Biology subjects

Doose, S.

Publications and source records attributed to Doose, S..

14 recordsLinked to original sources

bioRxiv2026

Programmable protein reference standards for benchmarking sub-10-nm fluorescence microscopy

Fluorescence microscopy is increasingly used to measure molecular organization at length scales where labeling, photophysics and sample preparation can dominate quantitative accuracy. Reference standards are therefore needed that combine defined nanoscale geometry with a protein-like environment and compatibility with biological imaging. Here we introduce circular tandem repeat protein as programmable protein standards for benchmarking sub-10-nm fluorescence microscopy. Using genetic code expansion and bioorthogonal labeling, we generated compact protein rings carrying up to six labeling sites. Photoswitching fingerprint analysis revealed geometry-dependent localization accumulation and blinking kinetics, demonstrating that short-range fluorophore interactions can be assessed as measurable benchmark parameters. DNA-PAINT confirmed accessible docking sites and programmed valency at the single-particle level. We further established recombinant tethering and genetically encoded membrane display, extending the standards to cellular environments. cTRP PicoRulers were also compatible with expansion microscopy. Together, cTRPs provide a modular protein-based platform for evaluating molecular-scale imaging performance in purified and cellular environments.

biophysics↗

bioRxiv2026

Two-dye-imager DNA-PAINT enables volumetric nanoscopy of expanded cells

Refined single-molecule localization microscopy methods demonstrated superior localization precisions on isolated sample but remain limited by labeling density and imaging speed in cells. Here we combine expansion microscopy (ExM) with two-dye-imager (TDI)-DNA-PAINT to resolve fine molecular details of protein assemblies in [~]8-fold expanded cells with nanometer resolution. Using lattice light-sheet (LLS) microscopy, Ex-TDI-DNA-PAINT provides a robust platform for three-dimensional (3D) volumetric nanoscopy of the molecular organization of cells.

biophysics↗

bioRxiv2026

Nanoscale imaging of expanded cells and proteins with spontaneously blinking dyes

Expansion microscopy (ExM) enables nanoscale imaging on standard microscopes, but combining ExM with single-molecule localization microscopy (SMLM) remains difficult, owing to the incompatibility of expanded hydrogels with photoswitching buffers. Here, we introduce a single-step expansion microscopy method that allows SMLM with spontaneously blinking dyes in 6-14x expanded samples, without re-embedding. We demonstrate nanometer-resolution imaging by resolving the organization of the nuclear pore complex (NPC) and the molecular structure of recombinant homotrimeric proliferating cell nuclear antigen (PCNA).

biophysics↗

bioRxiv2025

Single-molecule localization microscopy reveals the molecular organization of endogenous membrane receptors

Super-resolution microscopy in combination with genetic labeling methods allows imaging of single proteins in cells. However, visualizing endogenous proteins on primary cells remains challenging due to the use of sterically demanding antibodies for labeling. Here, we demonstrate how immunolabeling conditions and antibody crosslinking influence the quantification and identification of membrane receptor stoichiometry on cells using single-molecule localization microscopy. We developed an optimized immunolabeling and analysis protocol and demonstrate the performance of the approach by resolving the molecular organization of endogenous CD45, CD69, and CD38 on Jurkat T cells. To demonstrate the usefulness of the method for immunotherapy applications we investigated the interaction of primary multiple myeloma cells with the therapeutic monoclonal antibodies (mAbs) daratumumab and isatuximab, and a polyclonal anti-CD38 antibody. Our approach might lay the foundation for improved personalized diagnostics and treatment with therapeutic antibodies. One-Sentence SummarySingle-molecule localization microcopy quantifies the expression and resolves the stoichiometry of endogenous membrane receptors

