Essential Cell Division Protein FtsZ Assembles into One Monomer-thick Ribbons under Conditions Resembling the Crowded Intracellular Environment

Abstract

Experimental conditions that simulate the crowded bacterial cytoplasmic environment have been used to study the assembly of the essential cell division protein FtsZ from Escherichia coli. In solutions containing a suitable concentration of physiological osmolytes, macromolecular crowding promotes the GTP-dependent assembly of FtsZ into dynamic two-dimensional polymers that disassemble upon GTP depletion. Atomic force microscopy reveals that these FtsZ polymers adopt the shape of ribbons that are one subunit thick. When compared with the FtsZ filaments observed in vitro in the absence of crowding, the ribbons show a lag in the GTPase activity and a decrease in the GTPase rate and in the rate of GTP exchange within the polymer. We propose that, in the crowded bacterial cytoplasm under assembly-promoting conditions, the FtsZ filaments tend to align forming dynamic ribbon polymers. In vivo these ribbons would fit into the Z-ring even in the absence of other interactions. Therefore, the presence of mechanisms to prevent the spontaneous assembly of the Z-ring in non-dividing cells must be invoked. Experimental conditions that simulate the crowded bacterial cytoplasmic environment have been used to study the assembly of the essential cell division protein FtsZ from Escherichia coli. In solutions containing a suitable concentration of physiological osmolytes, macromolecular crowding promotes the GTP-dependent assembly of FtsZ into dynamic two-dimensional polymers that disassemble upon GTP depletion. Atomic force microscopy reveals that these FtsZ polymers adopt the shape of ribbons that are one subunit thick. When compared with the FtsZ filaments observed in vitro in the absence of crowding, the ribbons show a lag in the GTPase activity and a decrease in the GTPase rate and in the rate of GTP exchange within the polymer. We propose that, in the crowded bacterial cytoplasm under assembly-promoting conditions, the FtsZ filaments tend to align forming dynamic ribbon polymers. In vivo these ribbons would fit into the Z-ring even in the absence of other interactions. Therefore, the presence of mechanisms to prevent the spontaneous assembly of the Z-ring in non-dividing cells must be invoked. The FtsZ protein is conserved in most of the prokaryotic organisms and several organelles and plays a central role in microbial and organelle division forming a ring at the division site (1Bi E. Lutkenhaus J. Nature. 1991; 354: 161-164Crossref PubMed Scopus (1158) Google Scholar, 2Vitha S. McAndrew R.S. Osteryoung K.W. J. Cell Biol. 2001; 153: 111-119Crossref PubMed Scopus (235) Google Scholar). It is structurally related to the eukaryotic cytoskeletal tubulin (3Löwe J. Amos L.A. Nature. 1998; 391: 203-206Crossref PubMed Scopus (719) Google Scholar, 4Nogales E. Wolf S.G. Downing K.H. Nature. 1998; 391: 199-203Crossref PubMed Scopus (1808) Google Scholar), binds guanine nucleotides, and has GTPase activity (5de Boer P.A.J. Crossley R. Rothfield L.I. Nature. 1992; 359: 254-256Crossref PubMed Scopus (451) Google Scholar, 6Mukherjee A. Dai K. Lutkenhaus J. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 1053-1057Crossref PubMed Scopus (265) Google Scholar, 7RayChaudhuri D. Park J.T. Nature. 1992; 359: 251-254Crossref PubMed Scopus (341) Google Scholar). In the presence of GDP, the FtsZ monomers undergo a reversible non-cooperative magnesium-linked association to form linear oligomers (8Rivas G. López A. Mingorance J. Ferrándiz M.J. Zorrilla S. Minton A.P. Vicente M. Andreu J.M. J. Biol. Chem. 2000; 275: 11740-11749Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar, 9Sossong Jr., T.M. Brigham-Burke M.R. Hensley P. Pearce Jr., K.H. Biochemistry. 1999; 38: 14843-14850Crossref PubMed Scopus (66) Google Scholar), whereas in the presence of GTP, a variety of polymers are formed in vitro (10Erickson H.P. Taylor D.W. Taylor K.A. Bramhill D. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 519-523Crossref PubMed Scopus (410) Google Scholar, 11Löwe J. Amos L.A. EMBO J. 1999; 18: 2364-2371Crossref PubMed Scopus (180) Google Scholar, 12Lu C. Stricker J. Erickson H.P. Cell Motil. Cytoskeleton. 1998; 40: 71-86Crossref PubMed Scopus (165) Google Scholar, 13Mingorance J. Rueda S. Gómez-Puertas P. Valencia A. Vicente M. Mol. Microbiol. 2001; 41: 83-91Crossref PubMed Scopus (81) Google Scholar, 14Mukherjee A. Lutkenhaus J. J. Bacteriol. 