Circadian Conidiation Rhythm Research Articles

Activation and localization of protein kinase C in Neurospora crassa

The Neurospora crassa protein kinase C (NPKC) is reported to be a regulator of light responsive genes. It phosphorylates the light receptor WC-1 and regulates the levels of the circadian clock protein FRQ and transcription of the light-induced albino-2 gene. In mammals, the conventional and novel isoforms of PKC are activated by diacylglycerol (DAG), which induces PKC translocation from the cytoplasm to membranes. To investigate the interaction of NPKC and DAG in Neurospora, we constructed a strain that expresses a PKC-GFP fusion protein. We found that NPKC localizes to growing tips and sub-apical plasma membrane in actively growing hyphae, and actively participates in septum development. NPKC is activated by exogenous DAG and phorbol esters, and translocates to the plasma membrane from the cytoplasm. We have previously reported that choline depletion of the chol-1 mutant of Neurospora increases DAG levels and lengthens the period of the circadian rhythm of conidiation. We have found that the activity of NPKC is rhythmic, and that NPKC levels are increased on choline depletion. However, over-expression of NPKC did not lengthen the conidiation period, indicating that PKC in Neurospora may not be responsible for the lengthened period in low choline cultures.

Rhythmic Conidiation in Constant Light inVividMutants ofNeurospora crassa

In Neurospora crassa, a circadian rhythm of conidiation (asexual spore formation) can be seen on the surface of agar media. This rhythm has a period of 22 hr in constant darkness (D/D). Under constant illumination (L/L), no rhythm is visible and cultures show constant conidiation. However, here we report that strains with a mutation in the vivid (vvd) gene, previously shown to code for the photoreceptor involved in photo-adaptation, exhibit conidiation rhythms in L/L as well as in D/D. The period of the rhythm of vvd strains ranges between 6 and 21 hr in L/L, depending upon the intensity of the light, the carbon source, and the presence of other mutations. Temperature compensation of the period also depends on light intensity. Dark pulses given in L/L shift the phase of the rhythm. Shifts from L/L to D/D show unexpected after effects; i.e., the short period of a vvd strain in L/L gradually lengthens over 2-3 days in D/D. The rhythm in L/L requires the white collar (wc-1) gene, but not the frequency (frq) gene. FRQ protein shows no rhythm in L/L in a vvd strain. The conidiation rhythm in L/L in vvd is therefore driven by a FRQ-less oscillator (FLO).

The effects of temperature change on the circadian clock of Neurospora.

The phase resetting of the circadian oscillatory system by pulses of increased temperature (zeitgebers) and the temperature compensation of its period length during longer exposures are major features of the system, but are not well understood in molecular terms. In Neurospora crassa, the effects of pulses of increased temperature on the circadian rhythm of conidiation were determined and possible inputs to the oscillatory system tested, including changes in cyclic 3',5'-adenosine monophosphate (cAMP), inositol 1,4,5-trisphosphate and H+ concentrations, as well as changes of phosphorylation, synthesis and degradation of proteins. Following the kinetics of these parameters during exposure to increased temperature showed transient changes. Experimental manipulation of cAMP, Ca2+ and H+ levels, and of the synthesis and, possibly, degradation of proteins, resulted in phase shifts of the oscillatory system. It is assumed that the temperature signal affects the oscillator(s) by multiple pathways and shifts the whole state of the oscillatory system. Second messenger levels, protein synthesis and protein degradation show adaptation to longer exposures to elevated temperature which may be involved in the temperature compensation of the period length. The temperature compensation is also proposed to involve a shift in the state of all or most oscillator variables.

A new wc-1 mutant of Neurospora crassa shows unique light sensitivity in the circadian conidiation rhythm.

A new clock mutant ( rhy-2) was isolated by DNA insertion mutagenesis using a plasmid that contains a region located upstream of the cmd gene in the genome of Neurospora crassa. This mutant is arrhythmic with regard to conidiation in continuous darkness but rhythmic under a light-dark cycle. After plasmid rescue from genomic DNA of the rhy-2 strain, the insertion was localized to the gene white collar-1 ( wc-1). Plasmid DNA was inserted 3' to the sequence encoding the polyglutamine region of the WC-1 gene product, and an mRNA encoding a truncated WC-1 protein must be synthesized under the control of the cmd promoter. The new wc-1 mutant, rhy-2, is still sensitive to light, although only weakly. Since the circadian rhythm of conidiation in continuous darkness is eliminated in the mutant, the polyglutamine region in WC-1 may be essential for both clock function and light-induced carotenogenesis in Neurospora.

