A Self-Associating Protein Critical
for Chromosome Attachment, Division,
and Polar Organization in Caulobacter
Gitte Ebersbach,1Ariane Briegel,2Grant J. Jensen,2and Christine Jacobs-Wagner1,3,*
1Department of Molecular, Cellular and Developmental Biology, Yale University, New Haven, CT 06520, USA
2Division of Biology, California Institute of Technology, Pasadena, CA 91125, USA
3Howard Hughes Medical Institute
Cell polarization is an integral part of many unrelated
bacterial processes. How intrinsic cell polarization is
achieved is poorly understood. Here, we provide ev-
idence that Caulobacter crescentus uses a multi-
meric pole-organizing factor (PopZ) that serves as
a hub to concurrently achieve several polarizing
functions. During chromosome segregation, polar
PopZ captures the ParB?ori complex and thereby
anchors sister chromosomes at opposite poles.
This step is essential for stabilizing bipolar gradients
of a cell division inhibitor and setting up division near
and mediates the polar localization of the morphoge-
Polar accumulation of PopZ, which is central to its
polarizing activity, can be achieved independently
of division and does not appear to be dictated by
the pole curvature. Instead, evidence suggests that
localization of PopZ largely relies on PopZ multimeri-
zation in chromosome-free regions, consistent with
a self-organizing mechanism.
Bacterial cells display an intrinsic polarization affecting many as-
pects of their life (Bardy and Maddock, 2007; Ebersbach and
Jacobs-Wagner, 2007). Numerous functionally unrelated pro-
teins localize to the cell poles, affecting a wide variety of
processes including chemotaxis, signal transduction, polar mor-
phogenesis, and pathogenesis. Cell polarization also plays an
essential role during DNA segregation and cell division. Equal
partitioning of chromosomes and plasmids relies on sister DNA
origins localizing near opposite poles (Ebersbach and Gerdes,
2005; Thanbichler and Shapiro, 2006a) and the proper location
of cell division is governed by polar localization of cell division in-
hibitors (Lutkenhaus, 2007). In recent years, we have learned
more about how specific molecules or complexes are individu-
ally targeted or retained at a pole. However, it remains ill-defined
whether bacteria, like eukaryotic cells, use organizing factors to
govern multiple polarization events simultaneously.
We address this fundamental question in the a-proteobacte-
rium Caulobacter crescentus, whose life cycle is rich in
well-documented polarized events (Lawler and Brun, 2007).
Organelles such as stalks, flagella, and pili form at specific poles
during the cell cycle (Figure 1A). The coupling between polar
morphogenesis and the cell cycle is achieved through an intri-
cate regulatory network, including several histidine kinases
that exhibit polar localization during the cell cycle (Goley et al.,
2007). In C. crescentus, the origin of replication (ori) is located
at the ‘‘old’’ pole (that existed in the previous cell cycle)
(Figure 1A). The DNA partitioning protein ParB binds to a parS
centromeric sequence nearby ori (Mohl and Gober, 1997). After
initiation of DNA replication, one of the duplicated ParB?ori
complexes rapidly migrates toward the ‘‘new’’ pole (created by
the most recent division), in a process that requires the MreB
cytoskeleton (Gitai et al., 2005; Jensen and Shapiro, 1999).
What retains ParB?ori at the pole has remained mysterious.
Bipolar localization of ParB?ori is, however, crucial for setting
up division (Thanbichler and Shapiro, 2006b). MipZ, an inhibitor
of the FtsZ cytokinetic structure, binds to bipolar ParB?ori,
favoring FtsZ assembly in the central cell region where MipZ
concentration is low (Thanbichler and Shapiro, 2006b). Thus, in
C. crescentus, cell polarization is critical for temporal and spatial
execution of chromosome segregation, cell division, and polar
While cell polarization is clearly crucial for many functions in
C. crescentus, we know little about the mechanisms involved
in pole recognition and organization. Recently, TipN was identi-
fied as a spatial cue that specifies the site of the last division
(Huitema et al., 2006; Lam et al., 2006). In the absence of TipN,
several new-pole markers exhibit an abnormal frequency of
old-pole localization (Huitema et al., 2006; Lam et al., 2006).
How these markers localize to the wrong pole is unclear. More-
over, old-pole and bipolar markers appear largely unaffected by
TipN. Thus, additional mechanisms must be involved in spatially
organizing the cell.
In this study, we identify a self-assembling, multifunctional
protein that serves as a pole-organizing center to mediate
several polarizing functions important for chromosome attach-
ment, cell division, stalk morphogenesis and protein localization.
956 Cell 134, 956–968, September 19, 2008 ª2008 Elsevier Inc.
A Screen for Polarization Factors Involved in Cell
In bacteria, cell polarization plays an important role in cell divi-
sion site selection (Lutkenhaus, 2007). Consistently, overpro-
duction of the TipN polarity factor causes cell filamentation,
minicelling and cell branching in C. crescentus (Lam et al.,
2006). We exploited this observation to search for additional
polarity factors, and designed a screen for genes that cause
a division phenotype when overexpressed from a multicopy
plasmid (see Supplemental Data available online). From this
screen, we isolated the hypothetical gene cc_1319, now
renamed popZ (for pole-organizing protein that affects FtsZ;
see below). Overexpression of popZ from the xylose-inducible
promoter (Pxyl) on a multicopy plasmid caused a division defect
(Figure 1B). Time-lapse microscopy in the presence of xylose re-
vealed that PopZ overproduction initially resulted in one or sev-
eral divisions near the new pole (opposite the stalk), generating
minicells (Figure 1C). Over time, the cells stopped dividing ex-
tion (seen as FtsZ-YFP bands) in popZ-overexpressing cells was
consistent with this division pattern (Movie S1).
Based on its NCBI annotation, PopZ is a 177-residue protein
predicted to be cytoplasmic. While the PopZ sequence does
not match any known domain or motif, BLAST analysis showed
that PopZ homologs are widely present in a-proteobacterial
PopZ Exhibits Polar Localization
PopZ fused to a tetracysteine (TC) motif (encoded at the native
locus as the only copy) was found at one or both poles in an
asynchronous cell population (Figure S1A). Time-lapse experi-
ments with synchronized cells expressing a functional popZ-
yfp fusion from the native locus as the only copy of popZ (see
Supplemental Experimental Procedures) revealed that PopZ-
YFP localizes at the old pole of swarmer cells, later adopting
a bipolar localization (Figures 2A and S1B). Division breaks this
symmetry and yields daughter cells with PopZ-YFP at their old
The fact that the chromosomal ori exhibits a similar unipolar
localization after division and bipolar localization after duplica-
tion and segregation (Jensen and Shapiro, 1999) led us to simul-
taneously visualize PopZ-YFP and ori using CFP-ParB, which
binds to the parS sequence adjacent to ori (Mohl and Gober,
1997; Thanbichler and Shapiro, 2006b). This showed that
localization at about the time when the segregation of CFP-
ParB?ori is completed (Figure 2B).
