Weak Glycolipid Binding of a Microdomain-Tracer Peptide Correlates with Aggregation and Slow Diffusion on Cell Membranes

10.1371/journal.pone.0051222PLoS ONE712

Lipid dynamics in model lipid bilayers analysed by tirf-based single molecule tracking

Analysis of the dynamic behavior of membrane components in lipid membranes is essential for understanding plasma membrane structure. Single molecule tracking (SMT) can provide direct insight into diffusion characteristics of membrane probes, supplementing data acquired by ensemble averaging techniques. The effective use of SMT however requires not only careful data acquisition but also unbiased analysis to avoid artifacts and spurious or misleading results. Currently, such analysis can not be easily done by a non-expert due to a dearth in software availability. Many methods and algorithms have been described to deal with each level of SMT data processing, such as particle recognition and linking, data filtering, fitting using various methods, uncertainty calculation and data presentation. All of them however require implementation in the particular programming environment of choice prior to use. This complicated task is often circumvented by using archaic but readily available analysis methods, leading to unreliable, inadequate, biased, and/or non-quantitative results. In the course of our study of lipid bilayer dynamics, using a variety of membrane-integrating or –interacting probes with different lipid mixtures, TrackArt was designed to overcome these problems and integrate SMT data analysis methodology into one software ensemble. The resulting software package was then applied to the analysis of different membrane compositional scenarios and methods of bilayer preparation, where complex diffusion behavior was observed or influenced by experimental perturbation. This study presents major TrackArt features, which include not only data analysis, but also simulations of complex diffusion behavior in lipid bilayers. TrackArt functionality was demonstrated on in silico examples and real membranes, using a variety of different lipid mixtures and lipid and non-lipid probes of diffusion behavior. SMT on lipid bilayers supported on glass and mica revealed the single-molecule behavioral origins of diffusion differences between these substrates that have been previously reported by other (ensemble averaging) methods. Specific examples of diffusion applications where TrackArt was used to detect complex behaviors of membrane-interacting domain tracer molecules, or domain-disrupting molecules were 1) diffusion of the Alzheimer’s Aβ-derived sphingolipid binding domain (SBD) peptide, which was shown to be dependent on lipid bilayer composition, favoring sphingolipids and gangliosides. Here, a faster diffusion was seen in the presence of its characterized lipid targets in the membrane. 2) one of the membrane insertion molecules of the class polyphenylenevinylenes called COE1-5C, was shown by TrackArt analysis of SMT, to stabilize (de-fluidize) lipid bilayers composed of total E.coli lipid extract, and subjected to high butanol concentrations. Using these examples of practical applications, we demonstrate the effectiveness of the sufficiently complex, but user-friendly TrackArt software graphic user interface, where unbiased yet selective analysis of large data sets of individual molecule behavior are needed to gain an overall picture of the origins of different characteristic membrane dynamics and diffusion.​Doctor of Philosophy (SBS

Streptococcus pyogenes capsule promotes microcolony-independent biofilm formation

Biofilms play an important role in the pathogenesis of Group A Streptococcus (GAS), a gram-positive pathogen responsible for a wide range infections and significant public health impact. Although most GAS serotypes are able to form biofilms, there is large heterogeneity between individual strains in biofilm formation, as measured by standard crystal violet assays. It is generally accepted that biofilm formation includes initial adhesion of bacterial cells to a surface, followed by microcolony formation, biofilm maturation, and extensive production of extracellular matrix that links together proliferating cells and provides a scaffold for the three-dimensional biofilm structure. However, our studies show that for GAS strain JS95, microcolony formation is not an essential step in static biofilm formation, and instead, biofilm can be effectively formed from slow-growing or non-replicating late exponential or early stationary planktonic cells, via sedimentation and fixation of GAS chains into biofilms. In addition, we show that the GAS capsule specifically contributes to the alternative, sedimentation-initiated biofilms. Microcolony-independent, sedimentation biofilms are similar in morphology and 3-D structure to biofilms initiated by actively dividing planktonic bacteria. We conclude that GAS can form biofilms by an alternate, non-canonical mechanism that does not require transition from microcolony formation to biofilm maturation, and which may be obscured by biofilm phenotypes that arise via the classical biofilm maturation processes.NMRC (Natl Medical Research Council, S’pore)Accepted versio

Preparation of mica supported lipid bilayers for high resolution optical microscopy imaging

