Polymerization of FtsZ is essential for bacterial cell division. In this report, we detail simple protocols to monitor FtsZ polymerization activity and discuss the influence of buffer composition. The protocols can be used to study the interaction of FtsZ with regulatory proteins or antibacterial drugs that affect FtsZ polymerization.
Under bakteriel celledeling den væsentlige protein FtsZ samles i midten af cellen til dannelse af den såkaldte Z-ringen. FtsZ polymeriserer i lange filamenter i nærvær af GTP in vitro, og polymerisation er reguleret af flere accessoriske proteiner. FtsZ polymerisation er blevet grundigt undersøgt in vitro ved hjælp af grundlæggende metoder, herunder lysspredning, sedimentering, GTP hydrolyse-assays og elektronmikroskopi. Buffer forhold påvirker både polymerisationsaddukterne egenskaber FtsZ, og evnen til FtsZ at interagere med regulatoriske proteiner. Her beskriver vi protokoller for FtsZ polymerisationsteknikker studier og validere betingelser og kontrol ved hjælp af Escherichia coli og Bacillus subtilis FtsZ som model proteiner. En lav hastighed sedimentation assay indføres der tillader undersøgelse af interaktionen af FtsZ med proteiner, der sammensætter eller tubulate FtsZ polymerer. En forbedret GTPase assay protokol er beskrevet som tillader testningGTP hydrolyse over tid ved hjælp af forskellige betingelser i en 96-brønds plade opsætning med standardiserede inkubationstider der ophæver farvevariation udvikling i phosphat påvisningsreaktion. Forberedelsen af prøver til lysspredning studier og elektronmikroskopi er beskrevet. Adskillige buffere bruges til at etablere passende pH og saltkoncentration for FtsZ polymerisationsteknikker studier. En høj koncentration af KCl er bedst for de fleste af forsøgene. Vores metoder giver et udgangspunkt for in vitro karakteriseringen af FtsZ, ikke kun fra E. coli og B. subtilis, men fra en anden bakterie. Som sådan kan de metoder, der anvendes til undersøgelser af interaktionen af FtsZ med regulatoriske proteiner eller afprøvning af antibakterielle lægemidler, der kan påvirke FtsZ polymerisation.
Den væsentlige bakterielt protein FtsZ er bedst karakteriserede protein bakterielle celledeling maskiner. FtsZ er den prokaryote homolog af tubulin og polymeriserer in vitro i en GTP-afhængig måde. FtsZ er et meget attraktivt mål for nye antibiotika på grund af sin bevarede natur og unikke til bakterier 1,2. I begyndelsen af celledeling, FtsZ danner en cytokinetic ring på midcell, der tjener som et stillads til samling af andre celledeling proteiner. Dannelse af Z-ringen er af afgørende betydning for korrekt lokalisering af divisionen flyet. Monteringsvejledningen dynamik FtsZ er reguleret af flere accessoriske proteiner, såsom (afhængigt af bakteriearter) Minc, SepF, Zapa, UgtP og Ezra 2.. FtsZ polymerisering har været intensivt undersøgt in vitro og mange forskellige strukturer, herunder lige protofilamenter, buede protofilamenter, plader af filamenter, bundter af filamenter og rør af filamenter har været destilskrevet efter samling buffer, nukleotid og yderligere proteiner, der indgår i analysen 3. Arkitekturen i FtsZ protofilamenter in vivo er endnu ikke fuldt forstået, selvom elektron cryotomography eksperimenter i Caulobacter crescentus tyder på, at Z-ringen er samlet fra relativt korte, ikke sammenhængende enkelt protofilamenter uden omfattende bundling 4.
