Charged molecules modulate the volume exclusion effects exerted by crowders on FtsZ polymerization

14 p.-4 fig.-2 tab.We have studied the influence of protein crowders, either combined or individually, on the GTP-induced FtsZ cooperative assembly, crucial for the formation of the dynamic septal ring and, hence, for bacterial division. It was earlier demonstrated that high concentrations of inert polymers like Ficoll 70, used to mimic the crowded cellular interior, favor the assembly of FtsZ into bundles with slow depolymerization. We have found, by fluorescence anisotropy together with light scattering measurements, that the presence of protein crowders increases the tendency of FtsZ to polymerize at micromolar magnesium concentration, being the effect larger with ovomucoid, a negatively charged protein. Neutral polymers and a positively charged protein also diminished the critical concentration of assembly, the extent of the effect being compatible with that expected according to pure volume exclusion models. FtsZ polymerization was also observed to be strongly promoted by a negatively charged polymer, DNA, and by some unrelated polymers like PEGs at concentrations below the crowding regime. The influence of mixed crowders mimicking the heterogeneity of the intracellular environment on the tendency of FtsZ to assemble was also studied and nonadditive effects were found to prevail. Far from exactly reproducing the bacterial cytoplasm environment, this approach serves as a simplified model illustrating how its intrinsically crowded and heterogeneous nature may modulate FtsZ assembly into a functional Z-ring.This work was supported by Spanish government BIO2011-28941-C03 (GR and SZ) and BFU 2014-52070-C2-2-P (GR) (www.mineco.gob.es), European Commission HEALTH-F3-2009-223432 (GR) (http://ec.europa.eu/), and Human Frontiers Science Program RGP0050/2010-C102 (GR) (www.hfsp.org).Peer reviewe

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<p>(A) Effect of the specified crowders on FtsZ <i>Cc</i> of polymerization. Bars correspond to the average ± SD of the values determined for each specific crowder at that particular concentration (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0149060#pone.0149060.t001" target="_blank">Table 1</a>). The dashed line corresponds to the <i>Cc</i> value in the absence of crowder. Stars depict the theoretical <i>Cc</i> values for a pure volume exclusion behavior calculated for each crowder using their partial specific volumes as exclusion volume: υ_Dex = 0.61 mL/g [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0149060#pone.0149060.ref031" target="_blank">31</a>], υ_RNase = 0.703 mL/g [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0149060#pone.0149060.ref032" target="_blank">32</a>], υ_Ovo = 0.69 mL/g [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0149060#pone.0149060.ref033" target="_blank">33</a>], and considering tetramers in the particular case of RNase A (calculated value for RNase monomers is shown as a diamond). Ficoll simulated data were calculated with an exclusion volume of 0.96 mL/g. υ_FtsZ = 0.74 mL/g [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0149060#pone.0149060.ref003" target="_blank">3</a>]. (B) <i>Cc</i> values ± SD determined with 10 g/L Ficoll or ovomucoid and 11.8 g/L DNA. Dashed line as in A. (C) and (D) Structures induced on GTP-FtsZ polymers by the presence of RNase A (0.5 g/L FtsZ) and ovomucoid (0.25 g/L FtsZ), respectively. Crowder concentration was 150 g/L. Bar 200 nm. (E) Effect of different crowders (150 g/L) on kinetics of depolymerization of FtsZ (1.5 g/L). Profile in the absence of crowder is shown for comparison.</p

Effect of mixtures involving protein crowders on the polymerization of FtsZ.

<p>(A) and (B) <i>Cc</i> values determined in the presence of the specified crowders and their mixtures up to a total crowder concentration of 150 g/L (whole set of values in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0149060#pone.0149060.t002" target="_blank">Table 2</a>). In the x axis, F, O and R are Ficoll 70, ovomucoid and RNase A, respectively, and the numbers correspond to their concentration alone or in the mixtures, in g/L. Long and short dashed lines correspond to the <i>Cc</i> values in the presence of 150 g/L Ficoll (A and B) and ovomucoid (A) or RNase A (B), respectively. (C) <i>Cc</i> of FtsZ assembly in the presence of the individual crowders specified in the figure and their mixtures (50%). Values correspond to the average ± SD of at least three independent measurements.</p

Concentration dependent effect of Ficoll 70, ovomucoid and RNase A on the polymerization of FtsZ.

<p>Symbols correspond to the variation of FtsZ <i>Cc</i> with the crowders concentration ± SD, average of at least three independent measurements. Lines correspond to the simulations according to the volume exclusion model detailed in the main text. υ_FtsZ = 0.74 mL/g [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0149060#pone.0149060.ref003" target="_blank">3</a>], υ_Ficoll = 0.96 mL/g, υ_RNase = 0.703 mL/g [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0149060#pone.0149060.ref032" target="_blank">32</a>] (tetramers). For ovomucoid, υ_Ovo = 0.69 mL/g (dashed black, pure volume exclusion behavior), υ_Ovo = 1.61 mL/g (dotted black, repulsion with like molecules [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0149060#pone.0149060.ref036" target="_blank">36</a>]) and floating the parameter to υ_Ovo = 6.6 mL/g (solid black, accounting for additional effects). Arrow in the legend indicates the sense in which the exclusion volume increases.</p