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1. Reconstitution of circadian clock in synthetic cells reveals principles of timekeeping

   https://doi.org/10.1038/s41467-025-61844-5  

   Li, Alexander_Zhan_Tu; LiWang, Andy; Subramaniam, Anand_Bala (July 2025, Nature Communications)

   The cyanobacterial circadian clock maintains remarkable precision and synchrony, even in cells with femtoliter volumes. Here, we reconstitute the KaiABC post-translational oscillator (PTO) in giant unilamellar vesicles (GUVs) to investigate underlying mechanisms of this fidelity. We show that our encapsulation methodology replicates native protein variability. With long-term, single-vesicle tracking of circadian rhythms using fluorescent KaiB and confocal microscopy, we find that oscillator fidelity decreases with lower protein levels and smaller vesicle sizes. KaiB membrane association, observed in cyanobacteria, was recapitulated in GUV membranes. A mathematical model incorporating protein stoichiometry limitations suggests that high expression of PTO components and associated regulators (CikA and SasA) buffers stochastic variations in protein levels. Additionally, while the transcription-translation feedback loop contributes minimally to overall fidelity, it is essential for maintaining phase synchrony. These findings demonstrate synthetic cells capable of autonomous circadian rhythms and highlight a generalizable strategy for dissecting emergent biological behavior using minimal systems. 

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2. Connectomic analysis of the Drosophila lateral neuron clock cells reveals the synaptic basis of functional pacemaker classes

   https://doi.org/10.7554/eLife.79139  

   Shafer, Orie T; Gutierrez, Gabrielle J; Li, Kimberly; Mildenhall, Amber; Spira, Daphna; Marty, Jonathan; Lazar, Aurel A; Fernandez, Maria de (June 2022, eLife)

   Most organisms on Earth possess an internal timekeeping system which ensures that bodily processes such as sleep, wakefulness or digestion take place at the right time. These precise daily rhythms are kept in check by a master clock in the brain. There, thousands of neurons – some of which carrying an internal ‘molecular clock’ – connect to each other through structures known as synapses. Exactly how the resulting network is organised to support circadian timekeeping remains unclear. To explore this question, Shafer, Gutierrez et al. focused on fruit flies, as recent efforts have systematically mapped every neuron and synaptic connection in the brain of this model organism. Analysing available data from the hemibrain connectome project at Janelia revealed that that the neurons with the most important timekeeping roles were in fact forming the fewest synapses within the network. In addition, neurons without internal molecular clocks mediated strong synaptic connections between those that did, suggesting that ‘clockless’ cells still play an integral role in circadian timekeeping. With this research, Shafer, Gutierrez et al. provide unexpected insights into the organisation of the master body clock. Better understanding the networks that underpin circadian rhythms will help to grasp how and why these are disrupted in obesity, depression and Alzheimer’s disease. 

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3. Circadian Interactomics: How Research Into Protein-Protein Interactions Beyond the Core Clock Has Influenced the Model of Circadian Timekeeping

   https://doi.org/10.1177/07487304211014622  

   Mosier, Alexander E.; Hurley, Jennifer M. (August 2021, Journal of Biological Rhythms)

   The circadian clock is the broadly conserved, protein-based, timekeeping mechanism that synchronizes biology to the Earth’s 24-h light-dark cycle. Studies of the mechanisms of circadian timekeeping have placed great focus on the role that individual protein-protein interactions play in the creation of the timekeeping loop. However, research has shown that clock proteins most commonly act as part of large macromolecular protein complexes to facilitate circadian control over physiology. The formation of these complexes has led to the large-scale study of the proteins that comprise these complexes, termed here “circadian interactomics.” Circadian interactomic studies of the macromolecular protein complexes that comprise the circadian clock have uncovered many basic principles of circadian timekeeping as well as mechanisms of circadian control over cellular physiology. In this review, we examine the wealth of knowledge accumulated using circadian interactomics approaches to investigate the macromolecular complexes of the core circadian clock, including insights into the core mechanisms that impart circadian timing and the clock’s regulation of many physiological processes. We examine data acquired from the investigation of the macromolecular complexes centered on both the activating and repressing arm of the circadian clock and from many circadian model organisms. 

