MICROBIOLOGY AND MOLECULAR BIOLOGY REVIEWS, Dec. 2010, p. 504–528
Copyright © 2010, American Society for Microbiology. All Rights Reserved.
Vol. 74, No. 4
FtsZ in Bacterial Cytokinesis: Cytoskeleton and Force
Generator All in One†
Harold P. Erickson,* David E. Anderson, and Masaki Osawa
Department of Cell Biology, Box 3709, Duke University Medical Center, Durham, North Carolina 27710
LIGHT MICROSCOPY OF Z RINGS IN BACTERIA .........................................................................................504
Constriction of the Z Ring and Reassembly of New Z Rings ..........................................................................504
Two Kinds of Z-Ring Helices................................................................................................................................506
Assembly Dynamics of FtsZ In Vivo.....................................................................................................................506
SUBSTRUCTURE OF THE Z RING.......................................................................................................................507
Structure of the FtsZ Subunit and Protofilaments............................................................................................507
The Z Ring Modeled as Short, Overlapping Protofilaments............................................................................507
Tethering FtsZ to the Membrane.........................................................................................................................509
Lateral Bonds: Do They Exist?.............................................................................................................................510
Alternatives to Lateral Bonds...............................................................................................................................512
ASSEMBLY OF FtsZ IN VITRO...............................................................................................................................513
Buffer Conditions for In Vitro Studies.................................................................................................................513
Assembly Dynamics of FtsZ In Vitro....................................................................................................................514
Inhibition of FtsZ Assembly by SulA...................................................................................................................515
Inhibition of FtsZ Assembly by MinC.................................................................................................................515
FtsZ as a Target for Drugs ...................................................................................................................................516
Cooperative Assembly and Treadmilling of FtsZ...............................................................................................517
FtsZ AS A FORCE GENERATOR: BENDING PROTOFILAMENTS...............................................................518
The Z-Centric Hypothesis and Reconstitution of Z Rings in Liposomes.......................................................518
Two Different Curved Conformations of FtsZ Protofilaments.........................................................................519
Evidence That the Constriction Force Is Generated by Bending Protofilaments .........................................520
Incomplete FtsZ Rings Can Generate Constriction ..........................................................................................520
Finishing Division: Membrane Scission without FtsZ ......................................................................................522
FtsZ is the major cytoskeletal protein in the bacterial cyto-
kinesis machine. It forms a ring (the Z ring) under the mem-
brane at the center of the cell, and this Z ring constricts to
initiate division of the cell. In addition to FtsZ, there are a
dozen accessory proteins that are essential for cell division in
Escherichia coli. These are mostly transmembrane proteins
that are involved in remodeling the cell wall, and they will not
be discussed here. The present article focuses on FtsZ. For
more comprehensive reviews of bacterial cell division, includ-
ing the downstream proteins, see references 36, 59, 69, 111,
195, and 198. A recent review by Adams and Errington (2)
focuses on the proteins that interact directly with FtsZ and may
regulate its assembly. The evolutionary relationships of FtsZ to
tubulin and of MreB to actin have been discussed in two
articles, each providing some different perspectives (46, 102).
LIGHT MICROSCOPY OF Z RINGS IN BACTERIA
Constriction of the Z Ring and Reassembly of New Z Rings
The pioneering immunoelectron microscopy of Bi and Lut-
kenhaus (22) provided the first evidence that FtsZ is a cy-
toskeletal protein localized in a ring at the center of the cell.
They found that in a cell just starting septation, antibody-
coated gold beads localized near the membrane at the site of
constriction. Subsequently, Levin and Losick (97) used immu-
nofluorescence at the light microscope level and made the
surprising discovery that Z rings were present at the centers of
almost all cells, not just dividing ones. Wang and Lutkenhaus
(197) independently developed immunofluorescence technol-
ogy and discovered that Z rings were present in 50% of the
cells in a culture of the archaeon Haloferax volcanii. The frac-
tion of cells with Z rings is 85 to 95% in rapidly dividing
Bacillus subtilis and E. coli (3, 97). This means that FtsZ as-
sembles in the daughter cells very soon after division and
remains assembled in the cell for most of the cell cycle.
Green fluorescent protein (GFP) labeling of FtsZ was intro-
duced by Ma et al. (108), confirming the localization seen by
immunofluorescence. That study also found that FtsA-GFP
had a localization very similar to that of FtsZ. The powerful
* Corresponding author. Mailing address: Department of Cell Biol-
ogy, Box 3709, Duke University Medical Center, Durham, NC 27710.
Phone: (919) 684-6385. Fax: (919) 684-8090. E-mail: h.erickson
† Supplemental material for this article may be found at http://mmbr
advantage of GFP labeling is that the localization can be fol-
lowed over time in living bacteria. The Margolin lab later used
FtsZ-GFP to observe the dynamics of the Z ring throughout
the cell cycle and during constriction (186, 191). In those stud-
ies FtsZ-GFP was used as a dilute label in the presence of
wild-type FtsZ expressed from the genome. As long as the level
of FtsZ-GFP is less than ca. one-third of that of the wild-type
FtsZ, it labels the Z ring without introducing obvious defects in
division. Our lab has recently derived an E. coli strain that can
use FtsZ-YFP as the sole source of FtsZ (138). This strain has
a second-site mutation, whose nature is not known, somewhere
in the genome. This strain should be useful for future studies,
but for most observations, including those shown in Fig. 1, we
have used FtsZ as a dilute label.
Figure 1A shows our own use of FtsZ-GFP to follow the Z
ring through a division event. The typical appearance of the Z
ring is a pair of bright dots on either side of the cell, as seen in
the lower right Z ring in Fig. 1A. The Z ring is actually a
continuous circular filament of fairly uniform density. The
bright dots on the edges are due to the ring being more intense
when viewed in vertical projection. Some very bright rings do
not show the two-dot structure due to saturation of the image,
and in highly constricted cells the dots are too close to resolve.
We will look first at the constriction of the Z ring. In Fig. 1A,
the panel at 0:00 (min:s) shows the mother cell (arrow) with a
bright Z ring in the initial stage of constriction. This Z ring is
about half the diameter of the other two Z rings in the field, so
it began constriction at some undetermined earlier time. Com-
plete constriction of the mother Z ring took about another 4
min; the total time for constriction was probably 8 to 10 min
(about half of the 20-min cell cycle). As the Z ring constricted,
it disassembled completely. Several studies have noted that the
Z ring disassembles as it constricts (40, 120, 186). A recent
study reported that the density of FtsZ in the Z ring actually
increased during constriction (91). However, the single exam-
ple presented there measured FtsZ over a constriction of only
200 nm. This is less than the 250-nm resolution of the light
microscope and much less than the 1,000-nm diameter of an E.
coli cell. In our time-lapse movies, the Z ring decreased in
brightness during constriction and disappeared completely at
the end (Fig. 1A).
The mechanism by which the Z ring disassembles as it con-
stricts is not known. As discussed below, FtsZ rapidly cycles
between the Z ring and the cytoplasmic pool. The cycling
continues at the same rate when rings begin constricting (183).
However, it appears that something may block the return of
FtsZ to the Z ring, while still permitting its loss. In a temper-
ature shift experiment with FtsZ84, Addinall et al. noted that
Z rings rapidly disappeared when cells were shifted to 42°C
and rapidly reformed when cells were shifted back to 30°C (4).
There was one exception: sites with a visible constriction did
not reform a Z ring but rather assembled Z rings at the one-
and three-quarter positions, where the Z rings would assemble
in the daughter cells. This is consistent with some mechanism
that blocks return of FtsZ to the Z-ring site once constriction
Faint Z rings are already visible in the daughter cells in the
0:00 frame (Fig. 1). This confirms the observation of Sun and
Margolin (186) that the Z rings are able to initially assemble in
daughter cells before constriction of the mother cell is com-
FIG. 1. Time-lapse observation of Z rings in E. coli, using FtsZ-
GFP as a dilute label, expressed at about one-third the level of
genomic FtsZ. The cells were induced to produce FtsZ-GFP for about
1 h and then immobilized on an agar pad for time-lapse observation at
37°C. (A) Three cells. We will ignore the two on the bottom (with a
bright Z ring [left] and a dim Z ring [right]) and focus on the upper one
(arrow), which is undergoing division. The constriction of the Z ring,
its concurrent disassembly, and the assembly of new Z rings in the
daughter cells are described in the text. (B) A cell with three Z rings
(perhaps induced by excessive production of FtsZ-GFP). The upper Z
ring (arrow) alternately opens into a short-pitch helix and collapses
into an apparent circle. Frames from both panels A and B are taken
from Movie S1 in the supplemental material.
VOL. 74, 2010 FtsZ IN BACTERIAL CYTOKINESIS505
plete. However, this initial assembly is transient and seems to
have disappeared at 1:30 and 3:50. From 4:20 to 5:20 the FtsZ
in the daughter cells appears to form foci scattered throughout
the cell (discussed below). At 5:10 in the left-hand cell and 5:30
in the right-hand cell, the peripheral foci disappear and Z rings
are formed. The characteristic two-dot structure of the Z ring
is clearly seen in the right-hand cell at 5:30, and it becomes
brighter at 6:20 and 10:00.
Aarsman et al. (1) studied the assembly and maturation of
the Z ring over the course of the cell cycle. In LMC500 cells
grown with a doubling time of 40 min, the Z ring appeared
after 15% of the cell cycle. Proteins downstream of FtsK ap-
peared after a substantial lag, 49% of the cell cycle, and visible
constrictions appeared almost immediately after this. When
the cell cycle time was increased (slower growth), the Z ring
appeared later in the cycle and there was a further delay before
the downstream proteins assembled. It is likely that the Z ring
generates a constriction force as soon as it is assembles, but
constriction can begin only when all of the downstream pro-
teins are assembled to remodel the cell wall.
A number of studies agree that the constriction phase occu-
pies approximately the last half of the cell cycle. den Blaauwen
and colleagues (1, 40) found that constriction began at ?50%
into the 40-min cell cycle. Reshes et al. (158) used sophisti-
cated analysis of phase-contrast images to follow the course of
the constriction. Their analysis concluded that constriction be-
gan about half way through the 20-min cell cycle. Inoue et al.
(80) created a strain in which FtsZ-GFP was expressed from
the genome at a level ?10% that of wild-type FtsZ, giving a
steady level of fluorescence with no apparent aberration in cell
division. For a 100-min cell cycle they found 8 to 12% of cells
with a visible constriction, implying a constriction time of 8 to
12 min. This was a much shorter time for constriction than was
determined in other studies. The timing of when constriction
begins can be complicated by the different techniques used.
In a recent study, Stro ¨mqvist et al. (184) used fluorescence
recovery after photobleaching (FRAP) to measure diffusion of
GFP across dividing E. coli cells. From the rate of diffusion,
they calculated the radius of the constriction. Their analysis
was valid mostly for the later stages of constriction, which they
concluded proceeded at a linear rate on average.
Two Kinds of Z-Ring Helices
Ben-Yehuda and Losick (13) reported that when B. subtilis
was entering into sporulation, the single central Z ring spun off
helical structures in both directions, which then condensed into
two polar Z rings. Helical intermediates were also found to
precede the formation of sporulation Z rings in Streptomyces
coelicolor (62). Thanedar and Margolin (191) and Peters et al.
(143) analyzed time-lapse images of FtsZ-GFP in E. coli and B.
subtilis and reported localization to moving, membrane-bound
spots throughout the cell, especially prior to formation of the
Z ring. They used deconvolution microscopy to make a con-
vincing case that these spots were mostly projection views of
helices. The helices eventually collapsed toward the center of
the cell and condensed into the Z ring. Thanedar and Margolin
(191) noted two types of dynamic movement of these helices.
They changed position on a rapid time scale of a few seconds,
and on a longer time scale they oscillated from one side to the
other in elongated cells. This movement resembled the oscil-
lation of MinD, and indeed it was dependent on an operational
We will refer to this first type of helix as cytoplasmic or
long-pitch helices. Figure 1A shows a cell completing division
and assembling new Z rings in the daughter cells. The Z ring of
the mother cell constricts and disappears at 4:50 to 5:00. From
4:20 to 5:20 small patches of FtsZ are seen in the daughter
cells. These patches are similar to those resolved as long-pitch
helices by deconvolution microscopy (143, 191). These helices
then condense to form Z rings. From 5:40 each daughter cell
has a single Z ring, and the cytoplasmic patches/helices have
largely disappeared. It seems that the cytoplasmic helices are
the preferred FtsZ structure when there is no Z ring, but they
are incorporated into the Z ring once it forms; however, oc-
casional peripheral foci continue to appear and disappear even
in the presence of the Z ring (see Movie S1 in the supplemen-
tal material). The transition from diffuse helices to condensed
Z rings is also demonstrated in a recent study by Monahan et
al. (Fig. 6 in reference 120).
