Department of Microbiology and Molecular
Genetics, University of Texas Medical School, Houston, Texas 77030
The dynamics and assembly of bacterial cell division protein FtsZ
were monitored in individual, growing and dividing Escherichia coli cells in real time by microculture of a merodiploid strain expressing green fluorescent protein (GFP)-tagged FtsZ. Cells expressing FtsZ-GFP at levels less than or equivalent to that of
wild-type FtsZ were able to grow and divide over multiple generations, with their FtsZ rings visualized by fluorescence. During the late stages of cytokinesis, which constituted the last one-fourth of the
cell cycle, the lumen of the FtsZ ring disappeared as the whole
structure condensed. At this time, loops of FtsZ-GFP polymers emanated
outward from the condensing ring structure and other unstable
fluorescent structures elsewhere in the cell were also observed.
Assembly of FtsZ rings at new division sites occurred within 1 min,
from what appeared to be single points. Interestingly, this nucleation
often took place in the predivisional cell at the same time the central
FtsZ ring was in its final contraction phase. This demonstrates
directly that, at least when FtsZ-GFP is being expressed, new division
sites have the capacity to become fully functional for FtsZ targeting
and assembly before cell division of the mother cell is completed. The
results suggest that the timing of FtsZ assembly may be normally
controlled in part by cellular FtsZ concentration. The use of
wide-field optical sectioning microscopy to obtain sharp fluorescence
images of FtsZ structures is also discussed.
 |
INTRODUCTION |
FtsZ is one of several proteins
essential for cell division in Escherichia coli
(10) and acts at a very early stage of cytokinesis before
midcell constriction is visible (1; for three
excellent recent reviews, see references 8, 14, and
21). FtsZ is a GTPase (11, 24, 29) that
has significant structural similarity to tubulin (19). Like
tubulin, FtsZ can assemble into microtubule-like polymers (9, 15,
25). Recently, these polymers have been shown to have dynamic
properties in vitro, as they can grow from single nucleation sites and
eventually interconnect (34). Probably all prokaryotes,
including archaea and chloroplasts, contain FtsZ (5, 23, 27,
33). It is likely that FtsZ evolved into the three types of
tubulin proteins that are keystones of the eukaryotic cytoskeleton.
FtsZ localizes to the leading edge of the invaginating inner membrane
in E. coli and other prokaryotes and likely defines the cell
division plane (6, 20, 31). Based on immunoelectron microscopic data, the active assembled polymer is thought to exist as a
continuous ring. Therefore, the behavior of FtsZ seems to be more like
actin than like tubulin. FtsZ forms protofilament sheets and bundles in
vitro, suggesting that the FtsZ ring in vivo comprises such a
superstructure (15, 34). However, the FtsZ polymeric ring
has not been directly observed in electron microscopic sections. It is
also not known precisely how the FtsZ structure changes during the cell
cycle, particularly during invagination, and whether it is truly
dynamic in vivo. Recent results suggest that FtsZ has different
polymerization states (3, 22) and that the FtsZ structure
may be more unstable during the constriction process than before
constriction (2). A major question in the field is whether
FtsZ is actively involved in generating the cytokinetic force or is
simply the scaffold to which septum-synthesizing enzymes are recruited
and on which they exert inward pressure (14, 21, 30).
Previously, we showed that a fusion of FtsZ with green fluorescent
protein (GFP) was able to localize to the native FtsZ ring in E. coli cells (22). Here, by using three-dimensional image reconstruction of cells containing FtsZ-GFP, we show for the first time
that FtsZ dynamics can be visualized directly in individual, growing
E. coli cells throughout the entire cell cycle. The
advantage of monitoring individual cells is that rapid protein dynamics can be observed in real time.
| |
MATERIALS AND METHODS |
Bacterial strains and culture conditions.
