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COMMENTARY

How do bacteria localize proteins to the cell pole?

Geraldine Laloux1 and Christine Jacobs-Wagner2,3,4,*

ABSTRACT

It is now well appreciated that bacterial cells are highly organized,

which is far from the initial concept that they are merely bags of

randomly distributed macromolecules and chemicals. Central to

their spatial organization is the precise positioning of certain

proteins in subcellular domains of the cell. In particular, the cell

poles – the ends of rod-shaped cells – constitute important

platforms for cellular regulation that underlie processes as

essential as cell cycle progression, cellular differentiation,

virulence, chemotaxis and growth of appendages. Thus,

understanding how the polar localization of specific proteins is

achieved and regulated is a crucial question in bacterial cell biology.

Often, polarly localized proteins are recruited to the poles through

their interaction with other proteins or protein complexes that were

already located there, in a so-called diffusion-and-capture

mechanism. Bacteria are also starting to reveal their secrets on

how the initial pole ‘recognition’ can occur and how this event can be

regulated to generate dynamic, reproducible patterns in time (for

example, during the cell cycle) and space (for example, at a specific

cell pole). Here, we review the major mechanisms that have been

described in the literature, with an emphasis on the self-organizing

principles. We also present regulation strategies adopted by bacterial

cells to obtain complex spatiotemporal patterns of protein localization.

KEY WORDS: Bacterial cell cycle, Polar localization, Spatial

organization

IntroductionEvidence for the elaborate spatial organization of bacterial cells

started to accumulate more than two decades ago, challenging the

antiquated idea that bacteria were simple vessels of randomly

distributed macromolecules, far removed from organized,

compartmentalized eukaryotic cells. This misconception

naturally originated from the observation that the cytoplasm of

these tiny cells generally lacks intracellular membrane-enclosed

organelles in which biomolecules – hence cellular functions – can

be sorted. It is now well appreciated that cellular components of

various kinds (proteins, lipids, DNA, RNAs, ribosomes and

metabolites) can display subcellular arrangements in bacterial

cells. This spatial organization is critical for various aspects of

bacterial physiology and adaptation to diverse environments

(Matsumoto et al., 2006; Shapiro et al., 2009; Rudner and Losick,

2010; Campos and Jacobs-Wagner, 2013).

Within the bacterial cytoplasm, the cell poles – the roundedends of rod-shaped cells that are generated by each division event– constitute a domain where a subset of proteins specifically

localizes. Proteins that localize at the poles (referred to as ‘polarproteins’ hereafter) are numerous and vary widely in function.Localization of polar proteins, which is the focus of thisCommentary, is essential for a large number of important

cellular processes in bacteria, including cell cycle regulation,cell differentiation, virulence, chemotaxis, motility and adhesion(Bowman et al., 2011; Kirkpatrick and Viollier, 2011). Our goal

here is not to provide a survey of the large number of polarproteins described so far, many of which have been reviewedelsewhere (Ebersbach and Jacobs-Wagner, 2007; Dworkin, 2009;

Kirkpatrick and Viollier, 2011; Davis and Waldor, 2013). Instead,our objective is to review and illustrate the general principlesused by bacterial cells to localize proteins at the poles.

The patterns displayed by proteins inside bacterial cells can becomplex. Indeed, some proteins are known to change location

over time, such as during the course of the cell cycle; othersreproducibly accumulate at only one of the two cell poles. Thedynamics and asymmetry of polar localization underlie central

cellular processes in bacteria, yet the mechanisms that controlpolar localization in space and time are only starting to beuncovered. In this Commentary, we first summarize several polar

localization mechanisms that have been selected during bacterialevolution. Then, we examine possible strategies that control thesemechanisms to produce specific spatiotemporal patterns ofprotein localization.

How proteins localize at the cell poles: themes and variationsDiffusion and capture through protein–protein interactionMost of the polar proteins identified so far are simply recruited tothe cell poles through an interaction with a protein or proteincomplex already present at the pole (Fig. 1A). In this scenario,protein A diffuses in the cytoplasmic space until it encounters a

polar protein B for which it has a binding affinity. Because of thetransient nature of protein–protein interactions, protein A iscontinuously exchanged between the diffusing pool in the cell

body and the localized pool at the pole. The concentration ofprotein B at the poles and the rates of association and dissociationbetween the interacting partners A and B will determine the

extent of the polar accumulation of protein A at steady state. Notethat ‘diffusion and capture’ is a general mechanism that alsoexplains the subcellular localization of many non-polar proteins.

Remarkably, some proteins can serve as polar landmarks orhubs for multiple proteins, which can themselves recruit other

proteins, and so forth. These hub proteins tend to play importantroles in different pathways or cell cycle events. For example,DivIVA is involved in several cellular programs in Bacillus

subtilis through the recruitment of multiple proteins: thecompetence regulator ComN (dos Santos et al., 2012), thedivision inhibitor Maf in competent cells (Briley et al., 2011) and

the determinants of division site placement MinCD via MinJ

1de Duve Institute, Universite Catholique de Louvain, B-1200 Brussels, Belgium.2Department of Molecular, Cellular and Developmental Biology, Yale University,New Haven, CT 06520, USA. 3Howard Hughes Medical Institute, Yale University,New Haven, CT 06519, USA. 4Department of Microbial Pathogenesis, Yale Schoolof Medicine, New Haven CT 06511, USA.

