The Shiga toxin B subunit (STxB), which is involved in cell membrane attachment and trafficking of Shiga holotoxin, binds specifically to the glycosphingolipid Gb 3. In biological membranes, Gb 3 glycosphingolipids differ in their fatty acid composition and there is strong evidence that the fatty acid alters the binding behaviour of STxB as well as the intracellular routing of the Shiga toxin/Gb 3 complex. To analyse the binding of STxB to different Gb 3 s, we chemically synthesized saturated, unsaturated, a-hydroxylated Gb 3 s and a combination thereof, all based on a C 24-fatty acid chain starting from monosaccharide building blocks, sphingosine and the respective fatty acids. These chemically well-defined Gb 3 s were inserted into solid supported phase-separated lipid bilayers composed of DOPC/sphingomyelin/cholesterol as a simple mimetic of the outer leaflet of animal cell membranes. By fluorescenceand atomic force microscopy the phase behaviour of the bilayer as well as the lateral organization of bound STxB were analysed. The fatty acid of Gb 3 significantly alters the ratio between the ordered and disordered phase and induces a third intermediate phase in the presence of unsaturated Gb 3. The lateral organization of STxB on the membranes varies significantly. While STxB attached to membranes with Gb 3 s with saturated fatty acids forms protein clusters, it is more homogeneously bound to membranes containing unsaturated Gb 3 s. Large interphase lipid redistribution is observed for a-hydroxylated Gb 3 doped membranes. Our results clearly demonstrate that the fatty acid of Gb 3 strongly influences the lateral organization of STxB on the membrane and impacts the overall membrane organization of phase-separated lipid membranes.
When excited with rotating linear polarized light, differently oriented fluorescent dyes emit periodic signals peaking at different times. We show that measurement of the average orientation of fluorescent dyes attached to rigid sample structures mapped to regularly defined (50 nm) 2 image nanoareas can provide subdiffraction resolution (super resolution by polarization demodulation, sPod). Because the polarization angle range for effective excitation of an oriented molecule is rather broad and unspecific, we narrowed this range by simultaneous irradiation with a second, de-excitation, beam possessing a polarization perpendicular to the excitation beam (excitation polarization angle narrowing, exPAn). this shortened the periodic emission flashes, allowing better discrimination between molecules or nanoareas. our method requires neither the generation of nanometric interference structures nor the use of switchable or blinking fluorescent probes. We applied the method to standard wide-field microscopy with camera detection and to two-photon scanning microscopy, imaging the fine structural details of neuronal spines.In recent years the development of super-resolution techniques has had a profound impact on biology and other fields in which subdiffraction-limited resolution of fluorescently labeled samples is desired [1][2][3][4][5][6][7][8][9][10][11][12][13] . Prominent examples are stimulated emission depletion (STED) microscopy 1,4 , photoactivated localization microscopy (PALM) 3,5,6 and stochastic optical reconstruction microscopy (STORM) 2,7 . Generally, these techniques are based on reversible switching between two states. Whereas STED is based on a deterministic switching in a nanometric interference pattern, STORM and PALM are based on wide-field illumination and stochastic switching on the level of single isolated molecules, which are then localized. Other approaches, such as super-resolution optical fluctuation imaging (SOFI) 8 , reversible saturable optical fluorescence transitions (RESOLFT) microscopy 9,11 and saturated structured illumination microscopy (SSIM) 12,13 , are also based on stochastic or deterministic switching between two states and provide spatial resolution enhancement. Here we present an alternative approach that distinguishes adjacent molecules or nanoareas in the sample (arranged, for example, in a grid of 50 nm × 50 nm rectangular areas) by different average orientations of fluorescent dyes attached to rigid sample structures within these nanoareas. This is done by rotating the polarization of a wide-field excitation beam and detecting the periodic signals emitted with different phases from different nanoareas using wide-field camera detection (SPoD). We also show that the range of polarization angles that results in effective excitation of differently oriented molecules can be substantially narrowed by rotating a second wide-field de-exciting stimulated emission beam of a polarization perpendicular to the excitation beam polarization (ExPAN), resulting in better spatial resolution ...
Based on a combination of jump segmentation and statistical multiresolution analysis for dependent data, a new approach called J-SMURF to idealize ion channel recordings has been developed. It is model-free in the sense that no a-priori assumptions about the channel’s characteristics have to be made; it thus complements existing methods which assume a model for the channel's dynamics, like hidden Markov models. The method accounts for the effect of an analog filter being applied before the data analysis, which results in colored noise, by adapting existing muliresolution statistics to this situation. J-SMURF’s ability to denoise the signal without missing events even when the signal-to-noise ratio is low is demonstrated on simulations as well as on ion current traces obtained from gramicidin A channels reconstituted into solvent-free planar membranes. When analyzing a newly synthesized acylated system of a fatty acid modified gramicidin channel, we are able to give statistical evidence for unknown gating characteristics such as subgating.
We propose a new model-free segmentation method, JULES, which combines recent statistical multiresolution techniques with local deconvolution for idealization of ion channel recordings. The multiresolution criterion takes into account scales down to the sampling rate enabling the detection of flickering events, i.e., events on small temporal scales, even below the filter frequency. For such small scales the deconvolution step allows for a precise determination of dwell times and, in particular, of amplitude levels, a task which is not possible with common thresholding methods. This is confirmed theoretically and in a comprehensive simulation study. In addition, JULES can be applied as a preprocessing method for a refined hidden Markov analysis. Our new methodology allows us to show that gramicidin A flickering events have the same amplitude as the slow gating events. JULES is available as an R function jules in the package clampSeg.
Shiga toxin subunit B (STxB) binding to its cellular receptor Gb3 leads to the formation of protein-lipid clusters and bending of the membrane. A newly developed synthetic route allowed synthesizing the biologically most relevant Gb3-C24:1 2OH species with both, the natural (Gb3-R) as well as the unnatural (Gb3-S) configuration of the 2OH group. The derivatives bind STxB with identical nanomolar affinity, while the propensity to induce membrane tubules in giant unilamellar vesicles is more pronounced for Gb3-S. Fluorescence and atomic force microscopy images of phase-separated supported membranes revealed differences in the lateral organization of the protein on the membrane. Gb3-R favorably induces large and tightly packed protein clusters, while a lower protein density is found on Gb3-S doped membranes.
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