Calcium Inhibits Phase Shifting of the Circadian Conidiation Rhythm of <i>Neurospora crassa</i> by the Calcium Ionophore A23187

Nakashima, Hiroaki

doi:10.1104/pp.74.2.268

Cited by 37 publications

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“…1). It was previously suggested that DES shifts the phase by inhibiting some mitochondrial functions) (13); calcium transport in mitochondria was later suggested to be important for clock function in Neurospora (14). The respiratory inhibitors used in experiments presented here may affect such a process and cause phase shifting.…”

Section: Discussionmentioning

confidence: 73%

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

Nakashima¹

1984

Plant Physiol.

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Effects of respiratory inhibitors on the circadian clock, respiratory activity, and ATP content were examined in Neurospora crassa. AU 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.Circadian rhythms are observed in many organisms from lower eukaryotic plants to higher animals. However, the molecular mechanism ofthe clock which regulates these rhythms is still not known. Bunning (2) proposed that the clock is composed of two different phases: a physiological night phase which requires energy and a physiological day phase in which energy is not required. In fact, deprivation of oxygen for short times (1, 18) or treatment with respiratory inhibitors (3, 6, 9-11) resulted in phase shifting at specific phases in several organisms. These results suggest that certain phases of the clock, in particular the subjective night, may be energy dependent. Koyama and Feldman treated Neurospora with several respiratory inhibitors to examine the energy dependence of clock function (cited in Feldman and Dunlap [7]). They obtained phase-shifting data but did not measure inhibition ofrespiration or ATP levels, nor have such measurements been made in other organisms. The correlations such measurements might provide are needed before it can be concluded that energy depletion caused by these inhibitors results in phase shifting of the clock. In this report, both phase shifting by respiratory inhibitors and levels of the respiratory activity and ATP were measured. MATERIALS AND METHODSCulture Conditions and Analysis of the Conidiation Rhythm. The bd (band) strain of Neurospora crassa was used. Procedures for maintaining stock cultures and for liquid culture were the same as reported previously (12). Conidia (13 x 1O') suspended in distilled H20 were added to 25 ml ofliquid medium containing Fries' salts (8), 0.3% glucose, and 0.5% arginine. Conidial con-'Supported in part by the Japan-United States Cooperative Program. centration were determined by A at 480 nm. After culturing for 36 h in continuous light at 26C, discs were cut from the mycelial mats with a cork borer 11 mm in diameter. Six discs from each mat were transferred to a 125-ml Erlenmeyer flask with 25 ml of liquid medium containing Fries' salts, 0.03% glucose and 0.05% arginine and cultured in continuous darkness with reciprocal shaking (100 cycles per min) at 26C. The mediu...

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Section: Discussionmentioning

confidence: 73%

“…ATP was extracted and assayed as previously described (14). After treatment with inhibitors for 3 h, 18 discs were put into 4 ml of ice-cold 6% HC04 and homogenized in a glass homogenizer.…”

Section: Methodsmentioning

confidence: 99%

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

Nakashima¹

1984

Plant Physiol.

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“…Ca2+-sensitive mutants of yeast are defective in budding (7); sporulation in Penicillium is dependent on extracellular Ca2+ (8); circadian rhythms in Neurospora are reset by the Ca2+ ionophore A23187 (9); the Ca2+-binding protein calmodulin is present in Neurospora and yeast (10,11); inositol 1,4,5-trisphosphate (InsP3)-an established mediator of signal transduction in animal cells (12) and slime molds (13)-releases Ca2+ from vacuoles of Neurospora (14); and the precursor of InsP3 (phosphatidylinositol 4,5-bisphosphate) plays an essential role in yeast cell proliferation (15 (17,18). These reports are perhaps surprising, since hitherto the plasma membranes of fungi have proved to be excellent models for the study of transport in plant cells (19); yet Ca2+ transport across plant membranes is mediated by a Ca2+-ATPase (20).…”

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confidence: 99%

Cytosolic calcium homeostasis in fungi: roles of plasma membrane transport and intracellular sequestration of calcium.

