dynamic localization of the cyanobacterial circadian clock proteins

Please cite this article in press as: Cohen et al., Dynamic Localization of the Cyanobacterial Circadian Clock Proteins, Current Biology(2014), http://dx.doi.org/10.1016/j.cub.2014.07.036

Dynamic Localization of the

Current Biology 24, 1–9, August 18, 2014 ª2014 Elsevier Ltd All rights reserved http://dx.doi.org/10.1016/j.cub.2014.07.036

Article

Cyanobacterial Circadian Clock Proteins

Susan E. Cohen,1,2 Marcella L. Erb,2 Jangir Selimkhanov,3

Guogang Dong,1,2,5 Jeff Hasty,2,3,4 Joe Pogliano,2

and Susan S. Golden1,2,*1Center for Circadian Biology2Division of Biological Sciences3Department of Bioengineering and San Diego Center forSystems Biology4BioCircuits InstituteUniversity of California, San Diego, La Jolla, CA 92093, USA

Summary

Background: The cyanobacterial circadian clock system hasbeen extensively studied, and the structures, interactions,and biochemical activities of the central oscillator proteins(KaiA, KaiB, and KaiC) have been well elucidated. Despitethis rich repository of information, little is known about the dis-tribution of these proteins within the cell.Results:Herewe report that KaiA andKaiC localize as discretefoci near a single pole of cells in a clock-dependent fashion,with enhanced polar localization observed at night. KaiA local-ization is dependent on KaiC; consistent with this notion, KaiAand KaiC colocalize with each other, as well as with CikA, a keyinput and output factor previously reported to display unipolarlocalization. The molecular mechanism that localizes KaiC tothe poles is conserved inEscherichia coli, another Gram-nega-tive rod-shaped bacterium, suggesting that KaiC localizationis not dependent on other clock- or cyanobacterial-specificfactors. Moreover, expression of CikA mutant variants thatdistribute diffusely results in the striking delocalization of KaiC.Conclusions: This work shows that the cyanobacterialcircadian system undergoes a circadian orchestration of sub-cellular organization. We propose that the observed spatio-temporal localization pattern represents a novel layer ofregulation that contributes to the robustness of the clock byfacilitating protein complex formation and synchronizing theclock with environmental stimuli.

Introduction

Circadian rhythms, regulated by a 24 hr biological clock, arevitally important for controlling temporal programs of cellularphysiology to facilitate adaptation to daily environmentalchanges in diverse organisms [1]. Cyanobacteria are thesimplest organisms and only prokaryotes currently known topossess a circadian clock. Synechococcus elongatus PCC7942 has emerged as a premier model organism for studyingthe molecular details and regulation of the clock [2]. TheS. elongatus core oscillator, encoded by the kaiA, kaiB, andkaiC genes, regulates global patterns of gene expression[3, 4], the timing of cell division [5, 6], and compaction of thechromosome [7, 8]. Inactivation or overexpression of any ofthe kai genes stops the clock [4, 9]. KaiC is an autokinase,

5Present address: Joule Unlimited Technologies, Inc., Bedford, MA 01730,

USA

*Correspondence: [email protected]

autophosphatase, and ATPase whose daily rhythms of phos-phorylation at residues Ser431 and Thr432 and ATPase activityare key features of the timekeeping mechanism that drivescircadian rhythms in complex with KaiA and KaiB [10, 11].KaiA binds to the C-terminal segment of KaiC, referred to asthe ‘‘A loop,’’ promoting KaiC autokinase activity [12]. KaiB op-poses KaiA’s stimulatory activity by sequestering KaiA awayfrom the A loops, thereby promoting KaiC’s intrinsic autophos-phatase activity [12, 13]. These oscillations of KaiC phosphor-ylation can be reconstituted in vitro solely with purified KaiA,KaiB, KaiC, and ATP [14].These endogenously generated circadian rhythms are syn-

chronized with the environment, through a process referredto as entrainment, via an input pathway that monitors cellularredox state. Sensing of the cellular redox environment isachieved through input components such as CikA, as well asthe central oscillator component KaiA. Both proteins bind qui-nones, whose redox states vary as a function of light intensityin photosynthetic organisms [15, 16]. Moreover, changes in theATP:ADP ratio in the cell, which depends on photophosphory-lation, can reset the phase of KaiC phosphorylation directly[17]. The oxidation of the quinone pool may act in concertwith the changing levels of ATP:ADP to synchronize the circa-dian clock by signaling both the onset and duration of dark-ness [18].While cell division is not synchronous among cells in the

