Single molecules of the bacterial actin MreB
undergo directed treadmilling motion
in Caulobacter crescentus
So Yeon Kim*, Zemer Gitai†, Anika Kinkhabwala*, Lucy Shapiro‡§, and W. E. Moerner*§
*Department of Chemistry, Stanford University, Stanford, CA 94305;†Department of Molecular Biology, Princeton University, Princeton, NJ 08544;
and‡Department of Developmental Biology, Beckman Center, Stanford University School of Medicine, Stanford, CA 94305
Contributed by Lucy Shapiro, June 2, 2006
The actin cytoskeleton represents a key regulator of multiple
essential cellular functions in both eukaryotes and prokaryotes. In
eukaryotes, these functions depend on the orchestrated dynamics
of actin filament assembly and disassembly. However, the dynam-
ics of the bacterial actin homolog MreB have yet to be examined
in vivo. In this study, we observed the motion of single fluorescent
MreB–yellow fluorescent protein fusions in living Caulobacter cells
in a background of unlabeled MreB. With time-lapse imaging,
polymerized MreB [filamentous MreB (fMreB)] and unpolymerized
MreB [globular MreB (gMreB)] monomers could be distinguished:
gMreB showed fast motion that was characteristic of Brownian
diffusion, whereas the labeled molecules in fMreB displayed slow,
directed motion. This directional movement of labeled MreB in the
growing polymer provides an indication that, like actin, MreB
monomers treadmill through MreB filaments by preferential po-
lymerization at one filament end and depolymerization at the
other filament end. From these data, we extract several charac-
teristics of single MreB filaments, including that they are, on
average, much shorter than the cell length and that the direction
of their polarized assembly seems to be independent of the overall
cellular polarity. Thus, MreB, like actin, exhibits treadmilling be-
havior in vivo, and the long MreB structures that have been
visualized in multiple bacterial species seem to represent bundles
of short filaments that lack a uniform global polarity.
bacteria ? cytoskeleton ? single-molecule fluorescence
kinetic dynamics and ultrastructural architecture of actin’s po-
lymerized filaments has helped elucidate the mechanisms by
which eukaryotic actin functions. For example, high-resolution
imaging and the in vivo and in vitro dissection of the kinetics of
its assembly have demonstrated how actin polymerization at the
tips of a rigid, crosslinked actin meshwork can drive cell motility
at the leading edge of Dictyostelium (1, 2). In budding yeast, the
polarized assembly of actin cables provides both a road and
direction signs for the directed transport of proteins to the tip of
growing buds (3).
There are two known bacterial actin homologs, the widely
the plasmid-specific ParM family of proteins. ParM functions to
partition plasmid DNA by polymerizing in between two sister
plasmids, thereby generating a tension rod that physically pushes
them apart (4). MreB is essential in most bacteria and has been
shown to form a lengthwise spiral that contributes to cell shape,
chromosome segregation, and polar protein localization in mul-
tiple species, including Caulobacter crescentus, Escherichia coli,
and Bacillus subtilis (5–10). The mechanism by which MreB
executes its functions remains largely unknown (11).
In vitro studies of the dynamics of eukaryotic actin filament
assembly have demonstrated that actin polymerization is polar-
ized such that ATP-bound monomers preferentially polymerize
onto one filament end, hydrolyze their ATP to ADP while in the
n both eukaryotic and prokaryotic cells, actin mediates essen-
tial cellular processes. A quantitative understanding of the
filament, and then preferentially depolymerize from the oppo-
site filament end. Individual actin molecules thus appear to
directionally flow, or treadmill, through seemingly stationary
actin filaments (12–17). In contrast to actin’s polarized assembly,
the prokaryotic ParM protein polymerizes bidirectionally in vitro
and exhibits dynamic instability with periods of constant growth
interrupted by bursts of rapid depolymerization (18), a hallmark
of eukaryotic tubulin (19). Although the polarity of MreB
with MreB from the extremophilic bacterium Thermotoga ma-
ritima have raised the possibility that the elongation of MreB
polymers differs from actin, because MreB seems to require a
lower protein concentration for spontaneous polymerization
(critical concentration) and can polymerize in the presence of
either ATP or GTP (20, 21).