Strain WM720 was
made by transforming E. coli BSP853 (W3110
rnc40::
Tn10), obtained from R. Britton, with plasmid pZG (22), which contains FtsZ-GFP
under lac promoter control, and lacIq
in plasmid pBC (Stratagene). Cells were grown in Luria-Bertani medium
(10 g of tryptone per liter, 5 g of NaCl per liter, 5 g of
yeast extract per liter) at 30°C to early exponential phase (optical
density, 0.3) and then induced with 40 µM IPTG
(isopropyl-
-D-thiogalactopyranoside) for 40 min before
transfer to agar (see next section). Cells induced with this
concentration of IPTG exhibited normal FtsZ rings and cell division for
several hours after induction, indicating that the extra FtsZ-GFP did
not significantly perturb cell physiology during the time used for the
experiments.
Preparation of cells for fluorescence microscopy.
For vital
membrane staining, FM4-64 vital dye (Molecular Probes, Eugene, Oreg.)
was added to the culture at a final concentration of 30 µM. For
observation of GFP fluorescence in growing cells, a glass slide was
covered with a thin layer of Luria-Bertani medium plus 1.5% agar;
then, a drop of culture was added to the top of the agar and allowed to
dry at room temperature for 2 min, and a cover glass was placed over
the slide. Growth of the microcolonies was then monitored by light
microscopy at room temperature. Under the conditions of this assay, the
cells grew with an average generation time of about 2 h.
Immunofluorescence microscopy (IFM) with anti-FtsZ was performed on
fixed cells essentially as described previously (28).
Microscopy and image analysis.
For conventional fluorescence
microscopy, images were acquired with an Olympus BX60 microscope
equipped with a 100× oil immersion phase-contrast objective, a
standard fluorescein isothiocyanate (FITC) filter set for GFP, and an
Optronics Engineering DEI-750 24-bit color video camera. Images were
captured and digitized with a Scion LG3 framegrabber and manipulated
with Adobe Photoshop. Care was always taken to minimize exposure of the
bacteria to the blue excitation light to minimize photobleaching.
Three-dimensional image reconstruction of fluorescence was performed
with a Deltavision wide-field optical sectioning microscope (Applied
Precision, Issaquah, Wash.) equipped with a 100× oil-immersion
objective and visualized with a cooled charge-coupled device camera, an
FITC filter set (GFP), and a rhodamine filter set (FM4-64).
z-axis sections were taken at 0.1- to 0.2-µm intervals,
for a total of 12 to 20 sections. Since the cell diameters were less
than 1 µm, this oversampling ensured that image data would not be
omitted. Deconvolution of raw data was performed with five rounds of
iteration, and volume views were generated with the maximum-intensity
method at best quality.
To obtain fluorescence images of cell cross sections without using
deconvolution, it was necessary for bacteria to be simultaneously immobilized and perpendicular to the coverslip plane, which was not
possible by placing the cells on agar or by mixing them with agar. This
problem was circumvented by the following layering technique. A glass
slide was prewarmed at 37°C, and 20 µl of a logarithmic-phase cell
culture was dropped on it. Then, 20 µl of 3% low-melting-point
agarose was layered above the cells, followed immediately by 4 µl of
0.5% Casamino Acids layered on the top and center of the agarose. The
slide was then incubated at 37°C for 1 min. In order to avoid
crushing the solidified agarose, two strips of double-sided tape were
applied along the edge of the slide, upon which a cover glass was
carefully placed.
| |
RESULTS |
Normal cell division with fluorescent FtsZ.
To study FtsZ
dynamics in growing cells, we used merodiploid E. coli cells
expressing native FtsZ from the chromosome and an FtsZ-GFP fusion from
the lac promoter on a plasmid, as described previously
(22). Although it was reported previously (22) that this fusion failed to complement the ftsZ84
temperature-sensitive mutant, we subsequently found conditions, after
finely tuning expression levels with IPTG and glucose, that would allow
complementation of the mutant at the restrictive temperature (data not
shown). This indicated that the fusion protein was at least partially functional for septation, although the narrow concentration requirement for its activity suggested that it was somewhat less functional than the untagged protein.
