Lars Renner

Cardiolipin microdomains localize to negatively curved regions of Escherichia coli membranes

Many proteins reside at the cell poles in rod-shaped bacteria. Several hypotheses have drawn a connection between protein localization and the large cell-wall curvature at the poles. One hypothesis has centered on the formation of microdomains of the lipid cardiolipin (CL), its localization to regions of high membrane curvature, and its interaction with membrane-associated proteins. A lack of experimental techniques has left this hypothesis unanswered. This paper describes a microtechnology-based technique for manipulating bacterial membrane curvature and quantitatively measuring its effect on the localization of CL and proteins in cells. We confined Escherichia coli spheroplasts in microchambers with defined shapes that were embossed into a layer of polymer and observed that the shape of the membrane deformed predictably to accommodate the walls of the microchambers. Combining this technique with epifluorescence microscopy and quantitative image analyses, we characterized the localization of CL microdomains in response to E. coli membrane curvature. CL microdomains localized to regions of high intrinsic negative curvature imposed by microchambers. We expressed a chimera of yellow fluorescent protein fused to the N-terminal region of MinD—a spatial determinant of E. coli division plane assembly—in spheroplasts and observed its colocalization with CL to regions of large, negative membrane curvature. Interestingly, the distribution of MinD was similar in spheroplasts derived from a CL synthase knockout strain. These studies demonstrate the curvature dependence of CL in membranes and test whether these structures participate in the localization of MinD to regions of negative curvature in cells.

Measuring the stiffness of bacterial cells from growth rates in hydrogels of tunable elasticity

Although bacterial cells are known to experience large forces from osmotic pressure differences and their local microenvironment, quantitative measurements of the mechanical properties of growing bacterial cells have been limited. We provide an experimental approach and theoretical framework for measuring the mechanical properties of live bacteria. We encapsulated bacteria in agarose with a user‐defined stiffness, measured the growth rate of individual cells and fit data to a thin‐shell mechanical model to extract the effective longitudinal Youngs modulus of the cell envelope of Escherichia coli (50–150 MPa), Bacillus subtilis (100–200 MPa) and Pseudomonas aeruginosa (100–200 MPa). Our data provide estimates of cell wall stiffness similar to values obtained via the more labour‐intensive technique of atomic force microscopy. To address physiological perturbations that produce changes in cellular mechanical properties, we tested the effect of A22‐induced MreB depolymerization on the stiffness of E. coli. The effective longitudinal Youngs modulus was not significantly affected by A22 treatment at short time scales, supporting a model in which the interactions between MreB and the cell wall persist on the same time scale as growth. Our technique therefore enables the rapid determination of how changes in genotype and biochemistry affect the mechanical properties of the bacterial envelope.

MinD and MinE Interact with Anionic Phospholipids and Regulate Division Plane Formation in Escherichia coli

Background: Understanding the bacterial division machinery is essential to decoding cellular physiology. Results: The cell division proteins MinD/MinE bind tightly to anionic lipids, which reduces ATPase activity. Conclusion: MinD and MinE interact preferably with anionic lipids that are positioned at the cell pole. Significance: These results provide insight into a mechanism that regulates bacterial cell division and may present a useful target for antimicrobial development. The Min proteins (MinC, MinD, and MinE) form a pole-to-pole oscillator that controls the spatial assembly of the division machinery in Escherichia coli cells. Previous studies identified that interactions of MinD with phospholipids positioned the Min machinery at the membrane. We extend these studies by measuring the affinity, kinetics, and ATPase activity of E. coli MinD, MinE, and MinDE binding to supported lipid bilayers containing varying compositions of anionic phospholipids. Using quartz crystal microbalance measurements, we found that the binding affinity (Kd) for the interaction of recombinant E. coli MinD and MinE with lipid bilayers increased with increasing concentration of the anionic phospholipids phosphatidylglycerol and cardiolipin. The Kd for MinD (1.8 μm) in the presence of ATP was smaller than for MinE (12.1 μm) binding to membranes consisting of 95:5 phosphatidylcholine/cardiolipin. The simultaneous binding of MinD and MinE to membranes revealed that increasing the concentration of anionic phospholipid stimulates the initial rate of adsorption (kon). The ATPase activity of MinD decreased in the presence of anionic phospholipids. These results indicate that anionic lipids, which are concentrated at the poles, increase the retention of MinD and MinE and explain its dwell time at this region of bacterial cells. These studies provide insight into interactions between MinD and MinE and between these proteins and membranes that are relevant to understanding the process of bacterial cell division, in which the interaction of proteins and membranes is essential.

