Grzegorz Chwastek

Role of ceramide in membrane protein organization investigated by combined AFM and FCS

Ceramide-induced alterations in the lateral organization of membrane proteins can be involved in several biological contexts, ranging from apoptosis to viral infections. In order to investigate such alterations in a simple model, we used a combined approach of atomic force microscopy, scanning fluorescence correlation spectroscopy and confocal fluorescence imaging to study the partitioning of different membrane components in sphingomyelin/dioleoyl-phosphatidylcholine/cholesterol/ceramide supported bilayers. Such model membranes exhibit coexistence of liquid-disordered, liquid-ordered (raft-like) and ceramide-rich lipid phases. Our results show that components with poor affinity toward the liquid-ordered phase, such as several fluorescent lipid analogues or the synaptic protein Synaptobrevin 2, are excluded from ceramide-rich domains. Conversely, we show for the first time that the raft-associated protein placental alkaline phosphatase (GPI-PLAP) and the ganglioside GM1 are enriched in such domains, while exhibiting a strong decrease in lateral diffusion. Analogue modulation of the local concentration and dynamics of membrane proteins/receptors by ceramide can be of crucial importance for the biological functions of cell membranes.

Surface Topology Engineering of Membranes for the Mechanical Investigation of the Tubulin Homologue FtsZ

In spite of their small size, bacteria display highly organized cytoskeletal structures like coils, helices, or rings. Extensive mechanical modeling has been done to explain the occurrence of such specific structures within the small volume of bacterial cells. As they are difficult to image within cells, in vitro reconstitution provides a valuable approach to quantitatively analyze their properties under defined conditions. A particularly interesting cytoskeletal feature is the Z-ring, which plays a key role in cell division for many bacteria. It is composed of FtsZ, a tubulin homologue, and other components and has been implicated in constriction force generation. Mechanisms localizing FtsZ to the center of the cell are known, but how it takes the form of a functional helical or ring-like structure remains unclear. 6] We hypothesized that intrinsically curved FtsZ filaments should initially respond to the native shape of bacteria and align using geometric cues. Thus, we devised a controlled biomimetic platform with membrane-coated glass substrates mimicking biologically relevant curvatures, to elucidate the mechanical properties of membrane-associated FtsZ. We found that E. coli FtsZ is assembled into inherently curved and twisted filaments supporting a helical geometry, which showed preferential orientations at the native bacterial cell-like curvatures. Strikingly, the FtsZ did not recognize smaller curvatures in the same way, but rather oriented themselves at an angle in higher curvatures, which does not support the idea that FtsZ alone is able to exert a constriction force. In recent studies involving high-resolution imaging and cryo-electron microscopy, the “Z-ring” has generally been described as a helical structure. Purified FtsZ has been studied extensively by electron microscopy and atomic force microscopy. Consistently, the EM and AFM images from these studies show curved filaments. Cryo-EM on reconstituted FtsZ filaments in vitro seems to contradict the presence of any local spontaneous curvature. However, in a recent study, Osawa et al. showed the ability of FtsZ filaments with an artificially introduced membrane targeting sequence (MTS) to bend membranes, with an influence of the MTS placement in FtsZ on the membrane bending direction. They used an MTS from MinD at the C-terminus of FtsZ to mimic the recruitment of FtsZ to the membrane by adaptor proteins ZipA or FtsA. Upon shifting the MTS to the Nterminus, they find that the filaments bend the membrane in opposite directions. They interpret this to be caused by a constriction force produced by partial Z-rings. A dividing bacterial cell initially has a curvature of about 2 mm , but proceeds towards much higher curvature values as the cell progresses through division. It is unknown whether a bacterial membrane, fortified with many structural proteins, osmotic pressure, and a cell wall, would be as easily deformed. The spontaneous structure of FtsZ filaments may enable them to organize into highly curved suprastructures by sensing the inner cell-membrane curvature, but they may have to recruit other mechanically active factors for cytokinesis. The distortions observed in previous studies 18] could simply be caused by a bundle of curved filaments bending the flexible membrane towards their own curvature. We first repeated the experiments with MTS-FtsZ on freestanding giant unilamellar vesicle (GUV) membranes, and quantitatively evaluated the induced radii of curvature. We found that the filaments did not bend the membranes when the unilamellar vesicles were isotonic. They aligned into filament networks similar to those on planar supported bilayers (Figure 1 b). Changing the osmotic gradient across the membranes by adding 10 mm glucose decreased intravesicular pressure and relaxed the membrane surface tension. This resulted in a curved topology of the membrane as well as the filaments (Figure 1a,b). Only when the membrane tension was low, under hypertonic conditions, could the filaments [*] Prof. Dr. P. Schwille Dept. Cellular and Molecular Biophysics, Max Planck Institute of Biochemistry Am Klopferspitz 18, 82152 Martinsried (Germany) E-mail: schwille@biochem.mpg.de S. Arumugam, G. Chwastek, C. Ehrig Max Planck Institute for Cell Biology and Genetics Pfotenhauerstrasse 108, 01307 Dresden (Germany) and Biotechnology Center of the TU Dresden Tatzberg 47/51, 01307 Dresden (Germany)

Efficient Electroformation of Supergiant Unilamellar Vesicles Containing Cationic Lipids on ITO-Coated Electrodes

Giant unilamellar vesicles (GUVs) represent a versatile in vitro system widely used to study properties of lipid membranes and their interaction with biomacromolecules and colloids. Electroformation with indium tin oxide (ITO) coated coverslips as electrodes is a standard approach to GUV production. In the case of cationic GUVs, however, application of this approach leads to notorious difficulties. We discover that this is related to aging of ITO-coated coverslips during their repeated use, which is reflected in their surface topography on the nanoscale. We find that mild annealing of the ITO-coated surface in air reverts the effects of aging and ensures efficient reproducible electroformation of supergiant (diameter > 100 μm) unilamellar vesicles containing cationic lipids.