To monitor single cells in culture, exponentially growing cells were
subjected to a short-term, low-level induction with IPTG and then
transferred to thin agar overlays on a microscope slide beneath a
coverslip, and the structure and localization of FtsZ-GFP over time in
the microculture were monitored by fluorescence microscopy. The cells
were grown at room temperature, resulting in a generation time of about
2 h. The results of a typical time course experiment in which
cells were monitored through several cell division cycles are shown in
Fig. 1. One important conclusion to be
drawn from this data is that under these conditions, fluorescent
FtsZ/FtsZ-GFP rings were clearly functional through several cycles of
cell division, as they constricted and disappeared after cell
separation. This is consistent with the ability of FtsZ-GFP, when at
low levels in the cell, to colocalize with FtsZ (22) and
complement the ftsZ84 mutation. This result is also
consistent with the ability of purified FtsZ-GFP to bind and hydrolyze
GTP as efficiently as the untagged protein and to coassemble
homogeneously with the untagged protein into dynamic protofilament
bundles (34). Therefore, it is likely that FtsZ and FtsZ-GFP
coassembled to make a mixed ring, although it cannot be ruled out that
two adjacent rings, one with FtsZ and one with FtsZ-GFP, were being
assembled. Immunoblot analysis with anti-FtsZ showed that under these
growth conditions, the levels of FtsZ-GFP were between 50 and 100% of
the levels of FtsZ in the cell (data not shown).
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FIG. 1.
Growth and division of E. coli microcolonies
expressing FtsZ-GFP and FtsZ, showing that FtsZ rings containing
FtsZ-GFP function normally. Two consecutive cell division cycles are
shown, using conventional fluorescence microscopy. The first and last
panels are phase-contrast images; note that the last time point lacks a
fluorescence image. The other panels were obtained by a digital overlay
of phase-contrast and fluorescence images at the times shown. Times are
shown in hours and minutes. Note the simultaneous formation of daughter
FtsZ rings and contraction of the midcell ring at 0:15 in the bottom
cell and at 1:53 in two daughter cells of the original bottom cell.
|
|
Assembly of FtsZ rings at daughter division sites before
completion of mother cell division.
Interestingly, we found
that FtsZ/FtsZ-GFP rings assembled at one-fourth and
three-fourths of the cell length (1/4 and 3/4 positions) immediately
before the midcell site was fully constricted (Fig. 1, 0:15 time
point). This result demonstrates clearly that under our experimental
conditions, daughter division sites can become competent for FtsZ
assembly while the central septum is being formed, prior to the
complete division of the mother cell. The localization of FtsZ-GFP to
the 1/4 and 3/4 positions in dividing cells is consistent with the
placement of periseptal annuli in the periseptal annulus model
(30) as well as a previous proposal of such a possibility
based on FtsZ ring formation in cephalexin-treated cells
(28).
Our results differ from those of previous IFM experiments, in which
FtsZ was not observed to target daughter cell sites before completion
of the midcell septum in wild-type cells (1, 18) and newborn
cells almost always lacked FtsZ rings (28). To determine whether the expression of the FtsZ-GFP fusion was causing this localization of FtsZ to the quarter sites, we examined a large number
of cells with and without the fusion (WM720 and BSP853, respectively)
by IFM (Table 1). The high percentage of
cells containing FtsZ rings and the presence of FtsZ rings at quarter sites in a small fraction of dividing cells support our GFP data with
live cells. In addition, the more frequent quarter site localization in
cells expressing FtsZ-GFP suggests that our observations of very early
FtsZ targeting are probably due to a small excess of total FtsZ in the
cell because of the presence of the fusion protein. Although this
excess would be at most twofold, such an increase in FtsZ levels may be
sufficient to allow assembly at new division sites as soon as they are
ready, whereas normally some time after cell birth may be necessary to
build up FtsZ levels to a critical level.