Supported Lipid Bilayers on Spacious and pH-Responsive Polymer Cushions with Varied Hydrophilicity

We report the successful formation of supported multicomponent lipid bilayer membranes (sLBMs) on polymer cushions consisting of a set of alternating maleic acid copolymers. The formation of sLBMs was triggered by a transient reduction of the electrostatic repulsion between the polymer cushions and the lipid vesicles by lowering the solutions pH to 4. Upon formation, the stability of the sLBMs was not affected by subsequent variations of the environmental pH. The degree of hydrophilicity and swelling of the anionic polymer cushions was found to determine both the kinetics of the membrane formation and the mobility of the lipid bilayer with lipid diffusion coefficients in the range from 0.26 to 2.6 microm2s(-1). The introduced polymer cushion system is concluded to provide a versatile base for the integration of active transmembrane proteins in sLBMs.

Charging and structure of zwitterionic supported bilayer lipid membranes studied by streaming current measurements, fluorescence microscopy, and attenuated total reflection Fourier transform infrared spectroscopy.

The authors report on the characterization of the charge formation at supported bilayer lipid membranes (sBLMs) prepared from the zwitterionic lipid 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine on planar silicon dioxide substrates. The charging of the sBLMs was studied in KCl solutions of different ionic strengths between 0.1 and 10 mM by streaming current measurements. In addition, attenuated total reflection Fourier transform infrared spectroscopy and fluorescence microscopy were applied to determine the lipid concentration in the membrane and to study the influence of the harsh conditions (pH 9-2, shear forces) during the electrokinetic measurements on the membrane stability and the lipid diffusion coefficient. The sBLMs were found to be extremely stable. Isoelectric points of about 4 revealed that unsymmetrical adsorption of hydroxide and hydronium ions determined the charging of the outer leaflet of the membrane in the investigated pH range. The diffusion coefficients were found to be rather independent on the ionic strength at neutral and alkaline pH. However, significantly decreased lipid diffusion at pH<4 indicated a charge-induced transition of the fluidic bilayer into a gel/ordered-phase bilayer.

Studying Biomolecule Localization by Engineering Bacterial Cell Wall Curvature

In this article we describe two techniques for exploring the relationship between bacterial cell shape and the intracellular organization of proteins. First, we created microchannels in a layer of agarose to reshape live bacterial cells and predictably control their mean cell wall curvature, and quantified the influence of curvature on the localization and distribution of proteins in vivo. Second, we used agarose microchambers to reshape bacteria whose cell wall had been chemically and enzymatically removed. By combining microstructures with different geometries and fluorescence microscopy, we determined the relationship between bacterial shape and the localization for two different membrane-associated proteins: i) the cell-shape related protein MreB of Escherichia coli, which is positioned along the long axis of the rod-shaped cell; and ii) the negative curvature-sensing cell division protein DivIVA of Bacillus subtilis, which is positioned primarily at cell division sites. Our studies of intracellular organization in live cells of E. coli and B. subtilis demonstrate that MreB is largely excluded from areas of high negative curvature, whereas DivIVA localizes preferentially to regions of high negative curvature. These studies highlight a unique approach for studying the relationship between cell shape and intracellular organization in intact, live bacteria.