PopZ Captures the ParB?ori Complex and thereby
Anchors the Chromosomes at the Poles
A popZ deletion strain was viable but grew more slowly than
wild-type and exhibited defects in cell division placement, gen-
erating minicells and elongated cells (Figure 3A). Replication
and segregation of ori still occurred in DpopZ cells, asevidenced
by the presence of two or more CFP-ParB?ori foci (Figure 3B).
However, poles were often devoid of CFP-ParB foci. This was
not due to filamentation of DpopZ cells because filamentous
FtsZ-depleted cells showed bipolar localization of CFP-ParB in
addition to internal CFP-ParB foci (Figure 3B). Normally, the
origins remain at opposite poles after segregation (Jensen and
Shapiro, 1999; Viollier et al., 2004), as if attached (Figure 3C).
Figure 1. A Multicopy Plasmid Screen Identifies a Factor Involved
in Cell Division Placement
Pili, stalk and flagellum form at a defined pole, and segregation of the chromo-
somal origins of replication bound to ParB (ParB?ori) results in ParB?ori
complexes at opposite poles.
(B) DIC microscopy images of CB15N/pJS14Pxyl-PopZ cells growing in the
presence of glucose (no induction) or presence of xylose (which causes
popZ overexpression and cell filamentation).
(C) Time-lapse DIC microscopy of strain CB15N/pJS14Pxyl-PopZ. The slide
contained xylose to induce PopZ overproduction. Arrows indicate formation
The scale bars represent 2 mm.
Cell 134, 956–968, September 19, 2008 ª2008 Elsevier Inc. 957
Conversely, in DpopZ cells, CFP-ParB foci moved within a con-
strained region (Movies S2 and S3 and Figure 3D). This motion
was evident in kymograph analyses of CFP-ParB distribution
from time-lapse recordings (Figure 3E). In contrast, polar CFP-
ParB foci in FtsZ-depleted cells were held in place while internal
and Figure 3F). Since PopZ-YFP localized at both poles in FtsZ-
depleted cells (Figure 3G), our observations argued that PopZ
docks ParB?ori at the poles.
This notion was supported by the behavior of CFP-ParB in
a strain in which a xylose-inducible mYFP-PopZ fusion is the
only source of PopZ. After growth without xylose to obtain
a PopZ?phenotype, the cells were placed on a slide with xylose
microscopy (Movies S5and S6and Figure3H).At first,there was
no detectable mYFP-PopZ, and all CFP-ParB?ori foci displayed
motion as expected. Over time, mYFP-PopZ accumulated pri-
marily at the poles while the CFP-ParB?ori foci remained mobile,
as the polar localization of PopZ preceded that of CFP-ParB.
Eventually, during its motion, a CFP-ParB?ori focus would cross
the polar mYFP-PopZ site. From then on, this focus stopped
moving and remained colocalized with mYFP-PopZ, suggesting
a ‘‘capture’’ mechanism (Movies S5 and S6 and Figure 3H). On
few occasions, the newly synthesized mYFP-PopZ also accu-
mulated at an ectopic location along the cell length where it sub-
sequently captured a nearby CFP-ParB?ori complex (Figure 3H,
Movie S5), indicating that PopZ can recruit ParB?ori indepen-
dently of the poles.
Coimmunoprecipitation (Co-IP) experiments using anti-GFP
antibody (which recognizes YFP) and cell lysates of CB15N
popZ::popZ-yfp revealed that ParB is specifically pulled down
with PopZ-YFP (Figure 3I). Similar experiments with purified
poly-histidine-tagged ParB and PopZ (Figure S2) showed that
His6-PopZ coimmunoprecipitated with His6-ParB (Figure 3J),
suggesting a physical interaction between PopZ and ParB.
This is supported by the E. coli experiments presented below
PopZ-Mediated Docking of ParB?ori to the Poles Is
Required for Proper Temporal and Spatial Regulation
In C. crescentus, cell division and chromosome segregation are
connected, as the FtsZ assembly inhibitor MipZ binds to ParB
(Thanbichler and Shapiro, 2006b). The instability of ParB?ori in
DpopZ cells resulted in erratic movement of MipZ-YFP in DpopZ
cells (Movie S7), which likely decreased the probability of FtsZ
assembly at one specific location. Consistent with this, an
FtsZ-YFP reporter exhibited rapid movements in DpopZ cells
Figure 2. PopZ Colocalizes with ParB at the Poles
(A) Time-lapse microscopy of synchronized cells producing PopZ-YFP (CB15N popZ::pBGent-PopZ-YFP). Numbers indicate hr and min.
(B) Time-lapse microscopy of CB15N parB::cfp-parB popZ::pBGent-PopZ-YFP cells shows that PopZ-YFP and CFP-ParB bound to ori have similar polar local-
ization patterns. The second frame shows one of the duplicated CFP-ParB?ori migrating toward the new pole. The scale bar represents 2 mm.
958 Cell 134, 956–968, September 19, 2008 ª2008 Elsevier Inc.
and was able to form a stable and functional structure (seen as
a band) only on occasion and at aberrant places (Movie S8), re-
flecting the DpopZ division pattern described above (Figure 3A).
This argues that polar anchoring of ParB?ori is critical for proper
division by stabilizing MipZ gradients at the poles, thereby allow-
ing stable FtsZ assembly near midcell.
All together, our findings indicate that polar PopZ captures the
migrating ParB?ori complex during chromosome segregation,
which, in turn, affects where and when division occurs.
PopZ Self-Associates into Multimers
Since PopZ overproduction
(Figure 1B), we examined its distribution under this condition.
fusion to PopZ (GFP-PopZ or PopZ-TC) recapitulated the cell di-
vision phenotypes observed with untagged PopZ (Figures 1B
region of accumulation from the old pole extending into the cell
body (Figure 4A). Often, there was a second, weaker signal at
the opposite pole. The PopZ accumulation appeared homoge-
neous rather than concentrated at the membrane, suggesting
cytoplasmic accumulation. This was confirmed by 3D-recon-
struction of optical sections after 3D-deconvolution (data not
shown). The fact that excess PopZ did not disperse but parti-
tioned at the pole suggested that PopZ has a cohesive (i.e.,
Time-lapse recordings of GFP-PopZ during its overproduction
revealed that the biased old-pole accumulation is the result of
inheritance (Figure 4B). At first, GFP-PopZ progressively accu-
mulated at both poles. Division created new poles, and the
daughter cell that inherited the older pole had more GFP-PopZ
than its sibling because of its initial head start. Minicelling, which
typically occurred at the new pole, enhanced this asymmetry.
