Assay and Characteristics of Circadian Rhythmicity in Liquid Cultures of Neurospora crassa

Previous work on circadian rhythms of Neurospora crassa has been done almost exclusively with cultures expressing rhythmic conidiation and growing on solid agar medium. Such conditions severely restrict the kinds of biochemical experiments that can be carried out. We have now developed systems which allow indirect assay of circadian rhythmicity in liquid culture. Neurospora was grown in glucose and acetate liquid media under conditions which result in a range of growth rates and morphologies. Liquid media were inoculated with conidia and the cultures were grown in constant light for 33 or 48 hours, by which time floating mycelial pads had formed. Experimental pieces of mycelium then were cut and placed in fresh new liquid medium. As controls, other pieces of mycelium were cut and put directly on solid agar medium in race tubes. All cultures were transferred to constant darkness at this time. This light-to-dark transition set the phase of the circadian clock of both the liquid and solid cultures. At various times after the light-to-dark transition, the mycelial pieces in the liquid were transferred in the dark to solid medium in race tubes, where they grew normally and conidiated rhythmically. Comparison of the phase of the rhythm in these race tubes to the controls demonstrated that, under appropriate conditions, the circadian clock of the liquid cultures functions normally for at least two cycles in constant conditions. Using these culture systems, a significantly greater variety of biochemical studies of circadian rhythmicity in Neurospora is now possible.     

Photoperiod sensitivity of the Arabidopsis circadian clock is tissue-specific

Tissue-specific functions of the circadian clock in Arabidopsis have recently been revealed. The vasculature clock shows distinctive gene expression profiles compared to the clock in other tissues under light-dark cycles. However, it has not yet been established whether the vasculature clock also shows unique gene expression patterns that correlate with temperature cycles, another important environmental cue. Here, we detected diel phase of TIMING OF CAB EXPRESSION 1 (TOC1) expression in the vasculature and whole leaf under long-day light-dark cycles and temperature cycles. We found that the vasculature clock had advanced TOC1 phase under light-dark cycles but not under temperature cycles, suggesting that the vasculature clock has lower sensitivity against temperature signals. Furthermore, the phase advancement of TOC1 was seen only under long-day condition but not under short-day condition. These results support our previous conclusion that the circadian clock in vasculature preferentially senses photoperiodic signals.     

Changes in Intracellular pH Are Not Correlated with the Circadian Rhythm of Neurospora

Intracellular pH (pH_i) was measured during the circadian cycle of Neurospora. Internal pH of Neurospora cultures in liquid medium was assayed by the 5,5-dimethyl-2,4-oxazolidinedione method and gave values for pH_i which were similar to those previously obtained by other workers using pH-microelectrodes with agar-grown cultures. Cytoplasmic pH changed in liquid medium cultures, but these changes were not related to the circadian clock. Furthermore, treatments which raise or lower pH_i do not phase-shift the circadian rhythm. These results indicate that pH_i plays no specific role in regulating the circadian clock of Neurospora.     

Ultradian Growth in Prochlorococcus spp.

Species of the widespread marine prokaryote Prochlorococcus exhibited ultradian growth (faster than 1 division per day) both in situ and in culture, even though cell division is strictly phased to the light-dark cycle. Under optimal conditions a second DNA replication and cell division closely followed, but did not overlap with, the first division. The timing of cell cycle events was not affected by light intensity or duration, suggesting control by a light-triggered timer or circadian clock rather than by completion of a light-dependent assimilation phase. This mode of ultradian growth has not been observed previously and poses new questions about the regulation of cellular rhythms in prokaryotes. In addition, it implies that conclusions regarding the lack of nutrient limitation of Prochlorococcus in the open ocean, which were based on the appearance that cells were growing at their maximal rate, need to be reconsidered.     

