Non-Brownian diffusion in lipid membranes: Experiments and simulations

Biological membranes are tricky to investigate. They are complex in terms of molecular composition and structure, functional over a wide range of time scales, and characterized by nonequilibrium conditions. Because of all of these features, simulations are a great technique to study biomembrane behavior. A significant part of the functional processes in biological membranes takes place at the molecular level; thus computer simulations are the method of choice to explore how their properties emerge from specific molecular features and how the interplay among the numerous molecules gives rise to function over spatial and time scales larger than the molecular ones. In this review, we focus on this broad theme. We discuss the current state-of-the-art of biomembrane simulations that, until now, have largely focused on a rather narrow picture of the complexity of the membranes. Given this, we also discuss the challenges that we should unravel in the foreseeable future. Numerous features such as the actin-cytosk...

Multiscale Simulations of Biological Membranes: The Challenge To Understand Biological Phenomena in a Living Substance.

Lateral diffusion plays a crucial role in numerous processes that take place in cell membranes, yet it is quite poorly understood in native membranes characterized by, e.g., domain formation and large concentration of proteins. In this article, we use atomistic and coarse-grained simulations to consider how packing of membranes and crowding with proteins affect the lateral dynamics of lipids and membrane proteins. We find that both packing and protein crowding have a profound effect on lateral diffusion, slowing it down. Anomalous diffusion is observed to be an inherent property in both protein-free and protein-rich membranes, and the time scales of anomalous diffusion and the exponent associated with anomalous diffusion are found to strongly depend on packing and crowding. Crowding with proteins also has a striking effect on the decay rate of dynamical correlations associated with lateral single-particle motion, as the transition from anomalous to normal diffusion is found to take place at macroscopic time scales: while in protein-poor conditions normal diffusion is typically observed in hundreds of nanoseconds, in protein-rich conditions the onset of normal diffusion is tens of microseconds, and in the most crowded systems as large as milliseconds. The computational challenge which results from these time scales is not easy to deal with, not even in coarse-grained simulations. We also briefly discuss the physical limits of protein motion. Our results suggest that protein concentration is anything but constant in the plane of cell membranes. Instead, it is strongly dependent on proteins' preference for aggregation.

Anomalous and normal diffusion of proteins and lipids in crowded lipid membranes

In this study, we have imaged plasma membrane molecules labeled with quantum dots in live cells using a conventional wide-field microscope with high spatial precision at sampling frequencies of 1.75 kHz. Many of the resulting single molecule trajectories are sufficiently long (up to several thousand steps) to allow for robust single trajectory analysis. This analysis indicates that a majority of the investigated molecules are transiently confined in nanoscopic compartments with a mean size of (100–150 nm)2 for a mean duration of 50–100 ms.

Visualization of Plasma Membrane Compartmentalization by High-Speed Quantum Dot Tracking

Experimentalists are increasingly confronted with the demand to single out rare interaction events under a vast excess of non-interacting molecules. We recently presented an approach how to virtually dilute fluorescently labeled membrane constituents by combining photobleaching and single molecule microscopy, termed “Thinning Out Clusters while Conserving the Stoichiometry of Labeling” (TOCCSL; Moertelmaier et al., Appl. Phys. Lett., 2005, 87, 263903). Using this approach, single molecule microscopy can be performed even at arbitrarily high surface densities of fluorescent probe molecules. Here, we extended this method for two color microscopy. We provide a detailed statistical description of false positives and false negatives. In particular, we quantified the increase in sensitivity by tracking the colocalized objects over successive images. Proof of principle experiments were performed by measuring the interaction between Alexa647-labeled Cholera Toxin B (CTX-B-Alexa647) and Bodipy-labeled GM1 (Bodipy-GM1) diffusing in a fluid supported lipid bilayer. We directly observed single Cholera Toxin B molecules bound to Bodipy-GM1 and quantified their occupancy via brightness analysis. Each colocalized spot could be further analyzed with respect to its diffusion constant, yielding a clear anticorrelation between occupancy and mobility. We finally demonstrate that extremely low interaction probabilities of only 2.5% can be unambiguously identified.

Two-color single molecule tracking combined with photobleaching for the detection of rare molecular interactions in fluid biomembranes

In recent years increasing evidence has been reported for the inherent heterogeneity of cell populations. Cell-to-cell variability was particularly found when analyzing protein expression patterns or the responses of cells to different stimuli. Yet, structural features – in particular below the resolution limit of light microscopy – have so far eluded in-depth statistical analysis. We report here for the first time a detailed investigation of the variations in protein mobility between nominally identical cells. Our reasoning was that mobility probes nanometer-sized properties of the moving protein and its local environment, which may be subject to cell-to-cell variability. Single-molecule tracking was employed to characterize the diffusion constant of the glycosylphosphatidylinositol- (GPI-) anchored protein CD59 and the transmembrane protein CD147 in the plasma membrane of T24 cells. Automated and tailored data analysis routines allowed for the analysis of the required large data sets: ∼200000 trajectories obtained on ∼350 cells were analyzed in total. We found up to five-fold higher variability of the diffusion constant between cells compared to the uncertainty for the determination of the diffusion constant on a single cell.

Cell-to-cell variability in the diffusion constants of the plasma membrane proteins CD59 and CD147

Reproducibility and stability of C60 based organic field effect transistor

This paper describes a Power-On Reset (POR) circuit with very accurate threshold voltage levels. These voltage levels are achieved by calibrating an on-chip programmable voltage reference during the wafer sort or final test. Two different calibration methods are proposed. One which calibrates the reference voltage of a comparator and another method which calibrates the POR threshold voltage levels itself. Not only the variation of the POR threshold voltage levels can be reduced from 90mV to 7mV, but also the absolute value can be changed.

Calibration of a flexible high precision Power-On Reset during production test

Within the field of delta-sigma modulators utilizing switched-capacitor structures it is of great importance to use non-overlapping clocks. The circuit presented in this document generates these signals without the need of feedback loops. To verify the timing of non-overlapping clocks, an integrated circuit is introduced that is capable of sampling fast clock signals and forwards the processed data to the measurement equipment.

Michael Sams

Papers

Cell-to-cell variability in the diffusion constants of the plasma membrane proteins CD59 and CD147

Reproducibility and stability of C60 based organic field effect transistor

Calibration of a flexible high precision Power-On Reset during production test

Generation of non-overlapping clock signals without using a feedback loop