A second type of helical structure emerges from the Z ring
once it is formed. At 6:20 in the right-hand daughter cell, the
Z ring shows the characteristic structure of one dot on the
upper membrane and one on the lower. At 7:00 the upper dot
has separated into two, suggesting that the Z ring has split into
a helix. At 10:00 it has collapsed back into a ring. The separa-
tion of the Z ring into helices is seen in more detail in Fig. 1B,
which shows a segment of a cell that has elongated and assem-
bled three Z rings. The Z ring at the upper left appears as a
closed ring (a line or two bright dots on the sides of the cell) at
0:00, 4:20, and 9:40. At 0:30, 2:30, 3:30, and 4:00 the dots
separate into two, suggesting that this Z ring is actually a helix
of ?2 turns. This suggests that the Z ring is not a closed circle
but a helix. Most of the time the gyres are too close to be
resolved in the light microscope (?250 nm), but they occasion-
ally separate to ?350 nm apart. We refer to this as a short-
The two types of helices (diffuse and long-pitch helices over
the cytoplasm and short-pitch helices emerging from the Z
ring) point to a common feature of FtsZ assembly in the cell:
its tendency to form very long filamentous structures. If a long
filament is tethered to the membrane, its generic form will be
a helix. The shape of filaments tethered to the membrane and
generating bending forces in various directions was analyzed in
detail by Andrews and Arkin (9).
Assembly Dynamics of FtsZ In Vivo
As seen in Fig. 1A, Z-ring constriction and disassembly takes
place over several minutes, and condensation of the long-pitch
helices into definitive Z rings takes about 30 to 60 s. The
fluctuations between a ring and a short-pitch helix are on a
similar 30- to 60-s time scale (Fig. 1B). On a scale shorter than
?30 s, the Z ring appears to be static. However, FRAP (fluo-
rescence recovery after photobleaching) analysis showed that
the Z ring is much more dynamic. Recovery of a bleached spot
on the Z ring occurs with a half time of 8 to 9 s, which means
that subunits in the Z ring are exchanging with those in the
cytoplasm on this time scale (7). The turnover was very similar
in E. coli and B. subtilis, suggesting that it is a common feature
506 ERICKSON ET AL.MICROBIOL. MOL. BIOL. REV.
in bacteria. Turnover was only modestly altered by null muta-
tions in regulatory proteins MinCD, ZapA, and EzrA (7). A
subsequent study of Mycobacterium smegmatis, which has a
slower cell cycle, gave an average turnover half time of 34 s,
with a broad spread from 10 to 70 s (27).
In an independent study of E. coli, Geissler et al. (55) found
a turnover half time of 11 s for FtsZ, which is very similar to
our value. They also measured the turnover of FtsA. This was
16 s for FtsA-GFP, which was rather toxic when expressed with
the genomic FtsA. The turnover half time was 12 s for their
interesting mutant FtsA*-GFP, which was much less toxic. An
earlier study showed that ZipA, a transmembrane protein with
a cytoplasmic domain that binds FtsZ (121), was turning over
at a rate very close to that of FtsZ (183).
Niu and Yu studied FtsZ dynamics by tracking single mol-
ecules of FtsZ-GFP in E. coli (126). They were able to track for
only 1 to 2 s, but this was sufficient to define two classes of
molecules. FtsZ molecules in the center of the cell, presumably
those in the Z ring, were stationary over the 1 to 2 s. (Had they
been able to track molecules for 20 s, they would presumably
have seen the ?8-s turnover measured by FRAP.) FtsZ mol-
ecules outside the Z ring were highly mobile, with a diffusion
coefficient similar to that of monomeric membrane proteins.
This mobility is much faster than the movement of patches and
long-pitch helices (143, 191), an apparent contradiction that
remains to be resolved. Single-molecule tracking seems to be a
promising technique that should see important future applica-
tions to the Z ring.
Still unresolved is the oligomeric state of the FtsZ outside
the Z ring. For cells with a total concentration of 4 ?M FtsZ,
if 30% is in the Z ring, the cytoplasmic concentration should be
2.8 ?M. If the critical concentration is 1 ?M, one would expect
1.8 ?M FtsZ to be assembled into protofilaments. One might
expect these protofilaments to be tethered to the membrane,
but surprisingly, after the Z ring has formed, the cytoplasmic
FtsZ appears to be diffuse and not localized to the membrane
(Fig. 1). One possible explanation for the lack of membrane
attachment could be that all of the FtsA and ZipA are seques-
tered into the Z ring. Rueda et al. (162) determined for one
strain of E. coli that the total amount of FtsA plus ZipA was
about half the total amount of FtsZ, so it is possible that these
membrane-tethering molecules are mostly sequestered in the
Z ring. Unfortunately, there is presently no measure for what
fraction of FtsA and ZipA are in and outside the Z ring.
Another possible explanation is that the critical concentra-
tion in the cytoplasm may be higher than the 1 ?M measured
in dilute solution in vitro, perhaps due to negative regulatory
proteins. In this case the cytoplasmic FtsZ may not be assem-
bled into protofilaments. Monomeric FtsZ may not bind effi-
ciently to the membrane through the FtsA and ZipA tethers.
SUBSTRUCTURE OF THE Z RING
Structure of the FtsZ Subunit and Protofilaments
Figure 2 shows a cartoon image of an FtsZ protein subunit,
based on the crystal structure of FtsZ from Pseudomonas
aeruginosa (32), which is 67% identical to that of E. coli over
the globular domain. We use the amino acid numbering from
E. coli for this discussion. Figure 2A shows the view that we call
“front,” since it corresponds to the view of a tubulin subunit
from the outside of a microtubule. Figure 2B shows the FtsZ
subunit rotated 90 degrees and viewed from the left side.
The globular domain comprises two subdomains, which can
be expressed separately and are independently folding (133,
139). The N-terminal subdomain, colored dark blue, has the
structure of a Rossman fold and contains all the amino acids of
the GTP-binding site and the entire lower side of the interface
of the longitudinal protofilament bond. The C-terminal do-
main, colored cyan, contains all of the amino acids of the upper
side of the interface, including the “synergy” (T7) loop (dis-
cussed under “GTP Hydrolysis” below). The border between
the two subdomains is not completely clear. Oliva et al. (133)
terminated the N-terminal domain at amino acid 179 of the E.
coli sequence, placing the H7 helix entirely in the C-terminal
domain. Osawa and Erickson (139) suggested terminating the
N-terminal domain at amino acid 195, putting the first half of
helix H7 (which contains amino acids that contact the GTP and
make contact across the protofilament interface) in the N-
The C-terminal globular domain terminates at amino acid
G316. The following ?50 amino acids are highly divergent in
sequence across bacterial species, and this segment is invisible
in crystal structures. It is generally considered to be an unstruc-
tured peptide that can act as a flexible linker. In the absence of
an extension force, the peptide behaves as a worm-like chain
and collapses to an average end-to-end distance of 5 nm (130).
This segment is represented here as an irregular ribbon (ma-
genta) of alpha carbons. The N-terminal 10 amino acids are
also a disordered segment and are similarly represented. When
exerting a force upon the membrane, this 50-amino acid linker
could be extended to a maximum of 17 nm, its contour length.
The final ?17 amino acids are highly conserved across bac-
teria; this peptide binds to FtsA, ZipA, and several other
proteins (discussed below). It is shown in Fig. 2 in darker
purple as the extended beta strand and alpha helix that it
adopts when it binds ZipA (121).
FtsZ subunits assemble into protofilaments by stacking ver-
tically (Fig. 2C). This results in the GTP (shown in orange
space fill on the top) being sandwiched between its binding site
and the subunit above. The subunit above the GTP has three
highly conserved amino acids (N207, D209, and D212) that
play a critical role in GTP hydrolysis (45, 157, 168). Thus, FtsZ
is considered to act as its own GTPase-activating protein
(GAP), and hydrolysis occurs only after subunits come into
contact in the protofilament. The catalytic amino acid D212 is
shown in red space fill on the bottom of the model.
Figure 3 shows a negatively stained electron microscopy
(EM) image of FtsZ protofilaments assembled in vitro. These
protofilaments are one subunit thick and show little or no
tendency to associate laterally (78, 161).
The Z Ring Modeled as Short, Overlapping Protofilaments
FtsZ assembles in vitro into protofilaments that are one
subunit thick and average 120 to 200 nm long (30 to 50 sub-
units) (30, 78, 79, 161) (Fig. 2C and 3). Under some conditions
the protofilaments associate further into paired filaments or
larger bundles, but in dilute physiological buffers (100 to 300
mM potassium acetate [KAc], 5 mM Mg, pH 7.7) single pro-
VOL. 74, 2010 FtsZ IN BACTERIAL CYTOKINESIS507
tofilaments are the predominant form for E. coli FtsZ. It is
generally assumed that these protofilaments are the basic
structural unit and that they are somehow assembled further to
make the Z ring.
To propose a structure of the Z ring, we need to know how
much FtsZ it contains. A number of studies have used quan-
titative Western blotting to determine the number of mole-
cules per cell. These values, and in some cases the number of
FtsA molecules, are collected in Table 1. Most strains seem to
have 5,000 to 7,000 FtsZ molecules per cell, although some
have up to 15,000. An E. coli cell measuring 0.96 ?m in diam-
eter and 3.6 ?m long has a volume of 2.5 ?m3(89, 158). Six
thousand molecules per 2.5 ?m3gives a concentration of 4
?M; 15,000 molecules in the same volume would be 10 ?M.
These numbers are well above the ?1 ?M critical concentra-
tion, suggesting that most FtsZ in the cell is assembled into
We found by quantitative fluorescence imaging that only
30% of the total FtsZ was in the Z ring in both E. coli and
B. subtilis; the remaining 70% was cytoplasmic (7). Geissler
et al. (55) found that 40% of FtsZ was in the Z ring for their
strain of E. coli. For a cell with 6,000 FtsZ molecules, 2,100
FtsZ molecules in the Z ring would be sufficient to make a
total protofilament length of 8,400 nm (at 4 nm per subunit).
FIG. 2. (A) Structure of the FtsZ subunit. The globular domain, shown in cartoon format, comprises two subdomains colored blue (N-terminal)
and cyan (C-terminal). This is from the X-ray structure of P. aeruginosa FtsZ, PDB 1OFU (32). The GDP is shown in orange space fill, and the
synergy loop amino acid D212 (E. coli numbering) is in red. This view corresponds to that of a tubulin subunit seen from the outside of a
microtubule, and is designated the “front view.” A 10-amino-acid segment on the N terminus and a 50-amino-acid segment on the C terminus are
shown in magenta, each modeled as flexible peptides. Shown in dark purple are the extended beta strand and alpha helix formed by the C-terminal
17-amino-acid peptide when bound to ZipA (from PDB 1F47 ). The model was constructed using the program PyMol (39). (B) The FtsZ
subunit viewed from the side. This shows that the C-terminal peptide emerges from the front face and the N-terminal peptide from the back face,
?180 degrees away. (C) A protofilament is assembled by stacking subunits on top of each other so that the D212 of the upper subunit is just above
the GDP of the one below.
508 ERICKSON ET AL.MICROBIOL. MOL. BIOL. REV.
This would encircle a 1-?m-diameter cell two and a half
Figure 4A shows a model for how protofilaments might be
further assembled to make the Z ring. Since the protofilaments
are much shorter than the circumference of the bacterium,
they must be assembled with a staggered overlap. Support for
this kind of model came from a study of Caulobacter by
cryo-EM tomography (98). These images showed short fila-
ments scattered around the circumference of the cell a short
distance from the membrane. Control experiments, in which
FtsZ was depleted or overexpressed, provided evidence that
the filaments were in fact FtsZ.
An alternative model, discussed below, proposes that the
short protofilaments might anneal into one or a few longer
protofilaments (Fig. 4B).
Tethering FtsZ to the Membrane
One question raised by either model is how the protofila-
ments are attached to the membrane. This question has been
resolved by Pichoff and Lutkenhaus. They first showed that
FtsZ could assemble a Z ring if the cell had either FtsA or
ZipA, but not in the absence of both (147). ZipA is a trans-
membrane protein whose cytoplasmic domain is known to bind
the C-terminal peptide of FtsZ. Nuclear magnetic resonance
(NMR) and X-ray crystal structures show this peptide forming
a helix and a beta strand along a hydrophobic groove of the
cytoplasmic globular domain of ZipA (121, 122). This binding
therefore provides a tether of FtsZ to the membrane. The
same C-terminal peptide of FtsZ is known to bind FtsA (41,
109), and the binding site has been localized to a patch on
subdomain 2B (145). FtsA has long been considered a mem-
brane-associated protein, but the detailed mechanism for its
membrane binding was only recently discovered. Pichoff and
Lutkenhaus (146) showed that the C-terminal peptide of FtsA
forms an amphipathic helix that inserts into the lipid bilayer
and anchors FtsA to the membrane. Thus, FtsZ is also teth-
ered to the membrane by FtsA. Since ZipA is found only in
gammaproteobacteria and a gain-of-function point mutation in
FtsA can render ZipA nonessential in E. coli (14), we will
consider FtsA the primary tether to the membrane.