When carefully examined, the quantitative dynamics of eu-
karyotic actin assembly in vivo and in vitro have often differed.
The finding that the rate of actin depolymerization was far
greater in vivo than in vitro actually led to the prediction of the
existence of actin depolymerization factors and to the eventual
identification of actin depolymerizing factor?cofilin (22). Thus,
we sought to develop an in vivo method for characterizing the
assembly kinetics of MreB. We specifically focused on C. cres-
centus, because Caulobacter MreB is essential and regulates cell
morphology, chromosome segregation, and polar protein local-
ization (8–10). Caulobacter also has an inherently asymmetric
life cycle: With each cell cycle, it constructs a cellular extension
(known as a stalk) at one pole of the cell [stalked (ST) pole] and
a flagellum at the opposite pole (swarmer pole), such that
division gives rise to two daughter cells that differ in polar
morphology, size, and cell fate (23). With each cell cycle,
Caulobacter MreB forms a dynamic spiral that condenses into a
ring positioned at the future division plane and then expands
back into a lengthwise spiral (8).
In this study, we use quantitative imaging of single- molecule
fluorescence to assess the dynamics of MreB fused to a fluo-
rescent protein in living Caulobacter cells. Single-molecule im-
to various living cells to study intracellular dynamics (24–27).
This method allows classification of MreB–yellow fluorescent
protein (YFP) motion into both polymerized and unpolymerized
populations. Unpolymerized monomers move rapidly in a ran-
dom walk but appear to have a restricted rate of diffusion
compared with cytoplasmic proteins. By analyzing the rate,
distance, and direction of polymerized monomer motion, we
were able to demonstrate that MreB filaments indeed treadmill
Conflict of interest statement: No conflicts declared.
Abbreviations: fMreB, filamentous MreB; gMreB, globular MreB; YFP, yellow fluorescent
protein; MSD, mean square displacement; ST, stalked.
© 2006 by The National Academy of Sciences of the USA
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in vivo and thus assemble in a kinetically polarized fashion. From
these data, we also extracted the average MreB filament length
and the direction of its polarity with respect to the long axis of
the cell. Together, these results demonstrate that, in living cells,
MreB assembles in a manner similar to that of its eukaryotic
actin homolog, establishing the basis for understanding the
assembly, organization, and function of this central regulator of
bacterial cell biology.
Single MreB–YFP Molecules Can Be Observed in Vivo. To observe
MreB dynamics in vivo, we used single-molecule fluorescence
imaging of a fusion of MreB to YFP (MreB–YFP). Because this
method depended on the presence of a small number of MreB–
YFP molecules per cell, we constructed a merodiploid Cau-
lobacter strain containing a wild-type, unlabeled copy of MreB
under its endogenous promoter as well as a single copy of a
xylose-inducible MreB–YFP fusion integrated at the PxylX locus
(28). This strain was treated with varying concentrations of
xylose to find the optimal induction level for single-molecule
visualization. With 0.0006% xylose (1X), a Caulobacter cell
typically showed three to four discrete fluorescent molecules
that could be easily resolved from one another (Fig. 1A1; see also
Movie 1, which is published as supporting information on the
PNAS web site). To visualize the fluorescent molecules more
clearly, a smoothed version of the image was generated by
low-pass spatial filtering, as shown in Fig. 1A2. Several assays
confirmed that these fluorescent spots represent single MreB
molecules and not aggregates. The fluorescence signals showed
single-step digital photobleaching and the clear on–off blinking
behavior that is characteristic of single molecules (29) (see Fig.
4, which is published as supporting information on the PNAS
web site). In addition, the number of detected photons from a
single MreB–YFP before photobleaching (?91,000) was com-
parable to the literature value for single YFP molecules
(?140,000) (30) (data not shown). The fluorescent spots that we
observed did not appear to be free YFP that had been cleaved
from MreB, because the size of the fluorescent spot was com-
parable to that of a diffraction-limited spot (?240 nm in
diameter), whereas the rapid motion of the smaller free YFP
protein caused it to appear as a larger diffuse object, even on the
15.4-ms timescale (diffusion coefficient of ?7.7 ?m2?s) (31).
Thus, multiple criteria support the conclusion that we have
observed the fluorescence of single MreB–YFP molecules.
