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Characterization of Caulobacter crescentus FtsZ Protein Using Dynamic Light Scattering*

Open AccessPublished:May 09, 2012DOI:https://doi.org/10.1074/jbc.M111.309492
      The self-assembly of the tubulin homologue FtsZ at the mid-cell is a critical step in bacterial cell division. We introduce dynamic light scattering (DLS) spectroscopy as a new method to study the polymerization kinetics of FtsZ in solution. Analysis of the DLS data indicates that the FtsZ polymers are remarkably monodisperse in length, independent of the concentrations of GTP, GDP, and FtsZ monomers. Measurements of the diffusion coefficient of the polymers demonstrate that their length is remarkably stable until the free GTP is consumed. We estimated the mean size of the FtsZ polymers within this interval of stable length to be between 9 and 18 monomers. The rates of FtsZ polymerization and depolymerization are likely influenced by the concentration of GDP, as the repeated addition of GTP to FtsZ increased the rate of polymerization and slowed down depolymerization. Increasing the FtsZ concentration did not change the size of FtsZ polymers; however, it increased the rate of the depolymerization reaction by depleting free GTP. Using transmission electron microscopy we observed that FtsZ forms linear polymers in solutions which rapidly convert to large bundles upon contact with surfaces at time scales as short as several seconds. Finally, the best studied small molecule that binds to FtsZ, PC190723, had no stabilizing effect on Caulobacter crescentus FtsZ filaments in vitro, which complements previous studies with Escherichia coli FtsZ and confirms that this class of small molecules binds Gram-negative FtsZ weakly.
      Background: Self-assembly of the tubulin-homologue FtsZ is critical in bacterial cell division.
      Results: Dynamic light scattering (DLS) measurements provide insight into the kinetics and stable length of Caulobacter crescentus FtsZ in vitro.
      Conclusion: C. crescentus FtsZ forms short linear polymers in solution with the assembly rate depending on the concentrations of GTP and GDP.
      Significance: DLS is a valuable technique for studying the polymerization of cytoskeletal proteins.