Identification of a calcium/calmodulin-dependent protein kinase that phosphorylates the Neurospora circadian clock protein FREQUENCY.

Phosphorylation of circadian clock proteins represents a major regulatory step that controls circadian clocks. In Neurospora, the circadian clock protein FREQUENCY (FRQ) is progressively phosphorylated over time, and its level decreases when it is hyperphosphorylated. In this study, we showed that most of the kinase activity phosphorylating FRQ in vitro was calcium/calmodulin-dependent, and the endogenous FRQ in the Neurospora extracts was phosphorylated by a Ca/CaM-dependent kinase-like activity. From Neurospora cell extracts, an approximately 50-kDa Ca/CaM-dependent kinase (CAMK-1) that can specifically phosphorylate FRQ was purified. In vitro, this kinase accounts for near half of the FRQ kinase activity, and it can phosphorylate the FRQ region that contains the three known functionally important phosphorylation sites. To understand the function of camk-1 in vivo, it was disrupted in Neurospora by gene replacement. After germination from ascospores, the camk-1 null strains grew slowly, indicating that CAMK-1 plays an important role in growth and development of Neurospora. This phenotype was transient however, revealing redundancy in the system. Analysis of the camk-1 null strain revealed that the deletion of camk-1 affected phase, period, and light-induced phase shifting of the circadian conidiation rhythm. Taken together, our results suggest that multiple kinases may phosphorylate FRQ in vivo.

Vvd Is Required for Light Adaptation of Conidiation-Specific Genes of Neurospora crassa, but Not Circadian Conidiation

con-10 and con-6 are two of the conidiation (con) genes of Neurospora crassa that were identified based on their preferential expression during macroconidiophore development. They are also regulated by several other environmental stimuli independent of development, including a transient induction by light. We identified an allele of vivid (vvd) in a mutant screen designed to obtain strains with altered expression of con-10. vvd mutants display enhanced carotenoid pigmentation in response to light. In addition, con-10 and con-6 show a heightened response to photoinduction. We tested the function of the light-responsive circadian clock in the vvd mutant and found no major defect in the circadian rhythm of conidiation or light regulation of a key clock component, frequency (frq). We conclude that vvd is primarily involved in a process of light-dependent gene repression, called light adaptation. Although a number of gene products are known to control light induction in fungi, vvd is the first gene shown to have a role in adaptation to constant light.

Choline depletion, frq mutations, and temperature compensation of the circadian rhythm in Neurospora crassa.

In the fungus Neurospora crassa, the chol-1 mutation blocks the synthesis of the lipid phosphatidylcholine and also lengthens the period of the circadian rhythm of conidiation under conditions of choline depletion. The frq mutations, which have no known metabolic defect, affect both the period of the rhythm and temperature compensation. In this article, the chol-1 mutant strain has been further characterized with respect to its temperature compensation and its interactions with frq. Choline depletion of chol-1 abolishes good temperature compensation: Low temperatures lengthen the period under choline-depleted conditions, and low choline lengthens the period at any one temperature. Double-mutant strains carrying both chol-1 and one of a series of frq alleles demonstrate interactions between chol-1 and frq: On high choline, the periods of the double mutants are identical to the corresponding chol+ strains, whereas on low choline all double mutants display very long periods (greater than 50 h). Short-period frq mutations shorten the long period on low choline, whereas long-period frq mutations frq mutations have no effect. A null frq mutation in the chol-1 background is arrhythmic on high choline but is robustly rhythmic on low choline and has no effect on the long period. The interactions between frq and chol-1 are similar to the interactions between frq and cel, another lipid-deficient mutant. These results provide support for the hypothesis that membrane lipids may be involved in temperature compensation of the circadian rhythm. The possibility is discussed that the frq gene may not be required for circadian rhythmicity under some conditions and therefore may not be a central component of the circadian oscillator but rather a component of an input pathway.

