University of Manchester, School of Biological Sciences, 2.205 Stopford Building, Oxford Road, Manchester M13 9PT, UK
Correspondence
Stephen G. Addinall
stephen.addinall{at}man.ac.uk
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ABSTRACT |
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INTRODUCTION |
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There have been multiple studies of the dynamics of FtsZ polymerization in vitro. In the presence of GDP (Rivas et al., 2000, 2001
; Sossong et al., 1999
) or in the presence of GTP under particular conditions where only single protofilaments are formed (Romberg et al., 2001
), polymerization is proposed to be isodesmic. Under conditions where FtsZ forms more complex polymers, polymerization and the GTPase activity are co-operative (Caplan & Erickson, 2003
; Mukherjee & Lutkenhaus, 1999
; White et al., 2000
).
The technique of right-angled light-scattering has proved useful for real-time monitoring of FtsZ polymerization (Mingorance et al., 2001; Mukherjee & Lutkenhaus, 1999
; Romberg et al., 2001
; Scheffers et al., 2000
, 2002
; White et al., 2000
). In a typical assay using E. coli FtsZ, GTP provided at 40-fold molar excess is completely hydrolysed within 15 min, and depolymerization starts to occur within 8 min of addition of GTP (Mukherjee & Lutkenhaus, 1999
). Since GTP hydrolysis has been shown to occur immediately upon FtsZ polymerization (Scheffers & Driessen, 2002
), GDP levels will increase throughout the reaction, as GTP levels decrease. The effects of these changes in nucleotide concentration as the assay progresses are likely to be complex since GDP-bound and GTP-bound FtsZ molecules have different polymerization characteristics (Mukherjee & Lutkenhaus, 1999
; Rivas et al., 2000
, 2001
; Romberg et al., 2001
; Sossong et al., 1999
). Indeed, previous work has demonstrated that addition of GDP in at least 10 times excess (compared to GTP concentration) causes rapid depolymerization of FtsZ/FtsZ2 co-polymers which are otherwise relatively stable (Mukherjee et al., 2001
). It has also been demonstrated that FtsZ polymers formed in the presence of 10 mM calcium (which have reduced dynamic properties and reduced GTPase activity) are destabilized by addition of GDP (Scheffers et al., 2000
).
Observation of the dynamics of Z-rings suggests that the in vivo polymerization of FtsZ has different properties to that so far observed in vitro. Assembly of Z-rings can occur very quickly [e.g. less than 1 min (Addinall et al., 1997); 13 min (Sun & Margolin, 1998
)], indicating rapid initial polymerization. Subunits within the ring turn over with an approximate half-life of 30 s (Stricker et al., 2002
), thus the intact Z-ring is a very dynamic structure. However, Z-rings can be present for up to 50 min in actively dividing cells of E. coli K-12 (Den Blaauwen et al., 1999
) and Z-rings in ftsI temperature-sensitive filamentous cells (which do not constrict and can persist for multiple generations; Addinall et al., 1996
; Pogliano et al., 1997
) were shown to be equally as dynamic as those in dividing cells (Stricker et al., 2002
). Polymers in the Z-ring therefore undergo prolonged continuous, rapid turnover (Stricker et al., 2002
) in the presence of a constant, relatively high ratio of cellular GTP concentration ([GTP]) to GDP concentration ([GDP]) (Neuhard & Nygaard, 1987
).
In order to better understand the mechanism of in vivo polymerization of FtsZ, we have quantified the effect of alterations in the [GTP]/[GDP] ratio on dynamic FtsZ polymerization in vitro. We show that decreasing the [GTP]/[GDP] ratio reduces both the length of the FtsZ polymerization reaction (which we designate t0·5) and the initial light-scattering maximum (delta). Our approach to quantification of FtsZ polymerization monitored using light-scattering is further validated by our ability to detect lengthening of t0·5 in the presence of GTPS. We have used a GTP-regeneration system to prolong the polymerization steady-state period, allowing maximal GTPase activity and polymerization at constant GTP concentration for periods of up to 85 min, whilst maintaining the dynamic nature of the polymers. Our assay therefore more closely mimics the apparent in vivo situation.