Polymerized and Unpolymerized MreB Can Be Distinctly Visualized as
Two Separate Populations. With rapid continuous irradiation and
detection (65 frames per s, 15.4-ms integration per frame, and
total time of ?7 s), we observed two different classes of behavior
for single MreB–YFP molecules. Some of the molecules moved
rapidly to many locations, whereas others appeared to be
essentially stationary on this timescale. In Fig. 1A1, the arrow
points to a mobile molecule, and the arrowheads point to
molecules that are stationary during the total observation time.
The observed trajectory of the mobile molecule is shown in Fig.
1A3 as a white line. To contrast the two populations more
clearly, we summed all 450 images pixel by pixel (Fig. 1A4); the
spots from both the top and bottom molecules are visible
because they were not mobile, whereas the spot from the middle
(mobile) molecule disappears. Because MreB is an actin ho-
molog that can polymerize in vitro into filaments in the presence
of ATP (32), we hypothesized that the stationary molecules
represent MreB proteins whose diffusions are constrained by
being assembled into an extended polymer [filamentous MreB
(fMreB)], whereas the mobile molecules represent free, unpo-
lymerized MreB proteins [globular MreB (gMreB)]. To test this
hypothesis, we treated cells with A22 (33). A small-molecule
chemical inhibitor of MreB function (9), A22 is thought to
interact directly with the MreB ATP-binding pocket, leading to
disruption of MreB filaments and consequently diffuse MreB–
YFP fluorescence (9); moreover, A22 perturbs the in vitro
polymerization of an archaeal MreB homolog (34). When cells
were incubated with 10 ?g?ml A22, stationary molecules were
not observed (see Movie 2, which is published as supporting
information on the PNAS web site). However, fast, mobile spots
were observed regardless of the A22 treatment. Because the
disruption of MreB filaments specifically abolishes the station-
ary form of MreB–YFP single molecules, we conclude that the
stationary and mobile MreB populations that are observed at
rapid timescales indeed represent polymerized fMreB and un-
polymerized gMreB, respectively.
Analysis of the Motion of gMreB. The ability to discriminate
between fMreB and gMreB afforded us the capability to directly
examine the dynamics of each population. To characterize the
cell. White line shows the cell outline. (A1) Image showing three fluorescent molecules (MreB–YFP) in a cell at 15.4-ms integration time. The top and bottom
0.125, 0.25, 0.125, 0.0625, 0.125, and 0.0625). (A3) A representative trajectory of the mobile molecule (middle spot in A1). (A4) Summed image of 450 sequential
images. The fluorescence from the two stationary molecules is evident, whereas the middle molecule does not appear. (Scale bar, 1 ?m.) (B) MSD of fast-moving
MreB molecules versus time lag for both untreated and A22-treated cells (open circles and squares), with geometry-corrected data shown as filled circles and
squares. Solid lines represent a linear fit of the corrected data. (C) Distributions of diffusion coefficients from individual molecules, from trajectories truncated
to 10 time steps. MreB, n ? 81; MreB ? A22, n ? 84. Solid lines represent the error distribution (39), assuming a homogeneous underlying diffusion coefficient.
The arrow shows the expected diffusion coefficient of MreB (62 kDa) in cytoplasm.
Unpolymerized single MreB–enhanced YFP molecules exhibit random motion. (A) Fluorescence images of single MreB–YFP proteins in a Caulobacter
www.pnas.org?cgi?doi?10.1073?pnas.0604503103Kim et al.
behavior of the fast, mobile molecules, individual molecules
were tracked by recording their center position as a function of
time. One hundred eleven trajectories were obtained from
untreated cells (30 individual cells), and 132 trajectories (27
individual cells) were obtained for A22-treated cells. We tracked
tracked one molecule several times because of the blinking
behavior of the YFP).
