Flat-top TIRF illumination boosts DNA-PAINT imaging and quantification

Super-resolution (SR) techniques have extended the optical resolution down to a few nanometers. However, quantitative treatment of SR data remains challenging due to its complex dependence on a manifold of experimental parameters. Among the different SR variants, DNA-PAINT is relatively straightforward to implement, since it achieves the necessary 'blinking' without the use of rather complex optical or chemical activation schemes. However, it still suffers from image and quantification artifacts caused by inhomogeneous optical excitation. Here we demonstrate that several experimental challenges can be alleviated by introducing a segment-wise analysis approach and ultimately overcome by implementing a flat-top illumination profile for TIRF microscopy using a commercially-available beam-shaping device. The improvements with regards to homogeneous spatial resolution and precise kinetic information over the whole field-of-view were quantitatively assayed using DNA origami and cell samples. Our findings open the door to high-throughput DNA-PAINT studies with thus far unprecedented accuracy for quantitative data interpretation

Myosin motors fragment and compact membrane-bound actin filaments

Cell cortex remodeling during cell division is a result of myofilament-driven contractility of the cortical membrane-bound actin meshwork. Little is known about the interaction between individual myofilaments and membrane-bound actin filaments. Here we reconstituted a minimal actin cortex to directly visualize the action of individual myofilaments on membrane-bound actin filaments using TIRF microscopy. We show that synthetic myofilaments fragment and compact membrane-bound actin while processively moving along actin filaments. We propose a mechanism by which tension builds up between the ends of myofilaments, resulting in compressive stress exerted to single actin filaments, causing their buckling and breakage. Modeling of this mechanism revealed that sufficient force (∼20 pN) can be generated by single myofilaments to buckle and break actin filaments. This mechanism of filament fragmentation and compaction may contribute to actin turnover and cortex reorganization during cytokinesis

Design of biochemical pattern forming systems from minimal motifs

Although molecular self-organization and pattern formation are key features of life, only very few pattern-forming biochemical systems have been identified that can be reconstituted and studied in vitro under defined conditions. A systematic understanding of the underlying mechanisms is often hampered by multiple interactions, conformational flexibility and other complex features of the pattern forming proteins. Because of its compositional simplicity of only two proteins and a membrane, the MinDE system from Escherichia coli has in the past years been invaluable for deciphering the mechanisms of spatiotemporal self-organization in cells. Here, we explored the potential of reducing the complexity of this system even further, by identifying key functional motifs in the effector MinE that could be used to design pattern formation from scratch. In a combined approach of experiment and quantitative modeling, we show that starting from a minimal MinE-MinD interaction motif, pattern formation can be obtained by adding either dimerization or membrane-binding motifs. Moreover, we show that the pathways underlying pattern formation are recruitment-driven cytosolic cycling of MinE and recombination of membrane-bound MinE, and that these differ in their in vivo phenomenology

Mass-sensitive particle tracking to elucidate the membrane-associated MinDE reaction cycle

An iSCAT image processing and analysis strategy enables mass-sensitive particle tracking (MSPT) of single unlabeled biomolecules on a supported lipid bilayer. MSPT was used to observe the (dis-)assembly of membrane complexes in real-time. In spite of their great importance in biology, methods providing access to spontaneous molecular interactions with and on biological membranes have been sparse. The recent advent of mass photometry to quantify mass distributions of unlabeled biomolecules landing on surfaces raised hopes that this approach could be transferred to membranes. Here, by introducing a new interferometric scattering (iSCAT) image processing and analysis strategy adapted to diffusing particles, we enable mass-sensitive particle tracking (MSPT) of single unlabeled biomolecules on a supported lipid bilayer. We applied this approach to the highly nonlinear reaction cycles underlying MinDE protein self-organization. MSPT allowed us to determine the stoichiometry and turnover of individual membrane-bound MinD/MinDE protein complexes and to quantify their size-dependent diffusion. This study demonstrates the potential of MSPT to enhance our quantitative understanding of membrane-associated biological systems.We thank D. Bollschweiler (Cryo-EM MPIB Core Facility) for the initial introduction to the commercial Refeyn OneMP mass photometer, the MPIB Biochemistry Core Facility (Recombinant Protein Production)

124-Color Super-resolution Imaging by Engineering DNA-PAINT Blinking Kinetics

Optical super-resolution techniques reach unprecedented spatial resolution down to a few nanometers. However, efficient multiplexing strategies for the simultaneous detection of hundreds of molecular species are still elusive. Here, we introduce an entirely new approach to multiplexed super-resolution microscopy by designing the blinking behavior of targets with engineered binding frequency and duration in DNA-PAINT. We assay this kinetic barcoding approach in silico and in vitro using DNA origami structures, show the applicability for multiplexed RNA and protein detection in cells, and finally experimentally demonstrate 124-plex super-resolution imaging within minutes.We thank Martin Spitaler and the imaging facility of the MPI of Biochemistry for confocal imaging support

Measurement of the reaction \gamma p \TO K^ + \Lambda(1520) at photon energies up to 2.65 GeV