Mutation of the gene for the second-largest subunit of RNA polymerase I prolongs the period length of the circadian conidiation rhythm in Neurospora crassa.

The period length of the circadian conidiation rhythm was examined in a mutant strain of Neurospora crassa, un-18, that is temperature sensitive for mycelial growth. The un-18 mutant showed a temperature-sensitive phenotype with respect to both mycelial growth and the period length of the conidiation rhythm. Below 22 degrees C, the un-18 mutation did not affect the period length, but at temperatures between 22 degrees C and 32 degrees C, the period length of the un-18 mutant was approximately 2 h longer than that of the wild-type strain. The un-18+ gene was cloned and was found to encode the second-largest subunit of RNA polymerase I, which is involved in the synthesis of rRNA. These results indicate that a defect in ribosome synthesis, which must result in a lower rate of protein synthesis, lengthens the period of the circadian conidiation rhythm in Neurospora.

Effects of light-dark cycles on the circadian conidiation rhythm inNeurospora crassa

The effects of 24 hr light-dark cycles on the circadian conidiation rhythm inNeurospora crassa were compared among will-typefrq + and clock mutantsfrq +,frq 3,frq 7,frq 9 andfrq 11. The minimum length of the light period necessary for complete entrainment to the light-dark cycles was almost 2 hr infrq +,frq 3 andfrq 7 strains. The minimum duration of the dark period necessary for the appearance of circadian conidiation was almost 4 hr in all of the strains except thefrq 11 strain. The phase of the conidiation rhythm was dependent on the light to dark transition in thefrq 1 strain in all light-dark cycles examined and in thefrq + andfrq 3 strains when the light period was shorter than 16 hr. In contrast, the phase of thefrq 7 strain was dependent on the light to dark transition when the light period was shorter than 10 hr.

Mutation of the cys-9 gene, which encodes thioredoxin reductase, affects the circadian conidiation rhythm in Neurospora crassa.

Ten cysteine auxotrophs of Neurospora crassa were examined with regard to the period lengths of their circadian conidiation rhythms. One of the these cysteine auxotrophs, cys-9, showed dramatic changes in the circadian conidiation rhythm. At 10 microM methionine, the cys-9 mutant had a period length that was 5 hr shorter than that of the wild-type strain during the first 3 days after transfer to continuous darkness. At this concentration of methionine, the period length was unstable after the fourth day and varied widely from 11 to 31 hr. In contrast, other cysteine auxotrophs did not show such instability of the period length at any of the concentrations of methionine tested. Furthermore, only the cys-9 mutant exhibited partial loss of the capacity for temperature compensation of the period length. With regard to cold-induced phase-shifting of the circadian conidiation rhythm, the cys-9 mutant was more sensitive than the wild-type strain to low temperature. The cys-9+ gene was cloned and was found to encode NADPH-dependent thioredoxin reductase. These results indicate that mutation of the gene for thioredoxin reductase results in abnormal expression of the circadian conidiation rhythm in N. crassa.

Light-induced phase shifting of the circadian conidiation rhythm is inhibited by calmodulin antagonists in Neurospora crassa.

The effects of calmodulin antagonists and inhibitors of protein kinases and phosphatases on light-induced phase shifting were investigated in Neurospora crassa. Calmodulin antagonists, namely, trifluoperazine, chlorpromazine, and W-7, almost completely inhibited the light-induced phase shifting without having any effect on the circadian clock itself. Chlorpromazine was less effective in inhibiting the light-induced phase shifting than trifluoperazine. W-5, a dechlorinated analogue of W-7, failed to inhibit the light-induced phase shifting at the same concentration as that at which W-7 was effective. These results suggest that calmodulin is required during signal transduction from the light-perceiving system to the circadian clock in N. crassa. Inhibitors of protein phosphorylation did not inhibit the light-induced phase shifting, although these inhibitors completely inhibited mycelial growth. Trifluoperazine partially inhibited the phosphorylation of three proteins when phosphorylation was assayed in vitro.

Mutants with altered sensitivity to a calmodulin antagonist affect the circadian clock in Neurospora crassa.