population or with the circadian cycle, the circadian clock‘‘gates’’ the cell cycle, defining a portion of the 24 hr cycle inthe early night in which cell division is disallowed [5, 6, 19].Mutations that hyperstimulate KaiC phosphorylation and/ordownstream activity, including the loss of CikA or KaiB oroverexpression of KaiA, exaggerate the duration of theclosed-gate phase in the circadian cycle, resulting in elon-gated cells [6].The past several years have seen a revolution in our under-

standing of intracellular organization in bacteria. The bacterialcell is now known to possess a high degree of internal organi-zation that is of critical importance for a wide range of cellularactivities, including virulence, DNA replication, chromosomesegregation, cell division, chemotaxis, and gene transfer,among others (reviewed in [20]). Although it is not obviousthat specific localization should be necessary for clock func-tion, several pieces of evidence have suggested a nonrandomdistribution of clock gene products. The input and outputcomponent CikA is localized to one pole of the cell in a mannerthat depends on a specific quinone-binding domain [21], andCikA copurifies with at least two of the Kai proteins [22]. Arecent report shows that incorporation of the in vitro oscillatorinto liposomes affects circadian period and that KaiC associ-ates with the membranes, whereas KaiB is evenly distributed[23]. KaiC is also recovered from cells in a membrane fraction,specifically in samples collected during the subjective night[24]. Where the clock proteins are localized within the celland how this localization may change and contribute to clockfunction have not been previously addressed. In eukaryoticsystems, subcellular localization is an important feature oftimekeeping, and several clock components display rhythmsof nuclear localization [25, 26] and even rhythmic formationof subnuclear structures [27].

http://dx.doi.org/10.1016/j.cub.2014.07.036


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Figure 1. Spatiotemporal Localization of Circadian Clock Proteins

(A) Bioluminescence from strains that carry a PkaiB-luc reporter for circadian

rhythmicity. Blue, WT (kaiA+kaiC+), 25 hr period; purple, DkaiC, arrhythmic;

red, kaiA::Tn5, arrhythmic; orange, DkaiC expressing Ptrc-yfp-kaiC, 25 hr

period; and green, kaiA::Tn5 expressing PkaiA-kaiA-gfp, 27 hr period.

(B) 3D structured illumination microscopy (3D-SIM) micrograph of an indi-

vidual representative cell expressing YFP-KaiC in green, with autofluores-

cence in red.

Current Biology Vol 24 No 162


Here we show that the Kai complex interacts in a circadianmanner with CikA near the poles of cells, where enhanced po-lar localization of KaiC is observed at night or subjective night.This localization is preserved in Escherichia coli, suggesting aconserved targeting mechanism across Gram-negative bacte-ria. We provide additional evidence that polar clock proteinsrepresent a biologically active complex, as opposed to a pro-tein aggregate targeted for degradation. Together, these re-sults suggest that the proper timing of localization representsa previously unrecognized layer of regulation embedded intothe cyanobacterial circadian system that most likely contrib-utes to robustness and synchronization of the clock.

Results

KaiA and KaiC Localize to the Poles of CellsIn order to investigate where the clock proteins are localizedwithin the cell, we sought to create functional fluorescentfusion proteins. We identified fusions to both KaiA and KaiCthat restored rhythmicity to the respective null mutants andwere expressed as full-length fusion proteins, with no evi-dence of cleavage of the fusion protein, and at near wild-type (WT) levels as confirmed by immunoblot (Figure 1A andFigures S1A–S1C available online). Luciferase reportersconfirmed that both N- andC-terminal fusions of KaiC restoredrhythmicity to a kaiC-null strain (DkaiC). N-terminal fusions toyellow fluorescent protein (YFP) complemented the freerunning period as well as the WT (Figure 1A) and were usedpreferentially. C-terminal fusions to either YFP or enhancedcyan fluorescent protein (ECFP) resulted in a long periodphenotype, extended by w5 hr (Figure S1C), and were usedonly for colocalization experiments. C-terminal fusions toKaiA expressed from the native promoter (PkaiA) restoredrhythmicity to a kaiA::Tn5 insertion strain but displayedw2 hr period lengthening relative to WT strains (Figures 1A,S3A, and S4A). Despite the period lengthening observed withKaiA fusions, collectively, these tools allowed us to monitorthe subcellular localization of the clock proteins under physio-logical conditions in which the clock is functional and pheno-types that are observed can be attributed to the fusion protein.Although levels of the KaiA fusion protein appeared to beslightly elevated (Figures S1A and S4B), complementationstudies confirmed that the fusions are expressedwithin a func-tional range, as overexpression of any of the Kai proteins re-sults in apparent arrhythmia [4, 9]. We were not able to identifya fusion to KaiB, the smallest of the Kai proteins (11 KDa, aboutone-third of the size of GFP), which satisfied our rigorouscriteria.Fluorescence microscopy of these carefully vetted fusion

proteins indicated that KaiA and KaiC are localized as discretefoci at or near one pole of a subset of cells in the population

(C) Deconvolution fluorescence micrograph of a cell expressing KaiA-GFP,

for which autofluorescence was omitted to improve visualization of KaiA.