The observed mean square displacements (MSDs) as a
function of time lag (?t) for pooled data are shown in Fig. 1B
(open symbols). Even though the curve departs from linearity
at long times (considered below), we can extract an approxi-
mate diffusion coefficient D from the slope of the MSD plot
by using the first four points. The D values were 1.11 ? 0.18
?m2?s and 0.95 ? 0.14 ?m2?s in the absence or presence of
A22, respectively, exhibiting no significant difference. These
values are much smaller than the D value of 5.83 ?m2?s that
is expected for a cytoplasmic protein with the mass of MreB–
YFP (62 kDa), as estimated from D for cytoplasmic GFP (27
kDa; D ? 7.7 ?m2?s) (31), assuming a simple (mass)?1/3scaling
of the hydrodynamic radius. The fact that gMreB does not
diffuse like a cytoplasmic protein suggests that it may associate
with an additional factor(s).
An attractive explanation for the slower than expected move-
ment of gMreB and the curvature of MSD versus time lag is that
MreB associates with the plasma membrane. MreB has been
shown to biochemically associate with the Caulobacter mem-
brane (10), although it was not determined whether the mem-
brane-associated protein represented polymerized or unpoly-
merized MreB. To explore the possibility that membrane
association affects our measured diffusion coefficient, we mod-
account the capped-tube shape of the Caulobacter cell. As
described in detail in ref. 25, a simulation of 3D diffusional
movement on the cell surface allows correction of the MSD
values so that they apply to true 2D diffusion, effectively
flattening the cell into a plane (Fig. 1B, closed symbols). Using
this approach, we extracted corrected D values in the absence or
presence of A22 as 1.75 ? 0.17 ?m2?s and 1.55 ? 0.16 ?m2?s,
respectively. These values are consistent with observed D values
for membrane-bound proteins reported by others (26). There-
fore, it seems reasonable that membrane association could
contribute to our observed movement of gMreB.
To determine whether gMreB exhibits homogeneous diffu-
sion properties, distributions of measured diffusion coeffi-
cients from individual molecule trajectories at a time lag of
15.4 ms were examined for both untreated and A22 treated
cells (Fig. 1C). The average diffusion coefficient was 1.15 ?
0.05 ?m2?s for untreated MreB (n ? 81) and 1.03 ? 0.04 ?m2?s
for A22 treated MreB (n ? 84), comparable to the uncorrected
diffusion coefficients. The agreement between the data and
the expected error distribution (smooth curve) (35) shows no
evidence for heterogeneity.
Polymerized MreB Molecules Undergo Directional Movement. Al-
though the fMreB molecules did not appear to move on the
timescale of a few seconds with continuous imaging, we used
time-lapse imaging to explore the possibility that they might be
and 9.9 s) were inserted between acquisitions of fluorescence
images (100-ms exposure). By reducing photobleaching, this
time-lapse imaging allowed observation of the behavior for a
longer period than would normally be possible. We were easily
9.9-s dark intervals (Fig. 2A; see also Movies 3 and 4, which are
published as supporting information on the PNAS web site).
Motion of the entire cell was ruled out by observation of the
diffuse fluorescence from the fast-moving gMreB. Even though
the depth of focus was ?350 nm and the average cell thickness
was ?430 nm, in a few cases, we could observe the molecules
moving in a zigzag motion (the same molecule moved across the
cell and then back) (Figs. 2A and 3C). This movement is
reminiscent of the known helical distribution of Caulobacter
MreB filaments, which further suggests that the slow MreB
molecules are incorporated into polymerized filaments.
To quantify this motion, MSD values as a function of time lag
were calculated (Fig. 2B). We analyzed 120 of 174 trajectories,
because some of the molecular positions could not be fit to a 2D
Gaussian function because of the blinking of YFP. The plot of the
MSD of a particle moving in one direction is characterized by a
quadratic dependence on time lag (36–38), and our MSD of
polymerized MreB followed this behavior. The vertical scale in Fig.
2B shows that the slowly moving molecules did not move nearly as
far as the monomers discussed above, such that nonideal behavior
due to the nonplanar cell shape has less effect here. Nevertheless,
to guard against this concern, only separate linear portions of the
zigzag trajectories were included in the final analysis. The speed of
polymerized MreB motion was computed to be 2.9 ? 0.1 nm?s by
using a fit to the equation MSD ? 4Dt ? (Vt)2, where D is the
diffusion coefficient and V is the speed.
