Biology subjects

Jungmann, R.

Publications and source records attributed to Jungmann, R..

36 records · Page 2Linked to original sources

bioRxiv2021

Simultaneous multicolor DNA-PAINT without sequential fluid exchange using spectral demixing

Several variants of multicolor single-molecule localization microscopy (SMLM) have been developed to resolve the spatial relationship of nanoscale structures in biological samples. The oligonucleotide-based SMLM approach DNA-PAINT robustly achieves nanometer localization precision and can be used to count binding sites within nanostructures. However, multicolor DNA-PAINT has primarily been realized by Exchange-PAINT that requires sequential exchange of the imaging solution and thus leads to extended acquisition times. To alleviate the need for fluid exchange and to speed up the acquisition of current multichannel DNA-PAINT, we here present a novel approach that combines DNA-PAINT with simultaneous multicolor acquisition using spectral demixing (SD). By using newly designed probes and a novel multichannel registration procedure we achieve simultaneous multicolor SD-DNA-PAINT with minimal crosstalk. We demonstrate high localization precision (3 - 6 nm) and multicolor registration of dual and triple-color SD-DNA-PAINT by resolving patterns on DNA origami nanostructures and cellular structures.

biophysics↗

bioRxiv2021

Nanoscale organization of the endogenous ASC speck

The NLRP3 inflammasome is a central component of the innate immune system. Its activation leads to the formation of a supramolecular assembly of the inflammasome adaptor protein ASC, commonly referred to as the "ASC speck". Different models of the overall structure of the ASC speck, as well as the entire NLRP3 inflammasome, have been reported in the literature. While many experiments involve overexpression or in vitro reconstitution of recombinant ASC, the cytoplasmic endogenous ASC speck remains difficult to study due to its relatively small size and structural variability. Here, we use a combination of fluorescence imaging techniques including dual-color 3D super-resolution imaging (dSTORM and DNA-PAINT) to visualize the endogenous ASC speck following NLRP3 inflammasome activation. We observe that the complex varies in diameter between [~]800 and 1000 nm and is composed of a dense core from which filaments extend into the periphery. We used a combination of anti-ASC antibodies as well as much smaller nanobodies for labeling, and find that the larger antibodies reliably label the lower-density periphery whereas the nanobody, which has a lower binding affinity, is more efficient in labeling the dense core. Imaging whole cells using dSTORM, furthermore, allowed us to sort the imaged structures into a pseudo-time sequence suggesting that the endogenous ASC speck becomes denser but not much larger during its formation. The reported results add an important piece of information towards a comprehensive understanding of the supramolecular structure of the endogenous inflammasome complex. SignificanceThe assembly of ASC into a supramolecular complex referred to as the ASC speck is a critical step in the NLRP3 inflammasome response. Despite significance research, the organization and formation of the endogenous ASC speck remain elusive. We used a combination of differently sized ASC labels with super-resolution microscopy to address the organization of the ASC speck at the nanoscale. Our results allow inference of the supramolecular structure of the unperturbed, endogenous speck and characterize how it changes during ASC recruitment. We find that the complex varies in size and is composed of a dense core from which filaments extend into the periphery. Notably, our results further indicate that during inflammasome formation, ASC specks become denser but only slightly larger.

immunology↗

bioRxiv2021

Calibration free counting of low molecular copy numbers in single DNA-PAINT localization clusters

Single-Molecule Localization Microscopy (SMLM) has revolutionized light microscopy by enabling optical resolutions down to a few nanometer. Yet, localization precisions commonly not suffice to visually resolve single subunits in molecular assemblies or multimeric complexes. Since each targeted molecule contributes localizations during image acquisition, molecular counting approaches to reveal the target copy numbers within localization clusters have been continuously proposed since the early days of SMLM, most of which rely on preliminary knowledge of the dye photo-physics or on a calibration to a reference. Previously, we developed localization-based Fluorescence Correlation Spectroscopy (lbFCS) as an absolute ensemble counting approach for the SMLM-variant DNA-Points Accumulation for Imaging in Nanoscale Topography (PAINT), for the first time circumventing the necessity for reference calibrations. Here, we present a revised framework termed lbFCS+ which allows absolute counting of copy numbers for individual localization clusters in a single DNA-PAINT image. In lbFCS+, absolute counting in individual clusters is achieved via precise measurement of the local hybridization rates of the fluorescently-labeled oligonucleotides ( imagers) employed in DNA-PAINT imaging. In proof-of-principle experiments on DNA origami nanostructures, we demonstrate the ability of lbFCS+ to truthfully determine molecular copy numbers and imager association and dissociation rates in well-separated localization clusters containing up to six docking strands. We show that lbFCS+ allows to resolve heterogeneous binding dynamics enabling the distinction of stochastically generated and a priori indistinguishable DNA assemblies. Beyond advancing quantitative DNA-PAINT imaging, we believe that lbFCS+ could find promising applications ranging from bio-sensing to DNA computing.