*Author for correspondence (christine.jacobs-wagner@yale.edu)

� 2014. Published by The Company of Biologists Ltd | Journal of Cell Science (2014) 127, 11–19 doi:10.1242/jcs.138628

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(Bramkamp et al., 2008; Patrick and Kearns, 2008;Eswaramoorthy et al., 2011). Moreover, DivIVA attaches thechromosomal origin at the pole in the developing spore through aninteraction with the DNA-binding protein RacA (Ben-Yehuda et

al., 2003; Wu and Errington, 2003). In Caulobacter crescentus,PopZ, which shares no sequence or structural similarity withDivIVA, also anchors the chromosomal origin at the pole through

an interaction with the chromosome partition complex, andrecruits proteins that are involved in chromosome segregation andcell cycle control (Bowman et al., 2008; Ebersbach et al., 2008;

Bowman et al., 2010; Schofield et al., 2010). The partitioncomplex is composed of the parS sequences, located in thevicinity of the chromosomal origin, and the parS-binding protein

ParB. Upon origin replication, one of the duplicated ParB–parS

complexes remains at one pole while the other is segregatedtowards the opposite pole, followed by the rest of the sister

chromosome. Because both ParB–parS complexes also carry adivision inhibitor, their PopZ-dependent tight fastening at bothpoles after complete segregation is essential for the correctpositioning of the division site near the midcell, where the

inhibitor concentration is the lowest (Thanbichler and Shapiro,2006; Ebersbach et al., 2008). In Vibrio cholerae, HubP provides alink between chromosome segregation, chemotaxis and flagellum

synthesis by localizing three different ATPases that positionelements of these machineries at the pole (Yamaichi et al., 2012).

But what is the ultimate positional information that allows

these landmark proteins to ‘recognize’ the poles directly? Toaddress this question, one must examine what makes the cellpoles different from the rest of the cell. The poles have several

distinctive features, and perhaps not surprisingly, bacteria canexploit several of them to efficiently position proteins at the polesthrough a diversity of mechanisms, as described below.

A Diffusion and capture

Diffusing protein Polar protein

B Negative curvature

C =

1/R

C =

2/R

C =

2/R

C Nucleoid occlusion

Nucleoid(chromosomal DNAand proteins)

Oligomer of aself-assembling oraggregating protein

P

C

E

CM

OM

CM

P

C

OM

E

D Affinity for polar features of the cell envelope

Phospholipid

Anionic phospholipid enrichedat the poles (e.g. cardiolipin)

Peptidoglycan turnover

Peptidoglycan

Protein with affinity forcardiolipin

Lipopolysaccharide

PoleSidewall Key

Fig. 1. Localization of polar proteins through the recognition of polar features. (A) A protein (e.g. ParA1 in V. cholerae) diffusing in the cytoplasm(as indicated by single arrows) is trapped at the poles transiently (double arrows) through an affinity for a polar protein (e.g. HubP in V. cholerae) that is alreadylocalized at the cell poles. (B) Higher-order protein assemblies are favored in membrane regions of stronger curvature. Left: representation of a rod-shapedcell showing the radius of curvature (R) and the stronger negative curvature (C, curved arrows) at the cell poles (blue areas) compared with the sides of thecylinder (gray area), as described previously (Huang and Ramamurthi, 2010). Middle and right: formation of a higher-order protein assembly occurringpreferentially in membrane regions of stronger negative curvature (e.g. DivIVA in B. subtilis). Arrows indicate the free diffusion of oligomers. (C) Formation oflarge protein assemblies such as higher-order structures (e.g. PopZ in C. crescentus) or protein aggregates (e.g. misfolded proteins in E. coli) is energeticallyfavored outside the nucleoid region. (D) Differences in composition of the cytoplasmic membrane and peptidoglycan between cell poles and the rest of the cellenvelope can serve as cues for localization of polar proteins. The particular case of a protein (e.g. ProP in E. coli) that preferentially binds anionic phospholipidsenriched at the poles, such as cardiolipin, is depicted. E, extracellular space; OM, outer membrane; P, periplasmic space; CM, cytoplasmic membrane;C, cytoplasm. Note that the schematics in all figures are not to scale and do not reflect the structure of the illustrated proteins.