Another way to verify directional motion is to calculate the
velocity autocorrelation function CV(?) (39–41). For a random
a directed mover should have positive nonzero correlation due to
the tendency to keep moving in the same direction as before. Fig.
motion consistent with treadmilling. (A) Fluorescence images illustrating the
directional movement of fMreB. Time-lapse imaging with 10-s dark intervals,
100-ms exposures, black line showing the cell outline, and inverted contrast.
The molecule moves from left to right and then turns and moves right to left
autocorrelation for both gMreB (Inset) and fMreB. The autocorrelation of
gMreB dropped to near zero at the very first time lag, whereas that for fMreB
remained positive over at least 80 s. (D) Distribution of observed true irradi-
ation time of fMreB molecules. Inset shows the distribution of total emission
times before photobleaching of fMreB–YFP with continuous illumination
4.6 s (Inset), the average irradiation time of fMreB in the time-lapse experi-
ment was 0.8 s.
Polymerized single MreB-enhanced YFP molecules exhibit directed
Kim et al.
July 18, 2006 ?
vol. 103 ?
no. 29 ?
a drop of correlation to near zero at the first time step (Fig. 2C
Inset), whereas fMreB has positive correlation until ? ? 80 s. This
timescale is consistent with the MSD analysis as well as the average
we could observe fMreB molecules. Even though the autocorrela-
tion of fMreB dropped significantly at the first time lag of 10 s, this
drop can be explained by the fact that we conservatively recorded
the same position twice (i.e., zero velocity) when we saw any
blinking behavior. Together, these data demonstrate that gMreB
follows a fast random walk and that fMreB exhibits slow, directed
There are two possible explanations for the directional move-
ment of fMreB: Either the filaments into which these monomers
are incorporated are moving, or the monomers themselves are
treadmilling through largely stationary filaments. The latter
treadmilling motion is plausible because of the structural and
kinetic similarities between MreB and actin. Actin’s well docu-
mented treadmilling behavior results from polarized assembly
wherein monomers are preferentially polymerized on one fila-
ment end and depolymerized from the other filament end. Such
polarized assembly or treadmilling has yet to be documented for
MreB in vitro or in vivo.
If the motion of fMreB is due to whole-filament movement,
then the observation time for fMreB single molecules should be
limited by only the photobleaching rate of the YFP and should
thus be the same regardless of the experiment’s timescale. In
contrast, the observation times for monomers undergoing tread-
milling motion should be limited by both photobleaching and
depolymerization from the filament end. To quantify the pho-
tobleaching component, single molecules were observed with
continuous illumination until emission ceased for a long time
(photobleaching), ignoring short (one to two frame) blinking
events. The average emission time of single polymerized MreB
molecules was 4.6 s with continuous illumination (Fig. 2D Inset).
This average emission time under continuous illumination
should be compared with the distribution of true irradiation
times for the time-lapse experiments (10-s dark periods between
With these statistics, we concluded that most of the fluorescence
from the polymerized MreB molecules disappeared before they
photobleached, likely as a result of dissociation from the end of
the filament and onset of diffusive motion. These results argue
in favor of MreB molecule treadmilling through filaments with
fixed ends. Consequently, MreB appears to resemble actin not
only in structure but also in assembly dynamics.
In Vivo Assessment of Average MreB Polymerization Rates and Fila-
ment Lengths. Given that single fMreB molecules exhibit directed
motion, a speed value was extracted from each single-molecule
trajectory. The speed distribution for individual molecules is
shown in Fig. 3A. The average speed was 6.0 ? 0.2 nm?s (n ?
120, SEM). The slight difference between the average speed
value and the quadratic fit in Fig. 2B is likely due to small
changes in direction during each trajectory. The error bar on the
figure shows the error of determination of any one speed;
therefore, the excess width of the measured distribution suggests
the presence of heterogeneity.
We were able to estimate the steady-state rate of MreB
monomer polymerization and depolymerization by combining
treadmilling speed measurements with the known structural
from its crystal structure is 5.4 nm (32); in steady-state fixed
filaments, addition of each monomer at one end is accompanied
by release of a monomer at the other end. Thus, our observed
speed of 6.0 nm?s can be converted to a steady-state rate of
monomer addition of 1.2 s?1.
