Eugene P. Petrov

Translational Diffusion in Lipid Membranes beyond the Saffman-Delbrück Approximation

The Saffman-Delbrück approximation is commonly used in biophysics to relate the membrane inclusion size to its translational diffusion coefficient and membrane viscosity. However, this approximation has a restricted validity range, and its application to determination of inclusion sizes from diffusion data may in certain cases lead to unreliable results. At the same time, the model by Hughes et al. (Hughes, B. D., B. A. Pailthorpe, and C. R. White. 1981. J. Fluid Mech. 110:349-372.), providing diffusion coefficients of membrane inclusions for arbitrary inclusion sizes and viscosities of the membrane and surrounding fluids, involves substantial computational efforts, which prevents its use in practical data analysis. We develop a simple and accurate analytical approximation to the Hughes et al. model and demonstrate its performance and utility by applying it to the recently published experimental data on translational diffusion of micrometer-sized membrane domains.

Near-Critical Fluctuations and Cytoskeleton-Assisted Phase Separation Lead to Subdiffusion in Cell Membranes

We address the relationship between membrane microheterogeneity and anomalous subdiffusion in cell membranes by carrying out Monte Carlo simulations of two-component lipid membranes. We find that near-critical fluctuations in the membrane lead to transient subdiffusion, while membrane-cytoskeleton interaction strongly affects phase separation, enhances subdiffusion, and eventually leads to hop diffusion of lipids. Thus, we present a minimum realistic model for membrane rafts showing the features of both microscopic phase separation and subdiffusion.

The role of lipids in VDAC oligomerization.

Evidence has accumulated that the voltage-dependent anion channel (VDAC), located on the outer membrane of mitochondria, plays a central role in apoptosis. The involvement of VDAC oligomerization in apoptosis has been suggested in various studies. However, it still remains unknown how exactly VDAC supramolecular assembly can be regulated in the membrane. This study addresses the role of lipids in this process. We investigate the effect of cardiolipin (CL) and phosphatidylglycerol (PG), anionic lipids important for mitochondria metabolism and apoptosis, on VDAC oligomerization. By applying fluorescence cross-correlation spectroscopy to VDAC reconstituted into giant unilamellar vesicles, we demonstrate that PG significantly enhances VDAC oligomerization in the membrane, whereas cardiolipin disrupts VDAC supramolecular assemblies. During apoptosis, the level of PG in mitochondria increases, whereas the CL level decreases. We suggest that the specific lipid composition of the outer mitochondrial membrane might be of crucial relevance and, thus, a potential cue for regulating the oligomeric state of VDAC.

State of the Art and Novel Trends in Fluorescence Correlation Spectroscopy

This chapter presents a review of fluorescence correlation spectroscopy (FCS), an experimental technique with single-molecule sensitivity, which is based on the analysis of fluctuations of fluorescence intensity detected from a tiny volume. Correlation functions of fluorescence fluctuations can provide information on the translational and rotational diffusion of fluorophores, dynamics of singlet–triplet transitions, chemical reactions, flow, and active transport of fluorescent molecules. A detailed theoretical description of the fluorescence correlation and cross-correlation technique is followed by a discussion of various experimental aspects of FCS, including the choice of instrumentation and fluorophores, sample- and setup-related nonidealities and possible artifacts, statistical accuracy, and approaches to the analysis of FCS data. Additionally, some FCS application aspects are addressed, including the quantitative determination of translational diffusion coefficients and the use of FCS in studies of (bio)polymers and phospholipid membranes. The chapter concludes with an overview of both well-established and currently emerging varieties of FCS and related methods, including the use of two-photon excitation and the application of total internal reflection, nanoapertures, and stimulated emission depletion to confine the detection volume; the use of higher-order correlations; application of time-resolved and time-gated detection; multifocal and CCD-based FCS; and image correlation and scanning FCS techniques.

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.