(D) Heatmap of KaiC localization. The positions of >800 foci were deter-

mined relative to an ellipse drawn to fit the cell based on autofluorescence.

Colors represent number of foci present at each cellular position as indi-

cated.

(E) Strains that express YFP-KaiC were entrained to opposite light-dark

cycles and sampled every 2 hr in either light (ZT 0–12) or dark (ZT 12–24).

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 (red triangles) cells in a diurnal cycle for a representative

experiment.

Scale bars, 1 mm. See also Figures S1 and S2.

Subcellular Localization of the Kai Proteins3


(Figures 1B, 1C, S1D, and S1E). KaiC is an abundant protein inthe cell (5,000–15,000molecules/cell) [24], and foci were brightand easily observable, whereas KaiA expressed at nativelevels is much less abundant (w500 molecules/cell) and wasmore difficult to image. The occurrence of two foci per cell,one focus at each pole, was rarely observed (w1%). Fociwere occasionally found outside of the polar region of thecell; however, quantification showed that for KaiC, w94% offoci are indeed polar, with peak intensity lying within w15%of the curved region of the cell (Figures 1D and S1F–S1H).Together, these data are most consistent with an interactionwith the thylakoid membrane system. The remaining w6% offoci were almost exclusively localized in close proximity tothe thylakoid membrane, suggesting that association withthe membrane is important for KaiC polar localization.

Circadian Spatiotemporal Localization of KaiC

Because polar localization was observed in only a subset ofcells (Figures S1D and S1E), we asked whether KaiC localiza-tion changes, particularly over the course of the day. Sampleswere entrained with two cycles of 12 hr light followed by 12 hrdark (LD) to synchronize the clocks in the population. Prior toentrainment, the samples were split and subjected to LD cy-cles that were 12 hr out of phase from each other in order tocollect data for a single circadian cycle in 12 hr. Samplesfrom cultures that were maintained in a diurnal cycle (LD) orhad been released into conditions of continuous light (LL)were taken every 2 hr and fixed to preserve cellular architec-tures. By convention, Zeitgeber Time (ZT) refers to the timerelative to the light signal, such that the lights turn on at ZT0 with ZT 0–12 representing daytime and ZT 12–24 represent-ing nighttime. Under these conditions, KaiC polar localizationis enhanced at night (Figures 1E and S2A). Persistence incontinuous conditions is a fundamental property of circadianrhythms [1], and this spatiotemporal pattern of KaiC localiza-tion persists when cultures are released into LL (Figure S2B),albeit with oscillations that are not quite as striking as thoseobserved in LD.

Although KaiC is still capable of localizing to the poles in akaiA::Mu strain, the occurrence of foci no longer oscillates,and instead a low constitutive level of foci is observed (Fig-ure 1E). This YFP-KaiC is expressed from a Ptrc promoter (Fig-ure 1) rhythmically and in the same phase as the kaiBCpromoter, but it is not subject to feedback regulation fromthe clock [28, 29]. This approach allows us to distinguish theeffects of KaiA on subcellular localization from the dramaticreduction in KaiC levels that kaiAmutation causes when kaiBCis expressed from its native promoter. These data demon-strate that KaiC localization is subject to spatial and temporalregulation that is circadian in nature.

The spatiotemporal pattern of KaiC localization suggeststhat KaiC is in a diffuse state during the phosphorylation phasein the day or subjective day and highly localized in the dephos-phorylation phase at night or subjective night. In order toinvestigate whether a particular phosphorylation state ofKaiC is required to facilitate polar localization, we generatedphosphomimetic mutant variants representing each of thedifferent phosphorylation states in the YFP-KaiC fusionconstruct (Figure S2C). Although these phosphomimetic vari-ants are not perfect representations of each KaiC phosphory-lation state, they can shed light onto how the phosphorylationcycle of KaiC facilitates downstream processes [6]. The resultsdemonstrate that there is not a particular phosphorylationstate of KaiC that is essential for polar localization, as KaiC

localizes to some extent in all phosphomimetic mutants (Fig-ures S2C and S2D). However, strains expressing either AA orET phosphomimetic variants have the highest incidence ofpolar localization, which would correlate with the dephosphor-ylation phase of the cycle, consistent with the time-of-daydependence in a WT strain. Phosphomimetic variants thatlock the oscillator in all other states had a very low frequencyof localization.Additionally, we performed fluorescent recovery after pho-

tobleaching (FRAP) experiments to test the stability of KaiCpolar complexes. Very little recovery occurred after photo-bleaching, even when only one focus was bleached in therare case of a cell that possessed two foci, suggesting that po-lar KaiC complexes are stable when localized. In contrast, freeYFP recovered rapidly after a similar photobleaching event atthe pole (Figure S2E). Similarly, protein complexes in the thyla-koid membrane are relatively immobile, as diffusion is slowand restricted in cyanobacterial thylakoids [30, 31].