Repetition of this pattern in subsequent division cycles resulted
in expansion of the GFP-PopZ region at the older poles.
Freely diffusible GFP was evenly dispersed in cells overpro-
tains cytoplasmic content (Figure 4C). Even though PopZ was
untagged and therefore could not be visualized directly, we
were able to recognize the PopZ-rich region in DIC images
because of a small but reproducible shading difference between
the region of PopZ accumulation and the rest of the cell
Intrigued by the accumulation of PopZ and the optical effect it
generates, we examined popZ-overexpressing cells by electron
cryotomography. Tomograms of wild-type C. crescentus cells
have shown that the entire cytoplasm is packed with electron-
dense ribosomes (Briegel et al., 2006). In striking contrast, in
popZ-overexpressing cells, the PopZ-rich region was largely de-
pleted of ribosomes (Figure 4D). This provides an explanation for
the shading difference, as a ribosome-rich region should be op-
tically different than a ribosome-depleted region. Chromosomal
DNA was also excluded from the PopZ-rich region as visualized
by fluorescence microscopy using DAPI and FlAsH to stain DNA
and the PopZ-TC region, respectively (Figure 4E).
One possible interpretation of these data is that PopZ multi-
merizes into a matrix that excludes DNA and ribosomes (the
latter possibly because they are attached to DNA through
transcription and translation coupling) but notsmall components
(such as free GFP). Consistent with PopZ self-association, pu-
rified His6-PopZ, which has a predicted molecular weight (MW)
of 21 kDa (but runs as a 35–36 kDa protein by SDS-PAGE; Fig-
ure S2), migrates as a single band between the 480 and
720 kDa markers on a native gel (Figure 4F). To determine the
MW of the His6-PopZ multimer independently of its shape
(Folta-Stogniew, 2006), His6-PopZ was subjected to HPLC
size exclusion chromatography (SEC) coupled with UV, on-
line laser light scattering (LS) and refractive index (RI) detectors
(SEC-UV/LS/RI) (Figure 4G) . Measurements indicated that
His6-PopZ forms ordered structures between 125 and 175
kDa. The slow migration of His6-PopZ in the native gel and its
early elution position from the SEC column (ahead of the 475
kDa standard) indicate that His6-PopZ self-assembles into
a nonspherical oligomer.
Formation of Large PopZ Structures Affects the
Localization of ori, ParB, and MipZ
The cumulative profile of PopZ accumulation suggests that the
a large matrix that expands from the pole into the cell body. One
and sometimes two distinct ori were consistently found at the
border of the expanding PopZ regions (red arrow, Figure 4H;
data not shown), as visualized by the lacO array/LacI-CFP sys-
tem (Viollier et al., 2004). Thus, PopZ matrix expansion pushes
the anchored DNA origin farther from the pole. CFP-ParB was
found to ‘‘fill’’ the entire PopZ-rich region while also forming
a focus at its border (Figure 4I) where a DNA origin resides
(Figure 4H). Weak CFP-ParB foci were also occasionally
observed along the cell length, likely marking segregated ori
(Figure 4I). The retention of CFP-ParB in the DNA-free PopZ-
rich region is consistent with the observed affinity between
ParB and PopZ. MipZ-YFP was largely found at the border of the
PopZ-rich region and occasionally formed one or two weaker
foci along the cell length (Figure 4J). Since MipZ affects FtsZ as-
sembly, sequestration of MipZ at the PopZ matrix border may
cause the division defect observed in the PopZ overproduction
PopZ Achieves Multiple Polarizing Functions
PopZ plays a role in chromosome capture and cell division by
mediating the stable localization of ParB?ori, and thereby
MipZ, at the poles. This is reminiscent of the function of the
Bacillus subtilis DivIVA protein, a multimeric polar protein that
anchors the chromosomes at the poles during sporulation
through a (direct or indirect) interaction with origin-associated
RacA (Ben-Yehuda et al., 2003; Stahlberg et al., 2004;
Thomaides et al., 2001; Wu and Errington, 2003). During vegeta-
tivegrowth,DivIVA playsadistinct rolebymaintaining thecell di-
vision inhibitor complex MinCD at the poles (Marston and Erring-
ton, 1999). Despite their similar functional properties, PopZ and
DivIVA share no sequence similarity. Furthermore, DivIVA has
a largely coiled-coil structure (Edwards et al., 2000) whereas
PopZ has no predicted coiled-coil motifs. Another notable differ-
ence is that the two polarizing functions of DivIVA do not overlap
in time (normal growth versus sporulation) and are thought to be
competitive (Ben-Yehuda et al., 2003) whereas PopZ achieves
Cell 134, 956–968, September 19, 2008 ª2008 Elsevier Inc. 959
Figure 3. Deletion of PopZ Results in Minicelling, Cell Filamentation, and Failure of Polar Chromosome Attachment
(A) Time-lapse microscopy of strain CB15N DpopZ grown in PYE. Arrows show formation of minicells. Numbers indicate hr and min.
(B) Localization of CFP-ParB (red overlaid with DIC) in wild-type, DpopZ and FtsZ-depleted cells.
(C) Time-lapse images showing CFP-ParB localization (red) in a wild-type background (CB15N parB::cfp-parB).
(D) Time-lapse images (see Movie S2) showing CFP-ParB localization in DpopZ cells (CB15N DpopZ parB::cfp-parB).
(E) Kymograph analysis of CFP-ParB movement in DpopZ cells from a time-lapse recording (Movie S3). On the left is an overlay of CFP-ParB fluorescence (red)
and DIC images, corresponding to the first time point of the time-lapse sequence. On the right is kymographs showing the movement of two of the CFP-ParB foci
(one polar and one internal) over time within defined regions (indicated by dotted lines ‘‘a’’ and ‘‘b’’ in the overlay).
(F) Same as (E) for FtsZ-depleted cells (see Movie S4). While the internal CFP-ParB focus shows motion, the two polar foci display little, if any.
(G) Bipolar localization of PopZ-YFP in FtsZ-depleted cells.
