1,721,059 research outputs found
High-throughput AFM analysis reveals unwrapping pathways of H3 and CENP-A nucleosomes
Get PDFNucleosomes, the fundamental units of chromatin, regulate readout and expression of eukaryotic genomes. Single-molecule experiments have revealed force-induced nucleosome accessibility, but a high-resolution unwrapping landscape in the absence of external forces is currently lacking. Here, we introduce a high-throughput pipeline for the analysis of nucleosome conformations based on atomic force microscopy and automated, multi-parameter image analysis. Our data set of ∼10 000 nucleosomes reveals multiple unwrapping states corresponding to steps of 5 bp DNA. For canonical H3 nucleosomes, we observe that dissociation from one side impedes unwrapping from the other side, but in contrast to force-induced unwrapping, we find only a weak sequence-dependent asymmetry. Notably, centromeric CENP-A nucleosomes do not unwrap anti-cooperatively, in stark contrast to H3 nucleosomes. Finally, our results reconcile previous conflicting findings about the differences in height between H3 and CENP-A nucleosomes. We expect our approach to enable critical insights into epigenetic regulation of nucleosome structure and stability and to facilitate future high-throughput AFM studies that involve heterogeneous nucleoprotein complexes.sponsorship: We thank Philipp Korber and Felix Muller-Planitz for help with initial nucleosome reconstitutions, Herlinde De Keersmaecker for assistance with AFM imaging, and Thomas Nicolaus for help with sample preparation. This work was funded by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) through SFB863 - project ID 111166240. W. F., T. B., and S. D. F. acknowledge KU Leuven - internal funds (IDO) and Fund for Scientific Research (FWO). (Deutsche Forschungsgemeinschaft (DFG, German Research Foundation)|SFB863, Deutsche Forschungsgemeinschaft (DFG, German Research Foundation)|111166240, Fund for Scientific Research (FWO), KU Leuven - internal funds (IDO))status: Publishe
High speed multispectral fluorescence lifetime imaging
No full textWe report a spectrally resolved fluorescence lifetime imaging system based on time gated single photon detection with a fixed gate width of 200 ps and 7 spectral channels. Time gated systems can operate at high count rates but usually have large gate widths and sample only part of the fluorescence decay curve. In the system presented in this work, the fluorescence signal is sampled using a high speed transceiver. An error analysis is carried out to characterize the performance of both lifetime and spectral detection. The effect of gate width and spectral channel width on the accuracy of estimated lifetimes and spectral widths is described. The performance of the whole instrument is evaluated at count rates of up to 12 MHz. Accurate fluorescence lifetimes (error <2%) are recorded at count rates as high as 5 MHz. This is limited by the PMT performance, not by the electronics. Analysis of the large spectral lifetime image sets is challenging and time-consuming. Here, we demonstrate the use of lifetime and spectral phasors for analyzing images of fibroblast cells with 2 different labeled components. The phasor approach provides a fast and intuitive way of analyzing the results of spectrally resolved fluorescence lifetime imaging experiments
Jammed elastic shells-a 3D experimental soft frictionless granular system
No full textWe present a new experimental system of monodisperse, soft, frictionless, fluorescent labeled elastic shells for the characterization of structure, universal scaling laws and force networks in 3D jammed matter. The elastic shells in a jammed packing are deformed in such a way that at each contact one of the shells buckles with a dimple and the other remain spherical, closely resembling overlapping spheres. Using confocal microscopy, we obtained 3D stacks of images of shells at different volume fractions which were subsequently processed in ImageJ software to find their coordinates. The determination of 3D coordinates involved three steps: locating the edges of shells in all 2D slices, analyzing their shape and subsequently finding their 2D coordinates, and finally determining their 3D centers by grouping the corresponding 2D coordinates. From this analysis routine we obtained particle coordinates with sub-pixel accuracy. In a contact pair we also identified the shell that underwent buckling forming a dimple by analyzing the intensity profile of a line that connects the centers of particle pairs. The amorphous structure of the packing was analyzed as a function of distance to the jamming threshold by investigating the radial distribution function, bond order parameters, contact numbers and the number of dimples per particle (buckling number), which is a unique property of this system. We find that the power law scaling of the contact number with excess volume fraction deviated from theoretical and computer simulation predictions. In addition, the buckling number also showed a similar scaling as that of the contact number with distance to the jamming transition. This journal i