KaiA Localization Is Dependent on KaiCWe generated a strain in which the KaiA fusion protein couldbe overexpressed, as KaiA was difficult to observe whenexpressed at native levels. A KaiA-YFP fusion under the con-trol of an inducible Ptrc promoter overexpressed KaiA, asconfirmed by bright polar foci, elongated cell morphology (Fig-ure 2B), and high and arrhythmic expression from a PkaiB-lucreporter (Figure S3B), as well as by immunoblot analysis (Fig-ure S3C).When expressed at wild-type, noninduced levels, theKaiA-YFP fusion behaves as the GFP fusion expressed fromthe native promoter described in Figure 1 (see Figures S3Aand S3C). Despite overexpression, KaiA cannot localize as adiscrete focus in a DkaiBC background (Figure 2A). WhenkaiB or kaiC is added back individually to a strain that lackskaiBC, KaiA polar localization and elongated cell morphologyare restored only in the strain in which KaiC is addedback, indicating that KaiA polar localization is dependent onKaiC (Figures 2A–2D). Additionally, KaiA localization is notdependent on a particular phosphorylation state and, whenoverexpressed, forms bright foci in all kaiC phosphomimetic-expressing strains (Figure S3D).KaiA binds to the C-terminal A loop tails of KaiC, an action

that promotes KaiC autophosphorylation [12]. Truncation ofKaiC after residue 497 results in a mutant variant, kaiC497,which lacks the A loop. Expression of kaiC497 does not supportKaiA polar localization, where diffuse distribution is observed(Figure 2E), although the KaiC497 mutant itself remains local-ized to the pole (Figure S3E). In contrast, mutation of Glu 487to Ala (KaiCE487A) results in a mutant variant of KaiC with aconstitutively extended A loop [12]. Expression of kaiCE487A

supports the localization of both KaiC and KaiA (Figures 2Fand S3F). These data demonstrate that the A loops of KaiC,while not required for the localization of KaiC itself, are neces-sary to recruit and/or retain KaiA at the poles.In order to address whether the A loops are sufficient for

KaiA polar localization, we added the A loop to the C terminusof CikA, which is localized to the pole [21], and assayed forlocalization of KaiA. KaiA is known to bind to the A loop pep-tide alone in solution [12]. Addition of the A loops to the C ter-minus of CikA with a function-preserving ZsGreen (ZsG) tag atthe N terminus to monitor localization [21] (Figure S3G) doesnot alter CikA’s unipolar localization (Figure S3H). The CikA-Aloop fusion (without ZsG) complements a DcikA strain (Fig-ure S3G). However, this fusion did not recruit KaiA tothe pole, even when overexpressed, in a DkaiC mutant

Figure 2. KaiA Polar Localization Is Dependent on KaiC

(A and B) Fluorescence micrographs of overexpressed KaiA-YFP (green) as

the only source of KaiA overlaid onto autofluorescence (red) in DkaiBC

background (A) and DkaiBC background complemented with kaiBC+ (B).

(C–F) KaiA-YFP overexpression in a DkaiBC background expressing kaiB+

only (C), kaiC+ only (D), kaiBC497 mutant allele truncated to not express A

loops (E), and kaiBCE487A mutant allele, which expresses KaiC in which

the A loops are constitutively extended (F).

Scale bars, 2.5 mm. See also Figure S3.

Figure 3. Colocalization of Clock Components in S. elongatus

Fluorescence micrographs demonstrating colocalization of YFP-KaiC and

KaiA-ECFP (row 1), ZsG-CikA and KaiA-ECFP (row 2), and ZsG-CikA and

KaiC-ECFP (row 3). For pairs among KaiC, KaiA, and CikA, colocalization

is observed in essentially all cases (99%, n > 90). In contrast, KaiC does

not show a high degree of colocalization with ClpP2. Row 4 shows ClpP2-

YFP and KaiC-ECFP. Only 11.8% of KaiC foci were colocalized with ClpP2

focus (n > 224 foci). Autofluorescence is shown in red. Scale bars, 2.5 mm.

See also Figure S4.



background (Figure S3I). These results demonstrate that the Aloops are necessary, but not sufficient, to promote KaiA’s po-lar localization and suggest that perhaps another docking siteon KaiC is necessary to facilitate KaiA-KaiC interactions. Alter-natively, the A loops may not be accessible to KaiA in thisparticular CikA-A loop fusion.

