Rhythmic Conidiation in Circadian Experiments

In Neurospora, the conidial banding pattern is strikingly visual and can be accurately assayed using race tubes, which makes race tubes such a versatile tool to study circadian biology. A wide variety of circadian experiments can be performed in race tubes, as circadian parameters—for instance, the period and phase of the rhythm—are easily examined. For each experiment, of course, the exact protocol will differ. However, some general conditions and considerations regarding the inoculation, incubation, and analysis of race tubes are given under Subheadings 3.2.1. through 3.2.3., respectively. Subsequently, examples of "classical" circadian experiments—free-run, phase-shift, and en-trainment—are given (see Subheading 3.2.4.).

3.2.1. Inoculation of Race Tubes

1. Make sure to have one or two fresh slants (3-10 d old) for each Neurospora strain to be used (see Note 18).

For a small number of race tubes:

2. Inoculate each race tube at one end with a small number of conidia using a flamed loop or a large, sterile clinical swab (pre-wetted in sterile H2O). There is no need to flame the necks of the race tubes; just work cleanly and near a flame.

For a large number of race tubes:

3. Add 1 to 2 mL sterile H2O to each slant, replace cotton wool plug, and vortex vigorously. Take off the spore suspension and transfer to a 1.5-mL Eppendorf tube.

4. Using a sterile filtered tip add 10 to 20 ^L of spore suspension to one end of each race tube. Again, there is no need to flame the necks of the race tubes; just work cleanly and near a flame. Vortex or shake the spore suspension regularly to maintain even distribution of spores throughout the suspension.

5. To prevent the drop of spore suspension from running toward the middle of the race tube, place a pen under the opposite end of each stack to slightly tilt the race tubes. Keep tilted for the first day of incubation.

3.2.2. Incubation of Race Tubes

1. In general, all race tube experiments are started with a period of 24 h at 25°C under constant light. This allows spores to germinate and form a straight myce-lial growth front.

2. After this initial 24 h, mark the growth front with a permanent marker over the full width of each race tube to indicate the start of the experiment and change growth conditions to the experimental conditions — e.g., constant darkness (DD)—for free-run.

3. Incubate race tubes at temperatures between 18°C and 30°C.

4. Make sure that temperature and light conditions are constant within the incubator or be consistent regarding the position of the race tubes within the incubator.

5. Mark growth fronts regularly over half the width of each race tube (see Note 19). Marking at least once every 24 to 36 h is recommended (see Note 20).

3.2.3. Analysis of Race Tubes

Analysis of race tubes is generally performed after the experiment has finished and the fungus has grown the full length of the race tube. As interbands of undifferentiated mycelia (Fig. 2) will not fill in with conidia, the banding pattern is static once formed and can be thus assayed postexperiment. A more advanced and technically challenging analysis of rhythmic conidiation is the use of time-lapse video under constant red light (15,16), with very eye-pleasing results. Assuming a linear growth rate for Neurospora, the period and phase of the rhythm can be estimated using the 24-h marks. Although this key assumption has recently been proved to be incorrect (16), it has been used extensively (and still is used) in the majority of race tube analyses. Neurospora's growth rate can vary by up to twofold with the circadian cycle, causing an estimated maximal error of 1 to 2 h (16).

3.2.3.1. Using CHRONO Software (14) and Scanning Densitometry

1. Create a picture of the race tubes by means of scanning or digital photography.

2. Save each six-pack as a separate PICT file. Use grayscale and a resolution not greater than 150 dpi.

control

DD30

DD32

DD34

DD36 DD38 DD40 DD42

DD44 DD46 DD48 DD50 DD52

Fig. 5. Densitometry plots generated using the CHRONO software. Example of densitometry plots from race tubes used in a phase response curve experiment (data from ref. 18). Race tubes were grown at a constant temperature of 25°C for 24 h in constant light (LL) and then transferred to constant darkness (DD). At different times after lights-off (DD times indicated and represented by the black arrows) a 2-min saturating light pulse was given, resulting in strong clock delays and clock advances in the antisense frq-defective strain frq10frqccg-2 (18).

3. Import the .PICTfile into CHRONO.

4. Enter the exact time for all time marks (including the start of the experiment) for each individual race tube.

5. Double-check the peaks on the densitometry plot generated by the software for each individual race tube and adjust if necessary (see Note 21).

6. Period, phase, advances, and delays can be read or calculated. Densitometry plots can be exported for easy visualization of results (Fig. 5).

control

DD30

DD32

Fig. 6. Visualization of the period of the oscillator. Neurospora strains were grown for 24 h in constant light (LL) and then transferred to constant darkness (DD), allowing free-run of the clock. (A) The free-running period of the clock in different clock mutants is easily visualized and assayed when strains are grown in race tubes. Wildtype bd strain frq+ (22 h), short-period mutant frq2 (19 h), long-period mutant frq7 (29 h), null mutantfrq10 (arrhythmic). Two race tubes per strain are shown. Strains were grown at 25°C. (B) Rhythmic conidiation is temperature-dependent in antisense frq-defective strainfrq10frqccg-2 (18). (Pictures by S. K. Crosthwaite and C. Kramer, University of Manchester, UK.)

Fig. 6. Visualization of the period of the oscillator. Neurospora strains were grown for 24 h in constant light (LL) and then transferred to constant darkness (DD), allowing free-run of the clock. (A) The free-running period of the clock in different clock mutants is easily visualized and assayed when strains are grown in race tubes. Wildtype bd strain frq+ (22 h), short-period mutant frq2 (19 h), long-period mutant frq7 (29 h), null mutantfrq10 (arrhythmic). Two race tubes per strain are shown. Strains were grown at 25°C. (B) Rhythmic conidiation is temperature-dependent in antisense frq-defective strainfrq10frqccg-2 (18). (Pictures by S. K. Crosthwaite and C. Kramer, University of Manchester, UK.)

