J. Biomedical Science and Engineering, 2013, 6, 134-143 JBiSE
http://dx.doi.org/10.4236/jbise.2013.62017 Published Online February 2013 (http://www.scirp.org/journal/jbise/)
Spatial perturbation with synthetic protein scaffold reveals
robustness of asymmetric cell division
Jiahe Li1*, Pengcheng Bu2*, Kai-Yuan Chen2, Xiling Shen1,2#
1Department of Biomedical Engineering, Cornell University, Ithaca, USA
2School of Electrical and Computer Engineering, Cornell University, Ithaca, USA
Received 11 January 2013; revised 11 February 2013; accepted 18 February 2013
ABSTRACT
Asymmetric cell division is an important mechanism
for creating diversity in a cellular population. Stem
cells commonly perform asymmetric division to gen-
erate both a daughter stem cell for self-renewal and a
more differentiated daughter cell to populate the tis-
sue. During asymmetric cell division, protein cell fate
determinants asymmetrically localize to the opposite
poles of a dividing cell to cause distinct cell fate.
However, it remains unclear whether cell fate deter-
mination is robust to fluctuations and noise during
this spatial allocation process. To answer this ques-
tion, we engineered Caulobacter, a bacterial model for
asymmetric division, to exp ress synthetic scaffolds with
modular protein interaction domains. These scaffolds
perturbed the spatial distribution of the PleC-DivJ-
DivK phospho-signaling network without changing
their endogenous expression levels. Surprisingly, en-
forcing symmetrical distribution of these cell fate de-
terminants did not result in symmetric daughter fate
or any morphological defects. Further computational
analysis suggested that PleC and DivJ form a robust
phospho-switch that can tolerate high amount of spa-
tial variation. This insight may shed light on the pres-
ence of similar phospho-switches in stem cell asym-
metric division regulation. Overall, our study demon-
strates that synthetic protein scaffolds can provide a
useful tool to probe biological systems for better un-
derstanding of their operating principles.
Keywords: Caulobacter; Asymmetric Cell Division;
Protein Scaffold; Synthetic Biology
1. INTRODUCTION
In contrast to symmetric division, which produces two
equivalent daughter cells, asymmetric division produces
one daughter cell like the mother cell for self-renewal
and another daughter cell that takes on a different cell
fate. From unicellular bacteria to complex multicellular
organisms, cells perform asymmetric division to generate
diversity in a cell population [1-3]. In unicellular bacteria
and yeast cells, the diversity from asymmetric division
often provides a survival advantage. For instance, Bacil-
lus subtilis normally divides symmetrically but switches
to asymmetric division under nutritional stress to pro-
duce a single endospore that can survive unfavorable
environmental conditions. In higher-order, multi-cellular
organisms, stem cells often perform asymmetric division
to maintain a constant stem cell population while pro-
ducing more differentiated progenies for development
and tissue homeostasis [4]. In contrast, loss or defect of
asymmetric division has been associated with prolifera-
tion of cancer cells [5-9].
Caulobacter is a classical bacterial model for studying
asymmetric division and cell cycle [10-14]. A Caulo-
bacter swarmer cell swims around until finding a settle-
ment place, where it sheds its flagellum and grows a
stalk to attach to a surface. The stalked cell then asym-
metrically divides into a swarmer daughter and a stalked
daughter, and the swarmer daughter swims away to find
a new habitat. During cytokinesis (S-G2 transition), the
histidine kinase DivJ localizes to the stalked pole while
the histidine phosphatase PleC localizes to the swarmer
pole (PleC acts as a kinase during the G1-S transition but
switches to phosphatase activities during S-G2) [15].
Therefore, the single-domain response regulator DivK is
phosphorylated at the stalked pole and dephosphorylated
at the swarmer pole when cells are dividing [16] (Figure
1(a)). The distinct phosphorylation states of DivK at the
two poles contribute to daughter cell fate asymmetry, for
DivK functions through DivL to control CckA and con-
sequently the phosphorylation of CtrA, a master regula-
tor that determines the differential fate of the two daugh-
ter cells [17] (Figure 1(b)).
Cell fate determinants such as PleC and DivJ were
usually knocked out, constitutively expressed, or mutated
*Both authors contributed equally.
#Corresponding author.