Another possible explanation for our results is that FtsZ-GFP
expression slowed down the septation process, thus allowing enough
time for the new sites to become active. To test this, we measured the
lengths of WM720 and BSP853 cells in the same experiment and found that
their length distributions were superimposable (data not shown), with
an average cell length of 3.4 µm for both strains. These results rule
out a general cell cycle delay induced by expression of the fusion and
support the model invoking a threshold level of FtsZ as a prerequisite
for assembly at new sites. The low percentage of WM720 cells displaying
the quarter site localization is consistent with the transient nature
of the localization relative to the generation time. The fact that a
few BSP853 cells lacking the fusion also exhibited this localization
suggests that this strain has the capacity to target FtsZ to the
quarter sites during division on rare occasions, although it is
possible that these rare cells would have never divided.
Both the IFM experiments and the live-cell experiments failed to detect
FtsZ-GFP assembly at quarter sites prior to the development of a deep
midcell constriction (Fig. 1; also see below). This suggests that the
localization of FtsZ-GFP to quarter sites occurs simultaneously with
the normal disassembly of the medial ring after cytokinesis. Although
it appears that this phenomenon occurs mostly when FtsZ levels are
elevated, these results have allowed us to detect perhaps the earliest
time in the cell cycle at which division sites become competent to
assemble FtsZ. It is important to note that in steady-state
experiments, ZipA-GFP also seemed to target potential division sites
early, perhaps because of its similar ability to recognize the division
site as soon as it appears (17).
High-resolution microscopy of the FtsZ ring.
To obtain a more
detailed, three-dimensional picture of FtsZ structure throughout the
cell division cycle, we monitored FtsZ-GFP fluorescence in individual,
growing cells by using a wide-field optical sectioning technique
previously applied to cells in steady state (22). This
technique uses deconvolution algorithms to sharpen two-dimensional
images obtained with a conventional fluorescence microscope
(4). The deconvolved images are then rotated to make a
three-dimensional reconstruction. Figure
2 shows three cells; only the middle cell
is undergoing cytokinesis during the time course. The fluorescent FtsZ
structure can clearly be observed to contract to a dot and disappear.
This time course suggests three major points. (i) FtsZ initially
nucleates assembly at a point corresponding to the potential division
sites (the 1/4 and 3/4 positions), polymerizing outward along the cell
periphery to form arcs (Fig. 2, middle cell). (ii) The transition
between the 11- and 12-min time points for the middle cell is
equivalent to that in the 0:15 panel of Fig. 1 but with greater
resolution, showing simultaneous assembly of FtsZ at daughter sites and
disappearance at midcell. (iii) The assembly of the FtsZ structure is
detectably complete within 1 min. A more detailed time course and
discussion of the last step of septation are presented below.
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FIG. 2.
Three-dimensional image reconstruction of a shorter time
course showing a fluorescence image of three adjacent growing cells, in
vertical orientation, with the central cell undergoing complete
septation and cell separation. Cells in the left-hand panels are viewed
without rotation; the other panels show cells viewed with rotation to
highlight the FtsZ annular structure as it condenses and duplicates.
Times are shown in minutes; complete division of the central cell
occurred between the 12- and 22-min time points. Note the formation of
arcs in the 11-min panel that form nearly complete rings in the 12-min
panel. Also note the fluorescence at the bottom pole of the dividing
cell in the 11-min panel. Bar, 1.3 µm.
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|
Many of the images of the FtsZ rings obtained by deconvolution and
subsequent rotation appeared to exhibit discontinuities in their
fluorescence (Fig. 2), suggesting that the rings were not uniform. Even
rings from IFM experiments appeared to have segmented fluorescence
after deconvolution and rotation (data not shown). To independently
confirm whether this observation was true or was an artifact of the
optical sectioning, we obtained conventional fluorescence images of
cells containing FtsZ-GFP rings that were parallel to the plane of the
coverslip so that they could be viewed directly in cross section (see
Materials and Methods). It is clear from these images that the FtsZ
rings are uniformly fluorescent, with no detectable discontinuities (Fig. 3). Therefore, it is likely that
the gaps in fluorescence that we observed with rotated images from
deconvolution stem from z-axis resolution problems. Since
z-axis resolution is limited by the thickness of the optical
sections, when out-of-focus light is subtracted out by the
deconvolution algorithm, dropouts can occur. These dropouts are most
frequent at the top and bottom of the cell, which usually is captured
in only one optical section, whereas the sides of the cell are captured
in several optical sections. Therefore, despite the stunningly crisp
images delivered by deconvolution techniques, it is important not to
overinterpret the z-axis components of these images,
particularly in small cells such as bacteria and in cells with fine
structures such as FtsZ rings.