Controlled enhancement of transmembrane enzyme activity in polymer cushioned supported bilayer membranes

Reconstitution of transmembrane proteins into supported lipid bilayer (SLB) membranes has often been hampered by strong interactions of protein domains with the underlying solid support leading to loss of both activity and mobility within the plane of the lipid bilayer. Polymer cushioned SLBs can overcome this by avoiding direct contact with the support. To extend this approach we developed an anionic polymer cushion system that allows tunable lipid mobility as well as functional integration of the transmembrane protein β-amyloid precursor protein cleaving enzyme (BACE) into SLBs. Fluorescence recovery after photobleaching analysis revealed a homogeneous distribution and high lateral mobility of the reconstituted BACE in cushioned SLBs while an impaired mobility and inhomogeneous clustering of reconstituted BACE were found in SLBs on silicon oxide substrates. The cushioning of SLBs led to increased incorporation and enhanced enzymatic activity of the reconstituted BACE with a direct correlation between lipid mobility and BACE activity. The utilized polymer cushion system allows the successful reconstitution of transmembrane proteins within SLBs with tunable properties.

Friction-Controlled Traction Force in Cell Adhesion

The force balance between the extracellular microenvironment and the intracellular cytoskeleton controls the cell fate. We report a new (to our knowledge) mechanism of receptor force control in cell adhesion originating from friction between cell adhesion ligands and the supporting substrate. Adherent human endothelial cells have been studied experimentally on polymer substrates noncovalently coated with fluorescent-labeled fibronectin (FN). The cellular traction force correlated with the mobility of FN during cell-driven FN fibrillogenesis. The experimental findings have been explained within a mechanistic two-dimensional model of the load transfer at focal adhesion sites. Myosin motor activity in conjunction with sliding of FN ligands noncovalently coupled to the surface of the polymer substrates is shown to result in a controlled traction force of adherent cells. We conclude that the friction of adhesion ligands on the supporting substrate is important for mechanotransduction and cell development of adherent cells in vitro and in vivo.

Evaluation of the matrix effect of different sample matrices for 33 pharmaceuticals by post-column infusion.

Matrix effects that occur during quantitative measurement by liquid chromatography mass spectrometry specifically when using electrospray ionization are a widely recognized phenomenon. Sample matrix compounds affect the ionization process of the target analytes, lead to a low signal response, and flawed analytical results. How these matrix compounds directly influence the ionization process has not yet been completely understood. In the present study, we determined the matrix effect for 33 pharmaceutical substances in sample extracts of urine, plasma and wastewater. Most of the investigated substances were subject to a signal suppression effect. Only for a small subset of the compounds we detected a signal enhancement effect. We investigated the matrix effect profiles in detail to disentangle the influence of different matrices and to correlate the impact of specific components and groups of the analyzed extract in suppressing or enhancing effects in the profile. Most signal suppression effects were detected in the first half of the chromatographic run-time for the matrix extracts of urine and wastewater. The observed effects are caused by high mass flow of salts and other diverse matrix components that were contained in high concentrations in those biological matrices. We also found signal suppression in the matrix effect profile of plasma samples over a wide time range during the chromatographic separation that were associated with a high content of triglycerides of diverse carbohydrate chain lengths. Here, we provide a broader picture of how 33 substances were influenced during analysis. Our results imply that a high number of the investigated substances had comparable effects of matrix compounds, despite differences in their chemical structure.

Fluidity Modulation of Phospholipid Bilayers by Electrolyte Ions: Insights from Fluorescence Microscopy and Microslit Electrokinetic Experiments

Fluidity and charging of supported bilayer lipid membranes (sBLMs) prepared from 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine (DOPC) were studied by fluorescence recovery after photobleaching (FRAP) and microslit electrokinetic measurements at varying pH and ionic composition of the electrolyte. Measurements in neutral electrolytes (KCl, NaCl) revealed a strong correlation between the membrane fluidity and the membrane charging due to unsymmetrical water ion adsorption (OH(-) ≫ H(3)O(+)). The membrane fluidity significantly decreased below the isoelectric point of 3.9, suggesting a phase transition in the bilayer. The interactions of both chaotropic anions and strongly kosmotropic cations with the zwitterionic lipids were found to be related with nearly unhindered lipid mobility in the acidic pH range. While for the chaotropic anions the observed effect correlates with the increased negative net charge at low pH, no correlation was found between the changes in the membrane fluidity and charge in the presence of kosmotropic cations. We discuss the observed phenomena with respect to the interaction of the electrolyte ions with the lipid headgroup and the influence of this process on the headgroup orientation and hydration as well as on the lipid packaging.