FIG. 3. Electron micrograph of negatively stained protofilaments assembled in vitro from E. coli FtsZ (1 ?M FtsZ, 50 mM MES [morpho-
lineethanesulfonic acid] [pH 6.5], 100 mM KAc, 5 mM MgAc, 1 mM GTP). The bar is 100 nm. This specimen was prepared on a carbon film treated
with UV light and ozone to render it hydrophilic (24). With these carbon films we obtain protofilaments at lower FtsZ concentrations, and they
are longer than those previously reported. The protofilaments here are mostly straight, but some show a tendency to curve.
TABLE 1. Quantitation of FtsZ and FtsA in cells
No. of molecules/cell
aThe FtsZ number has been increased by 25% from the reported value to
account for the lower color of FtsZ relative to bovine serum albumin (BSA) in
bicinchoninic acid (BCA) and Bradford assays (106, 119, 203).
VOL. 74, 2010 FtsZ IN BACTERIAL CYTOKINESIS509
The C-terminal peptide that binds FtsA and ZipA is highly
conserved across bacteria, including some that have neither
FtsA nor ZipA. In B. subtilis, which has no ZipA, FtsA can be
eliminated and the cells are still viable, although with defects in
division variably described as severe or moderate (10, 85).
What tethers FtsZ to the membrane in the absence of FtsA
In Mycobacterium tuberculosis FtsW binds this peptide, but
this probably does not occur in other species, because the
binding site on FtsW is a C-terminal extension that is unique to
mycobacteria (38). Also, even in mycobacteria, FtsW is appar-
ently a late recruit to the Z ring (54, 155), so something else
must provide the primary membrane tether. Several other pro-
teins that regulate the Z ring (reviewed in reference 2) bind to
this C-terminal peptide of FtsZ: EzrA (175), SepF (176), ClpX
(25, 185), and the C-terminal domain of MinC (170). EzrA is
probably the best candidate for a tether, because it is a trans-
membrane protein with a topology like that of ZipA (96). Also
EzrA localizes to the Z ring early, along with FtsA and ZapA
(54). On the other hand EzrA is best characterized as a neg-
ative regulator of Z rings (66, 96), so its role as a tether is not
The C-terminal peptide is conserved in bacteria such as
Mycoplasma, which have no candidate protein for binding it
(194). We considered the possibility that these peptides might
bind the membrane directly, but several sequences that we
examined showed no features of an amphipathic helix. It is
attractive to think that this C-terminal peptide provides the
membrane tether in all species, but some additional binding
partners or mechanisms are waiting to be discovered.
Lateral Bonds: Do They Exist?
The Z ring appears by light microscopy as a very long fila-
ment of mostly uniform density. The model in Fig. 4A, where
the Z ring is made from overlapping short protofilaments,
raises the question of how the protofilaments are associated
with each other. One possibility is lateral bonds, which would
involve specific contacts between subunits in adjacent proto-
FIG. 4. (A) A model for how short protofilaments might be arranged to make the Z ring. The average 125-nm length is much shorter than the
3,000-nm circumference, so protofilaments would be arranged in a staggered overlap. (B) An alternative structure where the short protofilaments
are proposed to anneal into one or a few long protofilaments.
510ERICKSON ET AL.MICROBIOL. MOL. BIOL. REV.
filaments. This is the way that protofilaments are assembled to
make the microtubule wall.
If FtsZ formed lateral bonds like tubulin, protofilaments
should assemble into a two-dimensional (2-D) array with semi-
crystalline regularity. Sheets of protofilaments have been seen
under certain conditions for FtsZ from Methanococcus jann-
aschii (103, 104, 134) and from Thermotoga maritima (106).
FtsZ from M. tuberculosis forms long, two-stranded filaments,
in which the two strands must be connected by some type of
regular lateral bond (27, 199). FtsZ from most species, how-
ever, assembles one-stranded protofilaments that show no con-
sistent lateral association in physiological buffer.
In 10 mM Mg, protofilaments of E. coli FtsZ associate into
long, thin bundles that are several protofilaments thick (29, 35,
124). One study reported similar bundles in a more physiolog-
ical buffer (5 mM Mg) (116); however, other labs have re-
ported only one-stranded protofilaments under these condi-
tions. Ca at 10 mM produces bundles that are somewhat
thicker and quite long (202). When examined in cross section,
the Ca-induced bundles showed irregular profiles of protofila-
ments but no regular protofilament lattice (105). Ruthenium
red generated bundles similar to those in Ca (164).
The bacterial cytoplasm is a crowded environment due to
the high concentration of proteins and nucleic acids. Agents
such as Ficoll, polyvinyl alcohol, and methylcellulose, which
are thought to mimic the physical chemistry of crowding, have
a dramatic effect on FtsZ assembly, producing very large bun-
dles, ?100 nm in diameter and many that are micrometers long
(151). In the original study of FtsZ assembly in Ficoll, the
polymers were described as ribbons that were one subunit thick
(60). However, we have found by embedding and sectioning
that the bundles formed in a variety of crowding agents are
round (D.E.A. and H.P.E., unpublished observations). This is
also the interpretation from negatively stained specimens
(151). Under some conditions the bundles were straight, and
under others they curved into toroids or spirals. Importantly,
diffraction patterns of these bundles showed very poor order. A
diffuse equatorial spot indicated an average spacing of proto-
filaments of 6.8 nm, which is substantially larger than the width
of an FtsZ protofilament (maximum width of 4.5 to 5 nm). The
diffuseness of this spot and the lack of any second-order re-
flection suggested that the protofilaments are not spaced on a
lattice, as they would be if held by regular lateral contacts
between subunits, but are packed together with a liquid-crys-
Long filamentous bundles and toroids of FtsZ were also
formed when FtsZ-GFP was expressed in yeast cytoplasm,
which is a natural crowded environment (181). FRAP showed
that both the bundles and toroids were turning over subunits
with a half time of 11 s, similar to the 8-s turnover of the Z
ring in bacteria (7) and the 3.5 to 7 s for turnover of protofila-
ments in vitro (29). This is additional evidence that the proto-
filaments in the bundles are not associated by specific lateral
contacts, since lateral bonds would be expected to slow down
exchange. Protofilaments in the centers of the bundles appar-
ently are also turning over, which means that they have access
to subunits in solution.
Assembly in 1 M sodium glutamate produced very large
bundles that resembled the ones made under crowding condi-
tions (17). The GTPase was reduced only by about half in 1 M
glutamate, suggesting that FtsZ, even in the interiors of the
bundles, is rapidly exchanging with solution. This suggests a
very loose structure similar to that of the bundles generated in
the yeast cytoplasm. Bundles induced by Ca and Mg have
substantially lower GTPase (29 202; our unpublished observa-
tions), suggesting that they may involve lateral contacts that
inhibit subunit exchange.
Indirect evidence for bundling came from study of a tem-
perature-sensitive B. subtilis FtsZ mutant (120). At the non-
permissive temperature, the mutant formed long-pitch helices
in the cytoplasm but seemed to be incapable of collapsing them
to make a Z ring. That study found that wild-type FtsZ assem-
bled into toroid bundles in vitro at both 35 and 22°C. The
mutant FtsZ formed toroids at 22°C, but at 35°C it formed thin,
one-stranded protofilaments, suggesting that it was defective in
bundle formation at the higher temperature. Another line of
evidence for the bundling potential of FtsZ was its ability to
form elastic gels in vitro (35, 51). These were formed in buffers
with a low salt concentration, pH 6.5, and 10 mM Mg, so the
gel formation may be less under the physiological conditions of
Several division proteins interact with FtsZ and cause it to
form bundles or sheets. One of the first discovered was ZipA,
which bundles most efficiently at a pH of ?6 (67, 156). ZapA,
a protein that enhances Z-ring stability, generates a variety of
bundled forms (65, 101, 119, 178). ZapB, a small coiled-coil
protein that stimulates Z-ring assembly, induces FtsZ bundles
with a striking ?10-nm banding pattern. FtsA*, a gain-of-
function mutant of FtsA, caused FtsZ to assemble sheets of
protofilaments that appeared to adopt the ?200-nm-diameter
intermediate curvature (see below) (18). SlmA produced
curled sheets of FtsZ with sharp edges and a seeming regular
structure (15). It is surprising that SlmA induced polymers of
FtsZ, because SlmA was identified as a nucleoid occlusion
factor and was thought to inhibit Z-ring formation over the
nucleoid. The bonds between FtsZ and the bundling proteins
may involve electrostatic interactions (15) but are mostly un-
Large round bundles of protofilaments are probably not
relevant inside the bacterium. With only 5,000 to 7,000 mole-
cules of FtsZ in the typical bacterial cell (Table 1), there is only
enough FtsZ to make ?7 protofilaments 3 ?m long. In addi-
tion, because FtsZ protofilaments in the Z ring are tethered to
the membrane, they should be limited to a 2-D ribbon or sheet.
Expression of an FtsZ-GFP fusion with the C terminus of FtsZ
deleted did lead to formation of rods in the central cytoplasm
(108). When the C terminus is present, however, tethering to
the membrane and subsequent Z-ring assembly apparently
trump the tendency to form cytoplasmic bundles. Also, the
effects of crowding on FtsZ (or other) assembly may have been
overestimated. McGuffee and Elcock (112) have recently
shown that the steric effects of crowding, which are the main
ones considered in previous analyses, can be largely canceled
by the favorable ionic and hydrophobic interactions of (FtsZ)
subunits with the multiple other proteins in the cytoplasm.
That study suggests that polymerization of FtsZ in the bacterial
cytoplasm might be more similar to polymerization in dilute
buffer than to that in Ficoll and polyvinyl alcohol. Expression
of FtsZ in yeast did produce linear bundles and toroids, similar
VOL. 74, 2010 FtsZ IN BACTERIAL CYTOKINESIS511
to the structures in polyvinyl alcohol, but this might have in-
volved a high level of expression.
Hamon et al. (68) have obtained superb images of FtsZ
protofilaments by atomic force microscopy (AFM). They
found that adsorption to mica enhances the effective concen-
tration and can support protofilament assembly from solution
concentrations well below the critical concentration. Remark-
ably, that study found that tubulin also formed short, curved
single protofilaments when adsorbed onto mica from dilute
solution. In solution, tubulin protofilaments are unstable and
exist only when stabilized by lateral bonds to neighboring pro-
tofilaments (49). Adsorption to the 2-D mica surface appears
to provide the stabilization needed to observe these small in-
termediates. This technology has great promise for observing
polymer assembly reactions, including intermediates that are
too weak to be observed in bulk solution.
At higher FtsZ concentrations, protofilaments become very
crowded on mica and tend to associate in parallel sheets and
spiral structures (71–73, 141). The packing appears in places to
be just the result of the density of protofilaments, but the
authors have presented mathematical models in which lateral
bonds play an important role in determining the packing. The
lateral interactions between adjacent protofilaments are not
regular lateral bonds like those in the microtubule wall, be-
cause the protofilaments show irregular packing with variable
spaces. Instead the authors proposed a Lennard-Jones type
interaction, which is maximal when the protofilaments are
about 13 nm apart and becomes repulsive at closer distances
(discussed also in reference 48). If the Lennard-Jones attrac-
tion was independent of rotation around the protofilament
axis, it might be related to the round bundles formed under
crowding conditions. Still lacking, however, is a chemical basis
for the proposed lateral attractions that could explain the ap-
parent two-dimensional packing on mica and three-dimen-
sional packing in round bundles in solution.
In summary, lateral bonds between FtsZ protofilaments
probably do not exist as the kind of repetitive protein-protein
contacts that produce the microtubule wall. The association of
protofilaments into bundles seems to involve irregular packing
into a liquid-crystalline array. The protofilaments are not in
physical contact with each other but may interact through
electrostatic forces involving the ions between the protofila-
ments, as proposed for packing of actin bundles (150, 152,
190), or through other solvent effects.
Alternatives to Lateral Bonds
How might the long FtsZ filament, either the ring or the
extended helices, be constructed apart from lateral bonds?