1999; 181: 823-832Crossref PubMed Google Scholar, 15Romberg L. Simon M. Erickson H.P. J. Biol. Chem. 2001; 276: 11743-11753Abstract Full Text Full Text PDF PubMed Scopus (183) Google Scholar, 16Yu X.-C. Margolin W. 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Biochemistry. 1989; 28: 6513-6518Crossref PubMed Scopus (70) Google Scholar, 24Lindner R.A. Ralston G.B. Biophys. Chem. 1997; 66: 57-66Crossref PubMed Scopus (59) Google Scholar). In the case of FtsZ, it has been previously shown that crowding facilitates the formation of magnesium-dependent GDP-FtsZ oligomers (25Rivas G. Fernández J.A. Minton A.P. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 3150-3155Crossref PubMed Scopus (154) Google Scholar). We have investigated the effects of macromolecular crowding upon the more complex and physiologically relevant FtsZ assembly processes taking place in the presence of GTP, which is the most abundant form of the nucleotide in the cell. By means of analytical centrifugation, light scattering, optical, electron, and atomic force microscopy and assays of biochemical activity performed in solutions containing a suitable content of the main physiological osmolytes (see “Materials and Methods” (18Record Jr., T.M. Courtenay E.S. Cayley S. Guttman H.J. Trends Biochem. Sci. 1998; 23: 190-194Abstract Full Text Full Text PDF PubMed Scopus (202) Google Scholar)), we have found that high concentrations of unrelated macromolecules, resembling the crowded Escherichia coli cytoplasm, favor the formation of two-dimensional FtsZ polymer arrays (ribbons). This self-organization process, which is GTP-dependent, retards significantly the FtsZ GTPase activity, the polymer assembly/disassembly dynamics, and the nucleotide exchange within the polymer, when compared with the in the FtsZ filaments formed in We that in the bacterial interior FtsZ tend to into ribbons and that in the non-dividing their formation must be other mechanisms that Z-ring nucleotides, and GTP, from and The nucleotide used atomic force which more GTP, from from analytical from or Macromolecular and with of from used the The concentration of the solutions of and as the at of and of and Scholar). coli FtsZ the the described previously (8Rivas G. López A. Mingorance J. Ferrándiz M.J. Zorrilla S. Minton A.P. Vicente M. Andreu J.M. J. Biol. Chem. 2000; 275: 11740-11749Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar). The protein concentration the assay with FtsZ (see Ref. G. López A. Mingorance J. Ferrándiz M.J. Zorrilla S. Minton A.P. Vicente M. Andreu J.M. J. Biol. Chem. 2000; 275: 11740-11749Abstract Full Text Full Text PDF PubMed Scopus (150) Google and as which a of with Ref. C. Erickson H.P. 1998; PubMed Scopus Google Scholar). and Most of the in vitro of FtsZ in 17Addinall S.G. Holland B. J. Mol. Biol. 2002; 318: 219-236Crossref PubMed Scopus (115) Google and 2001; Scholar; see 12Lu C. Stricker J. Erickson H.P. Cell Motil. Cytoskeleton. 1998; 40: 71-86Crossref PubMed Scopus (165) Google Scholar, 13Mingorance J. Rueda S. Gómez-Puertas P. Valencia A. Vicente M. Mol. Microbiol. 2001; 41: 83-91Crossref PubMed Scopus (81) Google Scholar, 14Mukherjee A. Lutkenhaus J. J. Bacteriol. 1999; 181: 823-832Crossref PubMed Google have been in at concentrations that are at the or the physiological of of these have been done at not the main environment of the bacterial is at low concentration in the cytoplasm of E. coli. to high from to and to are in the cytoplasm of E. coli to prevent and (18Record Jr., T.M. Courtenay E.S. Cayley S. Guttman H.J. Trends Biochem. Sci. 1998; 23: 190-194Abstract Full Text Full Text PDF PubMed Scopus (202) Google Scholar). It has been that several of these physiological osmolytes are of in vitro (18Record Jr., T.M. Courtenay E.S. Cayley S. Guttman H.J. Trends Biochem. Sci. 1998; 23: 190-194Abstract Full Text Full Text PDF PubMed Scopus (202) Google Scholar, S. C. Cayley Biochemistry. 