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METHODS |
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For each experiment described in Table 2, reaction buffer and FtsZ were made up in a pool and then split into three. After a baseline had been obtained, either GTP or a mixture of GTP with another nucleotide phosphate was added, and the light-scattering was recorded as above. Thus each experiment had its own GTP-only (0·5 mM final concentration) control reaction. The baseline is the level of light-scattering before GTP is added and after all GTP has been hydrolysed (Mukherjee & Lutkenhaus, 1999
) and represents a further internal control. The delta is the maximal increase in light-scattering in response to addition of GTP (Mukherjee & Lutkenhaus, 1999
). The t0·5 is the time from addition of GTP to the point at which half-maximal light-scattering value is reached as the trace returns to baseline levels, and is a measure of the longevity of the polymerization reaction. Values for baseline, delta and t0·5 were obtained by plotting individual traces on identical axes and measuring these parameters by hand as illustrated in Fig. 1(b)
. This allowed any slight underlying slopes in the traces to be accurately accounted for. Baseline and delta values (measured in arbitrary light-scattering units) and t0·5 values (in seconds) represent the mean of three separate experiments (±standard error) and are expressed as a percentage of the control GTP-only reaction.
GTPase assay.
We routinely measured GTPase activity by TLC using [-32P]GTP (Amersham) essentially as described by RayChaudhuri & Park (1992)
and Yu & Margolin (1997)
. In the presence of acetyl phosphate, we used the malachite green assay for phosphate production which has been used previously to assay FtsZ GTPase (Rivas et al., 2001
; Romberg et al., 2001
). This was preferable to using radiolabelled GTP since, in the presence of the GTP-regeneration system, GDP molecules are recycled.
Negative stain electron microscopy.
FtsZ polymers were visualized by negative stain electron microscopy essentially as described previously (Lu & Erickson, 1998; Mukherjee & Lutkenhaus, 1998a
). Briefly, 10 µl polymerization reactions were applied to carbon-coated copper grids, left for 1 min, then blotted off. Negative staining was performed immediately by applying and blotting off 10 µl 2 % uranyl acetate, repeating this once then allowing the grids to dry. Grids were examined using a Philips 400 transmission electron microscope.
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RESULTS |
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The duration of FtsZ polymerization (t0·5) is highly sensitive to the [GTP]/[GDP] ratio. At a 1/1 [GTP]/[GDP] ratio, t0·5 was reduced to 67 % of the control (GTP only) level and at a 1/2 [GTP]/[GDP] ratio t0·5 fell to 47 % (Table 2). In contrast, ADP had no effect on t0·5 and ATP had minimal effect (74 % at 1/2 ratio; Table 2
). The value of the maximal increase in light-scattering (delta) was sensitive to addition of all nucleotide phosphates tested. Delta dropped to 7480 % of control levels when the ratio was 1/1 and 5269 % at a 1/2 ratio (Table 2
). Baseline light-scattering levels were constant throughout each experiment. Therefore, a decrease in the [GTP]/[GDP] ratio significantly decreases the total amount and the persistence of FtsZ polymers.
A slowing of FtsZ depolymerization due to GTPS has been demonstrated in the presence of 10 mM calcium (Scheffers et al., 2000
). Under these conditions, FtsZ polymers have significantly reduced GTPase activity and polymers are much more stable. Our approach to quantification of dynamic FtsZ polymerization is validated by our ability to detect a significant increase (13 %) in the t0·5 in the presence of GTP
S at a 1 : 1 ratio with GTP (Table 2
). Previous attempts to detect the effects of GTP
S under similar conditions were unsuccessful (Scheffers et al., 2000
).