Shlomovitz and Gov (173) have proposed a mechanism based
on forces transmitted across the membrane. In their model,
bending Z rings exert a constriction force that distorts the
membrane, and these membrane distortions interact when Z
rings approach each other. Their model predicts that adjacent
Z rings will undergo an initial attraction that would cause them
to slide together. The attraction would bring them to a certain
distance that is semistable, but eventually they would move
together and coalesce. This prediction fits the observed move-
ment of Z rings in tubular liposomes (137). This attraction was
mediated solely by the distortions of the membrane generated
by the two adjacent rings. The model assumed that the Z ring
was a uniform circle generating an inward constriction force. It
will be interesting to see if a future model can be applied to
scattered, short protofilaments. In particular, could short pro-
tofilaments with a preferential bend communicate through
membrane distortions to generate the uniform distribution of
filaments around the Z ring? An attractive feature of this
model is its agreement with the cryo-EM tomography (98),
which showed the FtsZ as short filaments not making contact
with each other.
Another possibility is that the protofilaments do not remain
as the short, ?30-subunit structures that assemble in solution
but that they anneal into much longer filaments (Fig. 4B).
Annealing has been visualized directly by AFM imaging of
FtsZ protofilaments adsorbed to mica (117). The protofila-
ments were able to diffuse on the 2-D mica surface, and ex-
amples of fragmentation and annealing were shown. In a re-
cent study, Chen and Erickson (29) reported indirect evidence
for annealing of protofilaments in bulk solution.
In the cell the protofilaments are tethered to the mem-
brane and are probably restricted to the center of the cell by
the Min and nucleoid occlusion systems. Surovtsev et al.
(187) proposed that FtsZ tethered to the membrane might
be confined to a distance 8 nm from the surface (a reason-
able estimate of the flexibility provided by the 50-amino-acid
tail of FtsZ [Fig. 2]) and to an axial zone 100 nm wide (a
reasonable estimate for the width of the Z ring). The volume
of this shell would be only 1/1,000 of the total cytoplasmic
volume. If the total cytoplasmic FtsZ is ?4 ?M, and 30% of
it is in the Z ring, the effective concentration of FtsZ in this
restricted volume would be 1,200 ?M. More importantly,
the concentration of filament ends would be 40 ?M, assum-
ing protofilaments 30 subunits long. Annealing should
therefore be much more favorable than adding single sub-
units from the 1 ?M cytoplasmic pool. This simple numer-
ical argument suggests that annealing could play a major
role in determining the structure of the Z ring. Of course,
the high concentration of protofilaments would also en-
hance any lateral interactions. A diagram of the Z ring as a
single long filament is shown in Fig. 4B.
One problem with an annealing model is to reconcile the
very long protofilaments with the rapid turnover of subunits
in the Z ring. In the 8-s half time for turnover (discussed
below), about 45 subunits could be added to a protofila-
ment, given the measured kinetics of assembly (7). This
turnover could be explained for a Z ring made from short
protofilaments averaging 30 to 50 subunits (Fig. 4A). How-
ever, if the Z ring was one long, continuous protofilament,
this exchange would have to occur from multiple interior
sites, which would involve frequent breakage and dissocia-
tion of subunits, followed by reassembly and reannealing.
The long protofilament produced by annealing would then
seem to be constantly breaking and reannealing. This fre-
quent breakage should also occur for a filament of short
protofilaments connected by lateral bonds. The rapid sub-
unit exchange in the Z ring is thus difficult to reconcile with
the evidence that the basic architecture of the Z ring is a
long, continuous filament, whether produced by annealing
or by lateral bonds.
The observation that the Z ring can separate into a short-
512 ERICKSON ET AL.MICROBIOL. MOL. BIOL. REV.
pitch, two-turn helix (Fig. 1B) is quite consistent with its struc-
ture being a long, continuous filament. If the Z ring consisted
of scattered short protofilaments, as depicted in Fig. 4A, lateral
drift should produce only a diffuse Z ring, not separate gyres of
ASSEMBLY OF FtsZ IN VITRO
Buffer Conditions for In Vitro Studies
Early in vitro studies of FtsZ assembly were done at pH
6.5, which appeared to give more robust assembly than a
higher pH (50, 124). However, the pH of E. coli cytoplasm
varies from 7.4 to 7.8 depending on the external pH (177).
Recent measurements showed a cytoplasmic pH of 7.7 for
an external pH of 7.5 (200). Potassium is the major cyto-
plasmic cation, and it varies from 0.14 to 0.76 M depending
on external osmolarity (26). The major anions in the cyto-
plasm are nucleic acids. The major monomeric anion is
glutamate, but its concentration is only about one-fifth that
of potassium (26). We generally use potassium acetate as
the salt, but we have found no effect on FtsZ assembly or
GTPase activity when the anion was changed to chloride.
(While the nature of the anion is probably not important,
that of the cation is. Substituting sodium for potassium
increased the critical concentration from 1 to 20 ?M .)
A KAc concentration of 350 mM is probably the best ap-
proximation to physiological salt, but for some assays, such
as negative-stain EM, 100 mM KAc gives better results.
There was only a small difference in the GTPase of E. coli
FtsZ between 100 and 350 mM KAc (29). The use of phys-
iological buffer for in vitro studies has been addressed pre-
viously (60, 106).
Magnesium is an important variable affecting FtsZ assembly
and dynamics. Various in vitro studies have used Mg concen-
trations of from 2.5 to 10 mM. Earlier work showed that at 2.5
to 5 mM Mg, FtsZ assembles into protofilaments that are one
subunit thick (78, 161). These studies were done at pH 6.5, but
we have repeatedly observed single protofilaments at pH 7.7
with these low Mg concentrations. With 10 mM Mg the pro-
tofilaments associate into long, thin bundles, several protofila-
ments thick, and the rates of GTP hydrolysis and subunit ex-
change are reduced 3-fold (29, 35). Mohammadi et al. (119)
recently undertook a comprehensive study of how pH and Mg
affect the interaction of FtsZ and ZapA. They found striking
differences between 5 and 10 mM Mg, as well as between pH
6.5 and 7.5.
The total cellular Mg has been measured to be 20 to 80 mM,
but it has long been known that most of this is bound by RNA
and other polyanions. How much Mg is unbound in the cyto-
plasm? The question was resolved recently using the fluores-
cent dye mag-fura to measure the concentration of free cyto-
plasmic Mg: it is 0.9 mM (53). This suggests that FtsZ assembly
in vitro should be studied at ?1 mM free Mg. In selecting an
Mg concentration for in vitro assembly experiments, it should
be kept in mind that GTP will chelate an equivalent amount of
Mg. If the reaction mixture contains 1 mM GTP, the buffer
should contain 2 to 2.5 mM Mg. We should also note that we
have not found any difference in assembly between 2.5 and 5
G proteins bind GTP in their active state, and when GTP is
hydrolyzed to GDP, they switch to an inactive state. The level
of intrinsic GTPase activity of G proteins is very low, and
hydrolysis is generated by a separate GTPase-activating pro-
tein (GAP), which binds to the G protein at the GTP-binding
pocket. The GAP provides catalytic side chains that contact the
GTP and initiate hydrolysis.
FtsZ, like tubulin, is its own GAP. Monomeric FtsZ hy-
drolyzes GTP very slowly, if at all. When assembled into a
protofilament, the bottom interface of one FtsZ makes con-
tact with the GTP pocket of the subunit below (Fig. 2). A
key player is the synergy or T7 loop, which contains the
sequence 207NxDFAD212 (E. coli sequence numbers). This
sequence is highly conserved in all FtsZ proteins and is
NxDxxE in all ?-tubulins. Mutation of the conserved amino
acids severely cripples the GTPase activity, leading to the pro-
posal that the synergy loop acts as a GAP to activate hydrolysis
of the GTP in the subunit below (45, 127, 157, 168). Details of
the mechanism have been imaged in a crystal structure (133).
The two aspartate residues are in good hydrogen-bonding po-
sitions to polarize a water molecule that attacks the gamma
phosphate of the GTP. The role of cations and pH has been
further explored by molecular dynamics (113).
FtsZ polymers contain a substantial amount of GTP,
which suggests that hydrolysis occurs with some lag follow-
ing assembly. Romberg and Mitchison measured the nucle-
otide content of protofilaments at steady state and found the
GDP/GTP ratio to be 20:80 (160). This is quite different
from the case for microtubules, where the nucleotide is
almost entirely GDP, with only a small cap of GTP at each
end. Chen and Erickson (29) repeated this assay for FtsZ
and found that the fraction of GDP increased as the external
concentration of GTP was increased from 20 to 120 ?M.
Above 100 ?M GTP, the GDP/GTP ratio plateaued at 50:
50. Total assembly and the rate of GTP hydrolysis at steady
state also increased at the higher external GTP concentra-
tions. The half maximum for these reactions (increasing
GDP in polymer, total assembly, and GTPase) occurred at
?50 ?M GTP. This is far above the binding affinity for GTP
and GDP nucleotides, which Huecas et al. (79) have found
to be in the 10 to 100 nM range (that reference should be
consulted for a comprehensive study of the thermodynamics
and kinetics of nucleotide binding to M. jannaschii apo-
FtsZ). In an earlier study, treating the GTPase reaction as a
classical Michaelis-Menten reaction, Sossong et al. (180)
determined a Kmof 82 ?M. From these studies it seems
clear that some aspect of FtsZ assembly is modulated by
GTP concentrations in the range of ?50 to 100 ?M. It is not
clear what mechanism is involved.
Earlier studies suggested that unlike microtubules, FtsZ pro-
tofilaments could exchange their nucleotide for GTP in solu-
tion (60, 116, 133, 189). Oliva et al. (133) supported this inter-
pretation with a crystal of M. jannaschii FtsZ that showed
dimers that were similar in structure to the ??-tubulin dimer.
These dimers showed a gap from which the GTP could exit,
and indeed the nucleotide pocket of the dimers could be filled
by soaking the crystals in GMPCPP.
Experimental evidence clarifying the exchange of nucleotide
VOL. 74, 2010 FtsZ IN BACTERIAL CYTOKINESIS513
into protofilaments was provided by Huecas et al. (79). They
showed that apo-FtsZ of M. jannaschii could assemble into
protofilaments and that GDP or mant-GTP could associate
into these polymers. However, when FtsZ was assembled with
nucleotide, the bound nucleotide was very slow to dissociate.
They concluded that the slow dissociation of bound nucleotide
presented a kinetic block to the exchange of nucleotide into
polymer. The data of Chen and Erickson agreed with this and
suggested that previous indications for nucleotide exchange
into polymer were probably due to the pool of subunits that
exchange without GTP hydrolysis (29). These studies now
agree that nucleotide exchange occurs only when the GTP-
binding pocket is exposed to the solvent, at the plus end of a
protofilament or on free monomers.
It is known that FtsZ-GDP can assemble but that it does so
much more weakly than FtsZ-GTP (79, 159, 180). However, it
is not known what happens following GTP hydrolysis in a
protofilament. One possibility is that the protofilament frag-
ments at the site of the GDP. This fragmentation would have
to follow a lag comparable to the lag for hydrolysis in order to
account for the 50:50 GDP/GTP ratio. Another possibility is
that fragmentation is minimal, and subunits dissociate primar-
ily from the ends of protofilaments. In this model the end
subunit would dissociate much faster if it was bound by a GDP
Finally, we note that GTP hydrolysis is not required for
assembly. Assembly occurs with GTP plus EDTA, which che-
lates Mg, but hydrolysis is completely blocked (28, 124). As-
sembly is well supported by the GTP analog GMPCPP, which
is hydrolyzed 50 times slower than GTP (179). When alumi-
num fluoride is added to FtsZ-GDP, the AlF binds to the GDP
and mimics the gamma phosphate, producing a nonhydrolyz-
able GTP analog. FtsZ assembled efficiently but with slower
kinetics in GDP-AlF (117). Two analogs that do not work are
GMPPNP and GTP?S (169). GMPPNP bound FtsZ very
weakly and could not compete GTP. GTP?S bound tightly, but
it could not support polymerization unless mixed with GTP.
GTP binding is not even required for assembly. Huecas and
Andreu showed that apo-FtsZ from M. jannaschii assembled as
well as FtsZ-GTP, with a similar critical concentration and
polymer morphology (77). GDP strongly destabilized the as-
sembly. The role of GTP therefore appears to be to provide a
means for destabilizing the polymer following hydrolysis, lead-
ing to a constant recycling of FtsZ subunits.