In vitro polymerisationen egenskaber FtsZ og interaktion FtsZ med regulatoriske proteiner er følsomme over for sammensætningen af reaktionsbuffer. For eksempel har vi for nylig beskrev samspillet site for SepF på FtsZ C-terminalen og viste, at en FtsZ B Δ16 C-terminal truncate ikke længere binder til SepF 5.. I en tidligere undersøgelse om SepF-FtsZ Bs interaktion, trunkere en lignende FtsZ B Δ16 stadig cosedimented med SepF, som foreslog, at SepF binder sig til et sekundært site på FtsZ <sup> 6. Forskellen mellem disse undersøgelser var sammensætningen af reaktionsbuffere-ved pH 7,5 var der ingen cosedimentation af SepF med FtsZ trunkat, hvorimod ved pH 6,5 var der cosedimentation. Gündoğdu et al. bemærkes, at SepF ikke er funktionelt, og præcipiterer ved pH 6,5 7, viser, at den observerede cosedimentation ved pH 6,5 forventes at være forårsaget af udfældning af SepF snarere end interaktion med FtsZ B Δ16 C-terminal truncate. Indflydelsen af pH og KCl-koncentration på polymerisationen af FtsZ er tidligere blevet undersøgt. Polymerer af E. coli FtsZ (FtsZ Ec) ved pH 6,5 er længere og mere rigelig end dem, der dannes ved neutral pH 8,9. Tadros et al. har studeret polymerisering af FtsZ Ec i overværelse af monovalente kationer bemærke, at K + binding er knyttet til FtsZ Ec polymerisation og er afgørende for FtsZ aktivitet 10. PH er mere critical, når interaktionen af FtsZ med andre proteiner er undersøgt, som det fremgår af det foregående eksempel SepF og pH-afhængigheden af den hæmmende virkning af Minc på FtsZ 11. Da både pH og saltkoncentration kan påvirke samspillet mellem FtsZ med andre proteiner, er det vigtigt at vælge de rigtige betingelser og kontrolforanstaltninger for FtsZ polymerisationsteknikker studier.
Her beskriver vi protokoller til at studere FtsZ polymerisation og GTPase aktivitet ved lysspredning, elektronmikroskopi, sedimentering og GTPase assays. Højre vinkel lysspredning er en standard metode til at studere FtsZ polymerisation i real tid 12. Vi introducerede et par forbedringer til sedimentering og GTPase assay. Vi præsenterer i detaljer, hvordan at forberede prøver til lysspredning og elektronmikroskopi. Adskillige buffere anvendt i litteraturen til at studere FtsZ polymerisation blev testet, og vi beskriver de bedste betingelser for hvert forsøg. Vi viser også, hvilke kontroller skalindført for at opnå de bedste data.
Disse metoder tillader en hurtig undersøgelse af FtsZ polymerisation, aktivitet og interaktion med andre proteiner ved hjælp af simple metoder og udstyr, som er tilgængelig i de fleste laboratorier. Der findes mere sofistikerede metoder til at studere FtsZ polymerisation, men kræver ofte adgang til mere specialiseret udstyr og / eller ændring af FtsZ med fluorescensmærker 8,13,14. De enkle metoder, der er beskrevet i dette papir illustreres ved hjælp FtsZ fra B. subtilis og E. coli, den mest almindelige Gram + og Gram-modelorganismer. Protokollerne kan tilpasses til enhver anden FtsZ protein. På grundlag af foreløbige analyser med disse nye FtsZs, små ændringer vedrørende tid, buffer eller inkubationstemperaturen kan være nødvendigt for et optimalt resultat. De her beskrevne eksperimenter skal hjælpe med at finde disse optimale betingelser.
We describe a set of methods that allows a quick analysis of FtsZ activity and its interaction with other proteins. Light scattering, sedimentation and GTPase assays as well as electron microscopy have been widely used to study FtsZ polymerization. We have made some improvements to existing protocols, we showed the influence of different conditions on FtsZ assembly, and we propose controls that should be included in FtsZ studies.
We introduce low speed centrifugation to distinguish large structures formed by the association between FtsZ and its interacting proteins from FtsZ polymers. This method shows two advantages over the standard sedimentation assay. First, no background is formed by the FtsZ polymers in the pellet fraction as they are not spun down at 24,600 x g. Second, the amount of FtsZ present in the structure formed with an interacting protein may be calculated from the gel. Two critical steps in this method are the incubation time and the GTP concentration. It is important to centrifuge the large protein structure when it is complete but before it disassembles when all GTP is hydrolyzed. The best control for this study is polymerization of FtsZ with GDP. There is one potential limitation of the assay. FtsZ forms a stable complex with SepF, which can easily be spun down at 24,600 x g. If the sedimentation with another activator or a drug that bundles FtsZ polymers is performed, it may be necessary to adapt the assay. It may be done by changing the incubation time, or increasing the speed of centrifugation.
