduced in the model. A second increase in light intensity results in a rapid increase of vvd RNA. The levels soon fall but remain at a higher level compared to levels at the lower light intensity. 100 data points per hour were plotted.
Figure 7.
(A) Plot of experimental data showing light-induced phase shifts [45]. Light pulses given during the late subjective night and early morning result in large phase shifts. (B) Light-induced phase shifts are reproduced in the model. 0.1 h light pulses (solid line) cause larger phase shifts than 0.01 h light pulses (dashed line). Large phase shifts occurred during the late subjective night and early morning. 20 and 200 data points/h were plotted for simulated 0.1 and 0.01 h light pulse respectively.
Figure 8.
Clock components levels at 21 and 28°C.
(A) Northern blot analysis of clock-specific transcript levels in strains grown in DD at either 21°C (left panel) or 28°C (right panel). Mycelial discs from wild-type or wc1-myc were grown in liquid culture at 21 or 28°C and tissue harvested at four-hour intervals in the first day of DD. Ethidium bromide stained gels were used to correct for loading. (B) The effects of temperature on clock-specific protein levels. Western blot analysis of clock-specific protein levels in strains grown in DD at either 21°C (left panel), 25°C (representative blot not shown) or 28°C (right panel). Amido black stained membranes were used to correct for loading. (C) Quantitative analysis of Northern blot data shown in A. Maximum transcript levels were set to 100%. (D) Quantitative analysis of western blot data shown in B. Maximum protein levels were set to 100%. The graphs in (C) and (D) represent three independent experiments. Error bars indicate ±1 standard error.
Figure 9.
Simulated results showing the clock period and frq RNA and FRQ protein levels between 20 and 30°C.
In (A) Only frq translation rate changes with temperature. In (B) translation of frq and the translocation of FRQ into the nucleus are temperature-dependent. (C) The translation of frq and the translocation of FRQ into the nucleus are temperature-dependent and frq translation has different activation energies above and below 25°C. Simulated period = closed green circles, experimentally derived period = open green circles. Peak levels of FRQ (closed red circles) and peak and trough levels of frq mRNA (closed black circles).