The reaction \gamma p \TO K^+\Lambda(1520) was measured in the energy range from threshold to 2.65 GeV with the SAPHIR detector at the electron stretcher facility ELSA in Bonn. The $\Lambda(1520)$ production cross section was analyzed in the decay modes $pK^-$, $n \bar{K}^0$, $\Sigma^{\pm}\pi^{\mp}$, and $\Lambda\pi^+\pi^-$ as a function of the photon energy and the squared four-momentum transfer $t$. While the cross sections for the inclusive reactions rise steadily with energy, the cross section of the process \gamma p \TO K^+\Lambda(1520) peaks at a photon energy of about 2.0 GeV, falls off exponentially with $t$, and shows a slope flattening with increasing photon energy. The angular distributions in the $t$-channel helicity system indicate neither a $K$ nor a $K^\star$ exchange dominance. The interpretation of the $\Lambda(1520)$ as a $\Sigma(1385)\pi$ molecule is not supported.Comment: 11 pages, 16 figures, 4 table

Toward Absolute Molecular Numbers in DNA-PAINT

Single-molecule localization microscopy (SMLM) has revolutionized optical microscopy, extending resolution down to the level of individual molecules. However, the actual counting of molecules relies on preliminary knowledge of the blinking behavior of individual targets or on a calibration to a reference. In particular for biological applications, great care has to be taken because a plethora of factors influence the quality and applicability of calibration-dependent approaches to count targets in localization clusters particularly in SMLM data obtained from heterogeneous samples. Here, we present localization-based fluorescence correlation spectroscopy (lbFCS) as the first absolute molecular counting approach for DNA-points accumulation for imaging in nanoscale topography (PAINT) microscopy and, to our knowledge, for SMLM in general. We demonstrate that lbFCS overcomes the limitation of previous DNA-PAINT counting and allows the quantification of target molecules independent of the localization cluster density. In accordance with the promising results of our systematic proof-of-principle study on DNA origami structures as idealized targets, lbFCS could potentially also provide quantitative access to more challenging biological targets featuring heterogeneous cluster sizes in the future

Measurement of gamma p --> K+ Lambda and gamma p --> K+ Sigma0 at photon energies up to 2.6 GeV

The reactions gamma p --> K+ Lambda and gamma p --> K+ Sigma0 were measured in the energy range from threshold up to a photon energy of 2.6 GeV. The data were taken with the SAPHIR detector at the electron stretcher facility, ELSA. Results on cross sections and hyperon polarizations are presented as a function of kaon production angle and photon energy. The total cross section for Lambda production rises steeply with energy close to threshold, whereas the Sigma0 cross section rises slowly to a maximum at about E_gamma = 1.45 GeV. Cross sections together with their angular decompositions into Legendre polynomials suggest contributions from resonance production for both reactions. In general, the induced polarization of Lambda has negative values in the kaon forward direction and positive values in the backward direction. The magnitude varies with energy. The polarization of Sigma0 follows a similar angular and energy dependence as that of Lambda, but with opposite sign.Comment: 21 pages, 25 figures, submitted to Eur. Phys. J.

Design Features to Accelerate the Higher-Order Assembly of DNA Origami on Membranes

Nanotechnology often exploits DNA origami nanostructures assembled into even larger superstructures up to micrometer sizes with nanometer shape precision. However, large-scale assembly of such structures is very time-consuming. Here, we investigated the efficiency of superstructure assembly on surfaces using indirect cross-linking through low-complexity connector strands binding staple strand extensions, instead of connector strands binding to scaffold loops. Using single-molecule imaging techniques, including fluorescence microscopy and atomic force microscopy, we show that low sequence complexity connector strands allow formation of DNA origami superstructures on lipid membranes, with an order-of-magnitude enhancement in the assembly speed of superstructures. A number of effects, including suppression of DNA hairpin formation, high local effective binding site concentration, and multivalency are proposed to contribute to the acceleration. Thus, the use of low-complexity sequences for DNA origami higher-order assembly offers a very simple but efficient way of improving throughput in DNA origami design.Published as part of The Journal of Physical Chemistry virtual special issue “W. E. Moerner Festschrift”

Communication

The geometry of reaction compartments can affect the local outcome of interface-restricted reactions. Giant unilamellar vesicles (GUVs) are commonly used to generate cell-sized, membrane-bound reaction compartments, which are, however, always spherical. Herein, we report the development of a microfluidic chip to trap and reversibly deform GUVs into cigar-like shapes. When trapping and elongating GUVs that contain the primary protein of the bacterial Z ring, FtsZ, we find that membrane-bound FtsZ filaments align preferentially with the short GUV axis. When GUVs are released from this confinement and membrane tension is relaxed, FtsZ reorganizes reversibly from filaments into dynamic rings that stabilize membrane protrusions; a process that allows reversible GUV deformation. We conclude that microfluidic traps are useful for manipulating both geometry and tension of GUVs, and for investigating how both affect the outcome of spatially-sensitive reactions inside them, such as that of protein self-organization.We acknowledge the MPIB Biochemistry Core Facility for assistance in protein purification