Accelerating in vitro studies on circadian clock systems using an automated sampling device.
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TLDR
A suite consisting of an automated sampling device equipped with an 8-channel temperature controller and accompanying analysis software and an independent software that helps users intuitively conduct a densitometric analysis of gel images in a short time with improved reliability is developed.Abstract:
KaiC, a core protein of the cyanobacterial circadian clock, is rhythmically autophosphorylated and autodephosphorylated with a period of approximately 24 h in the presence of two other Kai proteins, KaiA and KaiB. In vitro experiments to investigate the KaiC phosphorylation cycle consume considerable time and effort. To automate the fractionation, quantification, and evaluation steps, we developed a suite consisting of an automated sampling device equipped with an 8-channel temperature controller and accompanying analysis software. Eight sample tables can be controlled independently at different temperatures within a fluctuation of ±0.01°C, enabling investigation of the temperature dependency of clock activities simultaneously in a single experiment. The suite includes an independent software that helps users intuitively conduct a densitometric analysis of gel images in a short time with improved reliability. Multiple lanes on a gel can be detected quasi-automatically through an auto-detection procedure implemented in the software, with or without correction for lane ‘smiling.’ To demonstrate the performance of the suite, robustness of the period against temperature variations was evaluated using 32 datasets of the KaiC phosphorylation cycle. By using the software, the time required for the analysis was reduced by approximately 65% relative to the conventional method, with reasonable reproducibility and quality. The suite is potentially applicable to other clock or clock-related systems in higher organisms, relieving users from having to repeat multiple manual sampling and analytical steps.read more
Citations
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Journal ArticleDOI
Conformational rearrangements of the C1 ring in KaiC measure the timing of assembly with KaiB.
Atsushi Mukaiyama,Yoshihiko Furuike,Yoshihiko Furuike,Jun Abe,Shin-ichi Koda,Shin-ichi Koda,Eiki Yamashita,Takao Kondo,Shuji Akiyama,Shuji Akiyama +9 more
TL;DR: This study found that the C1 ring undergoes a structural transition, coupled with ATPase activity and the phosphorylation state, while maintaining its hexameric ring structure, which is a necessary event for recruitment of KaiB.
Journal ArticleDOI
Real-Time In Vitro Fluorescence Anisotropy of the Cyanobacterial Circadian Clock
TL;DR: By labeling one of the clock proteins with a fluorophore, in this case KaiB, the in vitro clock reaction can be monitored by fluorescence anisotropy on the minutes time scale for weeks.
Journal ArticleDOI
Room temperature magnetoresistance in an organic spin valve with an aromatic hydrocarbon macrocycle
Kazuya Z. Suzuki,Tomoo Izumi,X. M. Zhang,Atsushi Sugihara,Song Toan Pham,Hideo Taka,Sota Sato,Sota Sato,Hiroyuki Isobe,Hiroyuki Isobe,Shigemi Mizukami +10 more
TL;DR: In this paper, a spin valve with cyclo-2,7-naphthylene (CNAP), a type of aromatic hydrocarbon macrocycle, was studied and a magnetoresistance of approximately 1-2% at 300 K (4%-6% at 5 K).
Journal ArticleDOI
Development and Optimization of Expression, Purification, and ATPase Assay of KaiC for Medium-Throughput Screening of Circadian Clock Mutants in Cyanobacteria
Dongyan Ouyang,Yoshihiko Furuike,Yoshihiko Furuike,Atsushi Mukaiyama,Atsushi Mukaiyama,Kumiko Ito-Miwa,Takao Kondo,Shuji Akiyama,Shuji Akiyama +8 more
TL;DR: Through the present protocol, the time required for one KaiC mutant is reduced by approximately 80% (six-fold throughput) relative to the conventional protocol with reasonable reproducibility, and the high-performance liquid chromatography system equipped with a multi-channel high-precision temperature controller is designed.
Journal ArticleDOI
KaiC from a cyanobacterium Gloeocapsa sp. PCC 7428 retains functional and structural properties required as the core of circadian clock system.
TL;DR: A protocol to prepare KaiC from Gloeocapsa sp.
References
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Journal ArticleDOI
Reconstitution of circadian oscillation of cyanobacterial KaiC phosphorylation in vitro
Masato Nakajima,Keiko Imai,Hiroshi Ito,Taeko Nishiwaki,Yoriko Murayama,Hideo Iwasaki,Tokitaka Oyama,Takao Kondo +7 more
TL;DR: The self-sustainable oscillation of KaiC phosphorylation in vitro is reconstituted by incubating KaiC with KaiA, KaiB, and adenosine triphosphate and the enigma of the circadian clock can now be studied in vitro by examining the interactions between three Kai proteins.
Journal ArticleDOI
Peroxiredoxins are conserved markers of circadian rhythms
Rachel S. Edgar,Edward W. Green,Yuwei Zhao,Gerben van Ooijen,María Teresa Camacho Olmedo,Ximing Qin,Yao Xu,Min Pan,Utham K. Valekunja,Kevin A. Feeney,Elizabeth S. Maywood,Michael H. Hastings,Nitin S. Baliga,Martha Merrow,Andrew J. Millar,Carl Hirschie Johnson,Charalambos P. Kyriacou,John S. O’Neill,Akhilesh B. Reddy +18 more
TL;DR: It is shown that oxidation–reduction cycles of peroxiredoxin proteins constitute a universal marker for circadian rhythms in all domains of life, by characterizing their oscillations in a variety of model organisms and exploring the interconnectivity between these metabolic cycles and transcription–translation feedback loops of the clockwork in each system.
Journal ArticleDOI
Expression of a gene cluster kaiABC as a circadian feedback process in cyanobacteria.
Masahiro Ishiura,Shinsuke Kutsuna,Setsuyuki Aoki,Hideo Iwasaki,Carol R. Andersson,Carol R. Andersson,Akio Tanabe,Susan S. Golden,Carl Hirschie Johnson,Takao Kondo +9 more
TL;DR: A negative feedback control of kaiC expression by KaiC generates a circadian oscillation in cyanobacteria, and KaiA sustains the oscillation by enhancing kaiA expression.
Journal ArticleDOI
Circadian clocks in human red blood cells
TL;DR: It is found that peroxiredoxins, highly conserved antioxidant proteins, undergo ∼24-hour redox cycles, which persist for many days under constant conditions (that is, in the absence of external cues) and are entrainable and temperature-compensated, both key features of circadian rhythms.
Journal ArticleDOI
A sequential program of dual phosphorylation of KaiC as a basis for circadian rhythm in cyanobacteria.
Taeko Nishiwaki,Yoshinori Satomi,Yohko Kitayama,Kazuki Terauchi,Reiko Kiyohara,Toshifumi Takao,Takao Kondo +6 more
TL;DR: Double phosphorylation converted KaiC from an autokinase to an autophosphatase, whereas complete dephosphorylation had the opposite effect, and these mechanisms serve as the basis for cyanobacterial circadian rhythm generation.