Miller

Vogg

Sanders

1990

Proc. Natl. Acad. Sci. U.S.A.

100

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Cytosolic free calcium ([Ca2+]k) has been measured in the mycelial fungus Neurospora crassa with Ca2+-selective microelectrodes. The mean value of [Ca2+J is 92 ± 15 nM and it is insensitive to external pH values between 5.8 and 8.4. Simultaneous measurement of membrane potential enables the electrochemical potential difference for Ca2+ across the plasma membrane to be estimated as about -60 kJimol'-a value that cannot be sustained either by a simple Ca2+-ATPase, or, in alkaline conditions, by straightforward H+/Ca2+ exchange with a stoichiometric ratio of <5 H+/Ca2+. We propose that the most likely alternative mechanism of Ca2+ efflux is ATP-driven H+/Ca2+ exchange, with a stoichiometric ratio of at least 2 H+/Ca2 . In accord with this proposal, depletion of the ATP level from 2.5 to 0.5 mM by CN-elicits an increase in [Ca2+Jc, but only in alkaline conditions in which the putative H+/Ca2+-ATPase would be selectively stalled. The insensitivity of Ca2+ homeostasis to CN-in more acid conditions implies that the Km (ATP) of the transport system is 100 ,uM or less. The increase in [Ca2+1C in the presence of CN-at pH 8.4 (50 nM-min-') is compared with 45Ca2+ influx (0.62 mMniinif') under the same conditions. The proportion of entering Ca2+ remaining free in the cytosol is only 8 X 10-5, and since the concentration of available chelation sites on Ca2+-binding proteins is unlikely to exceed 100 ,uM, a major role for the fungal vacuole in short-term Ca2+ homeostasis is indicated. This notion is supported by the observation that cytosolic Ca2+ homeostasis is disrupted by a protonophore, which rapidly abolishes tbe driving force (a transmembrane pH difference) for Ca2+ uptake into fungal vacuoles.Cytosolic free calcium ([Ca2+]

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“…In Neurospora, we found that the calcium ionophore A23 187 and two calmodulin antagonists, W5 and W 12, shifted the phase of the circadian conidiation rhythm and that phase shifting by A23187 was inhibited by calcium ions. We concluded that the metabolism of calcium is important for clock functions from the late subjective day phase to the early subjective night phase (Nakashima, 1984(Nakashima, , 1986. Important roles for calcium in clock functions have also been indicated in other organisms, such as Aplysia (Eskin & Corrent, 1977) and Euglena (Goto et al, 1985;Tamponnet & Edmunds, 1991).…”

Section: Introductionmentioning

confidence: 99%

A mutant of Neurospora crassa that has a long lag phase in low-calcium medium

Ohnishi¹,

Cornelius²,

Nakashima³

1992

Journal of General Microbiology

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A mutant of Neurospora crassa that has a long lag phase when grown in medium that contains less than 10 PMCaCl, was isolated from UV-irradiated conidia. However, the rate of uptake of calcium ions by mycelium and the dependence on magnesium ions of the growth of this mutant were the same as those of the wild-type strain. The phase of the circadian clock, within the entire circadian cycle of the mutant, could not be shifted by exposure to fluorescent white light during growth in liquid medium. However, a brief incubation at high temperature resulted in a large shift in phase, and the phase response curve for the mutant was similar to that for the wild-type strain. The mutant strain had the same sensitivity to light as the wild-type strain with respect to carotenoid synthesis. The relevant mutation may affect the process involved in transduction of signals from the light-perception system to the clock mechanism and this process may be related to calcium homeostasis in Neurospora cells.

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Calcium Inhibits Phase Shifting of the Circadian Conidiation Rhythm of Neurospora crassa by the Calcium Ionophore A23187

Cited by 37 publications

References 10 publications

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

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

Cytosolic calcium homeostasis in fungi: roles of plasma membrane transport and intracellular sequestration of calcium.

A mutant of Neurospora crassa that has a long lag phase in low-calcium medium

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