Protein-membrane interactions: the virtue of minimal systems in systems biology

The plasma membrane of cells can be viewed as a highly dynamic, regulated, heterogeneous environment with multiple functions. It constitutes the boundary of the cell, encapsulating all its components. Proteins interact with the membrane in many ways to accommodate essential processes, such as membrane trafficking, membrane protrusions, cytokinesis, signaling, and cell–cell communication. A vast amount of literature has already fostered our current understanding of membrane–protein interactions. However, many phenomena still remain to be understood, e.g., the exact mechanisms of how certain proteins cause or assist membrane transformations. Systems biology aims to predict biological processes on the basis of the set of molecules involved. Many key processes arise from interactions with the lipid membrane. Protein interactome maps do not consider such specific interactions, and thus cannot predict precise outcomes of the interactions of the involved proteins. These can only be inferred from experimental approaches. We describe examples of how an emergent behavior of protein–membrane interactions has been demonstrated by the use of minimal systems. These studies contribute to a deeper understanding of protein interactomes involving membranes and complement other approaches of systems biology. WIREs Syst Biol Med 2011 3 269–280 DOI: 10.1002/wsbm.119

Asymmetry determines the effects of natural ceramides on model membranes

Ceramides can dramatically influence the lateral organization of biological membranes. In particular, ceramide-induced alterations of protein-lipid domains can be involved in several cellular processes, ranging from senescence to immune response. In this context, an important role is played by the length of the fatty acid bound to the sphingosine moiety. Asymmetric, heterogeneous ceramides, with more than 20 or less than 16 carbon atoms in the fatty acyl chain, in fact exert diverging effects in vivo if compared to their symmetric counterparts. In this work, we investigated the role of ceramide asymmetry and heterogeneity in model membranes showing raft-like phase separation, using a combination of fluorescence imaging, atomic force microscopy, fluorescence correlation spectroscopy and differential scanning calorimetry. We show that ceramide produced enzymatically from natural mixtures of sphingomyelin can dramatically alter the mixing behaviour of proteins and lipids in the membrane, inducing a homogenization of the bilayer. Furthermore, we characterized the physical properties of coexisting lipid phases at equilibrium in membranes with varying ceramide content, emphasizing the differences between symmetric-homogeneous and asymmetric-heterogeneous ceramides. While symmetric ceramides always produce enhanced order, asymmetric ceramides display a more complex behavior similar to that of cholesterol. Our results might help contribute to a more precise understanding of the rearrangements induced by different kinds of ceramide generation in cellular membranes.

Protein Patterns and Oscillations on Lipid Monolayers and in Microdroplets

Abstract The Min proteins from E.coli position the bacterial cell‐division machinery through pole‐to‐pole oscillations. In vitro, Min protein self‐organization can be reconstituted in the presence of a lipid membrane as a catalytic surface. However, Min dynamics have so far not been reconstituted in fully membrane‐enclosed volumes. Microdroplets interfaced by lipid monolayers were employed as a simple 3D mimic of cellular compartments to reconstitute Min protein oscillations. We demonstrate that lipid monolayers are sufficient to fulfil the catalytic role of the membrane and thus represent a facile platform to investigate Min protein regulated dynamics of the cell‐division protein FtsZ‐mts. In particular, we show that droplet containers reveal distinct Min oscillation modes, and reveal a dependence of FtsZ‐mts structures on compartment size. Finally, co‐reconstitution of Min proteins and FtsZ‐mts in droplets yields antagonistic localization, thus demonstrating that droplets indeed support the analysis of complex bacterial self‐organization in confined volumes.

The design of MACs (minimal actin cortices)

The actin cell cortex in eukaryotic cells is a key player in controlling and maintaining the shape of cells, and in driving major shape changes such as in cytokinesis. It is thereby constantly being remodeled. Cell shape changes require forces acting on membranes that are generated by the interplay of membrane coupled actin filaments and assemblies of myosin motors. Little is known about how their interaction regulates actin cell cortex remodeling and cell shape changes. Because of the vital importance of actin, myosin motors and the cell membrane, selective in vivo experiments and manipulations are often difficult to perform or not feasible. Thus, the intelligent design of minimal in vitro systems for actin‐myosin‐membrane interactions could pave a way for investigating actin cell cortex mechanics in a detailed and quantitative manner. Here, we present and discuss the design of several bottom‐up in vitro systems accomplishing the coupling of actin filaments to artificial membranes, where key parameters such as actin densities and membrane properties can be varied in a controlled manner. Insights gained from these in vitro systems may help to uncover fundamental principles of how exactly actin‐myosin‐membrane interactions govern actin cortex remodeling and membrane properties for cell shape changes.

Photoconversion of bodipy-labeled lipid analogues.

MOVING COLORS: Bodipy-labeled lipid analogues can change their photophysical properties and/or localization in the membrane upon light illumination. These changes are highly influenced by the lipid environment. This phenomenon can lead to lipid-environment-specific false positive signals in experimental techniques where spectral identity/separation is important.

A Monolayer Assay Tailored to Investigate Lipid-Protein Systems

Model membrane systems have become invaluable tools to investigate specific features of cellular membranes. Although a variety of different experimental assays does exist, many of them are rather complicated in their preparation and difficult in their practical realisation. Here, we propose a new simple miniaturised monolayer assay that can easily be combined with standard analytical techniques such as confocal fluorescence microscopy and fluorescence correlation spectroscopy (FCS).