biophysics↗

bioRxiv2025

Resolving protein organization in cells with nanometer resolution

Advances in super-resolution microscopy have outpaced molecular labeling methods, impeding nanometer-scale imaging of endogenous multiprotein complexes in cells. To overcome these limitations, we developed an expansion microscopy (ExM) approach based on double-homogenized hydrogels that enables direct stochastic optical reconstruction microscopy (dSTORM) of 7-8-fold expanded immunolabeled samples. The resulting [~]4-fold increase in effective immunolabeling density resolved the 8 nm distance between neighboring -tubulin molecules in microtubules and the polyhedral lattice in clathrin-coated pits with nanometer precision in cells. Two-color Ex-dSTORM further revealed the molecular organization of RIM scaffolding protein and Munc13-1, an essential synaptic vesicle priming protein, in ring-like structures with diameters of 40-45 nm at the presynapse in hippocampal neurons. Our results demonstrate that Ex-dSTORM resolves the molecular organization of endogenous multiprotein complexes with nanometer spatial resolution in genetically unmodified cells. Thus, it provides a versatile method for the investigation of molecular protein distributions in their physiologically relevant context. One-Sentence SummaryThe optimized combination of dSTORM and double TREx ExM resolves previously unresolvable protein assemblies in cells

biophysics↗

bioRxiv2025

RBM20-variants induce distinct calcium handling and metabolic phenotypes in patient-specific stem cell models of dilated and non-compaction cardiomyopathy

Background and aimMutations in the splice regulator RBM20 account for [~]3 % of genetic cardiomyopathies. In particular, the highly conserved RS domain is a hotspot for disease-associated mutations. Previously, mutations at same amino acid position 634 in the hotspot RS-domain were found to cause dilated cardiomyopathy (DCM) with left ventricular non-compaction (R634L) or without (R634W), but the pathophysiological mechanisms that govern the heterogeneity in phenotype presentation remained unknown. Here, we identify the molecular events caused by the distinct RBM20 mutations from DCM and left-ventricular non-compaction (LVNC) using patient-specific stem cell models. MethodsWe generated induced pluripotent stem cell-derived cardiomyocytes (iPSC-CM) of one LVNC- and two DCM-patients harboring the RBM20-mutations R634L (LVNC) or R634W (DCM). We investigated alternative splicing activity, RBM20 localization, sarcomeric regularity, cAMP level, kinase-specific phosphorylation of key Ca2+ handling enzymes, physiological cardiac functions as Ca2+ homeostasis, and metabolic activity on a patient-specific cardiomyocyte level. Force generation was analyzed in patient-specific engineered myocardial tissues. Isogenic rescue and mutation insertion lines were generated by CRISPR/Cas9 technology to analyze the direct impact of the RBM20 mutations on the cardiac phenotype. ResultsWe observed common splicing aberrations for LVNC- and DCM-CM in TTN and RYR2, RBM20 cytoplasmatic accumulation and irregular sarcomeric structure. LVNC-CM harboring the RBM20-p.R634L variant show distinct molecular, cellular and functional impairments that manifest in CAMK2D, TRDN and IMMT mis-splicing. Splicing defects in LVNC-CM correlate with elevated systolic Ca2+ and faster Ca2+ kinetics with elevated cAMP levels and PLN-hyperphosphorylation. An increased metabolic activity and mitochondrial membrane potential support the hyperactive LVNC-CM. By contrast, DCM-CM (RBM20-p.R634W) distinctly present with decreased systolic Ca2+ and increased SR Ca2+leak but unchanged Ca2+ kinetics and metabolic activity. Both mutations lead to severely reduced force of contraction in engineered myocardium. CRISPR/Cas9 gene-edited isogenic control lines of both described RBM20 mutations in LVNC and DCM demonstrated the causative nature of the two mutations and their diverging effects. Further, L-type Ca2+ channel blockade by verapamil ameliorates the Ca2+ cycling and leakage phenotypes in LVNC- and DCM-CM. ConclusionWe show the first comparative iPSC-model of splice-defect-associated RBM20-dependent LVNC-p.R634L and DCM-p.R634W. We found shared and variant-specific phenotypes on a patient-specific level. Our data demonstrate that the different RBM20 mutations manifest in distinct molecular aberrations in alternative splicing and RBM20 cytoplasmic accumulation that convey various physiological impairments in structure, Ca2+ handling, metabolism and contractile force.