J. H. Li et al. / J. Biomedical Science and Engineering 6 (2013) 134-143 135
Figure 1. Overview of asymmetric cell division and spatial perturbation of cell fate proteins in C. crescentus. (a) The spatial and
temporal dynamics of cell fate proteins in the bacterial cell cycle. The cell cycle begins with a swarmer cell with PleC, a phosphatase
localized at the flagellum pole. After entering DNA replication, S phase, the swarmer cell differentiates into a stalked cell. As cell
cycle progresses from S to G2 phase, dividing cells asymmetrically segregate DivJ, a histidine kinase and PleC to stalked and
swarmer cells, respectively; (b) PleC and DivJ play opposite roles in the dephosphorylation and phosphorylation of DivK,
dephosphorylated DivK triggers a signaling cascade leading to the accumulation of CtrA~P which blocks transition into S phase; (c)
Schematic design of co-localizing two different cell fate proteins at the same pole. Membrane-bound proteins PleC or DivJ are fused
with GBD peptide and cytoplasmic DivK is tagged with SH3 peptide. Scaffold proteins expressing SH3 and GBD domains are driven
by xylose-inducible promoter and therefore can be induced upon addition of xylose to culture media; (d) Four possible scenarios and
corresponding phenotypic outcomes after perturbation of cell fate proteins by scaffolds.
(e.g. for constitutive phosphorylation) in order to exam-
ine their roles in asymmetric division. These experiments
resulted in serious cell division defects, which confirmed
that the cell fate determinants are essential to asymmetric
division. For instance, a null mutation in DivJ resulted in
filamentous cells and mis-localized stalks [18]. However,
it is less clear how critical their spatial allocation and
localization is to asymmetric division. High-resolution
real-time imaging revealed that the localization dynamics
of these cell fate determinants are very noisy [19,20].
Nevertheless, temporary mis-localization seldom causes
asymmetric division defects. So we have to ask: is the
noisy localization dynamics an artifact from measure-
ment error, or cells indeed manage to tolerate spatial
uncertainty of these key cell fate determinants for robust
asymmetric division.
To answer this question, we need to devise a way to
spatially perturb the cell fate determinants without alter-
ing their overall expression levels. Since this cannot be
accomplished by existing genetic methods, we explored
techniques from the emerging field of synthetic biology.
Learning from naturally occurring scaffold proteins [21,

J. H. Li et al. / J. Biomedical Science and Engineering 6 (2013) 134-143 136
22], synthetic biologists have constructed synthetic scaf-
fold proteins with modular protein interaction domains to
make switches and logic gates [23], to redirect the MAP
kinase pathway [24] and to maximize metabolic fluxes
[25]. Can we build synthetic scaffold protein to recruit
asymmetrically localized cell fate determinant such as
PleC and DivJ to the opposite pole for spatial perturba-
tion?
During C. crescentus division, phosphatase PleC and
kinase DivJ asymmetrically localize to the swarmer pole
and the stalked pole, respectively. Dephosphorylated
DivK at the swarmer pole results in the accumulation of
phosphorylated CtrA (CtrA~P), a master regulator of cell
cycle. The presence of CtrA~P in the swarmer daughter
cell prevents the initiation of DNA replication and is
required for the expression of flagellum genes at the
swarmer pole [26]. In contrast, dephosphorylated CtrA is
inactive in the stalked daughter cell and subject to rapid
degradation by the ClpXP protease [27]. When synthetic
scaffolds interacting with both PleC and DivK force the
two determinants to co-localize to the same pole(s) (Fig-
ure 1(c)), there are two likely outcomes: 1) PleC recruits
most if not all DivK to the swarmer pole; and 2) DivK
recruits PleC to both poles (Figure 1(d)). In scenario (1)
we do not expect to observe any dividing defect because
most if not all DivK stay dephosphorylated at the swarmer
pole thus causing CtrA phosphorylation (CtrA~P) to
prevent DNA replication, whereas the absence of DivK
allows DNA replication in the stalked pole. However, in
scenario (2) PleC will keep DivK dephosphorylated at the
stalked pole, thus the accumulation of active CtrA~P to
inhibit DNA replication. Since DNA replication is cou-
pled to cell division in C. crescentus, we expect to see
cell growth arrest in this scenario [28].
Similarly, a synthetic scaffold interacting with both
DivJ and DivK will also generate two likely outcomes: 3)
DivJ recruits most if not all DivK to the stalked pole; and
4) DivK recruits DivJ to both poles (Figure 2(d)). We do
not expect to see swarmer cells in either scenario (3) or
(4), as the lack of dephosphorylated DivK in the swarmer
pole fails to activate CtrA signaling which causes defect
in flagellum assembly and additional DNA replication
[26].