Proper preparation of the sample is the most important for light scattering experiments. Proteins must be precleared by spinning and all the buffers should be filtered prior to use. If any aggregates are present in the sample, they will disrupt a stable signal obtained from FtsZ polymers. For the analysis of the FtsZ structures by electron microscopy, preparation of a grid is the main step. The time of sample incubation on the grid will have the effect of producing more or less compacted polymers. For bundles of FtsZBs, the time of incubation must be shorter than for FtsZEc and FtsZBs at high KCl concentration. We used a concentration of 12 µM for every sample to be able to compare the results. However, for FtsZBs at 50 mM KCl a lower FtsZ concentration should be used, as 12 µM resulted in a full saturation of the grid. This makes the polymers highly compacted and difficult to detect. Less compacted polymers are better to detect on EM.
The GTPase assay is the only experiment used to study the activity rather than the structures of FtsZ. Mg2+ is necessary for GTP turnover in FtsZ polymers. Thus, in the absence of Mg2+, FtsZ does not hydrolyze GTP. Therefore, a sample with no Mg2+ is the right control in this assay but cations of Mg are present in the FtsZ storage buffer. They may be removed by addition of 1 mM EDTA to the control sample. The critical step in this assay is the incubation time. It is important to stop FtsZ activity after a given time. This is achieved by transferring the FtsZ sample to a malachite green solution in a 96-well plate. However, development of the malachite green color is a continuous process. Thus the measurements must be taken at the same time for every sample. Using a well-planned GTP addition protocol with measurements taken each 30 sec apart in an established order, it is possible to obtain the same incubation and sample handling time for every time point. Another critical step is choosing the concentration of the protein for the experiment. In the experiment we used two different concentrations for FtsZEc and FtsZBs. GTP hydrolysis is much quicker for FtsZEc compared to FtsZBs. The GTPase activity of FtsZEc under chosen conditions and at 12 µM is linear only for maximum 5 min and after that time the hydrolysis rate plateaus. Thus, it is difficult to interpret data from the experiment when performed under these conditions. In this case FtsZEc must be used at lower concentration than FtsZBs to be able to compare activities of both proteins. The GTPase activity of FtsZs from different sources may vary. Thus, the right concentration must be chosen. The concentration for FtsZ polymerization should be well above the critical concentration (in general from 2.5-10 µM). The dynamics of FtsZ assembly and disassembly is also important. Some proteins show a significant lag in polymerization after addition of GTP, as shown for FtsZBs at 50 mM KCl. It is useful to perform the light scattering assay before the GTPase assay to approximate the time of assembly and disassembly of FtsZ polymers. After that, the time of incubation and concentration of protein may be chosen. Since the conditions chosen for FtsZ polymerization are crucial, it is important to use the right pH and KCl concentration in each method. In this work we studied 9 different buffers with pH ranging from 6.5-7.5 and KCl concentrations from 0 M to 300 mM. We noticed that the best condition to analyze FtsZs from B. subtilis and E.coli and their biological activity is at pH that is close to physiological level (7.5) together with a high KCl concentration. At a high KCl concentration, FtsZ has a higher GTPase activity and produces polymers that are better detectable by electron microscopy. We also confirmed that the physiological pH and a high KCl concentration are better for the study of the interaction between FtsZ and regulatory proteins than any other buffers mostly used to study FtsZ assembly. FtsZBs shows a similar activity to FtsZEc when studied at high KCl concentration. In addition, at low salt concentration the influence of pH is more visible than in the buffers with high salt concentration. FtsZ sometimes precipitates when using buffers without KCl, as a result, buffers without salt should be avoided. Sedimentation of FtsZ polymers is low when using buffers with high KCl concentrations. This may be an advantage when studying interactions between FtsZ and proteins that assemble FtsZ filaments such as SepF and ZapA as these higher order structures are easy to detect with centrifugation. In all our experiments we used MgCl2 at a 10 mM concentration. It was shown that a relatively high Mg2+ concentration stabilizes FtsZ polymers and reduces the GTPase activity of FtsZ. In Table 2 results from various studies are summarized describing FtsZ polymerization and GTPase activity at different Mg2+ concentrations using otherwise identical buffer conditions 27. The measured concentration of free cytoplasmic Mg2+ is 0.9 mM3. It should be noticed that GTP will chelate an equivalent amount of Mg2+. Thus, the optimal Mg2+ concentration for GTPase experiments is around 2-2.5 mM, which is close to physiological level3. However, in our experiments we used MgCl2 at a 10 mM concentration to obtain an easily detectable light scattering signal and to stabilize FtsZ polymers during the sedimentation assay.