Colocalization of Clock ProteinsCikA similarly shows unipolar localization [21] and has beenrecovered from cyanobacterial cells in a complex with theKai proteins [15]. Thus, we would expect CikA to colocalizewith the Kai proteins in vivo. Using a functional KaiA-ECFPfusion (Figure S4), we investigated the pairwise localizationof CikA, KaiA, and KaiC using YFP/ZsG and ECFP combina-tions. Indeed, we observed that KaiC and KaiA colocalizewith each other in essentially all cases (99%, n > 90), as doCikA/KaiA and CikA/KaiC pairs (Figure 3). In contrast, KaiCdoes not frequently localize with the protease subunit ClpP2(Figure 3), suggesting that the colocalization observed among

CikA, KaiA, and KaiC is specific. These data suggest that theclock proteins are interacting as a complex at the poles of cellsin a bona fide biological complex.

Clock Proteins Exhibit a Conserved Mechanism ofLocalization

To gain insights into themechanism of Kai protein localization,we expressed each of the fusion proteins in E. coli, which issimilar to S. elongatus in that it is a rod-shaped Gram-negativebacterium; however, E. coli is not photosynthetic and is notknown to possess a clock, nor does it encode homologs ofcikA or any of the kai genes. Fusions to CikA, KaiA, and KaiCwere expressed from a Ptrc promoter, under noninducing con-ditions in which low constitutive expression occurs. Both YFPand ECFP N-terminal fusions to KaiC behaved identically inE. coli and were used interchangeably. Strikingly, CikA andKaiC display a conserved mechanism of localization, as theyare able to independently localize to the poles of E. coli cells(Figure 4A), suggesting that their localization is not dependenton any photosynthetic or specific cyanobacterial factor. KaiA,on the other hand, is distributed diffusely rather than localizedto the poles in E. coli. However, if KaiC is coexpressed with

A

KaiC CikA KaiA

KaiC KaiA merge

KaiC CikA mergeB C co-localized

w/ IbpA (%) YFP-KaiC 4.6

ZsG-CIkA 4.8

Orf39-GFP 74.8 D

Figure 4. Clock Protein Localization Is Conserved between S. elongatus

and E. coli

(A) Fluorescence micrographs of E. coli MG1655 stained with vital mem-

brane stain FM4-64 (red) expressing only fluorescently labeled KaiC, CikA,

or KaiA (row 1); both ECFP-KaiC and KaiA-YFP, showing that KaiA polar

localization in E. coli is KaiC dependent (row 2); and both ECFP-KaiC and

ZsG-CikA, demonstrating that they colocalize when expressed in the

same cell (row 3).

(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 colocalized with a marker of inclu-

sion 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 colocalization.

(D) Representativemicrograph of cells expressing both YFP-KaiC and IbpA-

mCherry, with cell outlines drawn in white.

Scale bars, 2.5 mm. See also Figure S5.



KaiA, then KaiA colocalizes with KaiC at the poles (Figure 4A).Although KaiC and CikA are each capable of independentlylocalizing in E. coli, they colocalize when expressed in the

same cell, supporting the notion that the observed polar local-ization in both E. coli and S. elongatus functions to facilitate in-teractions among the clock components.Although KaiC displays polar localization in E. coli, the pat-

terns are not regulated as in S. elongatus. Primarily, KaiC local-ization is not sensitive to changes in the day-night cycle, butrather appears to be growth-phase dependent (Figure 4B).Cells have the highest percentage of localized KaiC in station-ary phase or in the lag phase, having just emerged fromstationary phase, irrespective of the lighting conditions. Thegrowth-phase dependence may merely reflect the accumula-tion of KaiC in older cells. Thus, while the mechanism of local-ization is conserved among the two organisms, KaiC localiza-tion is subject to additional regulation in S. elongatus. Thisfinding may suggest that polar localization of KaiC is thedefault and that mechanisms are in place in cyanobacteria tokeep KaiC from the pole during the day. Alternatively, a highlyconserved polar targeting factor may be required and is sub-ject to circadian regulation inS. elongatus, in which case activerecruitment would occur at night.

Polar Localization of KaiC Is Not Attributable to Formationof Inclusion Bodies

A major concern when expressing heterologous proteins inE. coli is that they may end up in inclusion bodies, which areaggregates of partially folded or misfolded proteins. This pos-sibility is especially a concern because inclusion bodies tendto display polar localization [32, 33]. In order test whether theobserved polar localization of KaiC and CikA is attributableto inclusion body formation in E. coli, we coexpressed amarker of inclusion bodies, ibpA-mCherry [34, 35]. We foundthat CikA and KaiC rarely localized with IbpA (w5%), whereasa protein that forms obvious inclusion bodies, Orf39 fromBacillus megatarium QMB155, colocalized with IbpA (w75%of the time in E. coli) (Figures 4C and 4D). These data supportthe premise that the polar clock proteins represent an activebiological complex.While no such marker of inclusion bodies exists for