Assembly Dynamics of FtsZ In Vitro
Several years ago we discovered a tryptophan mutant of
FtsZ that exhibited a 2.5-fold increase in fluorescence emission
upon assembly (28). This provided an important tool to assay
assembly, since the fluorescence was obtained in real time and
was directly proportional to the number of subunits forming
interfaces in protofilaments. This assay was used to measure
the kinetics of assembly initiated by adding GTP. The data
were interpreted with a model of cooperative assembly with a
weak dimer nucleus. The structural nature of this dimer is still
An alternative fluorescence assay was developed based on
fluorescence resonance energy transfer (FRET) (30). This
assay provided a way to measure the exchange of subunits
between protofilaments at steady state. Protofilaments were
preassembled from donor- and acceptor-labeled FtsZ and
then mixed. Initially there was no FRET, since the donor
and acceptor labels were on separate protofilaments. A
FRET signal developed as the protofilaments disassembled
and the subunits reassembled into mixed protofilaments.
The half time for subunit exchange was 7 s in the original
study at pH 6.5 and 100 mM KAc (30). At pH 7.5 the half
time for exchange was 3.5 s with 100 mM KAc and 17 s with
350 mM KAc (29).
This FRET assay was used to measure the protofilament turn-
over in a variety of different buffers and to compare the turnover
with the time required for hydrolysis of GTP (29). The half time
for subunit exchange varied from 3.5 to 35 s for the different
buffers. The time required for a subunit to hydrolyze a GTP
varied from 13 to 100 s. Over this range, the time for nearly full
subunit exchange (two times the half time) was generally twice
as fast as the time to hydrolyze a GTP. This suggested that
there are two mechanisms for subunit exchange from proto-
filaments. One is coupled to GTP hydrolysis and likely involves
exchange of one FtsZ for every hydrolysis event. The second
mechanism involves exchange of FtsZ-GTP subunits at the
ends of protofilaments, without GTP hydrolysis. These two
mechanisms contribute roughly equally to the overall ex-
Conditions resulting in slower exchange times and slower
GTP hydrolysis correlated with association of protofilaments
into bundles. (These bundles were similar to the Ca/Mg bun-
dles, rather than the large bundles of loose-packed protofila-
ments assembled in crowding agents, which maintain a rapid
exchange.) In the simplest model, once a subunit has hydro-
lyzed its GTP, it must dissociate to exchange GDP for GTP
before it can repolymerize and hydrolyze another. Although it
is possible that reagents such as calcium act to inhibit GTPase,
and this leads to bundling, we believe the opposite: that bun-
dling is the primary action and reduced GTPase is the second-
Two classes of dynamics are known for the eukaryotic cy-
toskeleton. Actin filaments undergo treadmilling at steady
state, where the filaments grow at one end and shorten at the
other. Microtubules undergo dynamic instability, where each
end switches between extended phases of growth and shorten-
ing. (Dynamic instability can be accompanied by treadmilling.)
Analysis of FtsZ cap mutants, which block either the top or
bottom assembly interface, gave evidence for treadmilling of
FtsZ, with subunits adding preferentially to the bottom end
(away from the GTP) (157).
Dynamic instability remains a possibility for FtsZ. Scheffers
et al. investigated the nonhydrolyzable analog GTP?S and
discovered an important clue to the mechanism of dynamics
(169). GTP?S could bind to FtsZ with an affinity similar to that
of GTP but could not support assembly on its own. When FtsZ
assembly was initiated with a limiting amount of GTP (20 ?M)
and no GTP?S, turbidity reached a peak and then fell as the
GTP was hydrolyzed (this assembly was in a pH 6.5 buffer with
10 mM Ca, which produces bundles and strong turbidity).
However, if 50 to 200 ?M GTP?S was included in the reaction,
the assembly was stabilized at the peak. This result is remark-
ably similar to the ability of nonhydrolyzable analogs to stabi-
lize microtubules assembled in limited GTP (129). It is likely
514 ERICKSON ET AL.MICROBIOL. MOL. BIOL. REV.
that the stabilization involves GTP?S coassembled with GTP
throughout the protofilaments, but the ratio and distribution
are not known. Further exploration may shed light on the
mechanisms of dynamics.
Inhibition of FtsZ Assembly by SulA
A number of proteins, called negative regulators, that ap-
pear to inhibit Z-ring assembly have been identified. The best
studied are SulA and MinC. The mechanisms by which these
proteins inhibit FtsZ are beginning to shed light on how FtsZ
assembles into protofilaments and the Z ring.
SulA is expressed during the SOS response to DNA damage.
It blocks cell division until the DNA is repaired, and SulA then
is degraded. Overexpression of SulA in E. coli was shown to
block Z-ring assembly (21, 37, 123). Two studies in 1998
showed that SulA inhibited both the GTPase activity and as-
sembly of FtsZ (123, 192). A recent study by Dajkovic et al.
(37) has made several important advances. An assay for the
critical concentration provided good evidence for a simple
mechanism. In the absence of SulA the critical concentration
was 0.9 ?M, and this increased to 4.3 and 5.9 ?M, respectively,
when 3.5 and 4.0 ?M SulA were present. The reaction behaved
as if the concentration of active FtsZ was equal to the total
FtsZ minus the concentration of SulA. This suggests that SulA
acts by binding free subunits of FtsZ and blocking their assem-
bly. A plot of GTPase versus FtsZ concentration was similarly
shifted, as if an amount of FtsZ equivalent to that of SulA was
sequestered from the reaction. An important extension of this
interpretation is that the affinity of SulA for FtsZ must be
higher than that of FtsZ for binding the end of a protofilament.
This has not yet been demonstrated directly.
This simple sequestration mechanism was predicted earlier
from the crystal structure of a SulA-FtsZ (32). SulA bound to
the bottom of FtsZ, near the T7/synergy loop. Bound SulA
would sterically block the formation of the protofilament in-
terface, and all FtsZ subunits with a bound SulA would be
essentially dead for the assembly reaction. Dajkovic et al. (37)
also found that Z rings disappeared in vivo at a SulA concen-
tration of around half that of FtsZ. The substoichiometric
activity probably reflects the critical concentration (one needs
to sequester only the FtsZ above the critical concentration to
block FtsZ assembly) and the fact that only 30 to 40% of the
cell’s FtsZ is in the Z ring.
The sequestration mechanism of SulA action would appear
to resolve this issue, were it not for a truly remarkable discov-
ery. Dajkovic et al. (37) found that FtsZ was no longer sensitive
to SulA when assembly was induced by GMPCPP, GTP plus
EDTA, or GDP plus AlF. Each of these nonhydrolyzable an-
alogs eliminates the cycling of subunits coupled to GTP hydro-
lysis. There is still an equilibrium exchange of subunits from
the ends of protofilaments (29), but it seems that the more
rapid cycling of subunits coupled to GTP hydrolysis is needed
for SulA to inhibit assembly. This is not consistent with a
simple sequestration mechanism.
Dajkovic et al. (37) suggested an explanation based on high-
and low-affinity conformations for FtsZ, which has been pro-
posed previously by three groups to explain cooperative assem-
bly (35, 79, 90, 118; see also reference 48). In the Dajkovic
model SulA was proposed to bind only the high-affinity con-
formation. A second requirement was that the off rate of FtsZ
from stabilized protofilaments (assembled in GMPCPP, GDP-
AlF, or EDTA) must be 1,000-fold lower than the off rate from
SulA and much lower than the off rate from Mg-GTP proto-
filaments. There is, however, contradictory evidence for both
points. First, there is no candidate for a high-affinity confor-
mation among the many crystal structures of FtsZ; they all
have the same conformation (135). Most important, in the
crystal structure of FtsZ bound to SulA (32), the conformation
of FtsZ was the same as in the other crystals. This conforma-
tion would presumably be the abundant, low-affinity confor-
mation. Second, the half time for dilution-induced disassembly
was 7 s with EDTA and 1.4 s with Mg (29). The off rate from
the EDTA protofilaments is at most 5 times lower than that
from Mg protofilaments. Although this particular explanation
appears to have problems, the mechanism of SulA inhibition
provides an important direction for new research. This phe-
nomenon may be related to the mechanism giving the appear-
ance of cooperative assembly (see below), and its study may
help unravel this enigma.
Inhibition of FtsZ Assembly by MinC
The MinCDE system helps localize the Z ring to the cell
center by inhibiting its assembly at the poles, where MinCD
has its highest average concentration (107). If the Min system
is deleted, division near the poles produces minicells. Although
most cells still have central septa, their placement was less
precise in cells lacking MinC (64). MinD and MinE operate to
localize MinC. MinC is the protein that interacts with FtsZ to
inhibit Z-ring assembly. Assembly of Z rings is blocked by
overexpression of MinCD in E. coli (75, 144).
In vitro analysis showed that MinC inhibited FtsZ polymer-
ization, as measured by a centrifugation assay, at approxi-
mately a 1:1 stoichiometry (75). Remarkably, however, the
GTPase was not inhibited even by a 2.5-fold excess of MinC.
This suggests that MinC does not inhibit the assembly of pro-
tofilaments but blocks their association into bundles. This is
also suggested by the specifics of the centrifugation assay used
to monitor polymers. The assembly was done in a pH 6.5 buffer
with 10 mM Mg, conditions that enhance the formation of
long, thin protofilament bundles. These bundles are pelleted
much more readily than short, one-stranded protofilaments.
This suggests that MinC inhibits Z-ring assembly not by affect-
ing the assembly of protofilaments but by inhibiting their as-
sociation into bundles.
Dajkovic et al. (35) supported this interpretation by use of
two different assays. They used a FRET assay (30) to show that
total polymer was not inhibited by MinC. They then explored
an assay measuring the elasticity of gels formed by FtsZ at high
concentrations (25 ?M). The elasticity is a measure of associ-
ation of protofilaments to form a three-dimensional gel. Ad-
dition of MinC to a preformed FtsZ gel, at a 1:2 or 1:1 MinC/
FtsZ ratio, resulted in loss of elasticity over 10 to 20 min. At
2:1, the elasticity was lost immediately. Most in vitro studies of
MinC have used the E. coli system, but Scheffers has recently
studied the interaction of FtsZ and MinC from B. subtilis (167).
He used a centrifugation assay that included DEAE-dextran,
which was needed to obtain FtsZ polymers large enough to
pellet. MinC inhibition was found to be pH sensitive. It was
VOL. 74, 2010 FtsZ IN BACTERIAL CYTOKINESIS 515
minimal at pH 6.5, but at pH 7.5 MinC inhibited assembly 80%
(although this required a 50% excess of MinC over FtsZ). As
in the E. coli system, MinC had no effect on GTP hydrolysis,
suggesting that it did not inhibit assembly of protofilaments but
blocked their association into DEAE-dextran bundles.
An intriguing observation in the study by Dajkovic et al. (35)
was that MinC was completely inactive against FtsZ polymers
assembled in GMPCPP or in GDP-AlF. This is remarkably
similar to the case with SulA, which inhibited assembly only
when GTP hydrolysis contributed to cycling of FtsZ subunits.
This is surprising, however, because SulA and MinC are sup-
posed to be operating by very different mechanisms (SulA
inhibits protofilament assembly, and MinC inhibits protofila-
ment bundling). Since MinC does not inhibit either the assem-
bly of protofilaments or GTP hydrolysis, it is difficult to see
how cycling of subunits would matter to the inhibition. Never-
theless, as with SulA, this observation seems of exceptional
importance and may lead to new understanding of FtsZ as-
sembly dynamics and cooperativity.
The essential role of GTP hydrolysis for sensitivity to MinC,
suggested by the in vitro experiments referenced above, is also
supported by in vivo observations. Three point mutants of
FtsZ, when expressed as the sole source of FtsZ, showed re-
sistance to overexpression of MinC: ftsZ2 (D212G, on the
bottom interface), ftsZ9 (an insert of VG between V18 and
G19, buried near the GTP in the Rossman fold), and ftsZ100
(A70T on the upper interface) (Fig. 5 in reference 144). The
locations of these mutations are scattered and do not suggest a
binding site. The mutants do, however, have one thing in com-
mon: their GTPase is greatly reduced or not detectable (34).
The resistance of these FtsZ mutants to MinC may be due
not to their inability to bind MinC but to their reduced
GTPase-dependent assembly dynamics.
Recent studies have suggested that the Min system in B.
subtilis may function other than by blocking Z-ring assembly at
the poles. Gregory et al. (63) expressed a functional MinC-
GFP from the chromosomal minCD locus, which should pro-
duce a normal cytoplasmic level of the fusion protein. They
found that MinC-GFP localized prominently to the Z ring late
in divisome assembly and prior to septation. MinC-GFP re-
mained transiently at the poles following septation, where it
appeared to destabilize newly forming Z rings and block their
maturation into division competency. The prominent polar
localization observed in previous studies was attributed to an
overexpression artifact. Bramkamp and colleagues (23, 193)
also found that MinC localized to Z rings late in the cycle, and
they concluded that “the main function of the Min system is to
prevent minicell formation adjacent to recently completed di-
vision sites by promoting the disassembly of the cytokinetic
ring” during constriction. These observations question the sim-
ple paradigm that MinCD acts simply to block FtsZ-ring as-
sembly at the poles and thereby localize it to the cell center.
