Large-scale modulation of reconstituted Min protein patterns and gradients by defined mutations in MinE’s membrane targeting sequence

<div><p>The <i>E</i>. <i>coli</i> MinDE oscillator is a paradigm for protein self-organization and gradient formation. Previously, we reconstituted Min protein wave patterns on flat membranes as well as gradient-forming pole-to-pole oscillations in cell-shaped PDMS microcompartments. These oscillations appeared to require direct membrane interaction of the ATPase activating protein MinE. However, it remained unclear how exactly Min protein dynamics are regulated by MinE membrane binding. Here, we dissect the role of MinE’s membrane targeting sequence (MTS) by reconstituting various MinE mutants in 2D and 3D geometries. We demonstrate that the MTS defines the lower limit of the concentration-dependent wavelength of Min protein patterns while restraining MinE’s ability to stimulate MinD’s ATPase activity. Strikingly, a markedly reduced length scale—obtainable even by single mutations—is associated with a rich variety of multistable dynamic modes in cell-shaped compartments. This dramatic remodeling in response to biochemical changes reveals a remarkable trade-off between robustness and versatility of the Min oscillator.</p></div

Asymmetric Supported Lipid Bilayer Formation via Methyl-β-Cyclodextrin Mediated Lipid Exchange: Influence of Asymmetry on Lipid Dynamics and Phase Behavior

Supported lipid bilayers (SLBs) are broadly used as minimal membrane models and commonly produced by vesicle fusion (VF) on solid supports. Despite its advantages, VF does not allow the controlled formation of bilayers that mimic the leaflet asymmetry in lipid composition normally found in biological systems. Here we present a simple, quick, and versatile method to produce SLBs with a desired asymmetric lipid composition which is stable for ca. 4 h. We apply methyl-β-cyclodextrin mediated lipid exchange to SLBs formed by VF to enrich the upper leaflet of the bilayer with sphingomyelin. The bilayer asymmetry is assessed by fluorescence correlation spectroscopy, measuring the lipid mobility separately in each leaflet. To check the compatibility of the method with the most common protein reconstitution approaches, we report the production of asymmetric SLBs (aSLBs) in the presence of a glycosylphosphatidylinositol-anchored protein, reconstituted in the bilayer both, via direct protein insertion, and via proteoliposomes fusion. We finally apply aSLBs to study phase separation and transbilayer lipid movement of raft-mimicking lipid mixtures. The observed differences in terms of phase separation in symmetric and asymmetric SLBs with the same overall lipid composition provide further experimental evidence that the transversal lipid distribution affects the overall lipid miscibility and allow to temporally investigate leaflet mixing

Mutation of MinE’s membrane targeting sequence leads to unusual dynamics and defects in gradient formation in cell-shaped compartments.

<p>WT and L3E panels show representative time-lapse images, kymographs along the compartment length as well as the time-averaged fluorescence intensity, which was measured along a compartment edge. The L3E mutant exhibited diverse dynamical modes observed in different compartments under the same experimental conditions. These mutant dynamics comprised (from left to right) bi- or unidirectional rotations, traveling waves and irregular pole-to-pole oscillations. All images at 1 μM MinD with 20% eGFP-MinD and 1 μM MinE. Scale Bar: 5 μm. The compartments were 35 μm long, 10 μm wide and 10 μm deep.</p

Truncation or mutation of MinE’s membrane targeting sequence decreases the lower limit of the length scale of Min protein patterns.

<p><b>(A)</b> Confocal images of self-organized WT and Δ(2–12), L3E, L4E, F6E, F7E mutant waves on flat membranes. WT MinE and mutant proteins were titrated from 0.5 to 5 μM (MinD at 1 μM with 20% eGFP-MinD). Scale Bar: 50 μm. Dependence of the mean <b>(B)</b> wavelength and <b>(C)</b> velocity of WT and mutant waves on MinE concentration (MinD at 1 μM). Error bars represent standard deviation (N ≥ 3) from at least three independent experiments.</p

Large-scale modulation of the Min oscillator by reducing MinE’s membrane affinity.