S. elongatus, we performed temperature-shift experiments totest whether KaiC’s polar localization is exacerbated by astress that is likely to promote unfolding and predicted toenhance inclusion body formation [36]. Strains expressingYFP-KaiC growing at 30�C were shifted from 30�C to 25�C,30�C, or 38�C and were monitored for focus intensity at 1 hrand 4 hr after the shift. Focus intensity did not change underany conditions tested (Figures S5A and S5B). Moreover, theYFP-KaiC fusion retained functionality at elevated tempera-tures (Figure S5C). Together, the E. coli and S. elongatusdata argue that the observed polar localization is not attribut-able to the formation of inclusion bodies.

KaiC Is Not Targeted to the Pole for Degradation

In Caulobacter crescentus, the ClpXP protease is dynamicallyand selectively localized to the stalked cell pole in a mannerthat is essential for cell-cycle progression [37, 38]. We hypoth-esized that a similar mechanism may target the clock proteinsto the pole in order to be degraded by the ClpXP protease atnight. Consistent with such a model, KaiC protein levels havebeen shown to decrease over the course of the night or subjec-tive night [24]. The S. elongatus genome encodes one ClpX ho-molog, an ATPase required for substrate recognition, andthree ClpP peptidase paralogs. ClpX, ClpP1, and ClpP2 areknown to affect the circadian clock as mutant alleles of clpX,P1, and P2 produce long-period rhythms [39, 40]. ClpP2 is of

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 (2) (n > 100). Cells were sampled dur-

ing the day (ZT 8) and night (ZT 20).

(B–D) CikA can affect KaiC localization. A graph demonstrating that reduced

YFP-KaiC polar localization is observed when mutant variants of CikA that

display diffuse distribution are expressed is shown (B). Representative

micrographs of YFP-KaiC fusion expressed in DcikA mutant background

(C), DcikA + cikADPsR (D), DcikA + cikADN (E), or DcikA + cikA-HA (F). Auto-

fluorescence is shown in red. Scale bars, 2.5 mm.



particular interest as it is the most similar to clpP of E. coli andis organized in an operon with ClpX.

We tested this hypothesis by investigating KaiC localizationin clpmutant strains and determined the localization of the Clpprotease in S. elongatus. Inactivation of clpXP in either E. coli(Figure S5D) or S. elongatus (Figure S5E) was not sufficient toperturb localization of KaiC or CikA, although clpX disruptionin S. elongatus dramatically affected cell division [39].Although the ClpXP protease of E. coliwas previously reportedto be localized to the cell pole, recent reports have suggestedthat this polar localization is not biologically relevant [41].

We identified a functional fluorescent fusion to ClpP2, whichis expressed as a full-length fusion protein and complementsthe long-period phenotype of a clpP2::Tn5 strain (FiguresS5F and S5G). Although the ClpP2-YFP fusion appears to beoverexpressed by immunoblot, it complements the null strain,and overexpression of clpP2 does not have any discernableeffects on rhythms of gene expression [39]. This ClpP2-YFPis localized to the pole similar to KaiC, KaiA, and CikA. How-ever, when ClpP2-YFP is coexpressed with KaiC-ECFP, only12% of KaiC foci colocalize with a ClpP2 focus (Figure 3).Together, these findings suggest that Clp-mediated proteoly-sis is not responsible for the localization or the function ofclock proteins at the pole.

Expression of cikA Mutant Variants Can Delocalize KaiCThe polar localization of CikA requires its C-terminal pseudor-eceiver (PsR) domain through which it binds quinones [15, 21].We tested a set of mutant cikA alleles to determine how dele-tion or alteration of each domain would affect CikA localiza-tion. We expressed each mutant variant with an N-terminalZsG tag in a DcikAmutant strain and checked localization pat-terns at either the ZT 8 (day) or the ZT 20 (night) time point in adiurnal cycle. Localization correlated with the ability of eachvariant to complement rhythmicity of a DcikA mutant. Full-length CikA and amutant variant that lacks the cryptic N termi-nus (DN) both fully complement rhythms and support polarlocalization (Figure 5A). In contrast, substitution of Ala for Hisin the histidine kinase domain (HA) or deletion of the PsRdomain (DPsR) abolishes the ability to restore these functions.Interestingly, a variant that lacks the GAF domain (DGAF),which does not complement the period defect of a DcikAmutant strain [21], displayed a split phenotype for localizationin which strong polar localization was observed only at nightand diffuse distribution was observed during the day. Thus,the histidine kinase and PsR domains are required for properCikA localization, whereas the GAF domain is specific for day-time localization. The GAF domain of CikA is similar to that ofphytochrome photoreceptors that typically bind bilin cofac-tors; however, the GAF of CikA lacks a conserved cysteine orhistidine to serve as a bilin-binding site [42].