Although we applied our protocols to FtsZ from the model organisms E. coli and B. subtilis, they can be adapted to FtsZ from any other organism. It has to be noted that the physiological pH, and concentrations of monovalent, and divalent cations differ among organisms. Thus, the optimal conditions for FtsZ polymerization may vary. Differences in doubling time and growth conditions of different bacteria may result in different assembly kinetics of FtsZ and optimal conditions of the experiments. However, our protocol provides a good starting point for the experiments with FtsZs from other organisms. The protocols should be useful for the study of FtsZ with regulatory proteins or the study of effects of small compounds and drugs on FtsZ.
Source | Polymerization [% of FtsZ sedimented] | GTPase [Pi/FtsZ/min] | Mg2+ concentration [mM] | FtsZ concentration [µM] | References |
FtsZEc | ~ 28% | ~ 2.1 | 10 | 12 | This work |
~ 50% | ~ 2.4 | 10 | 12.5 | 27 | |
~ 43% | ~ 3.5 | 5 | 12.5 | 27 | |
~ 27% | ~ 4.6 | 2.5 | 12.5 | 27 | |
ND | ~ 5.4 | 2.5 | 5 | 26 | |
FtsZBs | ~ 30% | ~ 0.8 | 12 | This work | |
~ 52% (with DEAE dextran) | ~ 0.5 | 10 | 10 | 11 | |
ND | ~ 2.25 | 2.5 | 5 | 26 |
Table 2. Effect on Mg2+ on FtsZ polymerization and GTPase. Results from this work compared to published data. All experiments were carried out in 50 mM MES/ NaOH, pH=6.5, 50 mM KCl.
The authors have nothing to disclose.
Work in our laboratory is funded by a VIDI grant from the Netherlands Organisation for Scientific research (to DJS). We thank Marc Stuart and the Department of Electron Microscopy at our university, for assistance with and providing access to the transmission electron microscope.
GTP | Roche | 10106399001 | Part 1, 2, 3, 4, 5, 6, 7 |
Thickwall Polycarbonate Tubes | Beckman Coulter | 343776 | Part 2 |
Optima MAX-XP Ultracentrifuge | Beckman Coulter | 393315 | Part 2, 3 |
Polyallomer Tube with Snap-on Cap | Beckman Coulter | 357448 | Part 3 |
AIDA Bio-package, 1D, 2D, FL | Raytest Isotopenmessgeräte GmbH | 15000001 | Part 4 |
Luminescence Image Analyzer LAS-4000 | Fujifilm | Part 4 | |
Thermo Spectronic AMINCO-Bowman Luminescence Spectrometer | Spectronic Instruments | Part 5 | |
Fluorescence Cell | Hellma Analytics | 105-250-15-40 | Part 5 |
Square 400 Mesh, Copper, 100/vial | Electron Microscopy Sciences | G400-Cu | Part 6 |
CM120 Electron Microscope Operating at 120 kV | Philips | Part 6 | |
96 ml x 0.2 ml Plate | BIOplastics | B70501 | Part 7 |
Malachite Green Phosphate Assay Kit | BioAssay System | POMG-25H | Part 7 |
PowerWave HT Microplate Spectrophotometer | BioTek | Part 7 |