Molecular structure, DNA binding mode, photophysical properties and recommendations for use of SYBR Gold
Get PDFSYBR Gold is a commonly used and particularly bright fluorescent DNA stain, however, its chemical structure is unknown and its binding mode to DNA remains controversial. Here, we solve the structure of SYBR Gold by NMR and mass spectrometry to be 2-N-(3-dimethylaminopropyl)-N-propylamino]-4-2,3-dihydro-3-methyl-(benzo-1,3-thiazol-2-yl)-methylidene-1-phenyl-quinolinium and determine its extinction coefficient. We quantitate SYBR Gold binding to DNA using two complementary approaches. First, we use single-molecule magnetic tweezers (MT) to determine the effects of SYBR Gold binding on DNA length and twist. The MT assay reveals systematic lengthening and unwinding of DNA by 19.1° ± 0.7° per molecule upon binding, consistent with intercalation, similar to the related dye SYBR Green I. We complement the MT data with spectroscopic characterization of SYBR Gold. The data are well described by a global binding model for dye concentrations ≤2.5~μM, with parameters that quantitatively agree with the MT results. The fluorescence increases linearly with the number of intercalated SYBR Gold molecules up to dye concentrations of ∼2.5~μM, where quenching and inner filter effects become relevant. In summary, we provide a mechanistic understanding of DNA-SYBR Gold interactions and present practical guidelines for optimal DNA detection and quantitative DNA sensing applications using SYBR Gold
Semidefinite programming for model-based sensorless adaptive optics
Get PDFWavefront sensorless adaptive optics methodologies are widely considered in scanning fluorescence microscopy where direct wavefront sensing is challenging. In these methodologies, aberration correction is performed by sequentially changing the settings of the adaptive element until a predetermined image quality metric is optimized. An efficient aberration correction can be achieved by modeling the image quality metric with a quadratic polynomial. We propose a new method to compute the parameters of the polynomial from experimental data. This method guarantees that the quadratic form in the polynomial is semidefinite, resulting in a more robust computation of the parameters with respect to existing methods. In addition, we propose an algorithm to perform aberration correction requiring a minimum of N+1 measurements, where N is the number of considered aberration modes. This algorithm is based on a closed-form expression for the exact optimization of the quadratic polynomial. Our arguments are corroborated by experimental validation in a laboratory environment.Delft Center for Systems and ControlMechanical, Maritime and Materials Engineerin
Patient autoantibodies induce platelet destruction signals via raft-associated glycoprotein Ibα and Fc RIIa in immune thrombocytopenia
No full textImmune thrombocytopenia (ITP) is an acquired immune-mediated disorder characterized by thrombocytopenia in the absence of an underlying cause.1 The patho-physiology of ITP is multifactorial and includes the development of autoantibodies that trigger abnormal thrombopoiesis, enhanced platelet destruction, complement activation and T-cell mediated effects.1–3 Platelet autoantibodies are detected in approximately 50% of patients4 and generally target the fibrinogen receptor αIIbβ3 or the receptor for von Willebrand factor (VWF), the glycoprotein (GP) Ib-V-IX complex. Anti-αIIbβ3 antibodies (70–80% of cases) are thought to induce thrombocytopenia through increased platelet clearance by Fcγ receptor-bearing macrophages. Autoantibodies against GPIb-V-IX (20–40% of cases) often induce a more severe fall in platelet count5 that is less responsive to standard therapies, such as intravenous immunoglobulin G (IVIG).6 Thrombocytopenia induced by GPIb-V-IX autoantibodies has not been characterized in great detail. Some monoclonal antibodies against GPIbα are known to induce platelet activation7 that may lead to accelerated platelet destruction in ITP patients8 with autoantibodies against this receptor. Here we report how an autoantibody against GPIbα, obtained from a patient with ITP, induces recognition signals for macrophages through interplay between glycoprotein Ibα and the low affinity IgG receptor FcγRIIa in lipid rafts
Platelet interaction with von Willebrand factor is enhanced by shear-induced clustering of glycoprotein Ibα
No full textInitial platelet arrest at the exposed arterial vessel wall is mediated through glycoprotein Ibα binding to the A1 domain of von Willebrand factor. This interaction occurs at sites of elevated shear force, and strengthens upon increasing hydrodynamic drag. The increased interaction requires shear-dependent exposure of the von Willebrand factor A1 domain, but the contribution of glycoprotein Ibα remains ill defined. We have previously found that glycoprotein Ibα forms clusters upon platelet cooling and hypothesized that such a property enhances the interaction with von Willebrand factor under physiological conditions. We analyzed the distribution of glycoprotein Ibα with Förster resonance energy transfer using time-gated fluorescence lifetime imaging microscopy. Perfusion at a shear rate of 1,600 s(-1) induced glycoprotein Ibα clusters on platelets adhered to von Willebrand factor, while clustering did not require von Willebrand factor contact at 10,000 s(-1). Shear-induced clustering was reversible, not accompanied by granule release or αIIbβ3 activation and improved glycoprotein Ibα-dependent platelet interaction with von Willebrand factor. Clustering required glycoprotein Ibα translocation to lipid rafts and critically depended on arachidonic acid-mediated binding of 14-3-3ζ to its cytoplasmic tail. This newly identified mechanism emphasizes the ability of platelets to respond to mechanical force and provides new insights into how changes in hemodynamics influence arterial thrombus formation