molecular biology↗

bioRxiv2023

Fast, whole-cell DNA-PAINT imaging with dual-labeled self-quenched imager probes

Elucidating the interaction between membrane proteins and antibodies requires fast whole-cell imaging at high spatiotemporal resolution. Lattice light-sheet (LLS) microscopy offers fast volumetric imaging but suffers from limited spatial resolution. DNA-PAINT achieves molecular resolution but is practically restricted to two-dimensional imaging due to long acquisition times. Here, we introduce two-dye imager (TDI) probes, manifesting negligible background and amplified fluorescence signal upon transient binding, enabling [~]15-fold faster imaging. Using a combination of TDI-DNA-PAINT and LLS microscopy on B cells, we reveal the oligomeric states and interaction of endogenous CD20 with the therapeutic monoclonal antibody rituximab (RTX), unperturbed by surface effects. Our results demonstrate that B cells become polarized, and microvilli stabilized by RTX binding. These findings, we believe, will aid rational design of improved immunotherapies targeting tumor-associated antigens.

biophysics↗

bioRxiv2023

CARs are organized in nanodomains in the plasma membrane of T cells that accumulate at tumor contact sites

Chimeric antigen receptors (CARs) are synthetic immune receptors that are expressed in T cells through genetic engineering. CAR-T cells have been successfully used to eradicate very advanced leukemias and lymphomas and their functional properties have been intensively studied. However, relatively little is known about the spatiotemporal expression and organization of CARs on the T-cell membrane and how this influences their efficacy. Here, we applied super-resolution microscopy to visualize CD19-, ROR1-, and ROR2-specific CARs in human CD4+ and CD8+ T cells that were engineered with lentiviral and transposon-mediated gene transfer. Our data show that the majority of CARs is organized in nanodomains virtually independent of the T cell type, CAR construct and expression level. Quantitative analyses revealed a slightly higher CAR density in transposon-engineered T cells correlating with higher antigen sensitivity and faster resolution of anti-tumor functions compared to lentivirally-engineered T cells. Live-cell fluorescence imaging revealed that both, CAR nanodomains and CAR monomers accumulate at tumor contact sites and form multifocal immunological synapses. Our study provides novel insights into the membrane organization of CARs with single-molecule resolution and illustrates the potential of advanced microscopy to inform the rational design of synthetic immune receptors for applications in immune cell therapy.

immunology↗

bioRxiv2023

Coronaviruses use ACE2 monomers as entry receptors

The angiotensin-converting enzyme 2 (ACE2) has been identified as entry receptor on cells enabling binding and infection with the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) via trimeric spike (S) proteins protruding from the viral surface1,2. It has been suggested that trimeric S proteins preferably bind to plasma membrane areas with high concentrations of preferably multimeric ACE2 receptors to achieve a higher binding and infection efficiency1,3. However, our current knowledge about the influence of ACE2 expression and organization in the plasma membrane on SARS-CoV-2 infection efficiency remains elusive. Here we used direct stochastic optical reconstruction microscopy (dSTORM) in combination with different labeling approaches to visualize the distribution and quantify the expression of ACE2 on different cells. Our results reveal that endogenous ACE2 receptors are present as monomers in the plasma membrane with densities of only 1-2 receptors m-2. In addition, binding of trimeric S proteins does not induce clustering of ACE2 receptors in the plasma membrane. Supported by infection studies using vesicular stomatitis virus (VSV) particles bearing S proteins our data demonstrate that a single S protein interaction per virus particle with a monomeric ACE2 receptor is sufficient for infection which attests SARS-CoV-2 a high infectivity.