biophysics↗

bioRxiv2021

Visualization of loop extrusion by DNA nanoscale tracing in single cells

The spatial organization of the genome is essential for its functions, including gene expression, DNA replication and repair, as well as chromosome segregation. Biomolecular condensates and loop extrusion have been proposed as the principal driving forces that underlie the formation of chromatin compartments and topologically associating domains, respectively. However, whether the actual 3D-fold of DNA in single cells is consistent with these mechanisms has been difficult to address in situ. Here, we present LoopTrace, a workflow for nanoscale 3D imaging of the genome sequence in structurally well-preserved nuclei in single human cells. Tracing the in situ structure of DNA in thousands of individual cells reveals that genomic DNA folds as a flexible random coil in the absence of loop extruding enzymes such as Cohesin. In the presence of Cohesin and its boundary factor CTCF, reproducibly positioned loop structures dominate the folds, while Cohesin alone leads to randomly positioned loops. The 3D structure and size variability of DNA loops we observe in a large number of single cells allow us to formulate a data-constrained computational model of genomic DNA folding that explains how sparse and dynamic loops in single cells lead to the emergence of compact topological domains in averages of cell populations.

cell biology↗

bioRxiv2021

Principles of RNA recruitment to viral ribonucleoprotein condensates in a segmented dsRNA virus

Rotaviruses transcribe eleven distinct protein-coding RNAs that must be stoichiometrically co-packaged prior to their replication to make an infectious virion. During infection, rotavirus transcripts accumulate in cytoplasmic ribonucleoprotein (RNP) condensates, termed viroplasms. Understanding the mechanisms of viroplasm assembly and RNA enrichment within is crucial to gaining greater insight into their function and stoichiometric assortment of individual transcripts. We analysed the subcellular distribution of individual RV transcripts and viroplasm transcriptome by combining multiplexed DNA-barcoded single-molecule RNA FISH of infected cells. Using DNA-PAINT microscopy, we provide evidence of the early onset of viral transcript oligomerisation that occurs prior to the formation of viroplasms. We demonstrate that viral sequences lacking the conserved terminal regions fail to undergo enrichment in rotavirus RNP condensates. We show that individual viral transcripts exhibit variable propensities to partition into viroplasms, irrespective of their absolute numbers in cells, suggesting a selective RNA enrichment mechanism distinct from other known cellular RNP granules. We suggest that rotavirus replication factories represent unique RNP condensates enriched in eleven types of cognate transcripts that may facilitate the assembly of a multi-segmented RNA genome.

biochemistry↗

bioRxiv2020

Rotavirus Replication Factories Are Complex Ribonucleoprotein Condensates

RNA viruses induce formation of subcellular organelles that provide microenvironments conducive to their replication. Here we show that replication factories of rotaviruses represent protein-RNA condensates that are formed via liquid-liquid phase separation. We demonstrate that rotavirus proteins NSP5 and NSP2 undergo phase separation in vitro and form RNA-rich condensates in vivo that can be reversibly dissolved by aliphatic diols. During infection, these RNA-protein condensates became less dynamic and impervious to aliphatic diols, indicating a transition from a liquid to solid state. Some aspects of assembly of rotavirus replication factories mirror the formation of cytoplasmic ribonucleoprotein granules, while the selective enrichment of viral transcripts appears to be a unique feature of these condensates. Such complex RNA-protein condensates that underlie replication of RNA viruses represent an attractive target for developing novel therapeutic approaches.

biophysics↗

bioRxiv2020

Direct Supercritical Angle Localization Microscopy for Nanometer 3D Superresolution

3D single molecule localization microscopy (SMLM) is an emerging superresolution method for structural cell biology, as it allows probing precise positions of proteins in cellular structures. Supercritical angle fluorescence strongly depends on the z-position of the fluorophore and can be used for z localization in a method called supercritical angle localization microscopy (SALM). Here, we realize the full potential of SALM by directly splitting supercritical and undercritical emission, using an ultra-high NA objective, and applying new fitting routines to extract precise intensities of single emitters, resulting in a four-fold improved z-resolution compared to the state of the art. We demonstrate nanometer isotropic localization precision on DNA origami structures, and on clathrin coated vesicles and microtubules in cells, illustrating the potential of SALM for cell biology.

biophysics↗

bioRxiv2020

DNA origami demonstrate the unique stimulatory power of single pMHCs as T-cell antigens