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4. Circadian clock proteins KaiB and Rbp2 of Synechococcus elongatus display oscillations in their subcellular localization patterns

   https://doi.org/10.1128/spectrum.01845-25  

   Bevir, Harry J; Hooper, Christopher C; Saikley, Parker; Chaljian, Tanya; Lin, Yahan; Cohen, Susan E (February 2026, Microbiology Spectrum)

   Kolodkin-Gal, Ilana (Ed.)

   ABSTRACT Circadian rhythms generated by molecular clocks time a diverse array of physiological cycles in many organisms. Cyanobacteria are currently the only prokaryotic system with a robust and rigorously tested circadian clock, which is carried out by KaiA, KaiB, and KaiC core oscillator proteins. This KaiABC oscillator drives global rhythms in gene expression, compaction of the chromosome, natural transformation, and timing of cell division in the model organismSynechococcus elongatusPCC 7942. The oscillator proteins have been previously shown to undergo changes in their subcellular localization patterns, where KaiA and KaiC are diffuse throughout the cell during the day and localize to or near one pole of the cell at night. However, functional fluorescent fusions of KaiB to track subcellular localization could not be obtained. More recently, we have described the identification of Rbp2 as a member of the extended clock network that associates with KaiC in a localized state. However, the subcellular localization patterns of KaiB and Rbp2 remain uncharacterized, and the mechanism driving the dynamic localization of circadian proteins remains undescribed. Here, we describe the patterns of KaiB and Rbp2 subcellular localization over the course of the day. Specifically, we demonstrate that KaiB and Rbp2 form polar foci and co-localize with KaiC at night using both traditional fusions to fluorescent proteins and endogenously expressed antibodies. We show that the RNA-binding activity of Rbp2 is necessary for the robust formation of Rbp2 foci. We propose a model by which Rbp2 drives circadian protein localization via its RNA binding activity.IMPORTANCECircadian rhythms driven by a circadian clock are required to time a wide range of physiological and metabolic processes. In cyanobacteria, the circadian clock is required for optimal fitness in environmental cycles. Where the clock proteins are located within the cell and how this helps them to synchronize with the environment and time rhythmic behaviors is incompletely understood. Here, we demonstrate that recently identified extended clock protein Rbp2 and core oscillator protein KaiB localize to the cell poles at night and co-localize with other members of the clock complex. We also demonstrate that the RNA binding activity of Rbp2 is necessary for the polar localization of Rbp2. Moreover, the use of endogenously expressed antibodies introduces a new and robust method for imaging challenging proteins in bacteria that is applicable across various fields. 

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5. Reconstitution of an intact clock reveals mechanisms of circadian timekeeping

   https://doi.org/10.1126/science.abd4453  

   Chavan, Archana G.; Swan, Jeffrey A.; Heisler, Joel; Sancar, Cigdem; Ernst, Dustin C.; Fang, Mingxu; Palacios, Joseph G.; Spangler, Rebecca K.; Bagshaw, Clive R.; Tripathi, Sarvind; et al (October 2021, Science)

   Circadian clocks control gene expression to provide an internal representation of local time. We report reconstitution of a complete cyanobacterial circadian clock in vitro, including the central oscillator, signal transduction pathways, downstream transcription factor, and promoter DNA. The entire system oscillates autonomously and remains phase coherent for many days with a fluorescence-based readout that enables real-time observation of each component simultaneously without user intervention. We identified the molecular basis for loss of cycling in an arrhythmic mutant and explored fundamental mechanisms of timekeeping in the cyanobacterial clock. We find that SasA, a circadian sensor histidine kinase associated with clock output, engages directly with KaiB on the KaiC hexamer to regulate period and amplitude of the central oscillator. SasA uses structural mimicry to cooperatively recruit the rare, fold-switched conformation of KaiB to the KaiC hexamer to form the nighttime repressive complex and enhance rhythmicity of the oscillator, particularly under limiting concentrations of KaiB. Thus, the expanded in vitro clock reveals previously unknown mechanisms by which the circadian system of cyanobacteria maintains the pace and rhythmicity under variable protein concentrations. 

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