The other physical property that can be extracted from our
treadmilling observations is the average length of the MreB
polymer filaments. With a fixed steady-state treadmilling fila-
ment, the total distance a molecule travels from the time it
polymerizes until the time it dissociates can be regarded as the
length of the polymer filament. We extracted the distribution of
the end-to-end contour length from our trajectories as shown in
Fig. 3B, using only newly polymerized molecules (that is, mol-
ecules that appeared as spots after the start of imaging). The
average MreB filament length is 392 ? 23 nm (n ? 128, SEM).
The error bars in Fig. 3 are the SDs of the distributions, which
estimate the error of single measurements. The observed aver-
age filament length is quite small compared with average cell
length (3.5 ?m), suggesting that the extended MreB helical
filaments rather than a few long filaments. It is plausible that by
observing only ST and predivisional cells (see Fig. 5, which is
published as supporting information on the PNAS web site), we
are sensing primarily the time in the cell cycle when the helix is
converting from an extended form (ST state) to a ring at the
divisional plane and when the new helix is not fully formed
(predivisional state). Because of the depth of focus, it is also
possible that we could not observe the entire trajectory in every
went beyond the edges of the focal plane. However, most of the
traces showing zigzag motions have a similar length regardless of
where they started, and many of the molecules moved less than
Global Cell Polarity. Treadmilling filaments are inherently polar-
ized, with a growing end and an end that shortens. Having
established that MreB treadmills, we take the direction of
single-molecule spot motion to indicate the local MreB filament
polarity. Caulobacter cells are polarized, most notably evidenced
by the presence of a stalk at one pole, and we calculated that the
average MreB filament length is quite short relative to the total
cell length. Thus, we were curious to see whether the multiple
filaments that must constitute the overall MreB helix have a
reproducible polarity with respect to the global cell polarity.
the movement of a single fMreB. The average length was 332 nm, which was
trajectories of fMreB filaments in a ST cell and in a predivisional cell. The
single-molecule trajectories were plotted on normalized cell shapes as de-
scribed in Supporting Text. Examples of global direction assignments are
shown as either ‘‘?’’ [toward the swarmer (SW) pole] or ‘‘?’’ (toward the ST
In vivo assessment of MreB polymer assembly rate, length, and
www.pnas.org?cgi?doi?10.1073?pnas.0604503103Kim et al.
To show the overall behavior more clearly, we projected some
of the trajectories from single polymerized MreB molecules onto
a normalized cell shape (colored lines in Fig. 3C; the method is
described in Supporting Text, which is published as supporting
information on the PNAS web site). Surprisingly, most of the
trajectories moved perpendicular to the cell long axis, along a
either pole. A relatively small number of the trajectories showed
oblique lines and zigzag shapes. To quantify the analysis for all
trajectories, a well defined procedure was used to determine
whether a polarized filament was oriented toward or away from
the ST end of the cell (see Supporting Text and Fig. 6, which is
published as supporting information on the PNAS web site). The
plus and minus labels in Fig. 3C show a few examples of this
determination. The results of this analysis yielded no significant
preference for local filament orientation toward or away from
the ST pole (see Table 1, which is published as supporting
information on the PNAS web site). In other words, the short
polymerized MreB segments have a polarity that seems to be
random relative to the overall cell polarity.
The actin homolog MreB has been shown to be essential for cell
viability, cell shape, polar protein localization, and chromosome
segregation in a wide array of bacterial species (5–10). These
central functions of MreB are thought to depend on its dynamic
ability to polymerize into filaments. In this study, we extend our
understanding of MreB assembly and function by reporting the
dynamic motion of single molecules of both gMreB and fMreB
populations in living Caulobacter cells.
Unpolymerized MreB Does Not Behave Like a Free Cytoplasmic Pro-
tein. Surprisingly, the diffusion coefficient that we calculated for
the unpolymerized MreB form was significantly slower than
expected for a free cytoplasmic protein of similar size. Such
slower diffusion indicates the presence of an as yet uncharac-
terized force that restricts the movement of unpolymerized
MreB. Our modeling suggests that the slower diffusion could be
explained by the association of unpolymerized MreB with the
cell membrane. Although polymerized MreB filaments are
found directly under the membrane and bulk MreB sediments in
the membrane-associated cell fraction (10), the MreB protein
sequence does not bear any motifs that are indicative of mem-
brane association. It will prove interesting to determine whether
unpolymerized MreB has an inherent affinity for the phospho-
lipid membrane or for specific membrane-bound proteins. Al-
ternatively, it remains a possibility that unpolymerized MreB is
not membrane-associated but instead interacts with a larger
protein complex that could function to sequester MreB or
exchange its nucleotide, much as CAP (cyclase-associated pro-
tein) or profilin function for eukaryotic actin (42).