A GTP-regeneration system can extend the steady-state period of FtsZ polymerization, without altering its dynamic properties
Having established that the [GTP]/[GDP] ratio has significant influence on dynamic FtsZ polymerization, we tested whether FtsZ polymerization could be extended by use of a GTP-regeneration system consisting of acetyl phosphate and acetate kinase (MacNeal et al., 1977; Purich et al., 1982
). In such a way, the [GTP]/[GDP] ratio could be maintained constant for long periods of time. Using FtsZ purified by the method of Mukherjee & Lutkenhaus (1998a)
, we found that addition of 0·013 U acetate kinase ml-1 and 0·5 mM acetyl phosphate increased the t0·5 of FtsZ polymerization approximately twofold (Fig. 2a
, open circles) compared to the control reaction (Fig. 2a
, closed circles). We were able to extend t0·5 up to approximately ninefold by adding increased amounts of acetyl phosphate, with the steady-state period of polymerization in the longest experiments lasting 45 and 85 min (2 and 4 mM acetyl phosphate, respectively; data not shown). Importantly, reactions extended by addition of the GTP-regeneration system returned to baseline levels in the same manner as the control (e.g. Fig. 2a, b
), indicating that the dynamic nature of the assay (see Methods) had not been compromised. We also found that the GTPase activity of FtsZ was not altered by the presence of the phosphate-donor compound (acetyl phosphate) of the GTP-regeneration system (data not shown; see Methods). Together these indicate that depolymerization occurs when GTP becomes limiting, exactly as in control reactions (Mukherjee & Lutkenhaus, 1998b
, 1999
), but that this occurs after exhaustion of acetyl phosphate.
Interestingly, we found that polymerization of this preparation of FtsZ was extended to a similar extent by addition of acetyl phosphate alone (Fig. 2a, filled triangles) and that the t0·5 of the polymerization was dependent upon the concentration of added acetyl phosphate (Fig. 2b
). This was evidently due to contamination of the FtsZ preparation with acetate kinase, since FtsZ purified from strain SGA79 [which has a Tn10 insertion in ackA (Barak et al., 1998
), the gene encoding acetate kinase] required addition of both components of the GTP-regeneration system for an increase of t0·5 (data not shown). As described in Methods, we have found that a combination of two previous FtsZ purification protocols (Lu & Erickson, 1998
; Mukherjee & Lutkenhaus, 1998a
) results in FtsZ with consistently high GTPase activity and no detectable acetate kinase contamination (data not shown).
FtsZ polymer morphology and the effects of calcium are not altered by the GTP-regeneration system
We wanted to establish whether use of the GTP-regeneration system altered certain well-established properties of FtsZ polymers. Firstly, we examined the morphology of FtsZ polymers using negative stain electron microscopy as described previously (Lu & Erickson, 1998; Mukherjee & Lutkenhaus, 1998a
). We found no significant difference in the types or apparent lengths of polymers formed in the presence or absence of the GTP-regeneration system (Fig. 3a, b
) or at different times after addition of GTP in the presence of the GTP-regeneration system (data not shown). Secondly, we examined the well-characterized effects of calcium on FtsZ polymerization. Addition of calcium at 10 mM to FtsZ polymerization assays inhibits the GTPase activity and causes bundling of polymers, resulting in hugely increased light-scattering (Mukherjee & Lutkenhaus, 1999
; Scheffers et al., 2000
; Yu & Margolin, 1997
). We demonstrate that calcium has the same effect on FtsZ polymers in the presence of the GTP-regeneration system (Fig. 3c
).
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DISCUSSION |
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GTP is hydrolysed immediately upon FtsZ polymerization while release of phosphate from the polymers lags behind (Scheffers & Driessen, 2002). FtsZ subunits within polymers have GDP plus phosphate at their active site and therefore are effectively in their GTP-bound configuration (Scheffers & Driessen, 2002
). Scheffers & Driessen (2001
, 2002)
have therefore invoked a GTP-cap model for FtsZ polymerization, similar to that for tubulin. In this model, exchange of guanine nucleotide phosphates takes place at the ends of polymers rather than by diffusion from within polymers. An explanation for the observed effect of GDP in our experiments would be that both GDP and GTP bind to FtsZ monomers and the end subunits of polymers with similar affinities. [Previous data show that GDP binds to FtsZ approximately half as well as GTP, and that GDP can compete with GTP for FtsZ binding (de Boer et al., 1992
; Mukherjee et al., 1993
; Scheffers et al., 2000
; RayChaudhuri & Park, 1992
).] Addition of GTP or GTP-bound FtsZ to the end of a polymer would allow polymerization to continue, whereas addition of GDP or GDP-bound FtsZ would promote depolymerization. Indeed, since in vivo experiments demonstrate continuous, rapid turnover of subunits within the Z-ring (Stricker et al., 2002
), addition of GDP or GDP-bound FtsZ to a growing polymer may cause events similar to microtubule catastrophe. Hence in our experiments, the dynamic polymerization reaction is effectively quenched by an increase in the concentration of GDP.