Circadian Clock Genes Contribute to the Regulation of Hair Follicle Cycling

Hair follicles undergo recurrent cycling of controlled growth (anagen), regression (catagen), and relative quiescence (telogen) with a defined periodicity. Taking a genomics approach to study gene expression during synchronized mouse hair follicle cycling, we discovered that, in addition to circadian fluctuation, CLOCK–regulated genes are also modulated in phase with the hair growth cycle. During telogen and early anagen, circadian clock genes are prominently expressed in the secondary hair germ, which contains precursor cells for the growing follicle. Analysis of Clock and Bmal1 mutant mice reveals a delay in anagen progression, and the secondary hair germ cells show decreased levels of phosphorylated Rb and lack mitotic cells, suggesting that circadian clock genes regulate anagen progression via their effect on the cell cycle. Consistent with a block at the G1 phase of the cell cycle, we show a significant upregulation of p21 in Bmal1 mutant skin. While circadian clock mechanisms have been implicated in a variety of diurnal biological processes, our findings indicate that circadian clock genes may be utilized to modulate the progression of non-diurnal cyclic processes.     

Human Gut Bacteria Are Sensitive to Melatonin and Express Endogenous Circadian Rhythmicity

Circadian rhythms are fundamental properties of most eukaryotes, but evidence of biological clocks that drive these rhythms in prokaryotes has been restricted to Cyanobacteria. In vertebrates, the gastrointestinal system expresses circadian patterns of gene expression, motility and secretion in vivo and in vitro, and recent studies suggest that the enteric microbiome is regulated by the host’s circadian clock. However, it is not clear how the host’s clock regulates the microbiome. Here, we demonstrate at least one species of commensal bacterium from the human gastrointestinal system, Enterobacter aerogenes, is sensitive to the neurohormone melatonin, which is secreted into the gastrointestinal lumen, and expresses circadian patterns of swarming and motility. Melatonin specifically increases the magnitude of swarming in cultures of E. aerogenes, but not in Escherichia coli or Klebsiella pneumoniae. The swarming appears to occur daily, and transformation of E. aerogenes with a flagellar motor-protein driven lux plasmid confirms a temperature-compensated circadian rhythm of luciferase activity, which is synchronized in the presence of melatonin. Altogether, these data demonstrate a circadian clock in a non-cyanobacterial prokaryote and suggest the human circadian system may regulate its microbiome through the entrainment of bacterial clocks.     

Improved automated monitoring and new analysis algorithm for circadian phototaxis rhythms in Chlamydomonas

Automated monitoring of circadian rhythms is an efficient way of gaining insight into oscillation parameters like period and phase for the underlying pacemaker of the circadian clock. Measurement of the circadian rhythm of phototaxis (swimming towards light) exhibited by the green alga Chlamydomonas reinhardtii has been automated by directing a narrow and dim light beam through a culture at regular intervals and determining the decrease in light transmittance due to the accumulation of cells in the beam. In this study, the monitoring process was optimized by constructing a new computer-controlled measuring machine that limits the test beam to wavelengths reported to be specific for phototaxis and by choosing an algal strain, which does not need background illumination between test light cycles for proper expression of the rhythm. As a result, period and phase of the rhythm are now unaffected by the time a culture is placed into the machine. Analysis of the rhythm data was also optimized through a new algorithm, whose robustness was demonstrated using virtual rhythms with various noises. The algorithm differs in particular from other reported algorithms by maximizing the fit of the data to a sinusoidal curve that dampens exponentially. The algorithm was also used to confirm the reproducibility of rhythm monitoring by the machine. Machine and algorithm can now be used for a multitude of circadian clock studies that require unambiguous period and phase determinations such as light pulse experiments to identify the photoreceptor(s) that reset the circadian clock in C. reinhardtii.     

Circadian-clock control of protein synthesis and degradation in Gonyaulax polyedra

In growing cultures of the dinoflagellate, Gonyaulax polyedra, total protein synthesis showed a circadian rhythm with a maximum during the phase of the cycle which corresponded to the previous darktime. The maximum coincided with the maximal phase shift of the glow rhythm caused by lower concentrations of the antibiotic anisomycin (Taylor, W., et al., 1982). J. Comp. Physiol. 148 B, 11–25. The dose reponses of inhibition of protein synthesis correlated well with the phase shifting by anisomycin. The amplitude and level of the total-protein synthesis rhythm increased with the growth rate, indicating that the majority of proteins controlled by the circadian clock were cell cycle-dependent. The degradation rate showed the same circadian rhythm as the synthesis rate. Slight variations in uptake and pool size of amino acids were not responsible for the rhythm in the protein-synthesis rate.     