MreB Assembles in a Polarized Fashion in Vivo. By observing the
behavior of polymerized MreB molecules, we were able to assess
determined that polymerized MreB molecules move in a direc-
tional manner and, by comparing their on-time persistence,
determined that this directed motion is likely to reflect tread-
milling of monomers through filaments (with fixed ends), rather
than wholesale filament translocation. The directed motion also
argues against the possibilities that filament segments might be
coming loose or that the filaments are coiling up and reanneal-
ing. This treadmilling behavior appears qualitatively similar to
eukaryotic actin treadmilling that has been reported both in vitro
(12–14, 43) and in vivo (15–17). The rate of monomer motion
also allowed us to calculate the steady-state rate of MreB
treadmilling to be 1.2 s?1. At steady state, the rate-limiting step
of actin treadmilling has been shown to reflect the rate of
monomer dissociation from the pointed end. The range of actin
treadmilling rates is reported to be 0.2–0.9 s?1in vitro, similar to
MreB’s in vivo treadmilling rates. In different contexts, 10- to
100-fold increases in the actin treadmilling rate could be ob-
served in vivo with the help of accessory proteins such as actin
depolymerizing factor?cofilin (22). This discrepancy between
the in vivo actin and MreB treadmilling rates could reflect either
the absence of an MreB depolymerization factor in Caulobacter
or inherent differences between actin and MreB polymerization.
It is also formally possible that the fusion of MreB to YFP affects
its dynamics, although the extremely low levels of MreB–YFP
expression used in this study make such effects unlikely. Inter-
estingly, another bacterial actin homolog, the plasmid-encoded
ParM protein, exhibits bidirectional, rather than polarized,
filament assembly (18). ParM functions by symmetrically extend-
ing from the cell center toward the two poles, whereas both actin
and MreB are involved in localizing asymmetrically distributed
macromolecules. Thus, polarized treadmilling may not be an
inherent feature of actin-like filaments; instead, it may reflect
the involvement of a filament in polarized processes.
Models for the Intracellular Organization and Activity of MreB. We
were able to explore additional aspects of the ultrastructural
organization of MreB filaments by quantitating several aspects of
the motion of polymerized MreB molecules. The distance traveled
by each polymerized MreB molecule allowed us to model the
average filament length, which we estimate to be ?400 nm. MreB
spiral structures have been observed traversing the cell from pole
to pole for lengths of several microns by both ensemble imaging of
GFP fusions and immunofluorescence (8, 10, 44, 45, 49). The fact
that we found individual filaments to be significantly shorter than
the overall MreB spiral suggests that MreB spirals consist of
multiple small filaments that are bundled together.
MreB has been shown to be a determinant of polar protein
localization and the translocation of chromosomal origins
toward cell poles in both Caulobacter and E. coli (6, 9, 46, 47),
leading to the hypothesis that MreB structures possess a
uniform polarity that can be interpreted by trafficking factors.
However, by using the direction of treadmilling as an assay for
the polarity of individual MreB filaments, we find a roughly
even distribution of filaments directed toward either pole in
every cell type and cell compartment examined. This hetero-
geneous filament polarity could indicate that the overall MreB
spiral does not have a uniform polarity. Because it is difficult
to understand how MreB could lead to directed macromolec-
ular trafficking in this scenario, such a model would support
less directed models for MreB’s involvement in such processes.
Alternatively, the heterogeneous filament polarity could re-
flect the presence of two separate spirals or a continuous
‘‘closed track’’ spiral, wherein each MreB bundle has a uniform
polarity but there exist bundles of both polarities in each cell,
allowing for directed trafficking to each pole. Higher resolu-
tion imaging (by cryoelectron tomography, for example)
should allow these possibilities to be distinguished.