Two newly isolated mutant strains of Neurospora crassa, cpz-1 and cpz-2, were hypersensitive to chlorpromazine with respect to mycelial growth but responded differently to the drug with respect to the circadian conidiation rhythm. In the wild type, chlorpromazine caused shortening of the period length of the conidiation rhythm. Pulse treatment with the drug shifted the phase and inhibited light-induced phase shifting in Neurospora. By contrast to the wild type, the cpz-2 strain was resistant to these inhibitory effects of chlorpromazine. Inhibition of cpz-2 function by chlorpromazine affected three different parameters of circadian conidiation rhythm, namely, period length, phase and light-induced phase shifting. These results indicate that the cpz-2 gene must be involved in or related closely to the clock mechanism of Neurospora. By contrast, the cpz-1 strain was hypersensitive to chlorpromazine with respect to the circadian conidiation rhythm.

Synthesis of mRNA is Required for Light-Induced Phase Shifting of the Circadian Conidiation Rhythm in Neurospora crassa

Synthesis of mRNA is Required for Light-Induced Phase Shifting of the Circadian Conidiation Rhythm in Neurospora crassa

Effects of Temperature on the Circadian Conidiation Rhythm of Temperature-Sensitive Mutants of Neurospora crassa

Effects of Temperature on the Circadian Conidiation Rhythm of Temperature-Sensitive Mutants of Neurospora crassa

Continuous shortening of the circadian conidiation rhythm inneurospora crassain short light‐dark cycles with enhanced light intensities

Abstract The role of light intensity in the entrainment (synchronisation) and the shortest entrainable period of the circadian conidiation rhythm of Neurospora crassa were tested by growing the fungus on solid media under different light‐dark‐regimes. The mutant strains bd, csp‐1‐bd and frq‐1‐bd could be entrained (synchronized) to a period of 12 h. The light phase of this period was inversely correlated with the log of the light intensity. Enhancing the irradiation to 3.65 W/m2 white light, the mutant csp‐l‐bd could be entrained by as short a period as 2: 2 h. An alteration in the effective wave lengths could not be observed. These results question a total identity between this rhythm and the biological clock found in other eucaryotic organisms.

BLUE LIGHT PHOTORECEPTION IN Neurospora ORCADIAN RHYTHM: EVIDENCE FOR INVOLVEMENT OF THE FLAVIN TRIPLET STATE

The mechanism of the photoreceptor acting on the circadian conidiation rhythm of Neurospora crassa was studied, with the following results: (1) the efficiency of 8-haloflavins as sensitizers increased with their triplet yields. (2) Phase shifts were not abolished by removal of oxygen prior to illumination. (3) Oxygen inhibited phase shifts when introduced into the cultures after light treatment. It is proposed that the blue light photoreceptor for the circadian clock of Neurospora crassa acts (1) from its triplet state, but (2) not via singlet oxygen; (3) signal transduction involves (an) oxygen-sensitive intermediate(s).

On the role of Ca2+-calmodulin-dependent and cAMP-dependent protein phosphorylation in the circadian rhythm ofNeurospora crassa

Pulses of some Ca2+ channel blockers (dantrolene, Co2+, nifedipine) and calmodulin inhibitors (chlorpromazine) lead to medium (maximally 5-9 h) phase shifts of the circadian conidiation rhythm of Neurospora crassa. Pulses of high Ca2+, or of low Ca2+, a Ca2+ ionophore (A23187) together with Ca2+, and other Ca2+ channel blockers (La3+, diltiazem), however, caused only minor phase shifts. The effect of these substances (A 23187) and of different temperatures on the Ca2+ release from isolated vacuoles was analyzed by using the fluorescent dye Fura-2. A 23187 and higher temperatures increased the release drastically, whereas dantrolene decreased the permeation of Ca2+ (Cornelius et al., 1989). Pulses of 8-PCTP-cAMP, IBMX and of the cAMP antagonist RP-cAMPS, also caused medium (maximally 6-9 h) phase shifts of the conidiation rhythm. The phase response curve of the agonist was almost 180 degrees out of phase with the antagonist PRC. In spite of some variability in the PRCs of these series of experiments all showed maximal shifts during ct 0-12. The variability of the response may be due to circadian changes in the activity of phosphodiesterases: After adding cAMP to mycelial extracts HPLC analysis of cAMP metabolites showed significant differences during a circadian period with a maximum at ct 0. Protein phosphorylation was tested mainly in an in vitro phosphorylation system (with 35S-thio gamma-ATP). The results showed circadian rhythmic changes predominantly in proteins of 47/48 kDa. Substances and treatments causing phase-shifts of the conidiation rhythm also caused changes in the phosphorylation of these proteins: an increase was observed when Ca2+ or cAMP were added, whereas a decrease occurred upon addition of a calmodulin inhibitor (TFP) or pretreatment of the mycelia with higher (42 degrees C) temperatures. Altogether, the results indicate that Ca2(+)-calmodulin-dependent and cAMP-dependent processes play an important, but perhaps not essential, role in the clock mechanism of Neurospora. Ca2+ calmodulin and the phosphorylation state of the 47/48-kDa proteins may have controlling or essential functions for this mechanism.