2. METHODS
2.1. Vectors
Annealed DNA oligos encoding SH3 peptide (PPPVPPRR)
and GBD peptide
(GLVGALMHVMQKRSRAIHSSDEGEDQAGDEDED)
flanked by 8 repeats of glycine-serine linkers were
cloned into pYFP-C4 and pCFP-C2, respectively through
KpnI and MluI restriction sites to generate pPB001 and
pPB002 [29]. cDNAs of DivJ and PleC were amplified
directly from genomic DNA of C. crescentus and in-
serted into N terminus of GBD peptide from construct
pPB002 through Nd eI and KpnI. Likewise, DivK was
inserted into N terminus of SH3 peptide from pPB001
through NdeI and KpnI (Figure 2(a)). To generate pro-
tein scaffold, GBD domain was amplified by PCR from
pJD791 and inserted into pXCHYC-1 (containing mcherry)
with Nde I and BglII. Subsequently, SH3 domains with 1,
4 and 8 repeats were removed from pJD766, pJD781 and
pJD787 with BglII and BamHI and inserted downstream
of GBD domain (Figure 2(b)). pJD791, pJD766, pJD781
and pJD787 were a kind gift from Prof. John Dueber
(University of California, Berkeley, CA).
(a)
(b)
Figure 2. Design of recombinant vectors. (a) Schematic design of scaffold proteins containing
fluorescent protein mCherry, one, four and eight SRC Homology 3 (SH3) domains and a single
guanosine triphosphatase (GTPase)-binding domain (GBD) under the control of xylose-inducible
promoter; (b) Schematic design of recombinant cell fate proteins PleC, DivJ and DivK. They are
fused with GBD peptide or SH3 peptide to interact with scaffold proteins. Cyan fluorescent protein
(CFP) and yellow fluorescent protein (YFP) are used for fluorescent microscopy.

J. H. Li et al. / J. Biomedical Science and Engineering 6 (2013) 134-143 137
2.2. Bacterial Strains and Growth Conditions
C. crescentus CB15N and its derivatives were grown in
peptone yeast medium (PYE) or M2-glucose (M2G) me-
dium at 30˚C. For cloning purposes, plasmids were am-
plified in Escherichia coli DH10B (Invitrogen) in Lu-
ria-Bertani medium at 37˚C. When appropriate, antibiot-
ics were added into media as the following concentra-
tions (liquid/solid media for C. crescentus; liquid/solid
media for E. coli; ug/ml): kanamycin (5/25; 50/50), gen-
tamicin (0.5/5; 15/20), spectinomycin (25/50; 50/100). C.
crescentus strains were induced with 0.3% xylose for 4
hours when OD600 reached 0.2 - 0.5. Plasmids were in-
troduced into C. crescentus and its derivatives by elec-
troporation. Transduction was achieved by Φ30 bacte-
riophage based on the published procedure [30]. The
bacterial strains generated in this work are listed in Ta-
ble 1.
2.3. Measurement of Bacterial Growth
Overnight culture of bacterial strains PB018, PB021,
PB022 and PB025 (Table 1) which had been induced to
express protein scaffolds with 0.3% xylose were diluted
to the optical density OD600 = 0.05 in 5 ml PYE media
containing 0.3% xylose. Bacterial cell growth was moni-
tored every 1.5 hours by measuring the optical density at
600 nm on aliquots of cell culture grown at 30˚C, 250
rpm in PYE.
Table 1. Caulobacter strains used in the
perturbation of cell fate proteins.