      Introduction

      FtsZ is a widely conserved protein that is found in many species of Eubacteria, Archaea, and higher plants (
      • Kiessling J.
      • Kruse S.
      • Rensing S.A.
      • Harter K.
      • Decker E.L.
      • Reski R.
      Visualization of a cytoskeleton-like FtsZ network in chloroplasts.
      ,
      • Paez A.
      • Tarazona P.
      • Mateos-Gil P.
      • Velez M.
      Self-organization of curved living polymers: FtsZ protein filaments.
      ,
      • Li Z.
      • Trimble M.J.
      • Brun Y.V.
      • Jensen G.J.
      The structure of FtsZ filaments in vivo suggests a force-generating role in cell division.
      ,
      • Margalit D.N.
      • Romberg L.
      • Mets R.B.
      • Hebert A.M.
      • Mitchison T.J.
      • Kirschner M.W.
      • RayChaudhuri D.
      Targeting cell division: small-molecule inhibitors of FtsZ GTPase perturb cytokinetic ring assembly and induce bacterial lethality.
      ,
      • Erickson H.P.
      • Anderson D.E.
      • Osawa M.
      FtsZ in bacterial cytokinesis: cytoskeleton and force generator all in one.
      ,
      • Chen Y.
      • Erickson H.P.
      Conformational changes of FtsZ reported by tryptophan mutants.
      ). FtsZ plays an important role in cell division in bacteria (
      • Romberg L.
      • Levin P.A.
      Assembly dynamics of the bacterial cell division protein FTSZ: poised at the edge of stability.
      ). During division, it forms polymers that assemble into a ring-like structure (the “Z-ring”) at the division plane (
      • Addinall S.G.
      • Lutkenhaus J.
      FtsZ-spirals and -arcs determine the shape of the invaginating septa in some mutants of Escherichia coli.
      ,
      • Bi E.F.
      • Lutkenhaus J.
      FtsZ ring structure associated with division in Escherichia coli.
      ,
      • Harry E.J.
      Bacterial cell division: regulating Z-ring formation.
      ,
      • Weart R.B.
      • Levin P.A.
      Growth rate-dependent regulation of medial FtsZ ring formation.
      ,
      • Quardokus E.M.
      • Brun Y.V.
      DNA replication initiation is required for mid-cell positioning of FtsZ rings in Caulobacter crescentus.
      ). The Z-ring lines the inner face of the cytoplasmic membrane and guides the localization of other cell division proteins to establish the site of division and to initiate cytokinesis (
      • Li Z.
      • Trimble M.J.
      • Brun Y.V.
      • Jensen G.J.
      The structure of FtsZ filaments in vivo suggests a force-generating role in cell division.
      ,
      • Goehring N.W.
      • Beckwith J.
      Diverse paths to midcell: assembly of the bacterial cell division machinery.
      ,
      • Reija B.
      • Monterroso B.
      • Jiménez M.
      • Vicente M.
      • Rivas G.
      • Zorrilla S.
      Development of a homogeneous fluorescence anisotropy assay to monitor and measure FtsZ assembly in solution.
      ). The assembly of FtsZ polymers is triggered by binding to GTP (
      • Nogales E.
      • Downing K.H.
      • Amos L.A.
      • Löwe J.
      Tubulin and FtsZ form a distinct family of GTPases.
      ). FtsZ polymerization is dynamic and terminates once free GTP is hydrolyzed to GDP (
      • Mukherjee A.
      • Lutkenhaus J.
      Analysis of FtsZ assembly by light scattering and determination of the role of divalent metal cations.
      ).
      As the earliest identified prokaryotic cytoskeletal protein (
      • Bi E.F.
      • Lutkenhaus J.
      FtsZ ring structure associated with division in Escherichia coli.
      ), FtsZ has been widely studied by the bacterial cell biology community, yet its polymer structure and in vivo mechanism of action are still not understood in detail. Real-time measurements of the kinetics of FtsZ polymerization and depolymerization may provide important mechanistic insight into the role of this protein in bacterial cell division.
      FtsZ homologues from different organisms share significant structural similarity yet have different biochemical and biophysical properties (
      • Andreu J.M.
      • Schaffner-Barbero C.
      • Huecas S.
      • Alonso D.
      • Lopez-Rodriguez M.L.
      • Ruiz-Avila L.B.
      • Núñez-Ramírez R.
      • Llorca O.
      • Martín-Galiano A.J.
      The antibacterial cell division inhibitor PC190723 is an FtsZ polymer-stabilizing agent that induces filament assembly and condensation.
      ,
      • Thanbichler M.
      • Shapiro L.
      MipZ, a spatial regulator coordinating chromosome segregation with cell division in Caulobacter.
      ,
      • Beuria T.K.
      • Krishnakumar S.S.
      • Sahar S.
      • Singh N.
      • Gupta K.
      • Meshram M.
      • Panda D.
      Glutamate-induced assembly of bacterial cell division protein FtsZ.
      ). For example, the small molecule PC190723 stabilizes FtsZ filaments from Staphylococcus aureus and Bacillus subtilis but not from Escherichia coli (
      • Andreu J.M.
      • Schaffner-Barbero C.
      • Huecas S.
      • Alonso D.
      • Lopez-Rodriguez M.L.
      • Ruiz-Avila L.B.
      • Núñez-Ramírez R.
      • Llorca O.
      • Martín-Galiano A.J.
      The antibacterial cell division inhibitor PC190723 is an FtsZ polymer-stabilizing agent that induces filament assembly and condensation.
      ). It has been proposed that the reduced antibiotic activity of this compound against Gram-negative bacteria arises from its weak binding to FtsZ in Gram-negative organisms.
      In this paper, we use dynamic light scattering (DLS)
      The abbreviations used are: DLS
      dynamic light scattering
      AFM
      atomic force microscopy
      SLS