A conformational transition of the D′D3 domain primes von Willebrand factor for multimerization
Get PDFVon Willebrand factor (VWF) is a multimeric plasma glycoprotein that is critically involved in hemostasis. Biosynthesis of long VWF concatemers in the endoplasmic reticulum and the trans-Golgi is still not fully understood. We use the single-molecule force spectroscopy technique magnetic tweezers to analyze a previously hypothesized conformational change in the D′D3 domain crucial for VWF multimerization. We find that the interface formed by submodules C8-3, TIL3, and E3 wrapping around VWD3 can open and expose 2 buried cysteines, Cys1099 and Cys1142, that are vital for multimerization. By characterizing the conformational change at varying levels of force, we can quantify the kinetics of the transition and stability of the interface. We find a pronounced destabilization of the interface on lowering the pH from 7.4 to 6.2 and 5.5. This is consistent with initiation of the conformational change that enables VWF multimerization at the D′D3 domain by a decrease in pH in the trans-Golgi network and Weibel-Palade bodies. Furthermore, we find a stabilization of the interface in the presence of coagulation factor VIII, providing evidence for a previously hypothesized binding site in submodule C8-3. Our findings highlight the critical role of the D′D3 domain in VWF biosynthesis and function, and we anticipate our methodology to be applicable to study other, similar conformational changes in VWF and beyond
Twisting DNA by salt
Get PDFThe structure and properties of DNA depend on the environment, in particular the ion atmosphere. Here, we investigate how DNA twist -one of the central properties of DNA- changes with concentration and identity of the surrounding ions. To resolve how cations influence the twist, we combine single-molecule magnetic tweezer experiments and extensive all-atom molecular dynamics simulations. Two interconnected trends are observed for monovalent alkali and divalent alkaline earth cations. First, DNA twist increases monotonously with increasing concentration for all ions investigated. Second, for a given salt concentration, DNA twist strongly depends on cation identity. At 100 mM concentration, DNA twist increases as Na(+) < K(+) < Rb(+) < Ba(2+) < Li(+) ≈ Cs(+) < Sr(2+) < Mg(2+) < Ca(2+). Our molecular dynamics simulations reveal that preferential binding of the cations to the DNA backbone or the nucleobases has opposing effects on DNA twist and provides the microscopic explanation of the observed ion specificity. However, the simulations also reveal shortcomings of existing force field parameters for Cs(+) and Sr(2+). The comprehensive view gained from our combined approach provides a foundation for understanding and predicting cation-induced structural changes both in nature and in DNA nanotechnology
A modified phasor approach for analyzing time-gated fluorescence lifetime images
No full textFluorescence lifetime imaging is a versatile tool that permits mapping the biochemical environment in the cell. Among various fluorescence lifetime imaging techniques, timecorrelated single photon counting and time-gating methods have been demonstrated to be very efficient and robust for the imaging of biological specimens. Recently, the phasor representation of lifetime images became popular because it provides an intuitive graphical view of the fluorescence lifetime content of the images and, when used for global analysis, significantly improves the overall S/N of lifetime analysis. Compared to time-correlated single photon counting, time gating methods can provide higher count rates (∼10 MHz) but at the cost of truncating and under sampling the decay curve due to the limited number of gates commonly used. These limitations also complicate the implementation of the phasor analysis for time-gated data. In this work, we propose and validate a theoretical framework that overcomes these problems. This modified approach is tested on both simulated lifetime images and on cells.We demonstrate that this method is able to retrieve two lifetimes from time gating data that cannot be resolved using standard (non-global) fitting techniques. The new approach increases the information that can be obtained from typical measurements and simplifies the analysis of fluorescence lifetime imaging data
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