Dynamic Localization of the Cyanobacterial Circadian Clock Proteins

Abstract

SUMMARY Background The cyanobacterial circadian clock system has been extensively studied and the structures, interactions and biochemical activities of the central oscillator proteins (KaiA, KaiB and KaiC) have been well elucidated. Despite this rich repository of information, little is known about the distribution of these proteins within the cell. Results Here we report that KaiA and KaiC localize as discrete foci near a single pole of cells in a clock-dependent fashion, with enhanced polar localization observed at night. KaiA localization is dependent on KaiC; consistent with this notion, KaiA and KaiC co-localize with each other as well as with CikA, a key input/output factor previously reported to display unipolar localization. The molecular mechanism that localizes KaiC to the poles is conserved in Escherichia coli, another Gram-negative rod shaped bacterium, suggesting that KaiC localization is not dependent on other clock- or cyanobacterial-specific factors. Moreover, expression of CikA mutant variants that distribute diffusely results in the striking de-localization of KaiC. Conclusions This work shows that the cyanobacterial circadian system undergoes a circadian orchestration of subcellular organization. We propose that the observed spatiotemporal localization pattern represents a novel layer of regulation that contributes to the robustness of the clock by facilitating protein complex formation and synchronizing the clock with environmental stimuli.

Circadian Rhythms in Cyanobacteria

“…The input/output protein CikA had been previously demonstrated to localize as a discrete focus at one pole of the cell in a manner that requires the quinone-binding PsR domain (73). More recently, we demonstrated by using fully functional fusions to fluorescent proteins that the oscillator components KaiA and KaiC similarly localize to a single pole of the cell (72,89). Whereas CikA is localized to the cell pole throughout the course of the day, KaiC exhibits spatiotemporal changes in subcellular localization, such that enhanced polar localization is observed at night in a clock-dependent fashion (72).…”

“…Whereas CikA is localized to the cell pole throughout the course of the day, KaiC exhibits spatiotemporal changes in subcellular localization, such that enhanced polar localization is observed at night in a clock-dependent fashion (72). Oscillations in KaiC localization that range from diffuse throughout the cell during the day to highly localized to the poles at night are observed under both diel and constant conditions and are dependent on the presence of a functional clock, demonstrating that regulation of these spatiotemporal changes is circadian in nature (72). CikA and KaiC are capable of independently localizing to the poles in Escherichia coli as well, suggesting that the mechanism of localization is not dependent on a cyanobacterial, clock, or photosynthetic factor.…”

“…More recently, we demonstrated by using fully functional fusions to fluorescent proteins that the oscillator components KaiA and KaiC similarly localize to a single pole of the cell (72,89). Whereas CikA is localized to the cell pole throughout the course of the day, KaiC exhibits spatiotemporal changes in subcellular localization, such that enhanced polar localization is observed at night in a clock-dependent fashion (72). Oscillations in KaiC localization that range from diffuse throughout the cell during the day to highly localized to the poles at night are observed under both diel and constant conditions and are dependent on the presence of a functional clock, demonstrating that regulation of these spatiotemporal changes is circadian in nature (72).…”

“…Remarkably, the addition of oxidized quinone (Q 0 ), proposed to serve as a proxy for darkness, applied during the subjective day is sufficient to induce phase shifts both in vivo and in vitro that mimic the effects of a true dark pulse in vivo (71). Both CikA and KaiA are peripheral membrane proteins that localize to the poles of cells, which may facilitate interactions with quinones in the thylakoid membranes (72,73). Moreover, CikA protein levels are subject to both circadian regulation and environmental changes, where CikA abundance is inversely proportional to light intensity and exhibits the highest levels in the dark (66).…”

Light Modulates the Biosynthesis and Organization of Cyanobacterial Carbon Fixation Machinery through Photosynthetic Electron Flow

“…We chose Synechococcus as the model organism due to its superior genetic tractability and proven suitability for fluorescence imaging (Savage et al, 2010;Liu et al, 2012;Cameron et al, 2013;Cohen et al, 2014). RbcL, the large subunit of Rubisco that resides in the b-carboxysome lumen, was tagged at the C terminus with enhanced GFP (eGFP) and was visualized under confocal fluorescence microscopy to characterize the formation and positioning of carboxysomes in vivo.…”