      static light scattering
      TEM
      transmission electron microscopy.
      to study the polymerization kinetics of FtsZ from Caulobacter crescentus, a Gram-negative bacterium that due to its unique biphasic life cycle and asymmetric cell division has developed into a model organism for cell biological studies (
      • Thanbichler M.
      • Shapiro L.
      MipZ, a spatial regulator coordinating chromosome segregation with cell division in Caulobacter.
      ). The C. crescentus cell carries a single, polar flagellum during the swarmer phase. After differentiation into a stalked cell, division produces a cell containing a flagellum and a daughter cell with a stalk. We anticipate that studies of the polymerization of C. crescentus FtsZ will enhance our understanding of Z-ring formation and cell division in this bacterium.
      DLS is a technique used to determine the distribution of the size of nucleotides and proteins (
      • Hou S.
      • Ziebacz N.
      • Wieczorek S.A.
      • Kalwarczyk E.
      • Sashuk V.
      • Kalwarczyk T.
      • Kaminski T.S.
      • Holyst R.
      Formation and structure of PEI/DNA complexes: quantitative analysis.
      ) through the temporal correlation of the fluctuating intensity of scattered light (
      • Koppel D.E.
      Analysis of macromolecular polydispersity in intensity correlation spectroscopy: the method of cumulants.
      ). We anticipated that DLS could provide quantitative data on the kinetics of FtsZ polymerization and depolymerization in vitro that are not measurable with many other techniques that are often used to study protein assembly, including static light scattering (SLS) (
      • Popp D.
      • Iwasa M.
      • Erickson H.P.
      • Narita A.
      • Maéda Y.
      • Robinson R.C.
      Suprastructures and dynamic properties of Mycobacterium tuberculosis FtsZ.
      ,
      • Small E.
      • Addinall S.G.
      Dynamic FtsZ polymerization is sensitive to the GTP to GDP ratio and can be maintained at steady state using a GTP-regeneration system.
      ,
      • Casini G.L.
      • Graham D.
      • Heine D.
      • Garcea R.L.
      • Wu D.T.
      In vitro papillomavirus capsid assembly analyzed by light scattering.
      ), transmission electron microscopy (TEM) (
      • Mukherjee A.
      • Lutkenhaus J.
      Analysis of FtsZ assembly by light scattering and determination of the role of divalent metal cations.
      ), and atomic force microscopy (AFM) (
      • Mingorance J.
      • Tadros M.
      • Vicente M.
      • González J.M.
      • Rivas G.
      • Vélez M.
      Visualization of single Escherichia coli FtsZ filament dynamics with atomic force microscopy.
      ). SLS encompasses both single angle light scattering and multiangle light scattering. Single angle light scattering measures the increase of the intensity of scattered light during polymerization, but this signal does not convey information about the order of polymerization of FtsZ polymers (
      • Small E.
      • Addinall S.G.
      Dynamic FtsZ polymerization is sensitive to the GTP to GDP ratio and can be maintained at steady state using a GTP-regeneration system.
      ). Multiangle light scattering can be used to detect protein assembly by monitoring the molecular mass of the resulting filaments (
      • Casini G.L.
      • Graham D.
      • Heine D.
      • Garcea R.L.
      • Wu D.T.
      In vitro papillomavirus capsid assembly analyzed by light scattering.
      ). It is best combined with off-line molecule fractionation techniques, such as capillary hydrodynamic chromatography and field flow fractionation methods, to resolve not only the average molecular mass of the assemblies but also their distribution (
      • Wyatt P.J.
      Submicrometer particle sizing by multiangle light scattering following fractionation.
      ). TEM (
      • Mukherjee A.
      • Lutkenhaus J.
      Analysis of FtsZ assembly by light scattering and determination of the role of divalent metal cations.
      ) and AFM (
      • Mingorance J.
      • Tadros M.
      • Vicente M.
      • González J.M.
      • Rivas G.
      • Vélez M.
      Visualization of single Escherichia coli FtsZ filament dynamics with atomic force microscopy.
      ) have been used to measure the length of FtsZ polymers adsorbed on surfaces. However, surface contact may enhance polymerization and bundle formation, so that the observed FtsZ filaments may not represent the actual length of polymers suspended in bulk fluids or in the cell (
      • Hamon L.
      • Panda D.
      • Savarin P.
      • Joshi V.
      • Bernhard J.
      • Mucher E.
      • Mechulam A.
      • Curmi P.A.
      • Pastré D.
      Mica surface promotes the assembly of cytoskeletal proteins.
      ). Hence, it would be helpful to supplement AFM and TEM studies of FtsZ with techniques that provide dynamic information. In this paper we describe the first analysis of C. crescentus FtsZ structure and dynamics using DLS and demonstrate the capabilities of this biophysical technique for studying self-assembling proteins in vitro.

      CONCLUSIONS

      We used DLS to study the polymerization and disassembly of FtsZ polymers. This technique enabled us to measure the increase of the intensity of scattered light and the characteristic decay time of the autocorrelation function and provided a quantitative value for the degree of FtsZ polymerization. We envision that the methodological framework established in this study has laid the foundation for the application of this technique to various other cytoskeletal proteins.

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