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FIG. 3.
Visualization of FtsZ ring cross sections in
immobilized, live cells. Shown are different cells, perpendicular to
the plane of the coverslip, in which the FtsZ-GFP fluorescence displays
a complete, uniform ring. Bar, 2 µm.
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Formation of an FtsZ spiral.
It was shown previously that
higher levels of FtsZ-GFP expression correlated with the generation of
FtsZ spirals (22). Such spirals appear to be nonfunctional
for cell division. Fluorescent spiral structures were also observed
when FtsZ was overexpressed in the presence of FtsA-GFP (22)
and ZipA-GFP (data not shown). Furthermore, FtsZ spirals were observed
by IFM of an ftsZ26 mutant, and these spiral structures can
invaginate to form dramatic spiral septa (3). We took
advantage of our ability to monitor FtsZ dynamics by examining the
formation of a spiral in an atypical cell with deconvolution imaging
(Fig. 4). Initially, the cell shown
contained a bright fluorescent midcell ring. However, instead of
constricting, this ring became distorted, and fluorescent polymers emanated outward from the central structure in a spiral (Fig. 4, 36-min
panel). It is likely that the formation of the spiral may have been a
result of higher levels of FtsZ-GFP relative to FtsZ in this particular
cell, which failed to divide within several hours after the images were
taken.
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FIG. 4.
Formation of a spiral FtsZ-GFP polymer at midcell. Shown
is a time course of a fluorescent FtsZ ring that ultimately fails to
participate in septation and instead forms a clear spiral that emanates
away from the midcell. The cell, expressing FtsZ-GFP, was grown in
microculture and failed to divide over the several hours of growth.
Times are shown in minutes. Images on the left for each time point are
unrotated, whereas images on the right are rotated to reveal the
cross-sectional structure. An arrow in the bottom panel highlights the
developing spiral.
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FtsZ dynamics during the final stages of cytokinesis.
Analysis
of the FtsZ-GFP structure in the later stages of cytokinesis, defined
as the last quarter or approximately 30 min before cell separation when
midcell constriction is easily detectable, revealed several interesting
structural changes. As the FtsZ-GFP structure condensed at the leading
edge of the cell invagination, the lumen of the ring could no longer be
resolved, and finally just a small dot of fluorescence remained (Fig.
2, 8- and 11-min time points; also data not shown). This constriction
at the leading edge of the invagination was also observed in the
original immunogold studies of FtsZ rings (6). A detailed
and high-resolution time course of an entire single cell undergoing
late cytokinesis is shown in Fig. 5A.
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FIG. 5.
Depolymerization and reorganization of FtsZ during the
last stages of septation, as shown by three-dimensional reconstruction.
(A) A single growing cell during cytokinesis. To aid visualization of
the cell outline, the membrane was stained with FM4-64 vital dye. Note
the disappearance of the loop formed at 20 min by the 25-min time
point, followed by reappearance at 28 min. Arrows point to putative
nucleation sites for FtsZ assembly in daughter cells. Also note that at
35 min, the FtsZ structures were formed at the future daughter cell
division sites while the old FtsZ structure was still located as a
highly condensed form at midcell. In order to show FtsZ rings in the
daughter cell more clearly, the cells at 35 and 37 min are shown at a
tilted angle. (B) A more detailed picture of a single growing cell,
showing dynamics of the FtsZ loops. The bright dot is the condensed
FtsZ ring, and the rings at the 6-min time point are formed in the
middles of daughter cells. The numbers throughout the figure represent
the time course, in minutes, after the first image was acquired. Bars,
1.3 µm.