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Direct sensing of enhanced curvatureA characteristic of the cell poles is their geometry. The total

curvature (C) of the cell envelope is stronger at the polesbecause of their shape: for example, in bacilli, C is equal to 1/Ralong the cylindrical cell body (where R is the radius of thecylinder), whereas C equals 2/R at the hemispherical poles of

the cell (Huang and Ramamurthi, 2010). An attractivehypothesis is that some proteins preferentially accumulate atthe poles through an affinity for the more concave membrane

curvature of the poles relative to the rest of the cell (Fig. 1B).An illustration of this mechanism is provided by the self-assembling protein DivIVA of B. subtilis, which preferentially

localizes in the most concave regions of the cell (Lenarcic et al.,2009; Ramamurthi et al., 2009). After cell division, DivIVAremains localized at the poles newly generated from the division

site (which are, at that time, the most curved regions in thecell), then mainly redistributes to a septal position when a newround of division is initiated (Ramamurthi et al., 2009;Eswaramoorthy et al., 2011). Whereas the polar localization of

DivIVA in B. subtilis derives mostly from its curvature-dependent accumulation at the septum, DivIVA initiallyconcentrates at the poles in outgrowing spores that are not yet

engaged in cell division (Hamoen and Errington, 2003; Harryand Lewis, 2003). This is consistent with the idea that DivIVAlocalizes where negative curvature is the strongest, that is,

primarily at the septum in actively dividing cells or at the polesin the absence of a septum. Experiments with B. subtilis cells inwhich cytokinesis is artificially blocked support this idea: as

cells become filamentous and do not form new septa, DivIVAprogressively relocalizes to the cell poles (Ramamurthi andLosick, 2009). In Streptomyces coelicolor hyphae, the DivIVAhomolog mainly localizes at future and emerging hyphal tips,

probably by sensing negatively curved surfaces (Flardh, 2003;Hempel et al., 2008; Holmes et al., 2013). These hyphal tips areformed de novo along the cell without a division event.

B. subtilis DivIVA spontaneously accumulates at the polesupon heterologous expression in very distant species that lackDivIVA homologs such as Escherichia coli and fission yeast

(Edwards et al., 2000). This supports the idea that DivIVAdirectly senses stronger concavity without the need of a proteinanchor. But how could a nanometer-sized protein sense acurvature difference that is negligible at the protein scale?

Indeed, a mathematical model shows that the average size of aprotein monomer is too small relative to the pole diameter to beable to detect any curvature change (Huang and Ramamurthi,

2010). Instead, the protein would need to assemble into largestructures to achieve cooperative, long-range sensing ofmembrane curvature (Lenarcic et al., 2009; Huang and

Ramamurthi, 2010) (Fig. 1B). Consistent with this notion,DivIVA oligomerizes in vitro and in vivo (Muchova et al.,2002; Stahlberg et al., 2004; Oliva et al., 2010), and these

oligomers can further assemble into larger structures (Stahlberg etal., 2004; Wang et al., 2009). A ‘molecular-bridging’ modelbased on Monte-Carlo simulations argues that the clustering ofDivIVA oligomers is favored at more-curved regions owing to the

enhanced possibility of stabilizing interactions with themembrane (Lenarcic et al., 2009). A recent structural studyshowed that the intrinsic curvature of a DivIVA tetramer is

distinct from the membrane curvature at the pole or septum(Oliva et al., 2010), further suggesting that a higher-orderassembly is required for the curvature-mediated localization of

DivIVA oligomers.

Self-assembly and nucleoid occlusionApart from their geometry, the cell poles are characterized by

being largely devoid of chromosomal DNA (nucleoid). Thisdistinguishing feature can also serve as a positional cue forprotein localization. The concept is based on the notion that theformation of big structures through protein self-assembly is

energetically more favorable outside bulky polymers such as thenucleoid because of volume-exclusion effects. Thus, large proteinclusters can be passively sorted to the poles by a process called

nucleoid occlusion (Fig. 1C). This polar localization mechanismwas first proposed for the self-assembling hub protein PopZ in C.

crescentus (Ebersbach et al., 2008). PopZ spontaneously interacts

with itself and the resulting oligomers further assemble in vivo

into a matrix that presumably enhances the availability of dockingsites for multiple partners (Bowman et al., 2008; Ebersbach et al.,

2008; Bowman et al., 2010). C. crescentus PopZ is also able toassemble into a polar matrix in the evolutionary divergent E. coli

(Bowman et al., 2008; Ebersbach et al., 2008; Laloux and Jacobs-Wagner, 2013), even though E. coli lacks PopZ homologs or any

known PopZ partners. PopZ multimerization is essential to itspolar localization (Laloux and Jacobs-Wagner, 2013; Bowman etal., 2013). This was shown not only in C. crescentus, but also in

E. coli, supporting the idea of a self-organizing localizationmechanism that is based on PopZ assembly (Laloux and Jacobs-Wagner, 2013). A bona fide PopZ matrix can form not only at the

poles but also in any non-polar DNA-free region, for example, inbetween segregated nucleoids along the filaments of a C.

crescentus mutant or E. coli cells in which cell division is

blocked (Ebersbach et al., 2008; Laloux and Jacobs-Wagner,2013). Thus, the polar localization of PopZ is independent ofcurvature, and instead appears to be determined by its preferentialself-assembly in regions of low DNA content (Ebersbach et al.,

2008; Laloux and Jacobs-Wagner, 2013).Misfolded proteins also passively aggregate at the cell poles in

E. coli as a result of nucleoid occlusion (Winkler et al., 2010;

Coquel et al., 2013). The principle is simple: misfolded proteinsfreely diffuse in the cytoplasm and tend to stick to each otherowing to the exposure of hydrophobic patches on their surface.