26: PubMed Scopus Google Scholar). Therefore, most of the described in work done in and which the in bacterial cytoplasm, with guanine as This with a high which the GTPase activity of E. coli FtsZ and the rate of of FtsZ filaments a recent review see Ref. 2001; Scholar), it to FtsZ polymers the of most of the performed in work done in the absence of crowding see In these the GTP to the solution an (1 from as from PubMed Scopus Google Scholar). these conditions FtsZ polymers in solution at at g/liter concentrations with FtsZ formation polymer are see and with in GTP a GTP more L. Simon M. Erickson H.P. J. Biol. Chem. 2001; 276: 11743-11753Abstract Full Text Full Text PDF PubMed Scopus (183) Google Scholar). of of FtsZ with the as FtsZ with and on FtsZ to the the protein previously at upon of and GTP (8Rivas G. López A. Mingorance J. Ferrándiz M.J. Zorrilla S. Minton A.P. Vicente M. Andreu J.M. J. Biol. Chem. 2000; 275: 11740-11749Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar). a of and the for at The in in with and the The of FtsZ and the of of of and in the presence of low concentrations of nucleotide performed in a as described in a study (8Rivas G. López A. Mingorance J. Ferrándiz M.J. Zorrilla S. Minton A.P. Vicente M. Andreu J.M. J. Biol. Chem. 2000; 275: 11740-11749Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar). The of the P. P. 2000; PubMed Scopus Google as in the FtsZ in the of Macromolecular and formation of FtsZ polymers in the absence of crowding light at A. Lutkenhaus J. J. Bacteriol. 1999; 181: 823-832Crossref PubMed Google a and to with of and reactions performed at FtsZ in or an in a of GTP and the reactions The formation of FtsZ filaments high at and the in an FtsZ in the of Macromolecular and low and of Biophys. J. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar), which the of FtsZ a of in g/liter the of a low to assay the formation of FtsZ ribbon polymers in the presence of of a concentration of FtsZ at low for at the in an The and the and the protein content in as as in the The protein and filaments but ribbons as the the FtsZ assembly in the absence of that under these conditions of FtsZ filaments in the assay between filaments and ribbons in a The FtsZ ribbons light described which the FtsZ filaments and ribbon and J.M. PubMed Scopus Google in an The is a for FtsZ ribbons of their in the presence of macromolecular but it not the assembly/disassembly of FtsZ filaments (8Rivas G. López A. Mingorance J. Ferrándiz M.J. Zorrilla S. Minton A.P. Vicente M. Andreu J.M. J. Biol. Chem. 2000; 275: 11740-11749Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar). 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Gómez-Puertas P. Valencia A. Vicente M. Mol. Microbiol. 2001; 41: 83-91Crossref PubMed Scopus (81) Google Scholar). FtsZ in in a to have studied the of physiological concentrations of osmolytes in the E. coli cytoplasm see “Materials and on the of E. coli In a the of GDP-FtsZ in a described a of FtsZ with a of the upon (8Rivas G. López A. Mingorance J. Ferrándiz M.J. Zorrilla S. Minton A.P. Vicente M. Andreu J.M. J. Biol. Chem. 2000; 275: 11740-11749Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar). and in the presence of the of the of association of FtsZ in upon protein The that the the concentration studied has an to an FtsZ with a to form and oligomers or protein In the of the of FtsZ (1 in is This to a with an of which is with an FtsZ (8Rivas G. López A. Mingorance J. Ferrándiz M.J. Zorrilla S. Minton A.P. Vicente M. Andreu J.M. J. Biol. Chem. 2000; 275: 11740-11749Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar). are in with a that, a of protein the from to the and of GDP-FtsZ (8Rivas G. López A. Mingorance J. Ferrándiz M.J. Zorrilla S. Minton A.P. Vicente M. Andreu J.M. J. Biol. Chem. 2000; 275: 11740-11749Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar). The upon protein concentration of the FtsZ in a The association that previously described the formation of FtsZ oligomers from monomers in see Ref. G. López A. Mingorance J. Ferrándiz M.J. Zorrilla S. Minton A.P. Vicente M. Andreu J.M. J. Biol. Chem. 2000; 275: 11740-11749Abstract Full Text Full Text PDF PubMed Scopus (150) Google in the work in but with a association in We that in solutions that the main FtsZ in has a to form high order FtsZ in the of GTP in into in FtsZ polymers formed upon of GTP as light the conditions of to polymerization the high concentration of in the the GTPase activity of FtsZ is the FtsZ polymers to disassemble GTP as in other (8Rivas G. López A. Mingorance J. Ferrándiz M.J. Zorrilla S. Minton A.P. Vicente M. Andreu J.M. J. Biol. Chem. 2000; 275: 11740-11749Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar, 14Mukherjee A. Lutkenhaus J. J. Bacteriol. 1999; 181: 823-832Crossref PubMed Google Scholar, A. Lutkenhaus J. 