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One surprising finding was that during the process of contraction,
loops of fluorescence often emanated from the central structure. Close
examination and rotation of these structures revealed that two FtsZ-GFP
loops emerged from the parent structure in a figure eight morphology
and were oriented such that they pointed away from the division site. A
detailed examination of one such loop is shown in Fig. 5B.
Interestingly, the loops were transient. As with the assembly of the
FtsZ ring, the dissipation of these loops did not appear to be uniform
and took less than 6 min under our experimental conditions (Fig. 5B).
In the whole-cell time course (Fig. 5A), the loop appears at the 6-min
time point and appears to change direction and intensity until it is
most obvious at 20 min. At the 25- to 33-min time points, the loop
appears to be collapsed; it then disappears when the medial ring
disappears. During the time interval represented in this series, we
also observed transient fluorescent segments in other parts of the
cell, including the cell poles and other locations not corresponding to
the 1/4 and 3/4 positions (Fig. 2, 11-min panel; Fig. 5A, 0- and 10-min panels). These may reflect a normal process of attempted transient assembly by FtsZ-GFP, perhaps due to the appearance of a division site
signal. These segments and the loops also may be artifacts of the GFP
tag or the presence of excess FtsZ in the cell, although the specific
locations of attempted assembly would argue that transient,
low-affinity division site signals may still exist. If the polar
fluorescence observed only at this time point represents FtsZ-GFP, then
it might reflect attempted polar assembly because of the natural
affinity of FtsZ for polar sites. Rapid inactivation of FtsZ assembly
could then occur via the min system (12).
As was shown above, during the very last stages of
cytokinesis the FtsZ loops disappeared at the same time that
new FtsZ rings were nucleating assembly at daughter cell division
sites. This nucleation was clearly visible as a dot at the 1/4 position
and another at the 3/4 position (Fig. 5A, 33-min panel). Interestingly, the dots appeared to be on opposite sides of the dividing cell.
| |
DISCUSSION |
In this work, we have monitored the movement of FtsZ-GFP in
individual, growing E. coli cells in real time over the
entire cell division cycle. We have shown that FtsZ-GFP is capable of rapid, dynamic assembly and disassembly. Our work with live cells complements a previous IFM study of fixed cells with a mutant FtsZ
protein (FtsZ84), in which the protein appeared to rapidly reassemble
and disassemble after shifts between nonpermissive and permissive
temperatures (2).
One major question in bacterial cell division is how assembly and
disassembly of FtsZ and the septation complex are regulated to occur
only once per cell cycle. The transient FtsZ-GFP loops that we observed
during the late stages of cytokinesis may reflect the loss of FtsZ from
the condensing, contracting central structure. The forces behind the
condensation of the FtsZ structure are not known, but one possibility
is that these loops represent FtsZ polymers that are released,
springlike, from the constraints of the condensing structure and that
the apparently repeated nature of loop formation could reflect the
continuous need to remove FtsZ subunits from the structure as it
contracts. Another possible explanation is that the loops are involved
in providing the cytokinetic force by pushing the daughter cell walls
apart. Since FtsZ-GFP is incorporated into the FtsZ structure, it is
possible that the loops normally do not exist but are a way to remove
some of the GFP-tagged protein. IFM analysis failed to reveal obvious
loops (32), but the background fluorescence in IFM is higher
and it is difficult to resolve such fine structures, even with
deconvolution.
Attempts by FtsZ-GFP to assemble elsewhere in the cell were also
apparent in the time-lapse analysis. Although it is possible that these
attempts also represent artifacts of the fusion protein, we favor the
possibility that these events are aborted assembly events at
low-affinity sites in the cell by higher-than-normal levels of FtsZ.
Such events might not occur at lower FtsZ levels, but they indicate
that assembly of unstable structures can occur at multiple locations.
Future IFM studies of cells with slightly elevated levels of FtsZ
should provide additional evidence for or against these possibilities.