As the amorphous aggregates grow by the addition of moremisfolded peptides, they are excluded from the nucleoid andaccumulate at the cell poles where they can expand further.

Supporting this model, in silico simulations demonstrate that

the passive diffusion of a particle, its intrinsic ability to multimerizeand the absence of nucleoid at the poles are sufficient to obtain apolar-localization pattern by entropy alone (Saberi and Emberly,

2010; Coquel et al., 2013; Saberi and Emberly, 2013). Note that thisprocess is spontaneous. However, as described in the second part ofthis Commentary, temporal and spatial control can be added to this

process through regulation of protein self-assembly.

Affinity for pole-specific elements of the cell envelopeAnother unique trait of the cell poles is the composition andmaturation of their cell envelope. Therefore, another possiblemechanism of polar localization relies on protein recognition ofpole-specific features of the cell envelope (Fig. 1D). For

example, some proteins interact preferentially with specificphospholipids such as cardiolipin (Mileykovskaya et al., 2003;Renner and Weibel, 2012) that are enriched in the cytoplasmic

membrane surrounding the poles of several bacterial species(Mileykovskaya and Dowhan, 2000; Kawai et al., 2004; Bernal etal., 2007). The polar enrichment of cardiolipin is driven by its

intrinsic curvature, which thermodynamically favors its insertion

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as clusters (‘microdomains’) in highly negatively curvedmembranes (Huang et al., 2006; Mukhopadhyay et al., 2008;

Renner and Weibel, 2011). At least two E. coli proteins, ProP andMscS, localize to the poles in a cardiolipin-dependent manner(Romantsov et al., 2007; Romantsov et al., 2010). The frequencyof polar localization in the cell population correlates with the

overall content of cardiolipin, although it is unknown whether thisis due to a direct protein–lipid interaction at the pole. It would beinteresting to see whether the polar enrichments of lipids and

proteins are also correlated at the single-cell level. Thisphenomenon is not unique: other proteins might also exploit theparticular lipid content at the poles for their local accumulation

(Renner and Weibel, 2012).Bacteria could also exploit the particular composition and

maturation of the peptidoglycan wall at the cell poles to localize

proteins there. Indeed, another particularity of the cell envelopeat the poles of most rod-shaped bacteria is the absence ofnew peptidoglycan synthesis. This is because insertion ofpeptidoglycan material in some bacteria such as E. coli is

redirected to the sidewall following cell septation (de Pedro et al.,1997). Therefore, polar peptidoglycan is considered less ‘active’in these species. It is conceivable that some proteins could

directly bind pole-specific peptidoglycan features and therebyaccumulate mainly at the cell poles. So far, a few proteins havebeen proposed to localize at the poles by this mechanism (Lawler

et al., 2006; Radhakrishnan et al., 2008; Wirth et al., 2012;Yamaichi et al., 2012). However, at this stage, the evidence ismostly correlative and a difference in chemical composition

between polar and non-polar peptidoglycan remains to bedetermined. The next challenge resides in advancing techniquesof peptidoglycan composition analysis, which currently lack thespatial resolution required to identify the key chemical elements

that serve as polar localization cues.

Growth-dependent transmission from the sidewallWhereas specificities of the polar envelope can be exploited as acue for protein localization (Fig. 1D), an alternative, less-directstrategy can use the lateral (non-polar) peptidoglycan wall as a

‘shuttle’ to passively direct proteins toward the poles (Fig. 2A). Ithas been proposed that the cell wall material is progressivelypushed towards the poles through continuous lateral insertion ofnew ‘building blocks’ of peptidoglycan over several rounds of

growth and division (Rafelski and Theriot, 2006). Accordingly,proteins that stably associate with cell wall constituents along thecell cylinder could be gradually guided toward the cell poles. This

multi-step mechanism has been proposed to explain the slowpolarization of the surface protein ActA of the Gram-positivepathogen Listeria monocytogenes (Rafelski and Theriot, 2006).

When this pathogen is inside mammalian cells, ActA induces thepolymerization of host actin at the cell pole, resulting in a cometactin tail that propels the bacterium inside the host cell and into

neighboring cells (Kocks et al., 1992; Kocks et al., 1993; Lacayoet al., 2012). The prevailing model suggests that upon entry of thepathogen into a host cell, ActA is secreted at discrete spots alongthe bacterium membrane, then associates with the peptidoglycan

(Garcıa-del Portillo et al., 2011) and eventually spreads uniformlyalong the entire cell length as a result of protein accumulation andcell wall growth. As the lateral wall grows, ActA is proposed to

follow the older peptidoglycan towards the poles. The cell wallturnover slows down as the cell pole ages, so that the pool ofActA becomes ultimately trapped at the older pole after a few

rounds of division (Rafelski and Theriot, 2006). Because

polarization of ActA – and hence, the formation of a propellingactin tail – depends on the growth rate of the bacterium, such a

localization mechanism might temporally connect the actin-powered motility of L. monocytogenes inside the host to thepathogen growth dynamics. Interestingly, IcsA, the functionalcounterpart of ActA in Shigella flexneri, appears to be directly

targeted to one pole before its secretion into the periplasm andinsertion in the outer membrane (Goldberg et al., 1993;Steinhauer et al., 1999; Charles et al., 2001). It remains unclear

how this initial positioning occurs, but the maintenance of IcsA atthe pole is achieved through specific proteolysis along the entirecell surface, preventing lateral diffusion of the protein away from

the pole (Steinhauer et al., 1999).