These studies have so far been done only with B. subtilis, so it
remains to be seen if similar phenomena are found in E. coli.
MinC has two domains. The N-terminal domain, MinC-N,
which is considered the primary inhibitory domain, blocked
Z-ring assembly when overexpressed in vivo, and it inhibited
FtsZ polymers in vitro (74, 171). The C-terminal domain,
MinC-C, mediates dimerization of MinC and its binding to
MinD (31, 74). Binding of MinC to MinD serves to localize it
to the membrane, and also enhances its association to the Z
ring and its disassembly activity. Shiomi and Margolin (172)
found that MinC-C could also inhibit Z-ring assembly, but this
required coexpression of MinD. A similar activation of MinC
is produced by its complex with DicB, a prophage protein
whose expression results in MinC-mediated destruction of Z
rings (86). There appear to be two steps in the activation of
MinC. Johnson et al. (87) showed that tethering MinC to the
membrane (the tether was a truncated ZipA comprising the
transmembrane domain and the ?160-amino-acid flexible P/Q
segment) rendered it toxic at a 9-fold-lower concentration than
when MinC was expressed as a soluble protein. The inhibitory
activity of tethered MinC was further increased by expression
of MinD, which also resulted in the MinC localizing to Z rings.
A simple summary of these results is that MinC itself can
inhibit Z-ring assembly at sufficiently high concentration; its
activity is enhanced by tethering it to the membrane and fur-
ther still by targeting it directly to Z rings. These last two steps
may be explained by an increase in effective concentration of
Shen and Lutkenhaus used random mutagenesis to identify
the sites on FtsZ that bind MinC-C and MinC-N (170, 171).
They found four mutations in FtsZ that rendered it insensitive
to MinC-C. These were all in the C-terminal peptide that binds
FtsA and ZipA, identifying this peptide as the binding site for
MinC-C. The same strategy was then used to find mutations of
FtsZ that would render it insensitive to MinC-N (171). Four
amino acid changes in FtsZ were found, and they clustered
into a small patch (a maximum of 14 Å apart) that probably
corresponds to the binding site for MinC-N. This patch shows
substantial overlap with the binding site for SulA, with two of
the amino acids (R271 and L205) being buried in the FtsZ-
SulA interface (32). In addition, all four correspond to amino
acids that are buried in the longitudinal interface of the ??-
tubulin protofilament (128). This suggests that the MinC-N
binding site is buried for all FtsZ subunits in the middle of
protofilaments. Its binding site will be exposed only on the
terminal subunit at the minus end of a protofilament. It has
been suggested that MinC-N might act by weakening the lon-
gitudinal interfaces in the protofilament (35), but this could
only happen if the interface were opened by bending. Even the
23-degree bend of the highly curved miniring conformation is
probably not enough to permit access of the globular MinC-N.
The affinity of MinC for binding to FtsZ has been measured
with a plasmon resonance assay, with a KD(equilibrium disso-
ciation constant) reported to be 6 ?M (171). This is surpris-
ingly weak compared to the 1 ?M critical concentration for
FtsZ assembly, suggesting that MinC would show minimal
binding to the pool of free FtsZ monomers in the cytoplasm. It
should also be noted that the concentration of MinC in E. coli
is only 400 molecules per cell (188). The weak binding affinity
might be compensated for by tethering MinC to the membrane
via MinD. However, the low stoichiometry relative to FtsZ
would seem to pose problems for models developed from in
vitro experiments at an equal or higher stoichiometry.
FtsZ as a Target for Drugs
FtsZ has long been recognized as an attractive target for
potential antibiotic drugs. A number of natural compounds
516ERICKSON ET AL.MICROBIOL. MOL. BIOL. REV.
with antibacterial activity have been shown to inhibit FtsZ (19,
42, 43, 76, 84, 154). Most of these studies are still preliminary,
and it is possible that some of the compounds target pathways
other than FtsZ. Two labs have studied GTP analogs for their
potential to inhibit FtsZ polymerization in vitro. C8 MeOGTP
inhibited polymerization of M. jannaschii FtsZ competitively,
with a 50% inhibitory concentration (IC50) of 15 ?M (in 60 ?M
GTP) (93). Compound 14 in another study showed a similar
inhibition in vitro and also showed substantial inhibition of
Staphylococcus aureus growth in an agar diffusion assay (142).
In contrast to these agents, which act by blocking FtsZ poly-
mer, the compound OTBA appeared to act by stabilizing FtsZ
polymer and causing its association into large bundles (20).
This is similar to the mechanism of PC190723, which is dis-
Several groups have used in vitro assays to screen libraries of
hundreds of thousands of compounds for inhibition of FtsZ.
One study used an assay involving assembly of fluorescent FtsZ
with DEAE-dextran to screen extracts of microbial fermenta-
tion broths and plants (196). They identified a viriditoxin, a
previously characterized metabolite of Aspergillus, as a prom-
ising lead candidate. Another study screened large chemical
libraries for compounds that inhibited the GTPase activity of
FtsZ, and those authors identified a half dozen compounds
that they called “zantrins” (110). The zantrins operated by two
different mechanisms. Some inhibited the formation of proto-
filaments, and others caused the protofilaments to associate
into bundles. Either mechanism could block the function of
FtsZ for cell division. The zantrins blocked Z-ring assembly in
E. coli and killed a range of bacterial species. Another study
used a cellular screening system to identify inhibitors of chro-
mosome partitioning in E. coli (82). Reasoning that some of
these might function by blocking FtsZ, this group screened 138
of the most active compounds for the ability to block assembly
and GTPase of FtsZ. They identified A189 as an attractive lead
Stokes et al. (182) developed a cell-based assay to screen
specifically for inhibitors of cell division. Their assay used a
reporter specific for the asymmetric cell division during sporu-
lation of B. subtilis. Out of 105,000 compounds screened, one
hit was selected for further study. The screen could have
picked up inhibitors of any of the seven essential cell division
proteins, but the selected hit compound was shown to target
FtsZ. It blocked assembly of the Z ring in cells, and the com-
pound inhibited pelleting of FtsZ and GTP hydrolysis in vitro.
Several point mutants of FtsZ that were resistant to the com-
pound were isolated.
Probably the most attractive compound targeting FtsZ is
PC190723. Its discovery originated with the compound 3-me-
thoxybenzamide, which was earlier shown to have anti-FtsZ
activity but only at high concentrations (132). Haydon et al.
(70) undertook a medicinal chemistry program and tested 500
modifications of the simple 3-methoxybenzamide. They iden-
tified one compound, PC190723, with vastly improved potency.
PC190723 was bactericidal against B. subtilis and all staphylo-
coccal strains tested, including methicillin-resistant S. aureus. It
was inactive toward Gram-negative bacteria, several Gram-
positive bacteria, and yeast. The compound was effective in
mice, providing 100% protection from a lethal dose of S. au-
reus. Several spontaneous mutations that caused resistance to
the compound arose in S. aureus. Each mutant had a single
amino acid change in FtsZ, at one of six positions. These
positions formed a cluster that was also identified as a binding
site for PC190723 by a docking program. The binding site was
equivalent to that for taxanes on ?-tubulin.
When B. subtilis was exposed to PC190723 the FtsZ-GFP
was mislocalized to discrete foci along the elongated cells,
rather than being completely disassembled as with some other
inhibitors. Andreu et al. have studied the interaction of
PC190723 with B. subtilis FtsZ in vitro, and they found that it
stabilized FtsZ protofilaments and caused them to associate
into protofilament bundles, toroids, and helical bundles (8). It
thus seems to block Z-ring function by sequestering FtsZ into
these inactive condensates.
Cooperative Assembly and Treadmilling of FtsZ
An enduring enigma is how FtsZ, which assembles into pro-
tofilaments that are one subunit thick, can exhibit features of
cooperative assembly. Three groups have suggested that the
appearance of cooperativity can be generated by postulating a
conformational change that favors assembly and is itself fa-
vored by the act of assembly (35, 79, 90, 118). However, no one
has yet suggested a possible model, at the level of atomic
structure, that could generate this enhancement.
An intriguing result with FtsZ interface mutants may be
relevant to cooperative assembly. Our lab prepared and char-
acterized a number of single-amino-acid mutants that targeted
the interface connecting subunits into the protofilament (157).
The original goal was to produce what we thought would be
“cap mutants” that would block either the top or bottom in-
terface. We speculated that a “bottom cap” mutant, with a
crippling defect on its bottom surface, would be able to bind to
the bottom of a growing protofilament and block further as-
sembly at that end.
We first tested the mutants in vivo. We found nine bottom
and eight top mutants that were unable to complement an FtsZ
null mutant. We then tested for dominant-negative effects,
expecting that some mutants might poison the wild-type FtsZ
substoichiometrically. Surprisingly, none of the mutants
showed dominant effects until they were expressed at levels 3
to 5 times that of the genomic wild-type FtsZ. If we assume an
FtsZ concentration of 4 ?M (discussed above) and a critical
concentration of 1 ?M, the mutant FtsZ needs to be present at
12 to 20 times the 1 ?M concentration of soluble wild-type
FtsZ. This indicates a surprisingly weak interaction.
Nine of 10 mutants on the bottom interface were dominant
negative at these high expression levels (157). A related study
examined various truncated FtsZ proteins (139). Truncation
after amino acid 195 permits the N-terminal domain to fold
and form a functional top interface, while the C-terminal do-
main (bottom interface) is nonfunctional. All such truncations,
with only the top interface functional, were dominant negative,
acting like bottom cap mutants (139).
The top mutants were even more surprising. Seven of eight
mutants showed no dominant-negative effects at the highest
levels tested. This suggests a directional effect equivalent to
treadmilling, where subunits assemble preferentially onto the
bottom of the protofilament and disassemble preferentially
from the top. When examined in vitro, bottom cap mutants
VOL. 74, 2010 FtsZ IN BACTERIAL CYTOKINESIS 517
inhibited the GTPase of wild-type FtsZ, but top cap mutants
did not (157).
Expression of wild-type FtsZ at comparable levels (3 to 5
times the genomic FtsZ) produced a 1.5-fold increase in the
length of cells growing in solution (139) and no reduction in
colony size (157). Although Dai and Lutkenhaus (33) reported
that cell division was inhibited by 3-fold overexpression of
FtsZ, they noted that “the filamentation … became less severe
after several passages.” Our observations on cell length were
made after growth to log phase in liquid culture from the
original colony. Filamentation due to excess FtsZ may depend
on strain or growth conditions.
One suggestion from the cap mutant study (157) is that a
point mutation on either the top or bottom interface surface
substantially weakens the ability of that FtsZ subunit to bind to
protofilaments with its supposedly good interface. This means
that the binding of a subunit to the end of a protofilament is
very weak unless it is itself able to bind the next subunit. This
may be related to the mechanism of cooperative assembly. In
particular, Miraldi et al. (118) pointed out that in order for
their conformational change to produce cooperativity, they
had to postulate that both the top and bottom surfaces had to
switch simultaneously to the high-affinity form. Our observa-
tion that crippling either interface cripples the binding affinity
of the other may be related to this, but it has not yet been
incorporated into any theory.
Martin-Galiano et al. (111a) have recently identified several
hinge points that would permit rotational movements of subdo-
mains of FtsZ. These bending mutations may be related to the
conformational changes needed for the high-affinity cooperative
binding (111a). The most important next step to advance the
conformational-change hypothesis will be to suggest specific con-
tact amino acids that might be involved in the proposed confor-
mational change and then to test them by mutagenesis.
FtsZ AS A FORCE GENERATOR: BENDING
The Z-Centric Hypothesis and Reconstitution of Z
Rings in Liposomes
In 1997 a “Z-centric hypothesis” proposed that FtsZ forms
the cytoskeletal framework of the Z ring and may also generate
the constriction force, all by itself (47). The rationale for this
second role was that (i) no motor molecules had been identi-
fied in bacteria, (ii) some bacteria lacked any homologs of the
dozen accessory division proteins identified in E. coli, and (iii)
FtsZ protofilaments underwent a conformational change from
a straight to a curved conformation that would be capable of
generating a force. The curved conformation will be discussed
in the next section.
A step toward confirming this hypothesis was the observa-
tion that divergent FtsZ from foreign bacteria could replace E.
coli FtsZ and function in cell division in E. coli (138). We
argued that, because of the extensive sequence divergence, the
FtsZ of B. subtilis would not be able to bind any E. coli division
protein. We found that B. subtilis FtsZ could function in divi-
sion in E. coli, but only if we gave it the E. coli C-terminal
peptide so it could bind ZipA and FtsA (which recruits the
downstream proteins). We also needed to generate a suppres-
sor mutation somewhere in the E. coli genome; these suppres-
sor mutations have not been identified, but they probably fa-
cilitate division by a partly crippled FtsZ. We concluded that
FtsZ needed to bind only itself and FtsA to function for cell
division. This would exclude any role for a motor molecule.