Total Internal Reflection Fluorescence Correlation Spectroscopy: Effects of Lateral Diffusion and Surface-Generated Fluorescence

Fluorescence correlation spectroscopy with total internal reflection excitation (TIR-FCS) is a promising method with emerging biological applications for measuring binding dynamics of fluorescent molecules to a planar substrate as well as diffusion coefficients and concentrations at the interface. Models for correlation functions proposed so far are rather approximate for most conditions, since they neglect lateral diffusion of fluorophores. Here we propose accurate extensions of previously published models for axial correlation functions taking into account lateral diffusion through detection profiles realized in typical experiments. In addition, we consider the effects of surface-generated emission in objective-based TIR-FCS. The expressions for correlation functions presented here will facilitate quantitative and accurate measurements with TIR-FCS.

Phase separation and near-critical fluctuations in two-component lipid membranes: Monte Carlo simulations on experimentally relevant scales

By means of lattice-based Monte Carlo simulations, we address the properties of two-component lipid membranes on the experimentally relevant spatial scales of the order of a micrometer and time intervals of the order of 1 s, using DMPC/DSPC lipid mixtures as a model system. Our large-scale simulations allowed us to obtain important results not reported previously in simulation studies of lipid membranes. We find that, for a certain range of lipid compositions, the phase transition from the fluid phase to the fluid–gel phase coexistence proceeds via near-critical fluctuations, whereas for other lipid compositions this phase transition has a quasi-abrupt character. In the presence of near-critical fluctuations, transient subdiffusion of lipid molecules is observed. These features of the system are stable with respect to perturbations in lipid interaction parameters used in our simulations. The line tension characterizing lipid domains in the fluid–gel coexistence region is found to be in the pN range. On approaching the critical point, the line tension, the inverse correlation length of fluid–gel spatial fluctuations and the corresponding inverse order parameter susceptibility of the membrane vanish. All these results are in agreement with recent experimental findings for model lipid membranes. Our analysis of the domain coarsening dynamics after an abrupt quench of the membrane to the fluid–gel coexistence region reveals that lateral diffusion of lipids plays an important role in the fluid–gel phase separation process.

Translational and rotational diffusion of micrometer-sized solid domains in lipid membranes

We use simultaneous observation of translational and rotational Brownian motion of domains in lipid membranes to test the hydrodynamics-based theory for the viscous drag on the membrane inclusion. We find that translational and rotational diffusion coefficients of micrometer-sized solid (gel-phase) domains in giant unilamellar vesicles showing fluid–gel phase coexistence are in excellent agreement with the theoretical predictions.

Cytoskeletal Pinning Controls Phase Separation in Multicomponent Lipid Membranes

We study the effect of a minimal cytoskeletal network formed on the surface of giant unilamellar vesicles by the prokaryotic tubulin homolog, FtsZ, on phase separation in freestanding lipid membranes. FtsZ has been modified to interact with the membrane through a membrane targeting sequence from the prokaryotic protein MinD. FtsZ with the attached membrane targeting sequence efficiently forms a highly interconnected network on membranes with a concentration-dependent mesh size, much similar to the eukaryotic cytoskeletal network underlying the plasma membrane. Using giant unilamellar vesicles formed from a quaternary lipid mixture, we demonstrate that the artificial membrane-associated cytoskeleton, on the one hand, suppresses large-scale phase separation below the phase transition temperature, and, on the other hand, preserves phase separation above the transition temperature. Our experimental observations support the ideas put forward in our previous simulation study: In particular, the picket fence effect on phase separation may explain why micrometer-scale membrane domains are observed in isolated, cytoskeleton-free giant plasma membrane vesicles, but not in intact cell membranes. The experimentally observed suppression of large-scale phase separation much below the transition temperatures also serves as an argument in favor of the cryoprotective role of the cytoskeleton.

DNA Origami Nanoneedles on Freestanding Lipid Membranes as a Tool To Observe Isotropic−Nematic Transition in Two Dimensions

We introduce a simple experimental system to study dynamics of needle-like nanoobjects in two dimensions (2D) as a function of their surface density close to the isotropic-nematic transition. Using fluorescence correlation spectroscopy, we find that translational and rotational diffusion of rigid DNA origami nanoneedles bound to freestanding lipid membranes is strongly suppressed upon an increase in the surface particle density. Our experimental observations show a good agreement with results of Monte Carlo simulations of Brownian hard needles in 2D.