(H) PopZ-dependent attachment of CFP-ParB?ori at poles. Strain CB15N DpopZ xylX::pXmYFP4-PopZ parB::cfp-parB was grown in PYE with glucose under
which conditions mYFP-PopZ (only source of PopZ) was depleted for 20 hr. These cells were then placed on an M2G agarose pad containing xylose in order
to restore mYFP-PopZ synthesis. Time-lapse imaging shows that emerging mYFP-PopZ foci (red) capture CFP-ParB?ori foci (green). Yellow arrows indicate
overlapping mYFP-PopZ and CFP-ParB foci.
(I) Immunoblotting of co-IP eluates from lysates of CB15N popZ::pBGent-PopZ-YFP cells and wild-type CB15N cells (negative control). The amount of cell lysate
loaded (two lanes to the right) was 1/100 of the amount used for the immunoprecipitates.
960 Cell 134, 956–968, September 19, 2008 ª2008 Elsevier Inc.
multiple polarizing tasks simultaneously as we show below. In
addition to chromosome capture and division, PopZ is involved
in polar stalk formation since stalks were undetectable when
DpopZ cells were analyzed by SEM (Figure S3). Moreover, the
polar localization of the histidine kinase DivJ, an old-pole marker
involved in polar morphogenesis and cytokinesis sensing (Ma-
piro, 1999), was disrupted in DpopZ cells (Figure 5A). In some
cells,DivJ-YFP showedsome accumulation ata pole(in addition
was unstable and transient (Movie S9). The cell cycle signaling
protein CckA, which exhibits unipolar and bipolar localization
during the cell cycle (Jacobs et al., 1999), was also dramatically
affected by the loss of PopZ function (Figure 5A). These defects
were probably not caused by the cell elongation phenotype of
some DpopZ cells as both DivJ and CckA retain a polar localiza-
tion in elongated FtsZ-depleted cells (Biondi et al., 2006; Ma-
troule et al., 2004).
The severe loss of polar localization for both DivJ and CckA
suggested that PopZ is required for the polar recruitment and/
or maintenance of these proteins. Consistent with this idea,
both CckA and DivJ coimmunoprecipitated with PopZ-YFP
(Figure 5B). In addition, membrane-bound CckA and DivJ were
found at the membrane periphery of the PopZ-rich region of
PopZ-overproducing cells (Figure 5C). In contrast, a GFP fusion
to the CheA chemotaxis protein formed a tight focus at the polar
tip of the PopZ-rich region (Figure S4), indicating that the locali-
zation pattern of CckA and DivJ is a result of a (direct or indirect)
interaction with PopZ, rather than the consequence of an expan-
sion of the polar region.
Thus, PopZ mediates several distinct polarizing functions at
the same time, suggesting that PopZ forms a multifunctional
platform that organizes the poles. PopZ also affects the localiza-
tion pattern of the TipN polarity factor. In wild-type cells, TipN
localizes at the new pole until the end of the cell cycle when it
delocalizes from the pole to accumulate at the site of division,
producing progeny with TipN at their new poles (Huitema et al.,
2006; Lam et al., 2006). DpopZ cells, on the other hand, often
failed to release TipN from the new pole, but still formed a focus
at the division site (Figure 5D). This resulted in a high frequency
(87%) of cells with bipolar TipN localization, which is not ob-
served in filamentous FtsZ-depleted cells (Huitema et al., 2006;
Lam et al., 2006). Unipolar components that are affected by
TipN (such as the FliG flagellar protein, the PleC histidine kinase,
CheA, and the CpaE pilus assembly protein) all remained polarly
localized in DpopZ cells but displayed varying degree of abnor-
malbipolar localization (TableS1), which may reflectthe effect of
PopZ on TipN localization. Thus, PopZ also affects several as-
pects of cellular asymmetry. Combining DpopZ and DtipN muta-
tions was synthetically lethal, and depletion of TipN in a DpopZ
PopZ Polarly Localizes in E. coli Where It Recruits ParB
ably, PopZ-TC retained this ability even when it was artificially
produced in the vastly divergent, g-proteobacterium E. coli
MC1000, which lacks PopZ homologs (Figure 6A). The localiza-
tion was mostly unipolar in minimal M9 medium whereas the
percentage of cells with bipolar localization increased in rich
LB medium (data not shown).
Since evidence suggests that ParB and PopZ interact without
the need of intermediate factors (Figure 3J), these proteins
should interact even when artificially produced in E. coli, which
lacks homologs of both. When popZ-tc expression was not in-
duced in E. coli, CFP-ParB had an expected diffuse distribution
in the cytoplasm given the absence of a parS binding sequence
(Figure 6B). However, when PopZ-TC synthesis was induced
with arabinose, CFP-ParB was recruited to the PopZ-TC pole
(Figure 6B). CFP-MipZ, used here as a control, retained a dis-
persed cytoplasmic distribution in E. coli cells even when
PopZ-TC was polarly present (Figure 6C). These data further
substantiate the notion of a physical association between
PopZ and ParB and provide evidence that PopZ is functional in
the heterologous E. coli system.
Polar Accumulation of PopZ Involves PopZ
Multimerization and DNA Occlusion
Pole recognition in bacteria is an unresolved question of funda-
mental importance. Typically, a polar ‘‘anchor’’ protein is in-
voked. Our findings strongly argue that PopZ plays such
a role for several polar components. But how does PopZ accu-
mulate at the poles? The fact that PopZ ‘‘recognizes’’ E. coli
poles despite the evolutionary distance between E. coli and
terialpoles. Sucha universal property is consistentwiththe pre-
vious finding that B. subtilis DivIVA localizes to the poles (and
septum) of E. coli (Edwards et al., 2000). Since poles originate
from division, PopZ may recognize a remnant of division. To
test this, we looked at PopZ in TipN-overproducing cells, which
produce ectopic poles independently of division events (Lam
et al., 2006). GFP-PopZ localized at the ectopic poles
(Figure 6D), indicating that cell division is not essential for
PopZ localization. A similar conclusion was reached for DivIVA
(Hamoen and Errington, 2003; Harry and Lewis, 2003). Another
popular hypothesis is that polar proteins are retained at the
poles through direct or indirect binding to the inert peptidogly-
can cell wall. We might then expect digestion of the peptidogly-
can to result in dispersion of the protein. However, PopZ still
formed tight foci in E. coli protoplasts obtained by lysozyme
treatment (Figure 6E).