T-cells detect with their T-cell antigen receptors (TCRs) the presence of rare peptide/MHC complexes (pMHCs) on the surface of antigen presenting cells (APCs). How they convert a biochemical interaction into a signaling response is poorly understood, yet indirect evidence pointed to the spatial antigen arrangement on the APC surface as a critical factor. To examine this, we engineered a biomimetic interface based on laterally mobile functionalized DNA origami platforms, which allow for nanoscale control over ligand distances without interfering with the cell-intrinsic dynamics of receptor clustering. We found that the minimum signaling unit required for efficient T-cell activation consisted of two ligated TCRs within a distance of 20 nanometers, if TCRs were stably engaged by monovalent antibody fragments. In contrast, antigenic pMHCs stimulated T-cells robustly as well-isolated entities. These results identify the minimal requirements for effective TCR-triggering and validate the exceptional stimulatory potency of transiently engaging pMHCs.

biophysics↗

bioRxiv2020

Tracking Single Particles for Hours via Continuous DNA-mediated Fluorophore Exchange

Fluorophores are commonly used to covalently label biomolecules for monitoring their motion in single particle tracking experiments. However, photobleaching is still a major bottleneck in these experiments, as the fluorophores finite photon budget typically limits observation times to merely a few seconds. Here, we overcome this inherent constraint via continuous fluorophore exchange based on DNA-PAINT, whereby fluorescently-labeled oligonucleotides bind to a 54 bp single-stranded DNA handle attached to the molecule of interest. When we assayed our approach in vitro by tracking single DNA origami, first surface-immobilized and subsequently diffusing on supported lipid bilayers, we were able to observe these origami for up to hours without losing their fluorescence signals. Our versatile and easily implemented labeling approach allows monitoring single-molecule motion and interactions over unprecedented observation periods, opening the doors to advanced quantitative studies.

biophysics↗

bioRxiv2020

Nanoscale pattern extraction from relative positions of sparse 3D localisations

We present a method for extracting high-resolution ordered features from localisation microscopy data by analysis of relative molecular positions in 2D or 3D. This approach allows pattern recognition at sub-1% protein detection efficiencies, in large and heterogeneous samples, and in 2D and 3D datasets. We used this method to infer ultrastructure of the nuclear pore, the cardiomyocyte Z-disk, DNA origami structures and the centriole.

cell biology↗

bioRxiv2019

Three dimensional particle averaging for structural imaging of macromolecular complexes by localization microscopy

We present an approach for 3D particle fusion in localization microscopy which dramatically increases signal-to-noise ratio and resolution in single particle analysis. Our method does not require a structural template, and properly handles anisotropic localization uncertainties. We demonstrate 3D particle reconstructions of the Nup107 subcomplex of the nuclear pore complex (NPC), cross-validated using multiple localization microscopy techniques, as well as two-color 3D reconstructions of the NPC, and reconstructions of DNA-origami tetrahedrons.

molecular biology↗

bioRxiv2019

Circumvention of common labeling artifacts using secondary nanobodies

The most common procedure to reveal the location of specific (sub)cellular elements in biological samples is via immunostaining followed by optical imaging. This is typically performed with target-specific primary antibodies (1.Abs), which are revealed by fluorophore-conjugated secondary antibodies (2.Abs). However, at high resolution this methodology can induce a series of artifacts due to the large size of antibodies, their bivalency, and their polyclonality. Here we use STED and DNA-PAINT super-resolution microscopy or light sheet microscopy on cleared tissue to show how monovalent secondary reagents based on camelid single-domain antibodies (nanobodies; 2.Nbs) attenuate these artifacts. We demonstrate that monovalent 2.Nbs have four additional advantages: 1) they increase localization accuracy with respect to 2.Abs; 2) they allow direct pre-mixing with 1.Abs before staining, reducing experimental time, and enabling the use of multiple 1.Abs from the same species; 3) they penetrate thick tissues efficiently; and 4) they avoid the artificial clustering seen with 2.Abs both in live and in poorly fixed samples. Altogether, this suggests that 2.Nbs are a valuable alternative to 2.Abs, especially when super-resolution imaging or staining of thick tissue samples are involved.

cell biology↗

bioRxiv2019

Sub-Nanometer Precision using Bayesian Grouping of Localizations

Single-molecule localization microscopy super-resolution methods such as DNA-PAINT and (d)STORM can generate multiple observed localizations over the time course of data acquisition from each dye or binding site that are not a priori assigned to those specific dyes or binding sites. We describe a Bayesian method of grouping and combining localizations from multiple blinking/binding events that can improve localization precision to better than one nanometer. The known statistical distribution of the number of binding/blinking events per dye/docking strand along with the precision of each localization event are used to estimate the true number and location of emitters in closely-spaced clusters.

biophysics↗

bioRxiv2019

A rationally designed and highly versatile epitope tag for nanobody-based purification, detection and manipulation of proteins