Strains Genetic features
PB003
p
le
C
: pleC-linker-gbdpep-linker-cfp;
PB004 divK: divK-linker-sh3pep-linker-yfp;
PB006 divJ: divJ-linker-gbdpep-linker-cfp;
PB015 xylX: PxylX-mcherry-sh3(1)-gbd;
PB016 xylX: PxylX-mcherry-sh3(4)-gbd;
PB017 xylX: PxylX-mcherry-sh3(8)-gbd;
PB018
p
le
C
: pleC-linker-gbdpep-linker-cfp;
divK: divK-linker-sh3pep-linker-yfp;
PB019
p
le
C
: pleC-linker-gbdpep-linker-cfp;
divK: divK-linker-sh3pep-linker-yfp;
xylX: PxylX-mcherry-sh3(1)-gbd;
PB020
p
le
C
: pleC-linker-gbdpep-linker-cfp;
divK: divK-linker-sh3pep-linker-yfp;
xylX: PxylX-mcherry-sh3(4)-gbd
PB021
p
le
C
: pleC-linker-gbdpep-linker-cfp;
divK: divK-linker-sh3pep-linker-yfp;
xylX: PxylX-mcherry-sh3(8)-gbd;
PB022 divJ: divJ-linker-gbdpep-linker-cfp;
divK: divK-linker-sh3pep-linker-yfp;
PB023
divJ: divJ-linker-gbdpep-linker-cfp;
divK: divK-linker-sh3pep-linker-yfp;
xylX: PxylX-mcherry-sh3(1)-gbd;
PB024
divJ: divJ-linker-gbdpep-linker-cfp;
divK: divK-linker-sh3pep-linker-yfp;
xylX: PxylX-mcherry-sh3(4)-gbd;
PB025
divJ: divJ-linker-gbdpep-linker-cfp;
divK: divK-linker-sh3pep-linker-yfp;
xylX: PxylX-mcherry-sh3(8)-gbd;
2.4. Fluorescence Microscopy
After induction with 0.3% xylose for four hours, cells
were immobilized on 1% agarose pads and imaged with
an upright Olympus BX-50 microscope equipped with a
100× oil objective, QImaging Retiga EXi cooled CCD
camera, filter sets (RFP, CFP and YFP) and MetaMorph
software. The exposure time was 3 to 5 seconds. Fluo-
rescence intensity analysis was conducted with Image J
software. Briefly, ten dividing cells were randomly picked
from images. In Image J, select “area”, integrated den-
sity” and “mean gray value” to quantify fluorescence in
the area of interest at the stalked and swarmer poles. The
background fluorescence was individually collected by
selecting a region right next to a cell. The corrected total
spot fluorescence (CTSF) can be calculated based on
CTSF = Integrated Density—Area of selected cell X
Mean fluorescence of background readings.
2.5. Scanning Electron Microscopy
After induction with 0.3% xylose for 4 hours, cells were
collected by spinning down at 12,000 × g and were fixed
in 2% glutaraldehyde in PBS buffer at 4˚C for 2 hours.
Cells were washed three times with PBS and then fixed
by OsO4 in PBS at 4˚C for 1 hour. Cells were transferred
to 0.2 μm filter membrane by vacuum and serially dehy-
drated with 25%, 50%, 70%, 95% and 100% ethanol fol-
lowed by critical point dry. After sputter coating with
gold-palladium, cell morphologies were observed using
LEICA-440 SEM operating at 22,000× magnification, 10
kV and 11 mm working distance.
3. RESULTS
3.1. Design of Synthetic Protein Interaction
Domains in Caulobacter
To bind and co-localize both determinants (PleC/DivK or
DivJ/DivK), the synthetic scaffolds must contain modu-
lar protein interaction domains that can bind to their
cognate peptide sequences, which are fused to the cell
fate proteins. In addition, the interaction domains and
peptides have to be orthogonal to endogenous Caulo-
bacter regulatory factors to avoid interfering with their
functions. Therefore, the synthetic scaffolds were con-
structed with single or repeated well-characterized
guanosine triphosphatase (GTPase)-binding domain (GBD)
from rat actin regulatory switch N-WASP and SRC Ho-
mology 3 (SH3) domains from Crk, an adaptor protein
found in mouse (Table 2). Whereas the localization of
protein scaffolds can be visualized with the fusion of
fluorescent protein mCherry, its expression can be tem-
porally controlled by a xylose-inducible promoter using
plasmids developed by Thanbichler et al. [29]. Since the
binding affinity (Kd) of GBD to GBD-binding peptide

J. H. Li et al. / J. Biomedical Science and Engineering 6 (2013) 134-143 138
Table 2. Components used in recombinant cell fate proteins
and scaffolds.