Inheritance from the division siteWhen rod-shaped bacteria divide, each daughter cell invariablyinherits an ‘old’ pole from its mother and a ‘new’ pole freshlyformed at the site of division. Therefore, a protein stably localizedat the division site before cytokinesis could result in localization

at a new pole after cell separation (Fig. 2B). The moststraightforward illustration of this mechanism is given bycomponents of the division machinery. In C. crescentus, the

tubulin homolog FtsZ, along with interacting partners, remainslocalized at the new cell poles after division (Thanbichler and

A Transmission from the sidewall

Growth anddivision

Peptidoglycan-associated protein

Displacement of peptidoglycan owingto lateral insertion of new material

Newpole

Newpole

Oldpole

Oldpole

B Inheritance from the site of division

Division

Protein localized at the new pole

Newpole

Newpole

Oldpole

Oldpole

Key

Fig. 2. Localization of polar proteins resulting from cell growth anddivision. (A) A protein associated with the peptidoglycan is progressivelydirected towards the poles as the cell proceeds through cycles of growth anddivision, as new peptidoglycan is inserted laterally and becomes more andmore inert during cell aging (e.g. ActA in L. monocytogenes). (B) Proteinsstably localized at the midcell before cell division remain associated with thenewly formed cell poles in the progeny (e.g. TipN in C. crescentus).

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Shapiro, 2006; Goley et al., 2011). Proteins that are not directlyinvolved in cell division can then couple their localization to

division cues, thereby ensuring their localization at the new polein the progeny. This is illustrated by the polar landmark TipN inC. crescentus (Huitema et al., 2006; Lam et al., 2006). At thebeginning of the cell cycle, the membrane-bound coiled-coil

protein TipN is present at the new pole where it acts as a beaconfor developmental and cell cycle programs such as the polarbiogenesis of a flagellum (Huitema et al., 2006; Lam et al., 2006).

TipN also promotes the unidirectional segregation of theduplicated ParB–parS partition complex toward the new pole(Ptacin et al., 2010; Schofield et al., 2010). In late predivisional

cells, TipN delocalizes from the new pole, possibly in a cell-size-dependent manner, and relocalizes at the midcell. This step isdependent on cell division and requires the Tol–Pal complex

(Huitema et al., 2006; Lam et al., 2006; Yeh et al., 2010), acomponent of the divisome in Gram-negative bacteria that isresponsible for outer membrane invagination during cytokinesis(Gerding et al., 2007). This way, each daughter cell inherits a

TipN marker at its newborn pole.In a variation of this mechanism, the midcell localization step

can occur spontaneously, independently of any division-

associated factor. This appears to be the case forchemoreceptors in E. coli. Arrays of chemoreceptors, which areamong the largest signaling structures in bacterial cells, display a

remarkable spatial organization at the membrane that is crucialfor their function in chemotaxis. Chemoreceptor clusters arepredominantly polar (Maddock and Shapiro, 1993), but a few are

regularly positioned along the cell body (Sourjik and Armitage,2010). This periodic arrangement is spontaneous: chemoreceptorsstochastically self-assemble in a concentration-dependent mannerto build new clusters or to expand old ones, and the formation of

new clusters is entropically favored far away from pre-existingones (Thiem et al., 2007; Thiem and Sourjik, 2008; Wang et al.,2008; Greenfield et al., 2009). As a consequence of this

organization, larger clusters appear confined to future divisionsites (Thiem et al., 2007). This progressively leads to their polaraccumulation and retention after several generations. Thus, two

recurrent keys for protein localization, spontaneous self-assemblyand inheritance at division, drive the polar clustering ofchemoreceptors in E. coli. Interestingly, the localization of themembrane-associated chemoreceptor clusters in Rhodobacter

spheroides has recently been shown to be independent of thecytokinetic FtsZ ring or the sites of future division. Instead, theseclusters diffuse freely in the membrane, excluding the area of the

FtsZ ring, and tend to accumulate at the poles possibly by adiffusion-and-capture process that could involve trapping of theclusters through negative-curvature sensing (Chiu et al., 2013).