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Chem. 2001; 276: 10577-10580Abstract Full Text Full Text PDF PubMed Scopus (1195) Google Scholar). We the of crowding in the absence of a under which FtsZ has been found to GTP but not J. Rueda S. Gómez-Puertas P. Valencia A. Vicente M. Mol. Microbiol. 2001; 41: 83-91Crossref PubMed Scopus (81) Google Scholar). FtsZ polymers not observed microscopy even in the presence of high concentrations of macromolecular This low and assays described in the presence of FtsZ formed filaments upon of GTP or The of the FtsZ observed in in the presence of high concentrations of or of g/liter of in the formation of of with a of that the of the FtsZ are (3Löwe J. Amos L.A. Nature. 1998; 391: 203-206Crossref PubMed Scopus (719) Google Scholar), these polymers would to a of when g/liter used to simulate crowding polymers observed when the in done in the presence of of GTP GTP a when the that are dynamic structures the of GDP-FtsZ into oligomers is macromolecular at (8Rivas G. López A. Mingorance J. Ferrándiz M.J. Zorrilla S. Minton A.P. Vicente M. Andreu J.M. J. Biol. Chem. 2000; 275: 11740-11749Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar, G. Fernández J.A. Minton A.P. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 3150-3155Crossref PubMed Scopus (154) Google Scholar), the of to the formation of FtsZ polymers at and in The FtsZ in the of force microscopy show that the of the FtsZ polymers crowding is to the association of and their is This is with two-dimensional structures with a of one We propose that the FtsZ polymers crowding are observed is to the of the polymers observed microscopy the between filaments found in the observed in the The FtsZ ribbons crowding to be observed under the when FtsZ used In the absence of FtsZ not form that be under the the of FtsZ from FtsZ of FtsZ filaments not significant in (8Rivas G. López A. Mingorance J. Ferrándiz M.J. Zorrilla S. Minton A.P. Vicente M. Andreu J.M. J. Biol. Chem. 2000; 275: 11740-11749Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar). to the formation of the FtsZ ribbons as a of protein concentration in the presence of In the presence of a or at concentrations between and the the formation of FtsZ ribbons found to be but to for related to the concentration of FtsZ at g/liter and at the of the concentration for FtsZ ribbon formation in the presence of The of ribbon at and g/liter in assay and light (see “Materials and a of g/liter at g/liter of ribbon to the of in the absence of macromolecular (see and several assays and we have that the concentration for the formation of FtsZ ribbons in the presence of macromolecular is to the of concentration for in the absence of that, within the E. coli physiological FtsZ concentration g/liter C. Stricker J. Erickson H.P. Cell Motil. Cytoskeleton. 1998; 40: 71-86Crossref PubMed Scopus (165) Google Scholar, S. Vicente M. Mingorance J. J. Bacteriol. PubMed Scopus Google Scholar)), macromolecular crowding, the assembly of FtsZ into filaments (see in promotes the of FtsZ filaments into ribbons in has been described for tubulin and actin polymerization (22Herzog W. Weber K. Eur. J. Biochem. 1978; 91: 249-254Crossref PubMed Scopus (32) Google Scholar, 24Lindner R.A. Ralston G.B. Biophys. Chem. 1997; 66: 57-66Crossref PubMed Scopus (59) Google Scholar)), and to volume D. Minton A.P. Biophys. Chem. 2002; 98: PubMed Scopus Google Scholar, D. Biophys. Chem. 2002; 98: PubMed Scopus Google Scholar, A.P. Mol. Biochem. PubMed Scopus Google Scholar), FtsZ formation must be in a crowded to the in the FtsZ concentration in the volume The of crowding on formation even in the absence of should be as is the low of under these conditions the of volume on ribbon formation is the in the absence of and presence of shown in we that, under these conditions, in which filaments be is assembly of FtsZ into order When filaments be assembly into ribbons upon the of concentrations of This is from of that the formation of FtsZ ribbons at a protein concentration (1 the concentration for formation a as low assays at concentrations of FtsZ as filaments and not form ribbons concentration of of FtsZ is found as ribbons when the concentration is Macromolecular the of the FtsZ the GTP and the of the presence of high concentrations of macromolecular the FtsZ more the FtsZ found to be dynamic It has been previously that high concentrations of the favor the formation of dynamic FtsZ polymer X.