We examined the effects of CikA mislocalization on KaiC byexpressing each mutant variant as the only source of CikAand testing its effect on KaiC localization. CikA is not requiredfor the polar localization of KaiC, consistent with the fact thatKaiC localizes inE. coli, which does not encode a cikA homolog(Figures 5B and 5C). However, we observed pervasive delocal-ization of KaiC in strains that express mutant variants of CikAthat do not localize to the poles (HA and PsR) (Figures 5B–5F),dropping from w50% localized in WT cells to <8% localizedin HA- and DPsR-expressing cells. These CikA variants maysequester KaiC in the cytosol or cause changes at the polethat affect KaiC localization. Expression of the CikA(DGAF)variant resulted in an intermediate frequency of KaiC

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 (gray shaded region),

Kai proteins colocalize 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 mole-

cules present in the cell.



localization, consistent with the split localization patternsobserved for that fusion. This finding favors a stoichiometriceffect of CikA on KaiC localization and also supports the hy-pothesis that CikA and KaiC physically interact, which has pre-viously been implied, but not yet demonstrated [15]. A cikAmutant displays a variety of phenotypes, including low ampli-tude and short period rhythms of gene expression, but itsmost striking phenotype is the inability to reset the phase ofthe oscillation after receiving a dark pulse [43]. Thus, theobserved localizationsmay contribute tomechanisms of circa-dian entrainment.

Discussion

Here we provide evidence that the spatiotemporal localizationof KaiA andKaiC represents a previously unrecognized layer ofregulation in the cyanobacterial circadian system. Cellularlocalization is not fundamental to the timekeeping mechanismitself because circadian oscillation of KaiC phosphorylationcan be reconstituted in vitro. However, the system is morecomplex in vivo because the central oscillator must integrateinput and output activities in order to regulate cellular func-tions. Spatial distribution of the clock components and the co-localization of factors that vary in space and time contribute tothis complexity. We propose that the dynamic changes in KaiCprotein localization patterns function to facilitate interactionswith metabolites of photosynthesis and protein complex as-sembly, which together contribute to the synchronizationand robustness of the circadian clock, as well as its integrationwith other cellular processes (Figure 6).

Why does KaiC localize to the bacterial cell pole? Photosyn-thesis in S. elongatus occurs in thylakoid membranes that arearranged peripherally and concentrically inside the innerplasma membrane [44]. The regulation of clock componentsby metabolites of photosynthesis that change with the light-dark cycle—KaiA and CikA by oxidized quinone [15, 16] andKaiC by the ratio of ATP:ADP [17]—provides a rationale forenrichment of clock protein complexes at a specific mem-brane location at night. The interaction of KaiA and CikA withquinones, in particular, would require close proximity to thethylakoid, where a quinone-binding site of the photosynthetic

cytochrome b6f complex is located close to the membrane-water interface, where protons are transferred to the aqueousphase [45]. Within the thylakoid membrane system, two keyrespiratory electron donors move from an even distributionin high light to discrete patches with an overrepresentationat the poles in low-light conditions [30]. Although the preciserelationship between photosynthetic and respiratory electrontransport chains in cyanobacteria remains unclear, some ele-ments are shared, including the pool of quinone electron car-riers. Thus, the quinone pool and potentially other metabolitesthat feed into the clock may be enriched at the poles at night.Maintaining the appropriate stoichiometries among the Kai

proteins is critically important for sustained rhythmicity in vivoand in vitro [46]. Whereas KaiB and KaiC protein levels havebeen found to change w3-fold over the circadian cycle, KaiAprotein levels remain constant or vary with low amplitude[24]. Enhanced polar localization is observed at a time whenKaiBCprotein levels are decreasing; in this case, randomdiffu-sion may not be sufficient to ensure proper complex formation(Figure 6). Others have previously reported the enhanced as-sociation of KaiA and KaiB in complex with KaiC at night orsubjective night [15, 47], consistent with our observed spatio-temporal localization patterns. In another study, cellular frac-tionation experiments recovered KaiB in association withmembrane at all circadian times tested [24], suggesting apossible constitutive membrane localization. This finding isconsistent with the premise that KaiB and KaiC would needto interact at night while KaiC is dephosphorylating, accordingto KaiB’s role in the cycle. Thus, the observed polar reorgani-zation of clock complexes may represent a mechanism to pro-mote appropriate stoichiometry.KaiC undergoes ‘‘monomer shuffling,’’ in which monomers