3.2.4. Examples of Circadian Experiments in Race Tubes

Below, some race tube examples are shown (Figs. 6-8) to emphasize the ease and versatility of race tube assays in general, which have contributed greatly to our current understanding of circadian rhythmicity in Neurospora.

When Neurospora strains carrying the bd mutation (3,12) are inoculated in race tubes and are left on the lab bench under ambient light/dark conditions, distinct bands of conidia are observed once every sidereal day—every 24 h. For the Neurospora strains in these race tubes, as in all circadian systems, the presence of light is the dominant zeitgeber (17). When cultures are transferred into DD (and under constant temperature conditions), conidial bands continue to appear once every circadian day — every 22 h (the free-running period of the Neurospora clock; see Fig. 6A; see also Fig. 2). The free-running period of the

Fig. 7. Visualization of the phase of the oscillator. Neurospora strains were grown for 24 h in constant light (LL) and then transferred to constant darkness (DD). (A) Rhythmic conidiation is delayed in antisense frq-defective strain frq10frqccg-2 (18). The light-to-dark transfer sets the clock to an earlier phase in this mutant strain compared with the wild-type bd strain frq+. Strains were grown at 25°C and 30°C, as indicated. (B) Examples of clock delay and clock advance responses of the antisense frq-defective strain frq10frqccg-2 (18), grown in race tube six-packs. At 26 h and 34 h after lights-off (DD26 and DD34) a 2-min saturating light pulse was given (indicated by "L"). Strains were grown at 25°C. The phases of the clock before and after the light pulse (old phase and new phase) are easily visualized and assayed in this way. (Pictures by C. Kramer and S. K. Crosthwaite, University of Manchester, UK. Pictures from Fig. 7A first published in ref. 18 and pictures from Fig. 7B first published online in ref. 26.)

Fig. 7. Visualization of the phase of the oscillator. Neurospora strains were grown for 24 h in constant light (LL) and then transferred to constant darkness (DD). (A) Rhythmic conidiation is delayed in antisense frq-defective strain frq10frqccg-2 (18). The light-to-dark transfer sets the clock to an earlier phase in this mutant strain compared with the wild-type bd strain frq+. Strains were grown at 25°C and 30°C, as indicated. (B) Examples of clock delay and clock advance responses of the antisense frq-defective strain frq10frqccg-2 (18), grown in race tube six-packs. At 26 h and 34 h after lights-off (DD26 and DD34) a 2-min saturating light pulse was given (indicated by "L"). Strains were grown at 25°C. The phases of the clock before and after the light pulse (old phase and new phase) are easily visualized and assayed in this way. (Pictures by C. Kramer and S. K. Crosthwaite, University of Manchester, UK. Pictures from Fig. 7A first published in ref. 18 and pictures from Fig. 7B first published online in ref. 26.)

Fig. 8. Entrainment of the oscillator. Neurospora strains were grown for 24 h in constant light and then transferred to constant darkness (DD). In DD and at a constant temperature of 25°C the clock is free-running, wild-type bd strain frq+ (22 h) and null mutant frq10 (arrythmic). In DD and under a temperature regime of 12 h at 25°C/12 h at 27°C, a 24-h rhythm of conidiation is observed in both frq+ and frq10. (Pictures by P. Gould and S. K. Crosthwaite, University of Manchester, UK.)

Fig. 8. Entrainment of the oscillator. Neurospora strains were grown for 24 h in constant light and then transferred to constant darkness (DD). In DD and at a constant temperature of 25°C the clock is free-running, wild-type bd strain frq+ (22 h) and null mutant frq10 (arrythmic). In DD and under a temperature regime of 12 h at 25°C/12 h at 27°C, a 24-h rhythm of conidiation is observed in both frq+ and frq10. (Pictures by P. Gould and S. K. Crosthwaite, University of Manchester, UK.)

clock in long-period, short-period, and arrhythmic clock mutant strains can easily be visualized and assayed when grown in race tubes (Fig. 6A). Whether conidiation appears to be arrhythmic on race tubes can sometimes be temperature-dependent (Fig. 6B).

3.2.4.2. Phase-Shift

Another important feature of the circadian clock, the phase of the oscillator, is also easily assayed when Neurospora is grown on race tubes. A light-to-dark transfer sets the clock, and hence the physiological state of the culture, to a defined time (6). In wild-type frq strains the peak of conidiation is observed approx 10 h after lights off, whereas in an antisense frq-defective strain (18) the appearance of the conidial band is significantly delayed (Fig. 7A), which means that the endogenous clock in this mutant strain is set to an earlier phase by a light-to-dark transfer. Deliberate phase-shifting of the Neurospora clock can be established by exposure to light (and also temperature and social cues). In all organisms, light pulses given at different times of the circadian day will have different effects on the clock (19). Clock delays and clock advances can be easily and accurately measured when Neurospora race tube six-packs are exposed to a short light pulse (Fig. 7B).

3.2.4.3. Entrainment

Apart from light, temperature is the other major zeitgeber for circadian oscillators (17). When Neurospora is grown in DD, the conidiation rhythm is in free-run (22 h; see Fig. 6A). However, when in DD the temperature is varied with 12-h alternating periods of cooler and warmer temperature, the conidiation rhythm has a period of 24 h (Fig. 8), the rhythm is entrained. Temperature entrainment offrq-null mutant strains has been suggested previously (20) (see also Fig. 8); however, a recent investigation revealed that rhythmic conidiation is characteristic of a driven rather than entrained rhythm (21).

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