PopZ localizes near the cell periphery, suggesting an affinity
for the membrane. Since cell poles exhibit a higher degree of
membrane curvature than the lateral sides, another attractive
hypothesis is that polar accumulation of proteins might rely on
geometric requirements, perhaps through interactions with
(J) Purified His6-ParB and His6-PopZ proteins coimmunoprecipitate. The presence of His6-ParB and His6-PopZ in the eluate was determined by immunoblotting
using a-Tetra-His antibodies. Controls lacking either His6-PopZ or His6-ParB were included. To identify the position of each protein after SDS-PAGE, samples of
His6-ParB and His6-PopZ were loaded directly onto the gel (two lanes to the right, 25 ng).
The scale bars represent 2 mm.
Cell 134, 956–968, September 19, 2008 ª2008 Elsevier Inc. 961
Figure 4. PopZ Multimerizes and during Overproduction, It Forms a Large Polar Accumulation that Excludes DNA and Ribosomes
(A) Microscopy images of strain CB15N/pJS14Pxyl-PopZ-TC grown with xylose for 6 hr to achieve overproduction of PopZ-TC.
(B) Time-lapse microscopy of strain CB15N/pJS14Pxyl-GFP-PopZ inducing GFP-PopZ (red) overproduction on the slide.
(C) Microscopy images showing uniform GFP localization in a strain (CB15N xylX::pXGFP4/pJS14Pvan-PopZ) overproducing untagged PopZ. Accumulation of
PopZ during overproduction creates a shading difference in DIC images (marked by arrows and broken line).
(D) Electron cryo-tomogram of CB15N/pJS14Pxyl-PopZ grown in the presence of xylose for 6.5 hr to overproduce PopZ. Left image: 15 nm thick slice from the
median filtered tomogram. Right image: 58 nm thick section of the segmentation including the region shown on the left. SL, surface layer; OM, outer membrane;
IM, inner membrane; Rib, probable ribosome (yellow in right panel). The scale bar represents 100 nm.
(E) Microscopy images showing FlAsH-stained PopZ-TC-rich region of a CB15N/pJS14Pxyl-PopZ-TC cell grown in the presence of xylose for 6 hr. DNA was
stained with DAPI. Broken line and arrows indicate the PopZ-rich region.
(F) Purified His6-PopZ forms a large complex. The protein (1 mg) was loaded onto a 4%–15% gradient native gel (Bio-Rad) together with a protein ladder.
insolutionundertheseconditions. Vertical arrowsatthebottomindicatethecolumnvoidvolumeand theelutionvolumeof4standards:TrypsinInhibitor (20kDa),
Bovine Serum Albumin (66 kDa), Beta Amylase (220 kDa) and Apo Ferritin (475 kDa).
962 Cell 134, 956–968, September 19, 2008 ª2008 Elsevier Inc.
high-curvature phospholipids that cluster at the poles (Howard,
2004; Huang et al., 2006). To explore the idea that PopZ cluster-
ing is dependent on pole curvature, we performed a series of
experiments using spherical cells obtained by treating E. coli
with mecillinam or A22, which inhibits the function of rod shape
determinants PBP2 or MreB, respectively (Gitai et al., 2005;
Iwai et al., 2002; Tamaki et al., 1980). Only the A22 experiments
will be presented as we obtained similar results with both drugs.
To test whether the formation of mYFP-PopZ foci is independent
of geometric constraints, cells were pre-treated with A22 until
a spheroid shape was achieved. These spheroid cells were
then placed on a slide containing arabinose and A22 to initiate
(H) Ori is located at the border of the PopZ-rich region in popZ-overexpressing cells. Strain CB15N Cori::Cori-lacOp-kan xylX::pHPV472/pJS14Pvan-PopZ was
grown in PYE and popZ overexpression (under the vanillate-inducible promoter) was induced with 0.5 mM vanillic acid for 8 hr. The location of ori was visualized
xylose for 1 hr prior to visualization. At the time of xylose addition, 50 mM IPTG was added in order to decrease potential side effects originating from binding of
LacI-CFP to its operator sites (Viollier et al., 2004). Broken line and arrows indicate the PopZ-rich region.
(I) CFP-ParB localizes to the PopZ-rich region of cells (CB15N parB::cfp-parB/pJS14Pxyl-PopZ) overproducing untagged PopZ for 8 hr. Broken line and arrows
indicate the PopZ-rich region. Red arrows indicate CFP-ParB foci at the border of the PopZ region and along the cell length.
(J) MipZ-YFP localizes at the border of the PopZ-rich region and occasionally along the cell filament in PopZ-overproducing cells (CB15N mipZ::mipZ-YFP/
pJS14Pxyl-PopZ). Broken line indicates the PopZ-rich region.
The scale bars represent 2 mm, except for (D).
Figure 5. PopZ Affects the Localization of CckA, DivJ, and TipN
(A) Microscopy images showing the localization of chromosome-encoded fusion proteins DivJ-YFP or CckA-mGFP in wild-type and DpopZ cells.
(B) PopZ-YFP was immunoprecipitated from cell lysates of strains CB15N popZ::pBGent-PopZ-YFP and CB15N (negative control). PopZ-YFP IP and co-IP of
crescentin (negative control), DivJ or CckA were analyzed by performing immunoblotting on the same membrane. The amount of cell lysate loaded (two lanes to
the right) was 1/100 of the amount used for the immunoprecipitates.
(C) Microscopy images showing the localization of DivJ-YFP or CckA-mGFP in cells overproducing PopZ from plasmid pJS14Pxyl-PopZ. PopZ-rich region is
shown by broken line.
(D) Microscopy images from a time-lapse sequence showing the localization of chromosome-encoded TipN-GFP in DpopZ cells during growth.
(E) Depletion of TipN in a DpopZ background results in a severe synthetic division phenotype. Micrographs of strains CB15N DtipN xylX::Pxyl-tipN and CB15N
DpopZ DtipN xylX::Pxyl-tipN grown in PYE-xylose (causing TipN synthesis) or PYE-glucose (causing TipN depletion) for 20 hr.
The scale bars represent 2 mm, except for (E).
Cell 134, 956–968, September 19, 2008 ª2008 Elsevier Inc. 963
Figure 6. PopZ Localizes to the Pole in E. coli
(A) Microscopy images showing polar localization of C. crescentus TC-tagged PopZ expressed in E. coli. Strain MC1000/pBAD33PopZ-TC was grown at 30?C.
PopZ-TC expression was induced by addition of arabinose for 1 hr and was visualized by staining with FlAsH for 30 min.