Pole-to-pole oscillations through nucleotide switch andmembrane bindingRemarkably, some proteins oscillate between the two cell poles(Lenz and Søgaard-Andersen, 2011). A well-studied case of pole-to-pole oscillation is the Min system in E. coli (Fig. 3), whichcontributes to the proper positioning of the division site (Raskin

and de Boer, 1999) and might facilitate chromosome segregation(Di Ventura et al., 2013). In this system, a pool of MinD proteinsrapidly oscillates between poles and carries along the division

inhibitor MinC. Because of continuous pole-to-pole oscillations,the concentration of the MinC division inhibitor is, on timeaverage, the lowest at the midcell, allowing division at that

location only. Experimental and modeling studies reveal that the

oscillatory behavior of Min is a multi-step self-organizing process(Shih and Zheng, 2013). Dimers of ATP-bound MinD

cooperatively bind the membrane in a zone that extends fromthe pole towards the center of the cell (Hu et al., 2002; Hu et al.,2003; Lackner et al., 2003). The MinE protein binds to MinD atthe edge of that zone and stimulates ATP hydrolysis (Hu and

Lutkenhaus, 2001), thereby releasing MinD monomers from themembrane into the cytoplasm. MinE then interacts with anddestabilizes the next MinD dimer at the membrane, and so on, in

a so-called ‘Tarzan of the jungle’ model (Park et al., 2011; Park etal., 2012). MinE eventually reaches the pole, dissociates anddiffuses freely in the cytoplasm before starting a new cycle on the

other side of the cell where MinD has re-assembled. Whattriggers the formation of a new MinD zone at the pole?Experiments in mutant round cells indicate that MinD can

localize anywhere on the membrane but preferentially re-assembles the furthest away from the MinE-destabilizing effect(Corbin et al., 2002). When cell poles are further away thannormal (e.g. in filamentous cells), the Min system adopts a striped

localization pattern (Raskin and de Boer, 1999), suggesting thatno polar feature is needed to obtain cycles of collectivemembrane binding and unbinding. Thus, the cell geometry and

the MinD–MinE interplay at the membrane might be sufficient toexplain pole-to-pole oscillations in vivo (Meinhardt and de Boer,2001; Howard and Kruse, 2005; Varma et al., 2008; Di Ventura

and Sourjik, 2011; Loose et al., 2011; Halatek and Frey, 2012).Consistent with this idea, Min proteins form spontaneous waveson artificial lipid layers in vitro (Loose et al., 2008). The

establishment of the MinD polar cap could be facilitated by a

Membrane-boundMinD-ATP (dimer)

Cytosolic MinD-ADP(monomer)

MinE (disassemblesMinD-ATP dimers)

Time (~1–2 minutes/cycle)

1

2

3

4

Key

Fig. 3. Pole-to-pole oscillation through nucleotide switch andmembrane binding. In E. coli, MinE binds MinD-ATP dimers at theedge of a membrane-bound MinD-ATP dimer zone and triggers ATPhydrolysis, which releases MinD-ADP monomers in the cytosol (Step 1,thin arrows). MinE then binds to the next MinD-ATP. Iterations of thisprocess lead to a progressive retraction of the MinD-ATP dimer zone atthe membrane (Step 2, thick arrow). Following ADP/ATP nucleotideexchange, MinD-ATP redimerizes and dimers collectively reassociatewith the membrane far from the MinE ring, i.e. at the opposite pole(Steps 2 and 3). After ‘running out’ of membrane-associated MinD-ATPdimers at one pole, MinE is released into the cytoplasm, diffuses until itassociates with the edge of the new MinD-ATP dimer zone at themembrane (Steps 3 and 4).

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specific polar-nucleation protein such as cardiolipin, to whichMinD preferentially binds (Mileykovskaya et al., 2003; Drew

et al., 2005; Cytrynbaum and Marshall, 2007; Renner and Weibel,2012), although the importance of such spatial cues is still underinvestigation (Halatek and Frey, 2012).

Control of polar localization in space and timePolar localization is often temporally and spatially regulated,which is essential for bacterial life and development (Shapiro et

al., 2009). Some proteins accumulate at one pole only or changetheir localization (e.g. from diffuse to polar or from unipolar tobipolar) as the cell cycle proceeds or in response to environmental

signals. The mechanistic basis underlying the various localizationdynamics observed in bacteria is currently a hot topic ofinvestigation. Below, we consider several regulatory strategies

that can modulate protein localization in time or space. Somehave concrete examples, whereas others are speculative.

Intrinsic temporal and spatial patterning through cell divisionSome polar localization mechanisms intrinsically yield cell-cycle-regulated patterns. For example, TipN only marks thenew pole of newborn C. crescentus cells because its polar

position is acquired from the division plane of the mother cell(Fig. 2B), which forms the new pole of the progeny (Huitemaet al., 2006; Lam et al., 2006). Along the same lines, cell-cycle-

associated alterations of the intracellular geometry can providespatio-temporal regulation to curvature-dependent polarlocalization. Provided that protein assemblies are dynamic

(i.e. exchanges exist between the cytoplasmic pool and thepolar cluster), the formation or disappearance of regions ofstronger curvature could redeploy protein enrichments fromone area of the cell to another. This is the case for DivIVA,

which redistributes itself between the poles or septumdepending on the initiation or block of cell division(Ramamurthi et al., 2009; Eswaramoorthy et al., 2011).

