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

Mandibular solitary plasmocytoma of the jaw : a case report

Plasma cell tumors are lymphoid neoplasms with an uncontrolled proliferation of B cells. These are divided into localized forms (solitary bone plasmocytoma -SBP- and extramedullary plasmocytoma -EP-) and disseminated forms (multiple myeloma-MM-). The SBP is a rare and controversial disease. The aim of this article is the analysis of this entity based on the presentation of a 64-year-old man without previous medical history, with a mass in the left mandibular angle extending to the parotid region on the same side. The panoramic radiography, computed tomography and magnetic resonance imaging showed an osteolytic lesion 6.5 x 5 x 6.7 cm in the mandibular angle with infiltration of the masticator space and left parotid region. The normality of the extension study, and histopathological examination confirmed the diagnosis of SBP. The patient received treatment with radiotherapy with good outcome. © Medicina Oral

Isolation, Characterization and Lipid-Binding Properties of the Recalcitrant FtsA Division Protein from Escherichia coli

We have obtained milligram amounts of highly pure Escherichia coli division protein FtsA from inclusion bodies with an optimized purification method that, by overcoming the reluctance of FtsA to be purified, surmounts a bottleneck for the analysis of the molecular basis of FtsA function. Purified FtsA is folded, mostly monomeric and interacts with lipids. The apparent affinity of FtsA binding to the inner membrane is ten-fold higher than to phospholipids, suggesting that inner membrane proteins could modulate FtsA-membrane interactions. Binding of FtsA to lipids and membranes is insensitive to ionic strength, indicating that a net contribution of hydrophobic interactions is involved in the association of FtsA to lipid/membrane structures

Control by Potassium of the Size Distribution of Escherichia coli FtsZ Polymers Is Independent of GTPase Activity

The influence of potassium content (at neutral pH and millimolar Mg(2+)) on the size distribution of FtsZ polymers formed in the presence of constantly replenished GTP under steady-state conditions was studied by a combination of biophysical methods. The size of the GTP-FtsZ polymers decreased with lower potassium concentration, in contrast with the increase in the mass of the GDP-FtsZ oligomers, whereas no effect was observed on FtsZ GTPase activity and critical concentration of polymerization. Remarkably, the concerted formation of a narrow size distribution of GTP-FtsZ polymers previously observed at high salt concentration was maintained in all KCl concentrations tested. Polymers induced with guanosine 5'-(¿,ß-methylene)triphosphate, a slowly hydrolyzable analog of GTP, became larger and polydisperse as the potassium concentration was decreased. Our results suggest that the potassium dependence of the GTP-FtsZ polymer size may be related to changes in the subunit turnover rate that are independent of the GTP hydrolysis rate. The formation of a narrow size distribution of FtsZ polymers under very different solution conditions indicates that it is an inherent feature of FtsZ, not observed in other filament-forming proteins, with potential implications in the structural organization of the functional Z-ringPeer Reviewe

MinC protein shortens FtsZ protofilaments by preferentially interacting with GDP-bound subunits

12 p.-8 fig.The interaction of MinC with FtsZ and its effects on FtsZ polymerization were studied under close to physiological conditions by a combination of biophysical methods. The Min system is a widely conserved mechanism in bacteria that ensures the correct placement of the division machinery at midcell. MinC is the component of this system that effectively interacts with FtsZ and inhibits the formation of the Z-ring. Here we report that MinC produces a concentration-dependent reduction in the size of GTP-induced FtsZ protofilaments (FtsZ-GTP) as demonstrated by analytical ultracentrifugation, dynamic light scattering, fluorescence correlation spectroscopy, and electron microscopy. Our experiments show that, despite being shorter, FtsZ protofilaments maintain their narrow distribution in size in the presence of MinC. The protein had the same effect regardless of its addition prior to or after FtsZ polymerization. Fluorescence anisotropy measurements indicated that MinC bound to FtsZ-GDP with a moderate affinity (apparent KD ∼10 μM at 100 mm KCl and pH 7.5) very close to the MinC concentration corresponding to the midpoint of the inhibition of FtsZ assembly. Only marginal binding of MinC to FtsZ-GTP protofilaments was observed by analytical ultracentrifugation and fluorescence correlation spectroscopy. Remarkably, MinC effects on FtsZ-GTP protofilaments and binding affinity to FtsZ-GDP were strongly dependent on ionic strength, being severely reduced at 500 mM KCl compared with 100 mM KCl. Our results support a mechanism in which MinC interacts with FtsZ-GDP, resulting in smaller protofilaments of defined size and having the same effect on both preassembled and growing FtsZ protofilaments.This work was supported in part by Human Frontier Science Program Grant RGP0050/2010 (to G. R. and W. M.), European Commission Contract HEALTH-F3-2009-223431 (to G. R.), and Spanish Government Grants BIO2008-04478-C03 (to G. R.), CSICPIE-201020I001 (to C. A.), BFU2010- 14910 (to S. Z.), and BIO2011-28941-C03 (to G. R. and S. ZPeer reviewe

Development of a homogeneous fluorescence anisotropy assay to monitor and measure FtsZ assembly in solution

We present here a fluorescence anisotropy method for the quantification of the polymerization of FtsZ, an essential protein for cytokinesis in prokaryotes whose GTP-dependent assembly initiates the formation of the divisome complex. Using Alexa 488 labeled wild-type FtsZ as a tracer, the assay allows determination of the critical concentration of FtsZ polymerization from the dependence of the measured steady-state fluorescence anisotropy on the concentration of FtsZ. The incorporation of the labeled protein into FtsZ polymers and the lack of spectral changes on assembly were independently confirmed by time-resolved fluorescence and fluorescence correlation spectroscopy. Critical concentration values determined by this new assay are compatible with those reported previously under the same conditions by other well-established methods. As a proof of principle, data on the sensitivity of the assay to changes in FtsZ assembly in response to Mg2+ concentration or to the presence of high concentrations of Ficoll 70 as crowding agent are shown. The proposed method is sensitive, low sample consuming, rapid, and reliable, and it can be extended to other cooperatively polymerizing systems. In addition, it can help to discover new antimicrobials that may interfere with FtsZ polymerization because it can be easily adapted to systematic screening assays. © 2011 Elsevier Inc. All rights reserved.Peer Reviewe

Secular mindfulness

<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