Clock Genes and Behavioral Responses to Light Are Altered in a Mouse Model of Diabetic Retinopathy

There is increasing evidence that melanopsin-expressing ganglion cells (ipRGCs) are altered in retinal pathologies. Using a streptozotocin-induced (STZ) model of diabetes, we investigated the impact of diabetic retinopathy on non-visual functions by analyzing ipRGCs morphology and light-induced c-Fos and Period 1–2 clock genes in the central clock (SCN). The ability of STZ-diabetic mice to entrain to light was challenged by exposure animals to 1) successive light/dark (LD) cycle of decreasing or increasing light intensities during the light phase and 2) 6-h advance of the LD cycle. Our results show that diabetes induces morphological changes of ipRGCs, including soma swelling and dendritic varicosities, with no reduction in their total number, associated with decreased c-Fos and clock genes induction by light in the SCN at 12 weeks post-onset of diabetes. In addition, STZ-diabetic mice exhibited a reduction of overall locomotor activity, a decrease of circadian sensitivity to light at low intensities, and a delay in the time to re-entrain after a phase advance of the LD cycle. These novel findings demonstrate that diabetes alters clock genes and behavioral responses of the circadian timing system to light and suggest that diabetic patients may show an increased propensity for circadian disturbances, in particular when they are exposed to chronobiological challenges.     

Cell and tissue-autonomous development of the circadian clock in mouse embryos

The emergence of the circadian rhythm is a dramatic and physiologically essential event for mammals to adapt to daily environmental cycles. It has been demonstrated that circadian rhythms develop during the embryonic stage even when the maternal central pacemaker suprachiasmatic nucleus has been disrupted. However, the mechanisms controlling development of the circadian clock are not yet fully understood. Here, we show that the circadian molecular oscillation in primary dispersed embryonic cells and explanted salivary glands obtained from mPER2^Luc mice embryos developed cell- or tissue-autonomously even in tissue culture conditions. Moreover, the circadian clock in the primary mPER2^Luc fibroblasts could be reprogrammed by the expression of the reprogramming factors. These findings suggest that mammalian circadian clock development may interact with cellular differentiation mechanisms.     

Circadian Clock Genes Are Essential for Normal Adult Neurogenesis,Differentiation, and Fate Determination

Adult neurogenesis creates new neurons and glia from stem cells in the human brain throughout life. It is best understood in the dentate gyrus (DG) of the hippocampus and the subventricular zone (SVZ). Circadian rhythms have been identified in the hippocampus, but the role of any endogenous circadian oscillator cells in hippocampal neurogenesis and their importance in learning or memory remains unclear. Any study of stem cell regulation by intrinsic circadian timing within the DG is complicated by modulation from circadian clocks elsewhere in the brain. To examine circadian oscillators in greater isolation, neurosphere cultures were prepared from the DG of two knockout mouse lines that lack a functional circadian clock and from mPer1::luc mice to identify circadian oscillations in gene expression. Circadian mPer1 gene activity rhythms were recorded in neurospheres maintained in a culture medium that induces neurogenesis but not in one that maintains the stem cell state. Although the differentiating neural stem progenitor cells of spheres were rhythmic, evidence of any mature neurons was extremely sparse. The circadian timing signal originated in undifferentiated cells within the neurosphere. This conclusion was supported by immunocytochemistry for mPER1 protein that was localized to the inner, more stem cell-like neurosphere core. To test for effects of the circadian clock on neurogenesis, media conditions were altered to induce neurospheres from BMAL1 knockout mice to differentiate. These cultures displayed unusually high differentiation into glia rather than neurons according to GFAP and NeuN expression, respectively, and very few BetaIII tubulin-positive, immature neurons were observed. The knockout neurospheres also displayed areas visibly devoid of cells and had overall higher cell death. Neurospheres from arrhythmic mice lacking two other core clock genes, Cry1 and Cry2, showed significantly reduced growth and increased astrocyte proliferation during differentiation, but they generated normal percentages of neuronal cells. Neuronal fate commitment therefore appears to be controlled through a non-clock function of BMAL1. This study provides insight into how cell autonomous circadian clocks and clock genes regulate adult neural stem cells with implications for treating neurodegenerative disorders and impaired brain functions by manipulating neurogenesis.     