Domains/Ligands Source Residues
(amino acids) Affinity (Kd)
GBD domain Rat N-WASP 196 - 274 -
GBD peptide Rat N-WASP 461 - 479 1 μM
SH3 domain Mouse Crk 134 - 191 -
SH3 peptide Synthetic peptidePPPVPPRR 10 μM
(GBDpep) is ten times that of SH3 to SH3-binding pep-
tide (SH3pep), we created three C. crescentus strains
(PB015, PB016, PB017) that express protein scaffolds
with one, four and eight SH3 binding domains and one
GBD domain connected by flexible glycine-serine (GS)
linkers (Table 2). After xylose induction, the scaffolds
were expressed and diffused throughout the entire cell
(Figures 3(b) and (c)). Furthermore, the scaffolds did
not cause any division defects (data not shown), which
suggests that the heterologous protein interaction do-
mains on the synthetic scaffold do not interfere with en-
dogenous cell cycle regulation.
Accordingly, to bind protein scaffolds DivK was fused
to SH3pep and yellow fluorescent protein (YFP) while
PleC and DivJ was fused to GBDpep and cyan fluores-
cent protein (CFP). The combination of YFP, CFP with
mCherry from scaffolds allows for simultaneous local-
ization of three different proteins. Through homologous
recombination mediated by phage Φ30, PleC-GBDpep-
CFP, DivK-SH3pep-YFP and DivJ-GBDpep-CFP replaced
their wild-type genes so that the resulting C. crescentus
strains (PB003, PB004 and PB006) express only recom-
binant proteins at their endogenous levels. These strains
divided without any defective phenotypes, indicating that
the inserted peptides do not disturb endogenous cell cy-
cle regulation. Furthermore, fluorescence microscopy
confirmed that the inserted SH3 and GBD peptides do
not alter localization patterns of these cell fate determi-
nants-PleC-GBDpep-CFP localizes at the swarmer pole,
DivJ-GBDpep-CFP localizes at the stalked pole, and
DivK-SH3pep-YFP localizes at both poles during early
stage of division (Figure 3(a)), exactly like their wild-
type counterparts.
3.2. Spatial Perturbation of Cell Fate Proteins in
Caulobacter
After confirming that individual scaffolds and recombi-
nant cell fate proteins do not interfere with normal Cau-
lobacter cell cycle regulation, the scaffolds were first
expressed in strains PB003, PB004 and PB006 integrated
with recombinant cell fate determinants (Table 1). Fluo-
rescence microscopy showed that PleC-GBDpep-CFP,
DivK-SH3pep-YFP and DivJ-GBDpep-CFP still local-
ized properly when each was co-expressed with scaffolds
Figure 3. Engineering modular and orthogonal interaction do-
mains for the perturbation of cell fate determinants. (a) The
localization patterns of recombinant DivJ, DivK and PleC dur-
ing C. crescentus division. Fluorescent microscopy showed that
their cellular localization are consistent with wild-type proteins;
(b) Western blot of scaffold proteins from C. crescentus lysate
after induction with 0.3% xylose for 4 hours. From left to right:
(1) wild-type strain, (2) strain expressing mCherry-SH3(1)-
GBD, (3) mCherry-SH3(4)-GBD and (4) mCherry-SH3(8)-GBD.
Star symbol indicates nonspecific band; (c) The cellular locali-
zatio n of protein scaffolds in C. crescentus. Fluorescent mi-
croscopy indicated cytoplasmic expression of scaffold proteins.
(Figure 4(a)). This suggests that the binding of synthetic
scaffolds does not seem to affect the ability of the cell
fate determinants to localize to the proper poles.
We then performed the spatial perturbation experi-
ments with the scaffolds. To force co-localization of
PleC and DivK, we constructed three additional strains
(PB019, PB020 and PB021), each of which has inte-
grated PleC-GBDpep-CFP, DivK-SH3pep-YFP, and one
of the scaffolds (one, four, or eight SH3 domains and one
GBD domain) (Table 1). Fluorescent microscopy re-
vealed that only the scaffold with eight SH3 domains
(PB021) was able to perturb the localization significantly,
probably due to the fact that the binding affinity (Kd) of
GBD domain is ten times that of SH3 domain（Figure
4(b)). Interestingly, DivK recruited PleC to both poles,
rather than PleC recruited DivK to the swarmer pole,
which fits Scenario (2) as described at the beginning of
the section (Figure 1(d)). This is a somewhat surprising
result, because PleC is a transmembrane histidine kinase
while DivK is a single-domain response regulator, so one
would have expected that PleC should recruit and relo-
cate DivK, not the other way around. Nevertheless, this
is consistent with a previous report that PleC molecules
diffuse without any directional biases in the cell and are
not actively transported [30]. Hence our observation
suggests that DivK is actively recruited to both poles,
pulling PleC along through the scaffold.