The Z-centric hypothesis was confirmed when we succeeded
in reconstituting Z rings in liposomes (137). For this we spliced
an amphipathic helix onto the C terminus of FtsZ so it could
tether itself to the membrane, eliminating the need for FtsA,
which normally provides the tether (146). This membrane-
targeted FtsZ, when incorporated inside tubular multilamellar
liposomes, assembled multiple Z rings. Figure 5 shows an ex-
ample where there were initially many dim Z rings, and they
generated no visible constrictions on the membrane. Over 6
min the Z rings slid back and forth, collided, and coalesced to
make brighter Z rings. The bright Z rings coincided with visible
constrictions in the wall of the liposome. Other examples
showed deeper constrictions. If the system was allowed to run
out of GTP, the constrictions suddenly relaxed (137). FtsZ can
therefore assemble a Z ring and generate a constriction force
without any other protein. It needs only to associate with itself
and be tethered to the membrane.
This reconstitution required two fortuitous events. First was
the formation of tubular liposomes. Initially the multilamellar
liposomes were mostly spherical, and they aggregated in
clumps. Applying the coverslip caused some of the liposomes
to transform into a tubular shape, probably by shearing forces.
Importantly, many had inside diameters of ?1 ?m, the same as
for E. coli. This diameter seems to be optimal for Z-ring as-
sembly. The cylindrical geometry is probably also essential,
since we have failed to generate Z rings inside spherical lipo-
somes. The second fortuitous event was that the FtsZ, which
was initially on the outside, became incorporated inside many
of the liposomes. We have found that about half of the tubular
liposomes are permeable or leaky to proteins, and this permits
FIG. 5. Z rings assembled in tubular liposomes from purified,
membrane-targeted FtsZ. The upper frame at the two times uses
Nomarski optics to show the profile of the liposome. The lower panel
shows FtsZ localized by yellow fluorescent protein (YFP) fluorescence.
At time zero (actually about 10 min after specimen preparation), there
are many dim Z rings. After 350 s, the dim Z rings have coalesced into
fewer bright ones, and these are generating constrictions in the lipo-
some. The full sequence may be seen in Movie S2 in the supplemental
material. (This figure is reprinted and Movie S2 is reproduced from
reference 137 with permission from AAAS.)
518 ERICKSON ET AL.MICROBIOL. MOL. BIOL. REV.
FtsZ and GTP to equilibrate inside. These and other details of
the liposome preparation have been described in detail (140).
Two Different Curved Conformations of FtsZ Protofilaments
The early rationale that FtsZ might generate a constriction
force all by itself was based on the observation that FtsZ
protofilaments had two conformations: straight and highly
curved. The highly curved protofilaments formed mini-rings or
helical tubes 24 nm in diameter (45a, 105). The miniring con-
tained 16 subunits with a 23-degree bend at each interface. The
straight conformation was thought to be favored by bound
GTP and the curved conformation by GDP (105). Thus, the
hydrolysis of GTP could power the conformational change and
generate a bending force.
Evidence has accumulated recently that there is a second
curved conformation of FtsZ protofilaments. These have an
intermediate curvature, corresponding to a 2.5-degree bend
between subunits and producing a curvature of ?200 nm di-
ameter. Mingorance et al. (117) obtained AFM images of FtsZ
protofilaments adsorbed to mica, showing a mixture of straight
and curved protofilaments. Hamon et al. (68) obtained AFM
images of protofilaments assembled on mica from very dilute
FtsZ solutions. The filaments were mostly straight when as-
sembled for only 5 s, but after 30 s they were mostly curved
with an ?160-nm diameter. At later times they formed multi-
turn spiral structures with an outer diameter of ?160 nm and
inner spirals as small as 80 nm.
Protofilaments with this intermediate curvature have been
seen by conventional negative-stain EM (28, 61, 78, 117).
These images show protofilaments with an extended contour of
uniform curvature, about 200 nm in diameter, mixed with
straight protofilaments. Sometimes the curved protofilaments
form closed circles. Under some conditions, especially with
crowding agents, the curved protofilaments form loose spiral
bundles or toroids with a diameter of ?200 nm (150, 151).
Similar toroids were assembled from B. subtilis FtsZ in the
presence of calcium (114) or in a low-ionic-strength buffer
(120). The antibiotic candidate PC190273 caused FtsZ from B.
subtilis and S. aureus FtsZ to associate into very similar toroids
and helical bundles (8). Beuria et al. (18) found the interme-
diate curved conformation when they mixed E. coli FtsZ with
a mutant of FtsA (FtsA*, which has enhanced activity). As-
sembly of this mixture produced protofilaments or sheets with
a striking ?200-nm-diameter curvature.
The intermediate curved conformation seems not to require
GTP hydrolysis. It was documented by both negative-stain and
atomic force microscopy for assembly supported by GDP-AlF,
a nonhydrolyzable analog (117). Circles were also formed by
the mutant L68W assembled in EDTA, which blocks GTP
hydrolysis (28), and we have found circles assembled by wild-
type FtsZ in EDTA (Y. Chen and H. P. Erickson, unpublished
It has been noted that the FtsZ mutant D212G, which hy-
drolyzes GTP at less than 1% the rate of the wild type, can
achieve cell division (36, 125). This requires generation of a
second-site mutation somewhere in the E. coli genome (138),
but the fact that the cells can divide with a GTPase-dead FtsZ
strongly suggests that the energy of GTP hydrolysis is not
needed to generate the constriction force (36). Two studies
showed that FtsZ-D212G plus DEAE-dextran assembled tubes
(125, 192). Since these tubes are a manifestation of the highly
curved miniring conformation (105), this suggests that this
conformation is not strictly determined by GTP hydrolysis. It
may be that the tilt giving this curved conformation can be
achieved by removing either the gamma phosphate of GTP or
the side chain of the D212 that makes contact with the GTP.
An important question is whether the intermediate curved
conformation represents a preferred and rigid conformation or
represents the bending by thermal forces of protofilaments
that are straight but very flexible. Two studies have assumed
the latter scenario and estimated a persistence length (Lp) of
0.18 ?m (35) or 0.054 nm (78). Lan et al. (91) noted that these
persistence lengths would mean a very flexible protofilament,
not strong enough to generate any significant bending force on
a membrane. Ho ¨rger et al. (72), in contrast, assumed the first
scenario and measured the curvature of protofilaments ad-
sorbed on mica, without a prior assumption that the preferred
conformation was straight. These filaments were predomi-
nantly curved, with a diameter ranging from 80 to 500 nm, with
an average of 200 nm. Importantly, these authors measured the
deviation from this average curvature and determined a per-
sistence length of ?4 ?m.
In a classic study of the flexural rigidity of protein polymers,
Gittes and Howard (57) measured the thermal bending of actin
and microtubules. They concluded that both of these polymers
had a Young’s modulus equivalent to that of hard plastic
(Plexiglas). By extension this Young’s modulus would apply to
globular proteins in general and their polymers. Mickey and
Howard (115) estimated the flexural rigidity for a single pro-
tofilament of a microtubule to be 1.2 ? 10?26nm2, which
corresponds to a persistence length of 2.9 ?m. This is remark-
ably close to the value determined experimentally by Ho ¨rger et
We have used the small-angle approximation of the beam
equation for a cantilevered beam (a rod with one end fixed) to
estimate the force that could be generated by a bending pro-
tofilament. Based on this approximation, the force needed to
bend the free end of the rod a distance y is given by F ?
(3EI/L3) ? y, where E is the Young’s modulus, I is the second
moment of inertia, and L is the length of the rod. To get an
idea of the forces required to bend a 130-nm protofilament
into (or out of) the ?200-nm-diameter intermediate curved
conformation, we can consider the protofilament to be fixed in
the middle; thus, each half can be modeled as a 65-nm canti-
levered beam (Fig. 6). Using 1.4 GPa for the Young’s modulus
(115), 8.4 ? 10?36m4for the second moment (6), 65 nm for
the length, and 20 nm for the deflection of the end, we estimate
a force on the end of each half-protofilament of approximately
2.6 pN. This is a significant amount of force for just one
protofilament and is in general agreement with estimations by
others that forces on the order of several pN would be required
to deform the bacterial membrane (92). This simple calcula-
tion is a rough approximation. A more complete model, which
would distribute the force to multiple attachment points along
the length of protofilament, is in progress.
Allard and Cytrynbaum (6) have formulated a model for
generation of a constriction force by protofilament bending,
using the flexural rigidity of Plexiglas and assuming that the
preferred curvature for FtsZ-GDP subunits was the 24-nm
VOL. 74, 2010FtsZ IN BACTERIAL CYTOKINESIS519
diameter of the miniring. They concluded that protofilament
bending could generate a substantial inward-directed constric-
tion force. Our simple analysis in Fig. 6 supports this interpre-
tation. Readers with a background in physics will be interested
in two recent papers that examine how protofilaments with an
intrinsic curvature are affected by applied bending forces, in-
cluding the thermal forces acting on a worm-like chain (12, 56).
Evidence That the Constriction Force Is Generated by
There are two general classes of models for how the Z ring
generates a constriction force (reviewed in reference 48). One
class is based on lateral bonds and proposes that if protofila-
ments can slide and increase the number of lateral bonds, this
would decrease the circumference and constrict the Z ring.
The second proposes that protofilaments exert a bending force
on the membrane as they are induced to a curved conforma-
tion. Recent experiments with liposomes support the bending
In those experiments, membrane-targeted FtsZ was applied
to the outside of liposomes, where it bound and produced
visible distortions. Initially the spherical liposomes present a
convex surface on the outside. The membrane-targeted FtsZ
distorted this surface into multiple concave depressions (Fig.
7A). The constriction force generating these concave depres-
sions is in the same direction as that of the Z rings inside the
liposomes, which bind to a concave surface and constrict to
make it more concave.
A very informative experiment was to switch the point of
attachment of the membrane-targeting amphipathic helix.
Normally the FtsA-binding peptide, which we replaced with an
amphipathic helix, is at the C terminus of FtsZ. This is at the
end of the 50-amino-acid linker (Fig. 2A), which is attached to
the globular domain in the center of the “front face,” equiva-
lent to the outside of a microtubule. The N terminus of FtsZ is
attached to the opposite face of the subunit, approximately 180
degrees away. We switched the amphipathic helix (and linker)
from the C to the N terminus and tested it on the outside of
liposomes. This construct formed convex protrusions on the
outside of liposomes (Fig. 7B), a bending opposite to the
concave depressions formed by the normal C-terminal attach-
ment (136). This is exactly what we would expect if the FtsZ
protofilaments have a fixed curvature, with the normal, C-
terminal attachment on the outside of the curve. When the
membrane attachment was switched to the inside of the curve,
it would produce convex protrusions. We do need to remind
the reader that the direction of curvature proposed from the
FtsZ experiments, where the front face (C terminus) is on the
outside of the curve, is the opposite of that proposed for
tubulin, where the front face is on the inside of the tubulin
rings (see reference 136 for references).
Constriction force models based on the other model, sliding
protofilaments, are difficult to reconcile with another aspect of
the liposome experiments. In order for sliding filaments to
generate a force on the membrane, they would need to be
anchored. One possible anchor would be to have the Z ring
form a complete circle, with the sliding generated where the
ends overlap. Another would be for shorter filaments to be
anchored to the rigid peptidoglycan wall. However, the con-
cave and convex distortions of liposomes are generated by
partial Z rings, and the fluid lipid bilayer would provide no
anchor. Protofilament bending seems the most attractive
mechanism for producing the concave depressions and convex
protrusions on the outside of liposomes and therefore the
constriction force of the Z ring.
Incomplete FtsZ Rings Can Generate Constriction
The initial assembly of FtsZ in newborn daughter cells pro-
duces wide-pitch helices, discussed above, but these helices
then collapse into a ring. The Z ring usually appears to be a
closed circle but occasionally separates into a short-pitch helix
(Fig. 1), showing perhaps its true structure. The density of the
ring is relatively uniform: if it is bright, it is bright all over; if it
is dim, it is dim all over. This applies to Z rings both in bacteria
(Fig. 1) and in liposomes (Fig. 5). This raises the question of
whether FtsZ needs to form a complete circle in order to
generate a constriction.