<p>Biochemical alterations, such as single mutations, in MinE’s membrane targeting sequence cause a marked reduction in the lower limit of the length scale of Min protein patterns along with unusual dynamic modes in cell-like geometry. Thus, the Min oscillator is both highly versatile and sensitive to biochemical changes. Scale Bars: 50 μm (left) and 5 μm (right). P_i: inorganic phosphate. Units and values on graphs are left out for simplicity (see Figs <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0179582#pone.0179582.g001" target="_blank">1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0179582#pone.0179582.g003" target="_blank">3</a> for data).</p

Reconstitution of a Reversible Membrane Switch via Prenylation by One-Pot Cell-Free Expression

Reversible membrane targeting of proteins is one of the key regulators of cellular interaction networks, for example, for signaling and polarization. So-called “membrane switches” are thus highly attractive targets for the design of minimal cells but have so far been tricky to reconstitute in vitro. Here, we introduce cell-free prenylated protein synthesis (CFpPS), which enables the synthesis and membrane targeting of proteins in a single reaction mix including the prenylation machinery. CFpPS can confer membrane affinity to any protein via addition of a 4-peptide motif to its C-terminus and offers robust production of prenylated proteins not only in their soluble forms but also in the direct vicinity of biomimetic membranes. Thus, CFpPS enabled us to reconstitute the prenylated polarity hub Cdc42 and its regulatory protein in vitro, implementing a key membrane switch. We propose CFpPS to be a versatile and effective platform for engineering complex features, such as polarity induction, in synthetic cells

High-Speed Atomic Force Microscopy Reveals the Inner Workings of the MinDE Protein Oscillator

The MinDE protein system from <i>E. coli</i> has recently been identified as a minimal biological oscillator, based on two proteins only: The ATPase MinD and the ATPase activating protein MinE. In <i>E. coli</i>, the system works as the molecular ruler to place the divisome at midcell for cell division. Despite its compositional simplicity, the molecular mechanism leading to protein patterns and oscillations is still insufficiently understood. Here we used high-speed atomic force microscopy to analyze the mechanism of MinDE membrane association/dissociation dynamics on isolated membrane patches, down to the level of individual point oscillators. This nanoscale analysis shows that MinD association to and dissociation from the membrane are both highly cooperative but mechanistically different processes. We propose that they represent the two directions of a single allosteric switch leading to MinD filament formation and depolymerization. Association/dissociation are separated by rather long apparently silent periods. The membrane-associated period is characterized by MinD filament multivalent binding, avidity, while the dissociated period is defined by seeding of individual MinD. Analyzing association/dissociation kinetics with varying MinD and MinE concentrations and dependent on membrane patch size allowed us to disentangle the essential dynamic variables of the MinDE oscillation cycle

Optical Control of Lipid Rafts with Photoswitchable Ceramides

Ceramide is a pro-apoptotic sphingolipid with unique physical characteristics. Often viewed as a second messenger, its generation can modulate the structure of lipid rafts. We prepared three photoswitchable ceramides, <b>ACe</b>s, which contain an azobenzene photoswitch allowing for optical control over the <i>N</i>-acyl chain. Using combined atomic force and confocal fluorescence microscopy, we demonstrate that the <b>ACe</b>s enable reversible switching of lipid domains in raft-mimicking supported lipid bilayers (SLBs). In the <i>trans</i>-configuration, the <b>ACe</b>s localize into the liquid-ordered (L_o) phase. Photoisomerization to the <i>cis</i>-form triggers a fluidification of the L_o domains, as liquid-disordered (L_d) “lakes” are formed within the rafts. Photoisomerization back to the <i>trans</i>-state with blue light stimulates a rigidification inside the L_d phase, as the formation of small L_o domains. These changes can be repeated over multiple cycles, enabling a dynamic spatiotemporal control of the lipid raft structure with light

Quantifying Lipid Diffusion by Fluorescence Correlation Spectroscopy: A Critical Treatise

Fluorescence correlation spectroscopy (FCS) measurements are widely used for determination of diffusion coefficients of lipids and proteins in biological membranes. In recent years, several variants of FCS have been introduced. However, a comprehensive comparison of these methods on identical systems has so far been lacking. In addition, there exist no consistent values of already determined diffusion coefficients for well-known or widely used membrane systems. This study aims to contribute to a better comparability of FCS experiments on membranes by determining the absolute diffusion coefficient of the fluorescent lipid analog 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindodicarbocyanine (DiD) in giant unilamellar vesicles (GUVs) made of dioleoylphosphatidylcholine (DOPC), which can in future studies be used as a reference value. For this purpose, five FCS variants, employing different calibration methods, were compared. Potential error sources for each particular FCS method and strategies to avoid them are discussed. The obtained absolute diffusion coefficients for DiD in DOPC were in good agreement for all investigated FCS variants. An average diffusion coefficient of <i>D</i> = 10.0 ± 0.4 μm² s^–1 at 23.5 ± 1.5 °C was obtained. The independent confirmation with different methods indicates that this value can be safely used for calibration purposes. Moreover, the comparability of the methods also in the case of slow diffusion was verified by measuring diffusion coefficients of DiD in GUVs consisting of DOPC and cholesterol

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