Cell division also inherently contributes to the transmission ofasymmetric polar patterns. For example, PopZ, which decoratesboth poles of predivisional C. crescentus cells, is invariably

transmitted to the old pole of each daughter cell (Fig. 4A)(Bowman et al., 2008; Ebersbach et al., 2008). This is particularlyimportant to maintain the spatial organization of the cell, becauseeach sibling receives a copy of the chromosome with a conserved

configuration, held in place by the ParB–parS partition complexthat is tethered at the old pole by a PopZ matrix. We describebelow how PopZ reverts to a bipolar localization at a later stage

of the cell cycle. In another scenario, proteins that are stablyconfined at one pole of the mother cell will be transmitted to theold pole of only one of the daughter cells. This is the case for

large aggregates of misfolded proteins in E. coli (Fig. 4A)(Winkler et al., 2010). Hence, the propagation of asymmetric (i.e.unipolar) polar localization patterns can be ‘encoded’ in the

mechanism that drives polar localization itself or can simplyderive from cell division.

Temporal and spatial modulation of protein assemblyAs described above, protein assembly into large complexes orstructures emerges as a recurrent key process for self-organizingmechanisms of polar localization based, for example, on

membrane curvature sensing (Fig. 1B) or nucleoid occlusion(Fig. 1C). Hence, a general strategy for modulating polar patternsmight be to facilitate or prevent protein assembly at given

locations and times.

Affecting self-assembly through post-translational modificationsA variety of post-translational modifications, such as changes of

phosphorylation state or nucleotide binding, control the complexintracellular distribution of several proteins that are involved incell cycle regulation, signal transduction, and polarized motilityand adhesion (Bulyha et al., 2011; Kirkpatrick and Viollier,

2012). Although most examples known to date concern proteinsthat are at some point recruited to the poles through protein–protein interactions (Fig. 1A), similar modifications could also

influence the ability of some proteins to multimerize, therebyimpacting their spontaneous polar accumulation. If the presenceor activity of the cognate kinases, phosphatases, GTPase-

activating proteins (GAPs), among others, is under temporalregulation, this would provide a way to regulate an otherwisespontaneous polar localization in time (Fig. 4B). Recent studies

suggest that this strategy can be exploited by bacteria. Forexample, in S. coelicolor, the increased phosphorylation ofDivIVA by the eukaryotic-like Ser/Thr protein kinase AfsK,which can be activated artificially or upon arrest of cell wall

synthesis, leads to the disassembly of DivIVA foci at the hyphaltips with dramatic effects on apical growth and hyphal branching(Hempel et al., 2012). Hence, phosphorylation of DivIVA could

modify its oligomerization state. In fast-growing Mycobacterium

tuberculosis cells, expression of kinases for the DivIVA homologWag31 is enhanced (Kang et al., 2005), which might result in an

increase in phosphorylation of Wag31. Phosphorylationstimulates Wag31 oligomerization and thereby its polarlocalization (Jani et al., 2010). Polar localization, in turn, would

promote Wag31-dependent synthesis of polar peptidoglycans(Jani et al., 2010).

Protein cleavage by specific proteases might also represent astrategy to modulate polar localization in space and time, as

proposed for the polar beacon PodJ. Conversion of PodJ from along (PodJL) to a shorter form (PodJS) by a cell-cycle-regulatedproteolytic sequence that eventually degrades PodJS ensures

proper localization and hence function of PodJ (Viollier et al.,2002; Chen et al., 2006). However, the precise mechanismwhereby both forms of PodJ differentially localize at the poles

remains to be determined.

Coupling to a cell cycle eventTemporal and spatial control of the localization of polar proteins

can also occur through coupling to a particular cell cycle event(Bowman et al., 2010; Iniesta et al., 2010; Ditkowski et al., 2013;Fernandez-Fernandez et al., 2013; Laloux and Jacobs-Wagner,

2013), and C. crescentus PopZ provides an example. Asmentioned above, daughter cells inherit a PopZ matrix at theold pole through division (Fig. 4A), but its chromosome-tethering

function requires that a second PopZ matrix forms at the newpole. Evidence from our work suggests that this unipolar-to-bipolar transition is directly linked to the accumulation of the

partitioning protein ParA at the new pole (Laloux and Jacobs-Wagner, 2013), which occurs during the segregation of the ParB–parS partition complex (Ptacin et al., 2010; Schofield et al., 2010;Shebelut et al., 2010). Because ParA interacts with PopZ

(Schofield et al., 2010; Laloux and Jacobs-Wagner, 2013), ourmodel (Fig. 4C) proposes that the accumulation of ParA at thenew pole results in a local accumulation of diffusing PopZ

oligomers at that location (Laloux and Jacobs-Wagner, 2013). Ahigher concentration of PopZ oligomers, in turn, promotes matrixformation at the right pole (new pole) and at the right time (when

tethering of the segregated ParB–parS complex is needed).