-C. Margolin W. EMBO J. 1997; 16: 5455-5463Crossref PubMed Scopus (220) Google Scholar). This assembly is GTP-dependent and the polymer We to the dynamics of the ribbons under conditions more to the bacterial the dynamic of FtsZ an to the of the on the of FtsZ ribbons in the presence of GTP shown the in macromolecular crowding the FtsZ the other the FtsZ as light scattering, in the absence of crowding The ribbon even more at low FtsZ concentrations The FtsZ GTPase activity of ribbons or when compared with FtsZ filaments substantially in a GTPase from the observed of crowding on the polymer dynamics, these effects at protein concentrations The rate of GTP exchange in the FtsZ ribbons a with The show that the rate in the ribbon is in the filaments GTP exchange in the ribbon even the in which significant GTPase activity observed This would be with the exchange of the nucleotide taking place of the GTP the polymer. We have a of experimental conditions that simulate the physiological bacterial environment more used in vitro This is a to study bacterial cell division at the Atomic force microscopy that under these in vitro conditions that the crowded environment of the cell cytoplasm, FtsZ into one subunit This is it the presence of that of volume a concentration of or the formation of FtsZ ribbons is the assembly and that filaments are not the the ring has been in the cell or to such a in under the have not Mingorance and M. a two-dimensional polymer, one subunit as the structures observed in would be to in observed microscopy when to the of a bacterial cell. The of FtsZ filaments to form order structures have been on J. Chem. 1996; PubMed Scopus Google Scholar), but the in two-dimensional ribbons and has several important the within a ribbon in the of be to to in would have to the would be to the or the cytoplasmic of the proteins in cell the in these ribbons would be to This means that the dynamics and the of the ring be at as FtsZ the polymer In a polymer, on the the of the would be and the in one of the would be to the that is to the of is are FtsZ in a bacterial cell to the of the of the cytoplasmic and as a ribbon into a The of FtsZ E. coli cell has been to from S. Vicente M. Mingorance J. J. Bacteriol. PubMed Scopus Google to C. Stricker J. Erickson H.P. Cell Motil. Cytoskeleton. 1998; 40: 71-86Crossref PubMed Scopus (165) Google in the the concentration of FtsZ not the E. coli cell S. Vicente M. Mingorance J. J. Bacteriol. PubMed Scopus Google the of FtsZ in cells is FtsZ polymerization a the FtsZ in the cell would be into and the of FtsZ concentration for assembly into in work and the for the of FtsZ S. Vicente M. Mingorance J. J. Bacteriol. PubMed Scopus Google we that of the FtsZ protein within a cell should be in the whereas the form of the polymers. an between FtsZ monomers of (see Ref. H.P. Taylor D.W. Taylor K.A. Bramhill D. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 519-523Crossref PubMed Scopus (410) Google Scholar), M. J. J. and J. M. J. Biol. Chem. and a cell of we an of FtsZ monomers in the form of the total found in would be to the cell which would be with the the form of a ribbon even when the of a Stricker J. P. Erickson H.P. Proc. Natl. Acad. Sci. U. S. A. 2002; PubMed Scopus Google that of is found in the This would be with a ribbon monomers the cell. is to that the of and FtsZ is favor that, in the crowded bacterial the main of FtsZ tend to be FtsZ and ribbons (see E. FtsZ in would have a to into high order oligomers and a more to form polymers. the is high under physiological conditions J. P. Escherichia coli and and for D. Scholar), FtsZ is to be found as the as FtsZ polymers. This in would the assembly into ribbons under crowded are observed in the of the cell S. Vicente M. Mingorance J. J. Bacteriol. PubMed Scopus Google that to ring formation the have to in the of the ribbon in vitro be at of the for in not in the absence of (see In vivo several mechanisms have been that prevent ring a that would assembly in the of the cell is the FtsZ protein Proc. Natl. Acad. Sci. U. S. A. 1999; PubMed Scopus Google Scholar, W. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar), which binds to FtsZ would play a role as the assembly described for tubulin Trends Cell Biol. 2000; Full Text Full Text PDF PubMed Scopus Google and for actin T. R. to the and in eukaryotic In E. coli are mechanisms that the of FtsZ and the spontaneous assembly of FtsZ into and the in J. Microbiol. 2002; PubMed Scopus Google and W. Microbiol. 2001; PubMed Scopus Google Scholar). and work FtsZ polymerization and ribbon We are to A. Minton for of the to J. M. for and to C. for