(B) Microscopy images showing PopZ-dependent localization of C. crescentus ParB to the pole of E. coli cells. Strain MC1000/pNDM220CFP-ParB/
pBAD33PopZ-TC was grown as described in (A). CFP-ParB was induced by addition of 1 mM IPTG for 4 hr. When indicated, PopZ-TC expression was
induced by adding arabinose for 2 hr. PopZ-TC was stained with ReAsH for 30 min to allow simultaneous visualization with CFP-ParB. It should be noted
that ReAsH only stained a small percentage of the cells. However, similar results were obtained with a strain expressing YFP-PopZ and CFP-ParB (data not
(C) Microscopy images showing a uniform localization of C. crescentus MipZ (used here as a control) in E. coli cells synthesizing PopZ-TC stained with ReAsH.
Strain MC1000/pNDM220CFP-MipZ/pBAD33PopZ-TC was grown as described in (A). The synthesis of CFP-MipZ and PopZ-TC were induced by addition of
1 mM IPTG for 4 hr and arabinose for 2 hr, respectively.
(D) Strain CB15N xylX::pXGFP4-PopZ/pJS14TipN was grown in PYE with 0.03% xylose. PopZ was found at the poles, including branched poles created by TipN
964 Cell 134, 956–968, September 19, 2008 ª2008 Elsevier Inc.
myfp-popZ expression while maintaining a spherical shape.
Time-lapse microscopy (Figure 6F) revealed that mYFP-PopZ
was first uniformly distributed, then formed small, mobile foci
that appeared and dissolved rapidly near the cell periphery. But
ultimately all foci coalesced into a single focus that displayed lit-
tle mobility and that grew in intensity. Note that the contrast of
the entire time-lapse sequence was greatly enhanced to permit
visualization of weak mYFP-PopZ signals in early time points. A
similar sequence of events was observed when untreated,
rod-shaped E. coli cells were used (Figure 6G and Movie
S10). Collectively, these experiments suggest that self-associa-
tion of PopZ plays a major role in protein clustering and that
specific membrane curvature is not a key factor in the clustering
Another common feature among bacterial poles is the ab-
sence of DNA, which suggested to us that PopZ complex forma-
tion may be facilitated in DNA-free regions. In agreement with
this idea, induction of mYFP-PopZ synthesis in filamentous
E. coli cells blocked for cell division by cephalexin generated
multiple mYFP-PopZ foci exclusively in chromosome-free re-
gions (based on DAPI staining), including between nucleoids
(Figure 6H). A DNA occlusion mechanism would be particularly
appropriate for C. crescentus, where the DNA fills virtually the
entire cell except for the polar tips (Viollier et al., 2004). Consis-
tent with a DNA occlusion mechanism, PopZ-YFP foci formed
largely outside the DAPI-stained DNA in regions corresponding
to the tip of the poles (Figure 6I).
If DNA spatially restricts PopZ multimerization to the poles in
C.crescentus,thenPopZ shouldbeableto formfociinany chro-
mosome-free regions, as we observed in filamentous E. coli
(Figure 6H). In C. crescentus, a block in division results in fila-
mentous cells with no obvious separation of nucleoids (Ward
and Newton, 1997). However, simultaneous inhibition of the
cell division protein FtsA and the topoisomerase IV subunit
ParE (using a ftsAtsparEtsdouble mutant at the restrictive tem-
perature) creates filamentous cells with large DNA-free regions
(Ward and Newton, 1997). Under these conditions, mYFP-PopZ
formed ectopic foci exclusively in the DNA-free regions (Fig-
ure 6J). This was also observed when popZ-yfp was expressed
under the native popZ promoter (Figure S5).
Collectively, the data strongly argue that the polar localization
of PopZ is achieved through PopZ multimerization in chromo-
some-free regions near the membrane.
the cell. One is to bind ParB?ori and anchor the chromosomes at
the poles. This plays a critical role in the spatio-temporal regula-
tion of cell division by stabilizing MipZ gradients at opposite
poles, favoring assembly of FtsZ near midcell (Figure 7A).
PopZ also mediates polar localization of DivJ and CckA while
maintaining its chromosome binding function. PopZ is also in-
volved in other polarized events because without PopZ, stalk
morphogenesis is impaired and the fidelity of asymmetric local-
ization of TipN (and TipN-controlled components) is compro-
mised. The molecular basis for these additional polarity defects
is less clear.
Polar accumulation of PopZ, which is clearly central to its
multifaceted polarizing activity, appears to rely on a possible
self-organizing mechanism. We propose that after nucleation
of PopZ at the membrane, self-association of PopZ into a large
multimer or matrix is favored in chromosome-free regions, as
shown in both E. coli and C. crescentus. The DNA polymer
may exert its inhibitory activity by steric hindrance or through
a general repulsion mechanism. During normal growth of
C. crescentus, localization of PopZ at the old pole is inherited
from the previous division cycle and provides a primer for further
region at the opposite pole results in a progressive accumulation
of PopZ at this location (Figure 7B). The PopZ multimer at the
new pole captures the segregated ParB?ori complex. Continued
synthesis and self-association of PopZ results in expansion of
the PopZ matrix. Covisualization of PopZ-YFP and CFP-
signal is often found overlapping with the pole-distal tip of the
PopZ signal (Figure 6K). This is because ParB is at the interface
between the growing PopZ matrix and the polymeric DNA. This
effect is amplified under popZ-overexpressing conditions when
a massive extension of the PopZ matrix progressively pushes
(E) Microscopy images showing the localization of mYFP-PopZ in E. coli protoplasts. Strain MC1000/pBAD33mYFP-PopZ was grown in LB at 37?C. Expression
of mYFP-PopZ was induced by addition of arabinose for 30 min.
(F) Selected frames from a time-lapse fluorescence microscopy experiment showing accumulation of mYFP-PopZ in a spheroid A22-treated E. coli cell. Strain
MC1000/pBAD33mYFP-PopZ was grown at 30?C. A22 was added for 4 hr before cells were mounted on a pad containing A22 and 0.2% arabinose to induce
mYFP-PopZ synthesis. Arrows show the appearance of mYFP-PopZ foci.
(G) Selected frames from a time-lapse fluorescence microscopy experiment showing accumulation of mYFP-PopZ in rod-shaped E. coli. Strain MC1000/
pBAD33mYFP-PopZ was grown at 30?C. The slide contained 0.2% arabinose to induce mYFP-PopZ synthesis. Arrows show the appearance of mYFP-
(H) Microscopy images of cephalexin-treated E. coli cells showing the formation of mYFP-PopZ foci (red) in chromosome-free regions including between
nucleoids (stained with DAPI; green). MC1000/pBAD33mYFP-PopZ cells were grown at 30?C and treated with cephalexin (2.5 hr) before adding arabinose
(1 hr) to induce expression of myfp-popZ.