Circadian Control of Dendrite Morphology in the Visual System of Drosophila melanogaster

Background

In the first optic neuropil (lamina) of the fly''s visual system, monopolar cells L1 and L2 and glia show circadian rhythms in morphological plasticity. They change their size and shape during the day and night. The most pronounced changes have been detected in circadian size of the L2 axons. Looking for a functional significance of the circadian plasticity observed in axons, we examined the morphological plasticity of the L2 dendrites. They extend from axons and harbor postsynaptic sites of tetrad synaptic contacts from the photoreceptor terminals.

Methodology/Principal Findings

The plasticity of L2 dendrites was evaluated by measuring an outline of the L2 dendritic trees. These were from confocal images of cross sections of L2 cells labeled with GFP. They were in wild-type and clock mutant flies held under different light conditions and sacrified at different time points. We found that the L2 dendrites are longest at the beginning of the day in both males and females. This rhythm observed under a day/night regime (LD) was maintained in constant darkness (DD) but not in continuous light (LL). This rhythm was not present in the arrhythmic per⁰¹ mutant in LD or in DD. In the clock photoreceptor cry^b mutant the rhythm was maintained but its pattern was different than that observed in wild-type flies.

Conclusions/Significance

The results obtained showed that the L2 dendrites exhibit circadian structural plasticity. Their morphology is controlled by the per gene-dependent circadian clock. The L2 dendrites are longest at the beginning of the day when the daytime tetrad presynaptic sites are most numerous and L2 axons are swollen. The presence of the rhythm, but with a different pattern in cry^b mutants in LD and DD indicates a new role of cry in the visual system. The new role is in maintaining the circadian pattern of changes of the L2 dendrite length and shape.     

Morphological changes of Campylobacter jejuni growing in liquid culture

Campylobacter jejuni growing in liquid culture was found to exhibit gross morphological changes with time. Exponentially growing cells showed typical short spiral forms. At mid-stationary phase the cells became approximately twice the length of the exponential forms. Late stationary/early decline phase cells were seen to be a mixture of coccal forms and cells which were between 3 and 4 times the length of exponentially growing cells. Continued incubation of cultures eventually resulted in a population largely of coccal forms. These morphological changes have not previously been observed when Camp. jejuni has been grown on agar-based solid medium. It is likely that such changes result from the differential expression of genes that control the timing of cell division.     

The S phase is discrete and is controlled by the circadian clock in the marine dinoflagellate Gonyaulax polyedra

Cloned cultures of the dinoflagellate Gonyaulax polyedra grown in a 12-h light-12-h dark cycle (LD 12:12) were synchronized to the beginning of G₁ by a two sequential filtration technique. After the second filtration, with the cultures growing in LD 12:12, not many cells had divided after 1 day, but approximately half underwent cell division after 2 days. Flow cytometric measurements of the cells revealed that there is one unique S phase starting about 12 h prior to cell division and lasting for less than 4 h. A majority of cells in cultures synchronized in the same way but maintained in continuous light (LL) after filtration also divided synchronously after 2 days. Just as for the cultures in LD 12:12, those in LL have a similar discrete DNA synthesis phase prior to division. It is concluded that the circadian control of cell division acts before the S phase, giving rise to a discontinuous DNA synthesis phased by the circadian clock.     

