Dynamic localization of the cyanobacterial circadian clock proteins

Abstract

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 colocalize with each other, as well as with CikA, a key input and 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 delocalization 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.

Figure 1

Spatiotemporal localization of circadian clock proteins. A) Bioluminescence from strains that carry a P_kaiB-luc reporter for circadian rhythmicity. WT (kaiA⁺kaiC⁺), blue/25 h period; ΔkaiC, purple/arrhythmic; kaiA::Tn5, red/arrhythmic; ΔkaiC expressing P_trc-yfp-kaiC, orange/25 h period; kaiA::Tn5 expressing P_kaiA-kaiA-gfp, green/27 h period. B) 3 Dimensional-Structured Illumination Microscopy (3D-SIM) micrograph of an individual representative cell expressing YFP-KaiC in green, autofluorescence in red C) Deconvolution fluorescence micrograph of a cell expressing KaiA-GFP, for which autofluorescence was omitted to improve visualization of KaiA. Scale bars = 1 micron. D) Heat map of KaiC localization. The positions of >800 foci were determined relative to an ellipse drawn to fit the cell based on autofluorescence. Colors represent number of foci present at each cellular position as indicated. E) Strains that express YFP-KaiC were entrained to opposite light-dark cycles and sampled every 2 hours in either light (ZT 0-12) or dark (ZT 12-24). Zeitgeber time (ZT) refers to the time relative to “lights on”. The fraction of the population that showed KaiC polar localization is plotted for WT (dark circles) or kaiA-disrupted cells (red triangles) in a diurnal cycle for a representative experiment.

Figure 2

KaiA polar localization is dependent on KaiC. Fluorescence micrographs of overexpressed KaiA-YFP (green) as the only source of KaiA overlaid onto autofluorescence (red) in A) ΔkaiBC background, B) ΔkaiBC background complemented with kaiBC⁺. C-F) KaiA-YFP overexpression in a ΔkaiBC background expressing C) kaiB⁺ only, D) kaiC⁺ only, E) kaiBC⁴⁹⁷ mutant allele, truncated to not express A-loops, F) kaiBC^E487A mutant allele, which expresses KaiC in which the A-loops are constitutively extended. Scale bars = 2.5 microns.

Figure 3

Co-localization of clock components in S. elongatus. Fluorescence micrographs demonstrating co-localization of Row 1: YFP-KaiC and KaiA-ECFP; Row 2: ZsG-CikA and KaiA-ECFP; Row 3: ZsG-CikA and KaiC-ECFP. For pairs among KaiC, KaiA and CikA, co-localization is observed in essentially all cases (99%, n>90). In contrast, KaiC does not show a high degree of co-localization with ClpP2. Row 4: ClpP2-YFP and KaiC-ECFP. Only 11.8% of KaiC foci were co-localized with ClpP2 focus n >224 foci. Autofluorescence in red. Scale bar = 2.5 microns.

Figure 4

Clock protein localization is conserved between S. elongatus and E. coli. A) Fluorescence micrographs of E. coli MG1655 stained with vital membrane stain FM4-64 (red) expressing Row 1: only fluorescently labeled KaiC, CikA or KaiA. Row 2: both ECFP-KaiC and KaiA-YFP, showing that KaiA polar localization in E. coli is KaiC dependent. Row 3: both ECFP-KaiC and ZsG-CikA, demonstrating that they co-localize when expressed in the same cell. B) KaiC localization in E. coli is not sensitive to a light-dark cycle. Closed symbols represent percentage of the population that possesses a KaiC focus (left Y-axis) and open symbols represent OD₆₀₀ (right Y-axis). C) Percentage of KaiC or CikA foci that co-localized with a marker of inclusion bodies (IbpA-mCherry) compared to an inclusion body positive control, ORF39 from large plasmid pMB400 of Bacillus megataruim QMB155. n > 100 foci were counted for IpbA co-localization. D) Representative micrograph of cells expressing both YFP-KaiC and IbpA-mCherry, cell outlines drawn in white.

Figure 5

CikA alters, but is not required for KaiC localization. A) Table representing how deletion or alteration of each CikA domain affects its localization, compared to the full-length (FL) protein. Symbols indicate whether polar localization was observed (+) or fluorescence was diffuse throughout all cells observed (−), n>100. Cells were sampled during the day (ZT 8) and night (ZT 20). B-D) CikA can affect KaiC localization. B) Graph demonstrating that reduced YFP-KaiC polar localization is observed when mutant variants of CikA that display diffuse distribution are expressed. Representative micrographs of YFP-KaiC fusion expressed in C) ΔcikA mutant background, D) ΔcikA + cikAΔPsR, E) ΔcikA + cikAΔN, or F) ΔcikA + cikA-HA. Autofluorescence in red. Scale bar = 2.5 microns.

Figure 6

Graphic representation of subcellular localization patterns of the clock proteins. KaiC and KaiA are predominantly distributed in the cytosol during the day (yellow shaded region), a time that corresponds to the phosphorylation phase of the cycle. During the night or subjective night (grey shaded region) Kai proteins co-localize with CikA at the poles of the cells in order to facilitate protein associations and interactions with metabolites of photosynthesis. KaiC protein abundance rhythms are depicted as numbers of KaiC molecules present in the cell.