In this study, we have provided evidence that MreB spirals
consist of bundles of multiple short filaments that each assemble
in a polarized manner. The similarities between MreB and actin
are thus kinetic as well as structural. This work should establish
a constructive framework for future efforts exploring the factors
that interact with MreB to influence its assembly and function as
well as its detailed, high-resolution architecture.
Materials and Methods
Bacterial Strains and Plasmid. A xylose-inducible, YFP-labeled
MreB fusion was introduced in single copy into the Caulobacter
chromosome to tightly control the expression of this fluorescent
fusion protein. The required Pxyl:efyp-mreB plasmid was prepared
as described in ref. 8. Importantly, these N-terminal YFP-fused
Kim et al.
July 18, 2006 ?
vol. 103 ?
no. 29 ?
MreB (10), and similar fusions to MreB homologs were functional
in other bacterial systems (48, 49). The movement of cytoplasmic
YFP proteins was observed with the previously described EJ153
the addition of xylose to the media.
Sample Preparation. Cells were grown overnight in PYE media at
30°C and then diluted into M2G minimal media with specific
concentrations of xylose (51). After the cells reached their loga-
added to a 1.5% agarose (A-0169, Sigma) pad slide along with 1 ?l
of a quantum dot solution (10 nM Qdot 565; Quantum Dot
Corporation, Carlsbad, CA), and covered with a coverslip for
room-temperature imaging as described in ref. 25. The quantum
dots were later used as fiduciary markers. Different xylose concen-
trations were used for the following experiments: (i) to track
xylose (1X); (ii) cytoplasmic YFP in EJ153 was induced with
0.006% xylose (10X); (iii) to track polymerized MreB–YFP with
time-lapse imaging, 0.003% xylose (5X) was used.
Single-Molecule Fluorescence Microscopy. Both white-light trans-
mission and epifluorescence images of single molecules were
acquired by using a Nikon TE300 inverted microscope. The
general experimental arrangement is described in ref. 25; for full
details, see Supporting Text.
To track fast- and slow-moving molecules, we used time-lapse
imaging by placing a variable-length dark interval between
exposure (integration) times. In cases of fast-moving molecules,
such as monomeric MreB and cytoplasmic YFP, samples were
illuminated with continuous laser light (no dark interval) with a
15.4-ms (65 Hz) integration time per frame. For slowly moving
polymerized MreB, images were recorded with 9.9-s dark inter-
vals (without laser illumination) between 100-ms exposures.
Lastly, the fluorescence on-time distribution of polymerized
MreB before photobleaching was measured with continuous
irradiation and a 100-ms integration time.
Analysis of Motion. For the fast-moving molecules, the center of
the spot in each image was determined manually, and an
estimated diffusion coefficient for each single-molecule trajec-
tory was computed by using the measured MSD for a 15.4-ms
time lag. The resulting distributions of diffusion coefficients
were compared with a theoretical distribution for observed D
values, which takes into account the finite trajectory length (35).
For polymerized MreB, a 2D Gaussian function was fit to each
single-molecule point-spread function to localize the position to
?15 nm without pixelation error by using the MATLAB function
FMINSEARCH. For measurements of positions as a function of
time, we also tracked fixed quantum dots imbedded in the
sample, which can be localized to ?4 nm under our imaging
conditions. To compensate for stage drifts during the time-lapse
imaging, the positions of the MreB molecules were determined
trajectory was determined by the average of the interframe
speeds for points along the trajectory.
For determinations of the velocity autocorrelation, molecules
were tracked by hand to 1-pixel accuracy to extract the velocity
v ?(t), and we used the expression CV(?) ? ?v ?(t)?v ?(t ? ?)?, where ??
indicates time average.
We thank Stefanie Nishimura for consultation regarding data analysis
and Patrick McGrath for suggesting polar coordinates (r, ?) to analyze
the direction of the polymerized MreB movements. This work was
supported by Department of Energy Grant DE FG02-04ER63777 (to
W.E.M.) and National Institutes of Health Grants 1P20-HG003638 (to
W.E.M.), 2R01C-M051426 (to L.S.), and 2R01C-M032506 (to L.S.).
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www.pnas.org?cgi?doi?10.1073?pnas.0604503103Kim et al.