Specialized epitope tags are widely used for detecting, manipulating or purifying proteins, but often their versatility is limited. Here, we introduce the ALFA-tag, a novel, rationally designed epitope tag that serves an exceptionally broad spectrum of applications in life sciences while outperforming established tags like the HA, FLAG or myc tags. The ALFA-tag forms a small and stable -helix that is functional irrespective of its position on the target protein in prokaryotic and eukaryotic hosts. We developed a nanobody (NbALFA) binding ALFA-tagged proteins from native or fixed specimen with low picomolar affinity. It is ideally suited for super-resolution microscopy, immunoprecipitations and Western blotting, and also allows in-vivo detection of proteins. By solving the crystal structure of the complex we were able to design a nanobody mutant (NbALFAPE) that permits efficient one-step purifications of native ALFA-tagged proteins, complexes and even entire living cells using peptide elution under physiological conditions.

biochemistry↗

bioRxiv2019

Direct visualization of single nuclear pore complex proteins using genetically-encoded probes for DNA-PAINT

The Nuclear Pore Complex (NPC) is one of the largest and most complex protein assemblies in the cell and - among other functions - serves as the gatekeeper of nucleocytoplasmic transport. Unraveling its molecular architecture and functioning has been an active research topic for decades with recent cryogenic electron microscopy and superresolution studies advancing our understanding of the NPC's complex architecture. However, the specific and direct visualization of single copies of NPC proteins and thus the ability to observe single-molecule heterogeneities of these complex structures is thus far elusive. Here, we combine genetically-encoded self-labeling enzymes such as SNAP-tag and HaloTag with DNA-PAINT microscopy. We employ the high localization precision in DNA-PAINT and molecular contrast of these protein tags to optically resolve single copies of nucleoporins in the human Y-complex in three dimensions with a precision of ~3 nm. This technological advancement now enables structural studies of multicomponent complexes on the level of single proteins in cells using optical fluorescence microscopy.

cell biology↗

bioRxiv2019

Localization microscopy at doubled precision with patterned illumination

MINFLUX offers a breakthrough in single molecule localization precision, but suffers from a tiny field-of-view and a lack of practical parallelism. Here, we combine centroid estimation and illumination pattern induced photon count variations in a conventional widefield imaging setup to extract position information over a typical micron sized field-of-view. We show a near twofold improvement in precision over standard localization with the same photon count on DNA-origami nano-structures.

molecular biology↗

bioRxiv2019

Bayesian Multiple Emitter Fitting using Reversible Jump Markov Chain Monte Carlo

In single molecule localization-based super-resolution imaging, high labeling density or the desire for greater data collection speed can lead to clusters of overlapping emitter images in the raw super-resolution image data. We describe a Bayesian inference approach to multiple-emitter fitting that uses Reversible Jump Markov Chain Monte Carlo to identify and localize the emitters in dense regions of data. This formalism can take advantage of any prior information, such as emitter intensity and density. The output is both a posterior probability distribution of emitter locations that includes uncertainty in the number of emitters and the background structure, and a set of coordinates and uncertainties from the most probable model.

biophysics↗

bioRxiv2017

Rapid Sequential In Situ Multiplexing With DNA-Exchange-Imaging

To decipher the molecular mechanism of biological function, it is critical to map the molecular composition of individual cells in the context of their biological environment in situ. Immunofluorescence (IF) provides specific labeling for molecular profiling. However, conventional IF methods have finite multiplexing capabilities due to spectral overlap of the fluorophores. Various sequential imaging methods have been developed to circumvent this spectral limit, but are not widely adopted due to the common limitation of requiring multi-rounds of slow (typically over 2 hours at room temperature to overnight at 4 {degrees}C in practice) immunostaining. DNA-Exchange-Imaging is a practical platform for rapid in situ spectrally-unlimited multiplexing. This technique overcomes speed restrictions by allowing for single-step immunostaining with DNA-barcoded antibodies, followed by rapid (less than 10 minutes) buffer exchange of fluorophore-bearing DNA imager strands. By eliminating the need for multiple rounds of immunostaining, DEI enables rapid spectrally unlimited sequential imaging. The programmability of DNA-Exchange-Imaging allows us to further adapt it to diverse microscopy platforms (with Exchange-Confocal, Exchange-SIM, Exchange-STED, and Exchange-PAINT demonstrated here), achieving highly multiplexed in situ protein visualization in diverse samples (including neuronal and tumor cells as well as fresh-frozen or paraffin-embedded tissue sections) and at multiple desired resolution scales (from ~300 nm down to sub-20-nm). Validation highlights include 8-target imaging using single-channel Exchange-Confocal in tens of micron thick retina tissue sections in 2-3 hours (as compared to days required in principle by previous methods using comparable equipment), and 8-target super-resolution imaging with ~20 nm resolution using Exchange-PAINT in primary neurons. These results collectively suggest DNA-Exchange as a versatile, practical platform for rapid, highly multiplexed in situ imaging, potentially enabling new applications ranging from basic science, to drug discovery, and to clinical pathology.

bioengineering↗