More surprisingly, scanning electron microscopy (SEM)
showed that the cells divided normally without any no-
ticeable morphological defect (Figure 4(c)). As scenario
(2) predicts that the cell division will be impaired as a

J. H. Li et al. / J. Biomedical Science and Engineering 6 (2013) 134-143 139
Figure 4. Perturbation of spatial localization of cell fate determinants by scaffold proteins. (a) Strains carrying DivK-SH3pep-YFP,
PleC-GBDpep-CFP or DivJ-GBDpep-CFP still localize properly when each is co-expressed with individual scaffolds (mCherry-
SH3(1)-GBD, mCherry-SH3(4)-GBD and mCherry-SH3(8)-GBD); (b) Co-localization of PleC with DivK at both poles in the strain
PB021 which expresses PleC-GBDpep-CFP, DivK-SH3pep-YFP and mcherry-SH3(8)-GBD; (c) Scanning electron microscopy
(SEM) revealed that the localization of PleC to both poles did not lead to morphological change in dividing C. crescentus as com-
pared to control strain PB018 which expresses only PleC-GBDpep-CFP and DivK-SH3pep-YFP; (d) Co-localization of DivJ with
DivK at both poles in strain PB025 which expresses DivJ-GBDpep-CFP, DivK-SH3pep-YFP and mcherry-SH3(8)-GBD; (e) SEM
revealed that the localization of DivJ to both poles did not lead to morphological change in dividing C. crescentus as compared to
control strain PB022 which expresses only DivJ-GBDpep-CFP and DivK-SH3pep-YFP; (f) Growth rate of different genetically
modified strains. The growth curve showed that the perturbation of cell fate proteins did not significantly alter growth rate. PB018:
PleC-GBDpep-CFP, DivK-SH3pep-YFP; PB021: PleC-GBDpep-CFP, DivK-SH3pep-YFP, mCherry-SH3(8)-GBD; PB022:
DivJ-GBDpep-CFP, DivK-SH3pep-YFP; PB025: DivJ-GBDpep-CFP, DivK-SH3pep-YFP, mCherry-SH3(8)-GBD. N = 3, scale bars
represent SEM.
croscopy revealed that only the scaffold with an 8:1
SH3-to-GBD ratio (PB025) was able to perturb the lo-
calization. Again, DivK recruited DivJ to both poles
(Figure 4(d)). Furthermore, the spatial perturbation to
DivJ failed to cause flagella loss in swarmer cells in con-
trary to our predictions from Scenario (4) in which the
DivK~P accumulated in the swarmer pole would lead to
inhibition of expression of key flagellum genes through
inactivation of CtrA signaling (Figure 4(e)). Similarly,
the scaffold did not affect growth rate when compared to
the control strain PB022 expressing only DivJ-GBDpep-
CFP and DivK-SH3pep-YFP (Figure 4(f)). This obser-
vation again attests the robustness of the Caulobacter
regulatory network.
result of the accumulation of CtrA~P at the stalked pole
through dephosphorylated DivK, we next compared
growth rates of different genetically modified strains.
Interestingly, the growth rate of PB021 measured by
monitoring cell density (OD600 ) was not affected when
compared to the control strain expressing PleC-GBDpep-
CFP and DivK-SH3pep-YFP but without the scaffold
(PB018) (Figure 4(f)). This remarkable robustness to-
wards spatial perturbations displayed by the Caulobacter
regulatory network provides an explanation to our pre-
vious question about noisy measurements of cell fate
determinant localization—asymmetric division is able to
tolerate occasional mis-localization of the cell fate de-
terminants.
Next, to induce co-localization of DivJ and DivK, we
constructed three more strains (PB023, PB024 and PB025),
each of which has integrated DivJ-GBDpep-CFP, DivK-
SH3pep-YFP, and a scaffold (Table 2). Consistent with
our observations withPleC-GBDpep-CFP, fluorescent mi-
3.3. Modeling of the PleC-DivJ-DivK
Phospho-Switch
It is intriguing how the Caulobacter regulatory network
achieves this remarkable level of robustness despite that

J. H. Li et al. / J. Biomedical Science and Engineering 6 (2013) 134-143 140
DivJ and PleC are essential to asymmetric division. It is
likely that the regulatory network has redundant mecha-
nisms to compensate for localization uncertainty. How-
ever, the PleC-DivJ-DivK phospho-signaling network
may also possess properties that enable it to be spatially
robust. To investigate the latter likelihood, we built a
simple ordinary differential equation (ODE)-based model
to analyze the network quantitatively (Supplementary
materials).