(I) PopZ-YFP localizes to chromosome-free regions at the poles of C. crescentus CB15N popZ::pBGent-PopZ-YFP. The image shows an overlay of PopZ-YFP
(red) and DNA (green).
(J) PopZ forms multiple foci exclusively in chromosome-free regions in a C. crescentus parEtsftsAtsdouble mutant grown at the restrictive temperature. CB15
divD308(Ts)::pDW110 (parEp) divE309(Ts) xylX::pXmYFP4-popZ cells were grown in PYE at 30?C and then shifted to 37?C for 6 hr. mYFP-PopZ synthesis (red)
was induced with xylose (0.3%) 1 hr before microscopy. DAPI stains the DNA (green).
(K) Images of CB15N parB::cfp-parB popZ::pBGent-PopZ-YFP cells grown in M2G medium show the overlap (yellow) between CFP-ParB signal (green) and the
pole-distal tip of the PopZ-YFP signal (red), consistent with PopZ forming a matrix under physiological conditions.
The scale bars represent 2 mm.
Cell 134, 956–968, September 19, 2008 ª2008 Elsevier Inc. 965
ParB, the attached DNA origin and the rest of the chromosome
away from the pole (Figure 4H–I). This is likely because PopZ
and the chromosome are both polymeric, and polymers cannot
mix well for steric reasons.
During the normal cell cycle, the PopZ matrix also binds, di-
(Figure 7B). The localization of ParB?ori at the poles closely par-
allels that of PopZ during the cell cycle, consistent with a simple
relationship. This is, however, not true for DivJ and CckA. While
PopZ is present at the poles where and when CckA and DivJ
localize, CckA and DivJ, which have distinct cell cycle patterns
of localization (Jacobs et al., 1999; Wheeler and Shapiro,
1999), are not always at the poles where and when PopZ is pres-
ent. This partial overlap indicates that PopZ does not dictate the
timing of CckA and DivJ localization. Other factors must achieve
this role by modulating the association of PopZ with CckA and
DivJ during the cell cycle.
PopZ homologs are widely found among a-proteobacteria,
feeding into the notion that cell polarization is an important cellu-
lar attribute in a-proteobacteria (Hallez et al., 2004), which form
relevance. Perhaps more importantly, this study provides mech-
Figure 7. Proposed Models for PopZ Func-
tion and Localization
(A)Model bywhichPopZ mediates the attachment
of ori at the poles and thereby regulates cell
division. Before DNA replication is initiated, the
chromosome is tethered at the old pole via an in-
teraction between PopZ and ParB bound to the
parS sequence nearby ori. ParB also associates
with MipZ and this association maintains FtsZ at
the opposite pole. After replication is initiated,
one of the duplicated ParB?ori complexes mi-
grates toward the new pole where PopZ accumu-
lates. PopZ captures the migrating ParB?ori and
the immobilization of ParB?ori at both poles via
PopZ generates stable bipolar accumulation of
MipZ activity, which is critical for stable assembly
of the FtsZ cytokinetic ring near midcell.
(B) Model by which PopZ achieves polar localiza-
tion and perform several polarizing activities. The
newborn swarmer cell inherits a PopZ self-assem-
bled matrix at the old pole from its mother cell.
Over time, PopZ also self-associates in the chro-
mosome-free space at the new pole where multi-
merization into a matrix is favored. The PopZ
matrix, which continues to grow in size, serves as
an organizing platform for the attachment of the
chromosome via a ParB interaction and for the
(direct or indirect) recruitment of CckA and DivJ.
anistic insights into how bacteria can or-
ganize their poles. Many unlinked bacte-
localization of various proteins. The iden-
tification of PopZ suggests that bacteria
can use multifunctional proteins that
serve as ‘‘hubs’’ to achieve and perhaps
coordinate multiple polarizing activities.
Such organizing centers may correspond to a primitive version
of the centralized polarity system of eukaryotic cells. Further-
more, our study suggests a mechanism of spontaneous self-or-
ganization for pole recognition, which relies on protein multime-
rization, membrane interaction and DNA occlusion. This
proposed mechanism might represent an important principle of
the multimeric DivIVA protein, the lattice-forming chemorecep-
tors or other proteins follow a similar principle of polar localiza-
Strains, Plasmids, and Growth Conditions
Strains and plasmids are listed in Table S2. Their construction is described
in the supplemental text. Transformations, conjugations and phage trans-
ductions were carried out as described (Ely, 1991). C. crescentus strains
were grown to log-phase at 30?C in PYE or M2G minimal medium (Ely,
1991). When needed, cell populations were synchronized as described
(Evinger and Agabian, 1977). E. coli strains used for microscopy experi-
ments were grown to log-phase at 30?C or 37?C in M9-minimal medium
supplemented with 0.2% glycerol and 0.1% casamino acids unless other-
wise stated. When required, gene expression was induced by adding
0.03%–0.3% xylose, 0.5 mM vanillic acid or 0.02% arabinose unless
966 Cell 134, 956–968, September 19, 2008 ª2008 Elsevier Inc.
otherwise stated.When needed, A22 (50mM) or cephalexin (10 mg/ml) were
used. E. coli protoplasts were generated as described (Dai et al., 2005).
Microscopy was performed by using a Nikon E1000 microscope and a Hama-
matsu Orca-ER LCD camera, or a Nikon E80i microscope and an Andor
iXonEM+ camera. Images were taken and processed with Metamorph 6.1r0
software. Samples were placed on agarose-padded slides containing medium
and xylose, vanillic acid, arabinose or IPTG when required. DNA and TC-
tagged proteins were visualized by using DAPI and FlAsH or ReAsH (Invitro-
Cells were harvested, washed twice in M2G, and resuspended in fixation so-
lution for 30 min at RT (5% glutaraldehyde, 4% formaldehyde in 0.08M sodium
phosphate buffer [pH 7.2]). Fixed cells were washed in PBS and mounted onto
poly-L-lysine coated coverslips. Cells were dehydrated through an ascending
series of ethanol baths ending in 100% ethanol, and were critical point dried
and gold-coated. Samples were examined by using a FEI XL-30 ESEM FEG
microscope (acceleration voltage: 10.0 kV; spot size: 3; working distance:
7.5 mm or 10 mm).
Data were collected on a FEI Polara? (FEI Company, Hillsboro, OR, USA),
300 kV FEG transmission electron microscope with a Gatan energy filter con-
figured with a slit width of 20 eV, on the lens-coupled 4k by 4k Ultracam CCD
(Gatan). Single axis tilt-series were recorded from ?60?to 60?with an incre-
ment of 1?at 10 or 12 mm underfocus, using UCSF-Tomo (Zheng et al.,
2007), with a pixel size of 0.96 nm and a cumulative dose of up to 200 e-/A2.