Supported lipid bilayers (SLBs) are widely used as a model for studying membrane properties (phase separation, clustering, dynamics) and its interaction with other compounds, such as drugs or peptides. However SLB characteristics differ depending on the support used. Commonly used techniques for SLB imaging and measurements are single molecule fluorescence microscopy, FCS and atomic force microscopy (AFM). Because most optical imaging studies are carried out on a glass support, while AFM requires an extremely flat surface (generally mica), results from these techniques cannot be compared directly, since the charge and smoothness properties of these materials strongly influence diffusion. Unfortunately, the high level of manual dexterity required for the cutting and gluing thin slices of mica to the glass slide presents a hurdle to routine use of mica for SLB preparation. Although this would be the method of choice, such prepared mica surfaces often end up being uneven (wavy) and difficult to image, especially with small working distance, high numerical aperture lenses. Here we present a simple and reproducible method for preparing thin, flat mica surfaces for lipid vesicle deposition and SLB preparation. Additionally, our custom made chamber requires only very small volumes of vesicles for SLB formation. The overall procedure results in the efficient, simple and inexpensive production of high quality lipid bilayer surfaces that are directly comparable to those used in AFM studies.Published versio

TrackArt: the user friendly interface for single molecule tracking data analysis and simulation applied to complex diffusion in mica supported lipid bilayers

BACKGROUND: Single molecule tracking (SMT) analysis of fluorescently tagged lipid and protein probes is an attractive alternative to ensemble averaged methods such as fluorescence correlation spectroscopy (FCS) or fluorescence recovery after photobleaching (FRAP) for measuring diffusion in artificial and plasma membranes. The meaningful estimation of diffusion coefficients and their errors is however not straightforward, and is heavily dependent on sample type, acquisition method, and equipment used. Many approaches require advanced computing and programming skills for their implementation. FINDINGS: Here we present TrackArt software, an accessible graphic interface for simulation and complex analysis of multiple particle paths. Imported trajectories can be filtered to eliminate spurious or corrupted tracks, and are then analyzed using several previously described methodologies, to yield single or multiple diffusion coefficients, their population fractions, and estimated errors. We use TrackArt to analyze the single-molecule diffusion behavior of a sphingolipid analog SM-Atto647N, in mica supported DOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine) bilayers. Fitting with a two-component diffusion model confirms the existence of two separate populations of diffusing particles in these bilayers on mica. As a demonstration of the TrackArt workflow, we characterize and discuss the effective activation energies required to increase the diffusion rates of these populations, obtained from Arrhenius plots of temperature-dependent diffusion. Finally, TrackArt provides a simulation module, allowing the user to generate models with multiple particle trajectories, diffusing with different characteristics. Maps of domains, acting as impermeable or permeable obstacles for particles diffusing with given rate constants and diffusion coefficients, can be simulated or imported from an image. Importantly, this allows one to use simulated data with a known diffusion behavior as a comparison for results acquired using particular algorithms on actual, “natural” samples whose diffusion behavior is to be extracted. It can also serve as a tool for demonstrating diffusion principles. CONCLUSIONS: TrackArt is an open source, platform-independent, Matlab-based graphical user interface, and is easy to use even for those unfamiliar with the Matlab programming environment. TrackArt can be used for accurate simulation and analysis of complex diffusion data, such as diffusion in lipid bilayers, providing publication-quality formatted results

Maritime Laser Scanning as the Source for Spatial Data

The rapid development of scanning technology, especially mobile scanning, gives the possibility to collect spatial data coming from maritime measurement platforms and autonomous manned or unmanned vehicles. Presented solution is derived from the mobile scanning. However we should keep in mind that the specificity of laser scanning at sea and processing collected data should be in the form acceptable in Geographical Information Systems, especially typical for the maritime needs. At the same time we should be aware that data coming from maritime mobile scanning constitutes a new approach to the describing of maritime environment and brings a new perspective that is completely different than air and terrestrial scanning

The composition and function of Enterococcus faecalis membrane vesicles.

10.1093/femsml/uqab002Microlife2uqab002

Ceramides And Stress Signalling Intersect With Autophagic Defects In Neurodegenerative Drosophila blue cheese (bchs) Mutants

10.1038/srep15926SCIENTIFIC REPORTS5

The question of the Eco-logical or Oikos-logical Architectural Space

Once all requirements of building in a sustainable way, cities or buildings, essentially guarantying less use of energy, only renewable materials, and all requirements of sustainability and so on, what has architecture itself, in his spatial structure, to do with ecology? This paper will present the requirements to architecture itself to be more then ‘sustainable’ but also ‘ecological’. The same question would be 'what is a non-anthropocentric architecture? So, we will make the difference between sustainable buildings or urbanism and ecological architecture (of buildings or of cities). The answer we would like to present is: We should orientate the mind in the direction of a non-anthropocentric structure. The structure of this anthropocentric mind is very centred on itself at the point that it presents an opposition between inside and outside; an opposition between finite and infinite; and an opposition between (sur)-face and depth. It is a closed mind. Eco-logical or Oikos-logical architecture or non-anthropocentric architecture is the architecture of a human which is not centered on itself in a closed mind, This human being has an open architecture in spatial non-opposition between inside and outside in spatial non-opposition between finite and infinite in spatial non-opposition between surface and depth We will present the scientific position of great psychologists and neurologists about the human being concluding that he cannot live centered on itself in anthropocentrism. - Great examples of Eco-logical or Oikos-logical architecture or non-anthropocentric architecture which gives aperture to the human being