The simulation suggests that the competition between
the phosphatase PleC and the kinase DivJ forms a sensi-
tive switch to regulate DivK phosphorylation (Figure
5(a), Supplement Figure 1). The steep transition from de-
phosphorylated DivK to phosphorylated DivK~P gener-
ates an abrupt change from largely DivK to largely DivK~P
over a very small change in the PleC/DivJ concentration
ratio ([PleC]/[DivK]). Therefore, unless spatial noise or
perturbation is able to cross the threshold and flip this
ratio, the phosphor-switch is able to function robustly.
More specifically, for the PleC and DivK co-localiza-
tion experiment (PB021 with PleC-GBDpep-CFP, DivK-
SH3pep-YFP, and the 8:1 scaffold), unless the activity
level of mis-localized PleC is higher than that of DivJ at
the stalked pole, the phospho-switch will function nor-
mally to produce DivK~P at the stalked pole so there will
not be any phenotypic division defects (Figure 5(b)),
Figure 5. Mathematical modeling of a robust PleC/DivJ switch. (a) Steady state analysis of DivK and DivK~P. The curves show that
PleC/DivJ regulation forms a sensitive switch to convert DivK and DivK~P concentration to two distinctly different stable states; (b)
and (c) are heat maps of DivK~P concentration level with PleC/DivJ regulation. Crosses represent wild type of swarmer cells
(lower-right) and stalked cells (upper-left). Circles shows the cells with spatial perturbations. The solid lines indicates the arbitrary
threshold that separate swarmer cell and stalked cell based on the concentration level of DivK~P; (b) Represents that PleC is re-
cruited to the stalked cell; and (c) Shows DivJ is recruited to the swarmer cell. Heatmaps show that DivK~P levels in both of stalked
and swarmer cells are changed with spatial perturbations, but the cell fates are not converted without strong enough effects; (d) The
ratio of fluorescent intensity of recombinant DivJ and PleC between stalked cells and swarmer cells. In strain PB021 (PleC-
GBDpep-CFP, DivK-SH3pep-YFP, mCherry-SH3(8)-GBD), the average ratio of mis-localized PleC in stalked cells and correctly
localized PleC in swarmer cells was 0.55:1, SEM = 0.05. In strain PB025 (DivJ-GBDpep-CFP, DivK-SH3pep-YFP, mCherry-
SH3(8)-GBD), the average ratio of correctly localized DivJ in stalked cells to mis-localized DivJ in swarmer cells was 2.2:1, SEM = 0.22.

J. H. Li et al. / J. Biomedical Science and Engineering 6 (2013) 134-143 141
which was what we observed. Similarly, for the DivJ and
DivK co-localization experiment (PB025 with DivJ-
GBDpep-CFP, DivK-SH3pep-YFP, and the 8:1 scaffold),
unless the activity level of mis-localized DivJ is higher
than that of PleC at the swarmer pole, the phospho-
switch will function normally to dephosphorylate DivK
at the swarmer pole so cells are still able to divide asym-
metrically as experimentally observed (Figure 5(c)).
The experimental observation that the spatial perturba-
tions did not cause division defects suggests that the ac-
tivity of mis-localized PleC or DivJ did not exceed their
correctly localized counterparts. To validate this conjec-
ture, we used Image J to quantify the fluorescence emit-
ted by localized PleC and DivJ from fluorescent images.
On average, the ratio between the correctly localized
determinants and mis-localized determinants is about 2:1
(Figure 5(d)), confirming that the spatial perturbations
by synthetic scaffold are not strong enough to reverse the
robust switch. Altogether, the bidirectional phosphatase/
kinase switch formed by spatially separated PleC and
DivJ form a robust switch to regulate asymmetric divi-
sion in the presence of noise and uncertainty.
4. DISCUSSION
Robustness is an essential feature of gene regulatory
networks in order to tolerate noise and stochastic fluctua-
tions. To this date, most work has been focusing on un-
derstanding how regulatory networks are resistant to
variation in molecule levels and timing of transitions
during the division of stem cells or stem-like bacteria.