Three-dimensional reconstructions were calculated using IMOD (Mastro-
narde, 1997). To visualize ribosome distribution, the template-matching pro-
gram Molmatch (Bohm et al., 2000; Ortiz et al., 2006) was run using the crystal
structures of the E. coli ribosome (PDB ID 2aw7, 2awb) as a template. The
cross-correlation peaks, selected by a combination of an automatic peak
search and manual interpretation, were visualized using the Amira software
package (Mercury Computer Systems).
In Vivo Co-IP
Cells were harvested, washed with buffer (20 mM HEPES [pH 7.5], 100 mM
NaCl, 0.05% TX100 containing antiprotease mix from Roche) and resus-
pended in IP1 buffer (20 mM HEPES [pH 7.5], 100 mM NaCl, 20% glycerol,
0.05% TX100 plus antiprotease mix). After lysozyme treatment (0.2 mg/ml)
and sonication, lysates were treated with DNaseI (30 U, Roche) and MgCl2
(10 mM) for 10 min on ice before centrifugation. Cleared lysate was mixed
with Protein A-agarose beads (Roche) that had been previously incubated
with a-GFP antibody (JL-8, Clontech) in IP1 buffer for 30 min at 4?C with gentle
shaking followed by 2 washes with IP1 buffer. After 3 hr of incubation (over-
night for ParB IP),the beadswere washed 6times withIP1 buffer. Thesamples
were subjected to SDS-PAGE and western blotting by using standard proto-
cols. For protein detection, the following antibodies and concentrations
were used: a-GFP, 1:10,000 for 1h; a-CckA:, 1:5,000 for 2 hr; a-DivJ,
1:10,000 for 2 hr; a-ParB, 1:4,000 for 1 hr; a-crescentin, 1:10,000 for 1 hr.
In Vitro Co-IP
Protein A-agarose beads were incubated with a-ParB antibody in IP2 buffer
(IP1 buffer without glycerol) at 4?C with gentle shaking. After 1 hr, purified
His6-ParB (1 mg) was added and the samples were left shaking overnight.
The beads were washed twice with IP2 buffer. BSA (0.25%) was added in
IP2 buffer for 1 hr at RT to block nonspecific binding. Finally, His6-PopZ
(1 mg) was added. After 5 hr of incubation at 4?C, the beads were washed 5
times with IP2 buffer. After SDS-PAGE, proteins were detected by immuno-
blotting using a-Tetra-His antibody (QIAGEN) 1:1,500 for 1 hr.
IPTG (0.1 mM) was added to LB cultures of BL21/pET28a-PopZ or BL21/
pET28a-ParB at OD600= 0.6 for 3 hr at 37?C. Cells were harvested, resus-
pended in wash buffer (50 mM phosphate buffer [pH 7.0, ParB] or [pH 8.0,
PopZ], 300 mM NaCl, antiprotease mix) containing DNaseI (30U) and MgCl2
(10 mM), and lysed by using a French press. His6-ParB and His6-PopZ were
purified from the supernatant and pellet, respectively, by using Talon metal
affinity resin (Clontech) and the standard batch/column protocol provided by
the manufacturer. All steps were performed in either 50 mM phosphate buffer
(pH 8.0), 300 mM NaCl, 6M urea (His6-PopZ) or 50 mM phosphate buffer (pH
7.0), 300 mM NaCl (His6-ParB). For washes, imidazole (20 mM for His6-PopZ
and 15 mM for His6-ParB) was added whereas 200 mM imidazole was used
for elution. Fractions were dialyzed against 5 mM Tris-HCl (pH 8.5) (His6-
PopZ) or 20mM HEPES (pH 7.6), 0.1 mM EDTA, 12.5 mM MgCl2,10%glycerol
Samples (pre-filtered through a 0.22 mm filter) were applied on a Superose 6,
10/30, HR SEC column (GE Healthcare) connected to HPLC Alliance 2965
(Waters Corp.) andequipped withanautosampler. Elutionfrom SEC wasmon-
itored by a photodiode array (PDA) UV/VIS detector (996 PDA, Waters Corp.),
differential refractometer (OPTI-Lab, or OPTI-rEx Wyatt Corp.), and static,
multi-angle laser light scattering detector (DAWN-EOS, Wyatt Corp.). The
SEC-UV/LS/RI system was equilibrated in 100 mM HEPES (pH 7.4), 150 mM
NaCl, 1 mM DTT buffer at a flow rate of 0.3 ml/min. The Millennium software
(Waters Corp.) controlled the HPLC operation and data collection from the
lected data from the refractive index detector, the light scattering detectors,
and recorded the UV trace at 280 nm sent from the PDA detector. Data collec-
tion and analyses were carried out at the Keck Foundation Biotechnology
Resource Laboratory, Yale University.
Supplemental Data include Supplemental Experimental Procedures, Supple-
mental References, five figures, and two tables and can be found with this
article online at http://www.cell.com/cgi/content/full/134/6/956/DC1/.
We thank M. Mooseker, E. Dufresne, T. Emonet, and J. Wolenski for valuable
discussions; K. Gerdes, J. Gober, A. Newton, N. Ohta, L. Shapiro, M. Than-
bichler, and P. Viollier for supplying strains or antibody; P. Angelastro, A. Jack-
son, H. Lam, and W. Schofield for construction of strains; H. J. Ding and
D. Rosenman for computational help; M. Mooseker, Z. Jiang, and G. Charbon
for assistance with EM; E. Folta-Stogniew and the Keck Foundation Biotech-
nology Resource Laboratory at Yale for the biophysical analyses, and the
Jacobs-Wagner laboratory and T. Emonet for critical reading of the manu-
script. G.E. was supported by postdoctoral fellowships from the Villum Kann
Rasmussen Foundation and the Danish Natural Science Research Council.
This work was funded in part by National Institutes of Health (GM065835 to
C.J.-W. and AI067548 to G.J.J.), gifts to Caltech from the Gordon and Betty
Moore Foundation and Agouron Institute (to G.J.J.) and the Pew Scholars
Program in the Biological Sciences sponsored by the Pew Charitable trust
(to C.J.-W.). C.J.-W. is a Howard Hughes Medical Institute investigator.
Received: November 26, 2007
Revised: April 18, 2008
Accepted: July 11, 2008
Published: September 18, 2008
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