Phase Determination of the Circadian Rhythm of Conidiation in Heterocaryons between two out-of-Phase Mycelia in Neurospora crassa

Neurospora grows vegetatively as a syncytium in which multiple nuclei exist within a connected cytoplasm. Because of the ability of separate and distinct mycelia to fuse, the possibility exists of generating heterocaryotic cultures in which the nuclei and cytoplasms of two different strains are comingled into the same syncytium. We have used such heterocaryons, in which the component parts differed with respect to their circadian clock phase, to examine whether or not clock-dominant phases exist in the circadian cycle. To this end, the phase subsequent to the formation of heterocaryons by pairs of mycelial discs that are initially at different circadian phases was examined in Neurospora crassa. The resulting phase was an average of the parent phases in many cases, but was sometimes observed to correspond more closely to just one of the original parental phases. In these cases, we did not observe any dominant phases in the circadian cycle; the phase of a particular parent disc was more dominant in the heterocaryon when the proportion of the nuclei from that parent was greater in the heterocaryon. In some instances, which occurred mostly when the difference in phase of the parental discs was large, the resultant phase could not be related in a simple way to the parental phases. An interpretation based on a limit cycle model of the circadian oscillation is possible.

The biological clock of neurospora in a microgravity environment

The circadian rhythm of conidiation in Neurospora crassa is thought to be an endogenously derived circadian oscillation; however, several investigators have suggested that circadian rhythms may, instead, be driven by some geophysical time cue(s). An experiment was conducted on space shuttle flight STS-9 in- order to test this hypothesis; during the first 7–8 cycles in space, there were several minor alterations observed in the conidiation rhythm, including an increase in the period of the oscillation, an increase in the variability of the growth rate and a diminished rhythm amplitude, which eventually damped out in 25% of the flight tubes. On day seven of flight, the tubes were exposed to light while their growth fronts were marked. Some aspect of the marking process reinstated a robust rhythm in all the tubes which continued throughout the remainder of the flight. These results from the last 86 hours of flight demonstrated that the rhythm can persist in space. Since the aberrant rhythmicity occurred prior to the marking procedure, but not after, it was hypothesized that the damping on STS-9 may have resulted from the hypergravity pulse of launch. To test this hypothesis, we conducted investigations into the effects of altered gravitational forces on conidiation. Exposure to hypergravity (via centrifugation), simulated microgravity (via the use of a clinostat) and altered orientations (via alterations in the vector of a 1 g force) were used to examine the effects of gravity upon the circadian rhythm of conidiation.

Comparison of phase shifting by temperature of wild typeNeurospora crassaand the clock mutant,frq‐7

Abstract Phase shifting of the circadian conidiation rhythm of Neurospora crassa by pulse treatments with different temperatures, which ranged from 10° to 35°, was compared for the wild type and the clock mutant, frq‐7, in liquid culture. Both strains showed similar phase response curves’ for high temperature (35°) but different phase response curves for low temperature (15°). In the wild type strain, pulses with temperatures higher than 25° caused small shifts in phase in the subjective day phase but pulses with temperatures lower than 25° caused large phase‐delay in the same phase. The clock mutant, frq‐7, showed similar responses to temperature pulses in phases other than the late subjective day phase (CT 9 to CT 14), during which frq‐7 was almost intensitive to pulses with temperatures lower than 25°. This phase (CT 9 to CT 14) coincided with the phase which has previously been proposed to be affected by mutations at the frq locus.