For example, we previously showed using hybrid control
theories and model checking that the Caulobacter cell
cycle master regulators are robust to temporal concentra-
tion variation [13]. However, compared to temporal ro-
bustness, spatial robustness is less explored partly due to
the lack of experimental techniques, even though single
molecule and time-lapse imaging demonstrated that the
diffusion and localization process is stochastic and noisy
[30]. Recently, C. Tropini and KC. Huang built a re-
action-diffusion model of PleC, DivJ and their cognate
response regulator DivK. The model predicts that the
system is remarkably robust to perturbation of the kinetic
parameters [31]. Consistent with this work, our protein
scaffolds have provided a unique experimental assay to
demonstrate that the asymmetric division of Caulobacter
is robust to spatial variations of cell fate determinants.
Moreover, our computational model suggests that a sharp
phospho-switch with spatially separated phosphatase and
kinase may partly contribute to this spatial robustness,
though redundant mechanisms yet to be discovered may
also play a role. Interestingly, phospho-switches also play
important roles in regulating stem cell asymmetric divi-
sion in various systems [1-3], so it is reasonable to an-
ticipate that those switches may also contribute to the
robustness of stem cell-mediated tissue homeostasis.
Lastly, this work demonstrates that forward engineer-
ing with synthetic protein scaffolds may provide new
capabilities to probe biological systems for better under-
standing of their operating principles. With advances in
systems biology and quantitative biology, our apprecia-
tion of the complexity of cell division and cell fate de-
termination keeps growing. Now we know that biologi-
cal systems have to evolve mechanisms with proper dy-
namic properties and deal with great uncertainty. Under-
standing of such mechanisms may eventually pave way
for stem cell engineering for tissue regeneration and
therapeutic purposes.
5. ACKNOWLEDGEMENTS
We thank H. McAdams, L. Shapiro, J. Dueber and A. Arkin for pro-
viding materials and advice. We thank members of the McAdams and
Shapiro laboratory, especially Dr. Grant Bowman, for discussion. We
thank J. Grazul and J. Hunt for assistance of SEM. This work was
supported by NIGMS R01GM95990.
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Supplemental Information:
Figure 1. Wiring diagram of PleC-DivJ-DivKphosphor-signal-
ing network.
REACTIONS:
1) DivK synthesis:
Θ→DivK
2) PleCdephosphorylateDivK~P:
DivK~P + PleC→DivK
3) DivJ phosphorylate DivK:
DivK + DivJ→DivK~P
4) DivK degradation:
DivK→Θ
5) DivK~P degradation:
DivK~P→Θ
EQUATIONS:
 



22
,, ,~,
22
22
~- -
d[ ]
d
~[]
s Divkd Divktrans DivkPtrans Divk
DivKP PleCDivKDivJ
DivK
tPleC DivJ
kkDivKkDivK PkDivK
J
PleCJ DivJ
 





 
22
,~, ,~
22
22
~- -
d[~ ]
d
~[]
trans DivkPtrans DivkdDivkP
DivKP PleCDivKDivJ
DivK P
tPleC DivJ
kDivKPkDivK kDivK
JPleC JDivJ
 

~P
STEADY STATE ANALYSIS:






2
,,~ 2
2
~-
2
2
2
-
,,
~
s DivktransDivkP
DivKP PleC
d Divktrans
DivK DivJ
Divk
PleC
kk DivK
JPleC
DivK
kDiv
JJ
kJ
Div





P





2
,2
2
-
2
,~ ,~2
2
~-
[]
~
trans Divk
DivK DivJ
dDivkPtrans DivkP
DivKP PleC
DivJ
kD
JDivJ
DivK PPleC
kk
ivK
J
PleC




PARAMETERS:
Parameters Value Unit
synthesis rate of DivK ,
s
Divk
k 0.0024 (min−1) [32]
degradation rate of DivK ,dDivk
k 0.002 (min−1) [32]
degradation rate of DivK ,~dDivk P
k 0.002 (min−1) [32]
phosphorylation rate of DivK ,trans Divk
k 0.15 (min−1) [32]
dephosphorylation rate of DivK ,~trans DivkP
k 0.6 (min−1) [32]
biding constant of DivK-DivJ -
D
ivK DivJ
J 0.3 (dimensionless)
binding constant of DivK~P-PleC ~-
D
ivKP PleC
J 0.3 (dimensionless)