Effects of Respiratory Inhibitors on Respiration, ATP Contents, and the Circadian Conidiation Rhythm of Neurospora crassa

Effects of respiratory inhibitors on the circadian clock, respiratory activity, and ATP content were examined in Neurospora crassa. All inhibitors, potassium cyanide, sodium azide, antimycin A, and carbonyl cyanide m-chlorophenyl hydrazone (CCCP), shifted the phase of the conidiation rhythm. All the phase response curves were similar and resembled that for cycloheximide, but were different from the phase response curve for light. Phase shifting by azide and CCCP was proportional to the lowering of respiratory activity and ATP content, but such a correlation was not observed for cyanide and antimycin A. In particular, cyanide at a concentration of 0.5 millimolar completely depleted ATP of the cultures but did not significantly shift their phase. Their results suggest that large shifts caused by these inhibitors are not due to a decrease in energy from respiratory activity.     

Diurnal Oscillations of Soybean Circadian Clock and Drought Responsive Genes

Rhythms produced by the endogenous circadian clock play a critical role in allowing plants to respond and adapt to the environment. While there is a well-established regulatory link between the circadian clock and responses to abiotic stress in model plants, little is known of the circadian system in crop species like soybean. This study examines how drought impacts diurnal oscillation of both drought responsive and circadian clock genes in soybean. Drought stress induced marked changes in gene expression of several circadian clock-like components, such as LCL1-, GmELF4- and PRR-like genes, which had reduced expression in stressed plants. The same conditions produced a phase advance of expression for the GmTOC1-like, GmLUX-like and GmPRR7-like genes. Similarly, the rhythmic expression pattern of the soybean drought-responsive genes DREB-, bZIP-, GOLS-, RAB18- and Remorin-like changed significantly after plant exposure to drought. In silico analysis of promoter regions of these genes revealed the presence of cis-elements associated both with stress and circadian clock regulation. Furthermore, some soybean genes with upstream ABRE elements were responsive to abscisic acid treatment. Our results indicate that some connection between the drought response and the circadian clock may exist in soybean since (i) drought stress affects gene expression of circadian clock components and (ii) several stress responsive genes display diurnal oscillation in soybeans.     

Signaling in the mammalian circadian clock: the NO/cGMP pathway

Mammalian circadian rhythms are generated by a hypothalamic suprachiasmatic nuclei (SCN) clock. Light pulses synchronize body rhythms by inducing phase delays during the early night and phase advances during the late night. Phosphorylation events are known to be involved in circadian phase shifting, both for delays and advances. Pharmacological inhibition of the cGMP-dependent kinase (cGK) or Ca2+/calmodulin-dependent kinase (CaMK), or of neuronal nitric oxide synthase (nNOS) blocks the circadian responses to light in vivo. Light pulses administered during the subjective night, but not during the day, induce rapid phosphorylation of both p-CAMKII and p-nNOS (specifically phosphorylated by CaMKII). CaMKII inhibitors block light-induced nNOS activity and phosphorylation, suggesting a direct pathway between both enzymes. Furthermore, SCN cGMP exhibits diurnal and circadian rhythms with maximal values during the day or subjective day. This variation of cGMP levels appears to be related to temporal changes in phosphodiesterase (PDE) activity and not to guanylyl cyclase (GC) activity. Light pulses increase SCN cGMP levels at circadian time (CT) 18 (when light causes phase advances of rhythms) but not at CT 14 (the time for light-induced phase delays). cGK II is expressed in the hamster SCN and also exhibits circadian changes in its levels, peaking during the day. Light pulses increase cGK activity at CT 18 but not at CT 14. In addition, cGK and GC inhibition by KT-5823 and ODQ significantly attenuated light-induced phase shifts at CT 18. This inhibition did not change c-Fos expression SCN but affected the expression of the clock gene per in the SCN. These results suggest a signal transduction pathway responsible for light-induced phase advances of the circadian clock which could be summarized as follows: Glu-Ca2+-CaMKII-nNOS-GC-cGMP-cGK-->-->clock genes. This pathway offers a signaling window that allows peering into the circadian clock machinery in order to decipher its temporal cogs and wheels.