Another new observation presented here is the appearance, in wild-type
cells expressing FtsZ-GFP, of new FtsZ rings precisely when the old
FtsZ ring has successfully completed its task and is disassembling. The
most likely explanation for the differences between our results and
previous findings is the small excess of FtsZ in the form of FtsZ-GFP.
Normally, there is only enough FtsZ for one ring per cell
(13), so that newborn cells, despite having competent
midcell division sites, must wait until FtsZ levels are above a
critical concentration. Such a concentration may be related to the 2 µM concentration required for in vitro assembly of FtsZ protofilament
bundles (34). Under our experimental conditions,
twofold-higher FtsZ/FtsZ-GFP levels would result in FtsZ in excess of
what is required at midcell to assemble at new division sites as soon
as they become active. Since FtsZ-GFP assembly was never observed at
quarter sites earlier than the late midcell constriction stage, one
possibility is that this is the earliest that the new division sites
become active for FtsZ recruitment. Another possibility is that the
division sites are present even earlier but that FtsZ can assemble
there only when the central structure is disassembling, releasing large
amounts of free FtsZ. Interestingly, much of this released protein may
immediately participate in the assembly process at new sites. The
important conclusion from our data is that in our system, the elusive
signal for forming a new division site occurs not in newborn cells, as
would be predicted by existing models, but instead earlier in the cell
cycle, in the oldest cells undergoing cytokinesis. The localization of
periseptal annuli (30), as well as F plasmid, to the 1/4 and
3/4 sites of predivisional cells (16, 26) implies that
structures that may be prerequisites to the division site are already
in place and active at these positions. This possibility is supported
by recent studies showing that FtsZ or FtsZ84 localizes to the 1/4 and
3/4 sites in cells that fail to divide at midcell (1, 2, 28,
35), which is also true for a GFP fusion to FtsK in nondividing cells (35). However, the important distinction between our
present results and these is that our cells with FtsZ-GFP divided
normally, without any obvious cell cycle delay. Therefore, the
localization of FtsZ-GFP to the quarter sites in our experiments is
clearly not due to premature disassembly of the medial FtsZ ring but
rather is likely due to excess FtsZ that is recruited to the new
division sites. The fact that we never saw FtsZ localize to these sites until septation was nearly complete argues that FtsZ targeting requires
sufficient cellular FtsZ levels and perhaps an additional assembly
factor that might be released upon segregation of the daughter
nucleoids.
FtsZ appears to nucleate at new division sites as a fluorescent dot,
followed by what seems to be an arc of fluorescence that quickly
becomes a complete ring. This process is very difficult to see, even in
our real-time studies with deconvolution. However, these results
strongly support recent work by Addinall and Lutkenhaus on a spherical
mutant of E. coli in which FtsZ appeared to nucleate at a
point and then polymerize as an arc (3). Therefore, it is
likely that the model proposed by Addinall and Lutkenhaus can be
generalized to normal, rod-shaped E. coli cells.
It will be interesting to find out if the dynamic and structural
behavior of FtsZ observed here is also conserved in more distantly
related prokaryotic species, such as gram-positive bacteria, mycoplasmas, and archaea. Moreover, it would be interesting to study the dynamics of FtsZ assembly in strains carrying mutations in
ftsW, which appears to destabilize the FtsZ ring
(7). The future use of in vivo reporters with different
emission wavelengths, combined with deconvolution imaging of individual
wild-type and mutant cells, should allow a more detailed study of the
structure and function of the prokaryotic cell division machine as it
functions in real time.
We thank X. Yu, D. Ehrhardt, P. Christie, D. Hereld, P. Zuber,
and F. Cabral for valuable discussions and T. Vida for reagents.
This work was supported by grants from the NSF (MCB-9513521) and the
Texas Advanced Research Program (011618-016) to W.M.
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Temperature shift experiments with an ftsZ84(Ts) strain reveal rapid dynamics of FtsZ localization and indicate that the Z ring is required throughout septation and cannot reoccupy division sites once constriction has initiated.
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Addinall, S. G., and J. Lutkenhaus.
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Abstract
Introduction