An early indication that closed circular rings are not essen-
tial was the study of the ftsZ26 mutant (5). This mutant forms
normal-looking Z rings about 50% of the time, but the other
50% are distinctly spirals. When examined by scanning EM,
many bacteria showed spiral constrictions, suggesting that the
spiral Z rings were capable of generating a constriction that
matched their shape. Similar twisted septa have been shown
recently for the B. subtilis mutant TS1 (120). The earlier study
also showed some examples of partial Z rings or arcs in cells
that had a mutation causing them to grow as spheres. A recent
study of MreBCD depletion mutants growing as large spheres
provides convincing evidence for asymmetric constriction, with
the constriction located at an arc of FtsZ (11).
FIG. 6. Scale model of protofilament bending. A 130-nm-long pro-
tofilament (solid line) is shown fixed at the center (dot) in the straight
and the intermediate curved conformations. The two 65-nm halves
behave as individual cantilevered beams as the protofilament bends to
the radius of the intermediate curved conformation. Arrows show the
direction of force (lighter central arrows represent the opposite force
that would necessarily push back against the membrane if the proto-
filament were free).
520 ERICKSON ET AL.MICROBIOL. MOL. BIOL. REV.
A dramatic example of asymmetric septation is seen in the
division of the cyanelle, a primitive plastid of the protist Cya-
nophora paradoxa (165, 166). The Z ring extends from one-
fourth to one-half way around the cell and is located over a
very asymmetrically forming septum (Fig. 8).
In all of these examples, including the cyanelle, there is a
peptidoglycan cell wall, and the generation of a constriction
force could involve coupling of the Z ring to the rigid wall.
However, the concave depressions on the outside of liposomes
demonstrate that peptidoglycan is not necessary for generating
FIG. 7. FtsZ on the outside of liposomes produces distortions. (A) Normal membrane-targeted FtsZ, with the membrane tether on the C terminus,
forms concave depressions. (B) When the tether is switched to the N terminus, it forms convex protrusions. (C) Diagram showing the curvature and two
sides for attaching the membrane targeting sequence (MTS). (Reprinted from reference 136 with permission from Macmillan Publishers Ltd.)
FIG. 8. Asymmetric division of isolated cyanelle plastids. (A) Three cyanelles imaged by scanning EM, showing furrows progressing from
shallow (arrow) to deep (double arrowhead). (B to D) Increasingly deep furrows correspond to increasing length of the FtsZ arc. Row 1 shows
Nomarski images, row 2 shows the cytoplasm imaged by autofluorescence of chlorophyll and phycobilin, row 3 shows immunofluorescence staining
of FtsZ, and row 4 shows overlap of rows 2 and 3. (Reprinted from reference 165 with kind permission from Springer Science?Business Media.)
VOL. 74, 2010 FtsZ IN BACTERIAL CYTOKINESIS521
force on the membrane. This simple system shows that an
incomplete arc of protofilaments is capable of generating a
bending force on a fluid membrane.
Finally, we should note one major problem for a simple
model of bending protofilaments. These are thought to be
tethered to the membrane by attaching to FtsA (Fig. 9A and B)
and ZipA (not shown). In both cases the C-terminal peptide
that binds FtsA and ZipA is at the end of a 50-amino-acid
segment that is thought to be a flexible linker (Fig. 2A). This
has a contour length of 17 nm and a relaxed end-to-end dis-
tance of 5 nm. ZipA has an additional 150-amino-acid tether
between the FtsZ-binding domain and the membrane (131). If
these tethers are flexible, then why would a short protofila-
ment, with a preferred curvature, not simply roll 90 degrees
and curve in the plane of the membrane? Some additional
structural feature is needed to limit the rolling of the proto-
filament and maintain its plane of curvature perpendicular to
Finishing Division: Membrane Scission without FtsZ
It is now thought that the FtsZ protofilament has three
preferred conformations: straight, intermediate curved
(200-nm diameter, 2.5-degree bend per subunit) and highly
FIG. 9. Bending protofilaments can only constrict so far. (A) An FtsZ protofilament is shown tethered to the membrane of an undivided
cell. The blue FtsZ subunits are connected into a protofilament that has a preferred bend. About one out of five FtsZ proteins are shown
tethered to an FtsA, indicated by an orange circle. An amphipathic helix extends from FtsA into the membrane bilayer. The FtsZ tether is
shown extended to ?10 nm at the ends of the protofilament, where the bending toward the cell center is maximal. Near the middle of the
protofilament it is pushing outward on the membrane, and the tethers are compressed. (B) When fully in the curved conformation, the 24-nm
miniring plus an ?10-nm tether plus FtsA could constrict the membrane to about a 57-nm outside diameter. (C) The diameters of the
undivided cell, the intermediate curved conformation, and the miniring are compared.
522 ERICKSON ET AL.MICROBIOL. MOL. BIOL. REV.
curved (24-nm diameter, 23-degree bend per subunit). Al-
though we initially focused on the highly curved (miniring)
conformation, which seemed to be coupled to GTP hydrolysis
(50, 105), there is accumulating evidence that the intermediate
curved conformation may play the most important role. How-
ever, neither curved conformation can pull the membrane to a
complete scission. We suggest here that the scission might be
completed by membrane fluctuations.
The membrane of an undivided cell has a diameter of 1,000
nm. A protofilament attached to this slightly curved membrane
would need a bend of 0.5 degrees per FtsZ, which is close to
the straight conformation. A straight protofilament would tend
to align along the axis of the cell, but if it had any inherent
curvature it would tend to align circumferentially (9). Initially
the protofilaments would not be able to bend because they are
tethered to the approximately straight membrane (Fig. 9A).
However, if their preferred conformation is curved, the proto-
filaments will generate a bending force on the membrane. As
long as the membrane is attached to the rigid peptidoglycan
wall, the force will not result in constriction. However, as the Z
ring recruits transmembrane proteins that remodel the wall, it
will be able to follow the constricting membrane, initiating
The highly curved conformation could pull the septum to a
diameter of ?50 nm (24 nm for the outside of the ring plus ?8
to 12 nm on each side for the 50-amino-acid tether plus 5 nm
for FtsA [Fig. 9B]). If the intermediate curved conformation is
producing the force, it could pull the septum to a diameter of
?250 nm. In neither case would the curved FtsZ protofila-
ments be able to pull the membrane to complete closure and
scission. How is the division process completed?
One possibility is that the remodeling of the cell wall, which
initially followed the membrane invagination produced by
FtsZ, eventually becomes a positive force pushing the septum
toward closure. Favoring this possibility, Joseleau-Petit et al.
(88) reported that unstable L forms of E. coli, which they could
generate by overnight growth in cefsulodin, retained about 7%
of the wild-type level of peptidoglycan. Moreover, they re-
quired peptidoglycan synthesis to grow and divide. This sug-
gested that the remodeling of the peptidoglycan might be pow-
ering division, at least partly, by pushing from without.
Another possibility is that the constriction of the Z ring is
regulated by feedback from the transmembrane proteins of
In contrast to these L forms, Leaver et al. (95) created L
forms of B. subtilis and provided convincing evidence that
peptidoglycan was completely absent. One might have hoped
to use these L forms to observe how FtsZ could divide cells
having only a plasma membrane. However, Leaver et al. went
on to show that FtsZ was not used for division and was com-
pletely dispensable for growth of the L forms. The cells some-
times appeared to divide by extruding membrane buds from
the large spherical mother cell. Another mechanism, probably
related, involved extrusion of a pseudopod-like extension,
which then fragmented into small spherical bodies, with at
least some presumably containing a complete nucleoid. The
authors suggested that extrusion likely involved a force pro-
duced by a cytoskeletal element; an active chromosome segre-
gation system was one possibility. This force would form the
extended pseudopod, and resolution might then be achieved by
collapse and resealing of membranes.
Perhaps related to the mechanism of division of this L form
is the existence of some bacteria that can divide without FtsZ.
The entire phylum of Crenarcheota has no FtsZ, but these
archaea are now known to use a completely different system,
which is related to the eukaryotic ESCRT system (99, 163).
However, there are no ESCRT proteins in eubacteria. The
bacterial phyla Chlamydiaceae and Planctomycetes have no
FtsZ. Pilhofer et al. (148) have noted that several genes of the
dcw cluster are present in various species of these phyla, and
they suggested that these organisms descended from an ances-
tor that had FtsZ and have lost it. At least two Mycoplasma
species, Ureaplasma urealyticum (58) and Mycoplasma mobile
(83), have no FtsZ. A recent study showed that Mycoplasma
genitalium can grow and divide when its ftsZ is deleted (48a,
100). These organisms grow as gliding cells attached to a sub-
strate and apparently divide by “traction-mediated cytofis-
sion,” where a cell develops two gliding organelles that move in
opposite directions and pull the cell apart. Mycoplasma species
without ftsZ probably use the same mechanism.
If these bacteria and the B. subtilis L form can divide without
FtsZ, perhaps the same mechanism might apply to the normal
process of completing scission. More specifically, we suggest
that membrane collapse and scission may occur as natural
fluctuations of the membrane, independent of force from cy-
toskeleton or motor molecules (48a). In support of this, Ben-
dezu ´ and de Boer (11) reported that defects in the mre or mrd
pathways caused cells to lose their rod shape and become
round, and the round cells accumulated membrane-enclosed
vesicles inside. The large round cells had an excess of lipid
bilayer, and the formation and excision of vesicles might occur
to accommodate the excess. We suggest that related mem-
brane fluctuations may drive the final steps of septum closure
and scission. FtsZ protofilament curvature would then be re-
sponsible for bringing the septum to a 200- or 50-nm diameter,
with the final step of scission completed by membrane fluctu-
In a theoretical study, Zhang and Robinson (204) concluded
that differential tension and pressure of the membrane at the
poles and center of the cell can drive constriction in the com-
plete absence of a contractile machine. In an in vitro system,
Yanagisawa et al. (201) showed that liposomes prepared with
lipids with different melting temperatures could be induced to
form different shapes in response to osmotic pressure. Phase
separation following a temperature downshift could cause
them to bud spherical vesicles. In one example, a long tubular
liposome resolved into a necklace of beaded domains, resem-
bling somewhat the resolving pseudopod of the B. subtilis L
form (95). Finally, we should note the progress of Szostak and
colleagues in creating artificial “protocells” (205). Fatty acid
vesicles fed with micelles developed an elongated tubular mor-
phology. Then, upon mild shear, the tubules divided into small
spherical vesicles. The growth and division could be repeated,
mimicking the growth and division of cells. These in vitro re-
sults show that membrane growth and fluctuations can gener-
ate effects related to cytokinesis without the involvement of
proteins. Similar mechanisms may play a role in cytokinesis in
cells, especially the final scission event.
VOL. 74, 2010 FtsZ IN BACTERIAL CYTOKINESIS523
The field needs a more definitive model for the substructure
of the Z ring. At present there is support for a model of
overlapping short protofilaments (Fig. 4A) but also some in-
dication that protofilaments may anneal into much longer fil-
aments (Fig. 4B). A promising new approach is superresolu-
tion light microscopy, especially photoactivation localization
microscopy (PALM) (16, 174). PALM can achieve a resolution
of 20 to 30 nm, which might be sufficient to resolve the proto-
filaments in the Z ring. A particular advantage of PALM is that
the protofilaments are colored red by the fused fluorophore,
and there is no noise from unrelated cytoplasmic structures.
There are many technical hurdles, but the study of the struc-
ture of the Z ring would seem to be one of the most attractive
applications for PALM.
An enduring enigma from in vitro studies is how the one-
subunit-thick protofilaments can exhibit cooperative assembly.
The proposals for a conformational change seem attractive,
but we now need suggestions for specific amino acids or do-
mains that might be involved. The inhibition of assembly by
SulA and MinC may be related to the mechanism of cooper-
ative assembly. SulA and MinC now bring their own enigma,
i.e., that they inhibit assembly only when subunits are hydro-
lyzing GTP and undergoing the GTPase-dependent cycling.
An important step in understanding the mechanisms will be to
determine the association and kinetic constants for FtsZ bind-
ing to these inhibitors.
We know very little about the mechanism of GTP hydrolysis
and related subunit exchange. Does hydrolysis in the middle of
a protofilament lead to fragmentation, or do GDP subunits
dissociate only when they are at the end? What are the time
course and molecular mechanism for GTP hydrolysis after a
subunit enters a protofilament? Do protofilament dynamics
involve a mechanism like dynamic instability, where the pro-
tofilament has excursions of growth and disassembly? The
mechanism of microtubule dynamic instability is thought to be
explained by a GTP cap, but we really do not know what
determines the dynamics inside the cap. There is some hope
that understanding the dynamics of FtsZ may explain the GTP
cap of microtubules.
Single-molecule fluorescence microscopy of FtsZ protofila-
ments may be a useful approach, but the small size of the
protofilaments, which is less than the resolution of the light
microscope, will pose formidable challenges.
This work was supported by NIH grant GM66014.
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