An alternative explanation for decrease in both t0·5 and delta in the presence of increased GDP could be that mean polymer length is reduced. The concentration of polymer ends would be elevated and therefore GTP would be hydrolysed more quickly. As yet, there is no established method for measurement of FtsZ polymer lengths in such dynamic assays; however, we do not favour this explanation because GTPase activity is not increased in assays with added GDP (data not shown).
Control experiments were performed using ATP or ADP in place of GDP. It has previously been demonstrated that ATP does not bind to FtsZ and cannot compete out binding of GTP to FtsZ (de Boer et al., 1992; Mukherjee et al., 1993
; RayChaudhuri & Park, 1992
). Therefore, we were surprised to find that, although neither ATP or ADP affected the duration of FtsZ polymerization, both had negative effects on the delta value similar to that of GDP. It is possible that ADP and ATP can occupy the nucleotide-binding site on FtsZ transiently and that this is enough to interfere with the very fast initial polymerization (the kinetics of which are largely undescribed). Nevertheless, we note that GDP and GTP
S, both of which are known to bind to FtsZ, had the most severe effects on delta.
Acetate kinase and acetyl phosphate represent a highly efficient system for GTP regeneration which maintains the GTP to GDP ratio at 200 to 1 or greater, even when 90 % of acetyl phosphate has been converted to acetate (Purich et al., 1982). In this study, we have shown that acetate kinase and acetyl phosphate do not significantly alter the dynamic properties of FtsZ characterized thus far in vitro (Mukherjee & Lutkenhaus, 1998b
, 1999
). In the light of our results which demonstrate that FtsZ polymerization is sensitive to the [GTP]/[GDP] ratio, and since binding of GDP by FtsZ favours bending and depolymerization of polymers (Lu et al., 2000
), a regeneration system is preferable to adding high concentrations of GTP to a polymerization reaction. The latter prolongs the polymerization steady state to some extent (Mukherjee & Lutkenhaus, 1999
) but GDP will still accumulate. Other methods which prolong the existence of FtsZ polymers in vitro, such as addition of 10 mM calcium or reduction of magnesium concentration (Mukherjee & Lutkenhaus, 1999
; Scheffers et al., 2000
), serve to reduce the FtsZ GTPase activity and thus the dynamic nature of the polymers.
The intracellular [GTP]/[GDP] ratio in Salmonella typhimurium is approximately 7/1 (Neuhard & Nygaard, 1987) and is likely to be similar in E. coli. Since levels of GTP are unlikely to be affected (even locally at the cell centre) by the strong GTPase of FtsZ (Stricker et al., 2002
), the Z-ring therefore must assemble, constrict and disassemble in conditions of consistently high [GTP]/[GDP] ratio. Consequently, models for mechanisms of initiation of Z-ring assembly and initiation of Z-ring constriction must take this into account, together with the observed constant turnover within the Z-ring (Stricker et al., 2002
). We assert that the gradual build-up of GDP in in vitro polymerization assays used so far is unlike the in vivo situation. We therefore present the FtsZ polymerization in the presence of a GTP-regeneration system as a more in vivo-like assay for FtsZ polymerization which will be useful to identify and characterize proteins which affect critical aspects of the control of cell division.
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ACKNOWLEDGEMENTS |
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Received 14 November 2002;
revised 5 March 2003;
accepted 20 May 2003.