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Consistent with this model, the unipolar-to-bipolar localizationpattern of PopZ could be recapitulated in a ParA-dependent

fashion in a heterologous E. coli system (Laloux and Jacobs-Wagner, 2013).

Other self-assembling proteins might similarly rely on aninteracting factor that is present at a specific pole at a specific

time during the cell cycle to modulate their concentration in timeand space, thereby modulating their propensity to self-assembleand localize at the poles. Bactofilins, which self-assemble in vitro

without any cofactor and whose low cytoplasmic concentrationmight not allow a spontaneous polymerization, could be goodcandidates for such a spatiotemporally regulated polar

accumulation (Kuhn et al., 2010).

Conclusion and perspectivesBacteria use a diversity of mechanistic principles to drive proteinlocalization at the cell poles (Figs 1–3). Molecular dynamics andasymmetries inherent to specific cell cycle events orphysiological responses can be exploited in order to bring

spatial and temporal regulation to otherwise spontaneouslocalization processes (Fig. 4). Although exciting progress hasbeen made in the past few years, it will take a concerted effort

from both experimentalists and theorists to understand theintricacies of each localization mechanism. We also anticipatethat additional mechanisms await discovery as the list of

identified polar proteins keeps increasing.Essential to the investigation of protein localization is the

tracking of proteins inside live bacterial cells. The development of

less-bulky fluorescent tags and membrane-permeable organic dyesfor protein visualization in bacteria (Griffin et al., 1998; Charbon etal., 2011), combined with near-native or adjustable expressionlevels, should ensure more physiologically relevant observations of

subcellular distributions in the future. Currently, artefactuallocalization patterns – often polar accumulations – remain aconcern as they can arise upon overexpression, misfolding or

fusion to some fluorescent tags, depending on the protein beingvisualized (Winkler et al., 2010; Landgraf et al., 2012; Margolin,2012; Swulius and Jensen, 2012). In addition, the quantification of

subcellular patterns should become an integral part of anylocalization study. In that respect, recent post-imaging softwarepackages are becoming essential tools to quantify proteinlocalization dynamics – at both population and single-cell levels

– that cannot be inferred from simple observations or manualmeasurements (Guberman et al., 2008; Sliusarenko et al., 2011;van Teeffelen et al., 2012). Importantly, their automation precludes

the unconscious biases inherent to visual inspections. The field willalso benefit from novel in vitro and in vivo assays based on thereconstruction of minimal cells with variable shape or composition

(Renner and Weibel, 2011; Renner and Weibel, 2012).

AcknowledgementsWe are grateful to the members of the Jacobs-Wagner lab for critical reading ofthe manuscript. We apologize to all authors whose work could not be cited owingto space limitations.

Competing interestsThe authors declare no competing interests.

FundingWork in the Jacobs-Wagner lab is funded by the National Institutes of Health[grant number RO1 GM065835]. G.L. was the recipient of a fellowship from theResearch Department of the French-speaking Community of Belgium. C.J.-W isan investigator of the Howard Hughes Medical Institute. Deposited in PMC forrelease after 12 months.

C Coupling with a cell-cycle-associated asymmetry

B Post-translational modificationsSignal

Post-translationalmodification

Self-assembling protein

A Asymmetry generated by cell division

Old pole

Old pole

New poleNew pole

Old pole

Old pole

Step 1 Step 2

?

Proteins forming stablemultimeric structures oramorphous aggregates

Difffusingoligomer

Protein withasymmetricdistribution

Fig. 4. Possible strategies for spatial and temporal regulationof polar localization. (A) Asymmetric polar patterns can benaturally produced by a cell division event. Left: bipolar to old-polelocalization (e.g. PopZ in C. crescentus). Right: propagation of anold-pole accumulation (e.g. protein aggregates in E. coli). In the caseof protein aggregates, misfolded proteins produced in the progenyaccumulate onto the existing polar aggregate. Eventually, de novo

polar accretions can appear in progeny that did not acquire a polarfocus (top cell), for example after new protein synthesis. (B) Theability of some proteins to self-assemble and thereby to localize at thepoles (e.g. Wag31, the DivIVA homolog in M. tuberculosis) could beinfluenced by modifications such as phosphorylation upon a specificsignal (e.g. expression of the kinases of Wag31 in exponential phasein M. tuberculosis may increase Wag31 phosphorylation). Thequestion mark indicates a hypothetical step. (C) The concentration ofa self-assembling protein or oligomer (e.g. PopZ oligomer in C.

crescentus), and thereby its propensity to multimerize, can be modifiedlocally through protein–protein interaction with a partner whosesubcellular distribution is asymmetric (e.g. ParA during DNA segregationin C. crescentus). In Step 1, the protein (blue) has an asymmetricdistribution inherent to a cell cycle event. Concentration of the diffusingprotein oligomer (red) increases locally owing to interaction with theasymmetric protein. In Step 2, the self-assembly of a protein oroligomer leads to the formation of a large structure at the pole. Thisprovides spatial and temporal regulation to a multimerization-dependentpolar localization.

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