are exchanged among KaiC hexamers. Monomer shuffling oc-curs during the dephosphorylation phase of the cycle and con-tributes to synchronization and stability of the Kai oscillator[48, 49]. The aggregation of KaiC hexamers at the pole duringthe dephosphorylation phase of the cycle may also facilitatemonomer shuffling. Thus, polarity may serve as a generalfeature to facilitate protein association and assembly at giventimes during the circadian cycle.How does KaiC localize to the bacterial cell pole? While a

wide variety of cellular components in diverse bacteria aretargeted to cell poles, the mechanisms that establish polarrecognition are not well understood (reviewed in [50, 51]). Inknown cases, the majority of proteins are recruited to thepole by specific interactions with a complex already atthe pole by a defined diffusion-and-capture mechanism. Thedependence of KaiA localization on KaiC is consistent withsuch a model, and unknown polar elements may captureKaiC and CikA, neither of which has intrinsic membrane-asso-ciating regions. Because KaiC and CikA localize to the polesin E. coli, their polar targeting mechanisms are not dependenton any photosynthetic, cyanobacterial, or circadian-specificfactor. The polar determinant would most likely be an essen-tial protein in E. coli, because knockout of nonessential polarlandmark proteins and performance of exhaustive randommutagenesis have not yet identified such a factor. Compo-nents of the electron transport chain that are shared betweenrespiration and photosynthesis would satisfy these criteria inthat they represent membrane proteins with a high degree ofconservation. Moreover, these proteins would facilitate inter-actions with the quinone pool or other metabolites synthe-sized or localized to the membrane that could feed into theclock.



Cell curvature is a feature of the bacterial cell poles thatdistinguishes them from the rest of the cell. Specifically, DivIVAof B. subitilis localizes to parts of the cell where negativecurvature is the strongest [52, 53]. Because E. coli andS. elongatus are both rod-shaped cells, cell curvature repre-sents a potentially conserved mechanism to recruit KaiC tothe poles. However, other mechanisms would need to be inplay because both CikA and KaiC localize to the same singlepole. In cases where poles could be distinguished, KaiC local-ized to the old cell polew65% of the time (n = 316), suggestingthat KaiC does not distinguish between the old and new cellpole.

Expression of CikA variants that display diffuse distributioncan sequester KaiC in the cytosol, suggesting either that CikAcan keep KaiC from the pole directly or that these variants cancause changes at the pole that affect KaiC localization. CikAvariants that have altered kinase [42] or phosphatase activitiesmay modify a scaffolding protein such that it can no longersupport KaiC polar localization. The identification of polardeterminants for localizing KaiC would provide insights notonly into how the clock proteins are specifically targeted tothe pole, but also the consequences that underlie polarlocalization and how they affect downstream targets of theclock.

This work shows that the cyanobacterial circadian systemundergoes a circadian orchestration of subcellular organiza-tion in addition to the known levels of regulation through pro-tein abundance and phosphorylation. Thus, the central oscil-lator proteins may also need to be at the right place at theright time for the clock to function properly. We propose thatthese dynamic changes in subcellular localization function infacilitating interactions with other clock components andmetabolites of photosynthesis, contributing to the robustnessof the circadian clock.

Experimental Procedures

Strains and plasmids used in this study are listed in Tables S1–S3. A detailed

description of procedures including circadian bioluminescence monitoring,

immunoblot analysis, fluorescence microscopy, and image analysis can be

found in the Supplemental Experimental Procedures.

Supplemental Information

Supplemental Information includes Supplemental Experimental Proce-

dures, five figures, and three tables and can be found with this article online

at http://dx.doi.org/10.1016/j.cub.2014.07.036.

Author Contributions

S.E.C., M.L.E., G.D., J.P., and S.S.G. designed research; S.E.C. and M.L.E.

performed research; J.S. and J.H. contributed new analytic tools; S.E.C.,

M.L.E., J.P., and S.S.G. analyzed data; and S.E.C. and S.S.G. wrote the

paper.

Acknowledgments

We thank B. Trial, A. Taton, and S. Zou for technical support, A. Ma and

J. Bordowitz for critical reading of the manuscript, and A. Derman for

sharing unpublished results and pOrf39-GFP. We also thank A. Clarke for

the gift of ClpP2 antiserum, I. Fung for contribution to the graphical abstract,

and J. Santini for help with the OMX. The OMX and Light Microscopy Facility

at UCSD is funded by grant NS047101. This work was supported by NIH

grant GM100116 to J.P. and S.S.G. J.S. is supported by San Diego Center

for Systems Biology NIH grant GM085764 and a UCSD Chancellor’s Collab-

oratories Program for Interdisciplinary Scholarship to J.H. and S.S.G. S.E.C.

is supported by American Cancer Society Postdoctoral Fellowship PF-12-

262-01-MPC.

Received: June 10, 2014

Revised: July 4, 2014

Accepted: July 14, 2014

Published: August 7, 2014

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