molecular biology↗

bioRxiv2022

Sub-10 nm fluorescence imaging

Advances in superresolution microscopy demonstrated single-molecule localization precisions of a few nanometers. However, translation of such high localization precisions into sub-10 nm spatial resolution in biological samples remains challenging. Here, we show that resonance energy transfer between fluorophores separated by less than 10 nm results in accelerated fluorescence blinking and consequently lower localization probabilities impeding sub-10 nm fluorescence imaging. We demonstrate that time-resolved fluorescence detection in combination with photoswitching fingerprint analysis can be used advantageously to determine the number and distance even of spatially unresolvable fluorophores in the sub-10 nm range. In combination with genetic code expansion (GCE) with unnatural amino acids and bioorthogonal click-labeling with small fluorophores photoswitching fingerprint analysis enables sub-10 nm resolution fluorescence imaging in cells.

biophysics↗

bioRxiv2021

Dynamic remodeling of ribosomes and endoplasmic reticulum in axon terminals of motoneurons

In neurons, endoplasmic reticulum forms a highly dynamic network that enters axons and presynaptic terminals and plays a central role in Ca2+ homeostasis and synapse maintenance. However, the underlying mechanisms involved in regulation of its dynamic remodeling as well as its function in axon development and presynaptic differentiation remain elusive. Here, we used high resolution microscopy and live cell imaging to investigate rapid movements of endoplasmic reticulum and ribosomes in axons of cultured motoneurons after stimulation with Brain-derived neurotrophic factor. Our results indicate that the endoplasmic reticulum extends into axonal growth cone filopodia where its integrity and dynamic remodeling are regulated mainly by actin and its motor protein myosin VI. Additionally, we found that in axonal growth cones, ribosomes assemble into 80S subunits within seconds and associate with ER in response to extracellular stimuli which describes a novel function of axonal ER in dynamic regulation of local translation. Summary statementIn axonal growth cones of motoneurons, dynamic remodeling of ER is coordinated through drebrin A-mediated actin and microtubule crosstalk and contributes to local translation as response to BDNF stimulation.

neuroscience↗

bioRxiv2021

Bioorthogonal labeling of transmembrane proteins with non-canonical amino acids allows access to masked epitopes in live neurons

Progress in biological imaging is intrinsically linked to advances in labeling methods. The explosion in the development of high-resolution and super-resolution imaging calls for new approaches to label targets with small probes. These should allow to faithfully report the localization of the target within the imaging resolution - typically nowadays a few nanometers - and allow access to any epitope of the target, in the native cellular and tissue environment. We report here the development of a complete labeling and imaging pipeline using genetic code expansion and non-canonical amino acids in primary neurons that allows to fluorescently label masked epitopes in target transmembrane proteins in live neurons, both in dissociated culture and organotypic brain slices. This allowed us to image the differential localization of two glutamate receptor auxiliary proteins in complex with their partner with a variety of methods including widefield, confocal, and dSTORM super-resolution microscopy.

cell biology↗

bioRxiv2019

Active zone compaction for presynaptic strength

Brain function relies on neurotransmission which is stabilized by presynaptic homeostatic potentiation (PHP). PHP operates on time scales ranging from minute- to life-long adaptations and likely involves reorganization of presynaptic active zones (AZs). At Drosophila melanogaster neuromuscular junctions, earlier work ascribed AZ enlargement by incorporating more Bruchpilot (Brp) scaffold protein a central mechanistic role in PHP. We used localization microscopy (direct stochastic optical reconstruction microscopy, dSTORM) and hierarchical density-based spatial clustering of applications with noise (HDBSCAN) to study AZ plasticity during PHP. We found that both acute, philanthotoxin (PhTx)-induced and chronic, genetically-induced PHP lead to compaction of individual AZs without altering Brp copy numbers per AZ. This compaction even occurs within Brp subclusters of the AZ scaffold which also move towards AZ centers. Furthermore, lowering imaging resolution revealed how AZ compaction in PHP translates into apparent increases in AZ area and Brp protein content as implied earlier. Our results suggest AZ compaction in PHP as an effective mechanism to raise presynaptic protein density and transmitter release. SIGNIFICANCE STATEMENTHomeostatic plasticity stabilizes chemical synaptic transmission in multiple organisms ranging from insects to humans. Changes in active zones (AZs), membrane specializations of the presynapse where synaptic vesicles are discharged, are thought to be crucial in homeostatic adaptations. AZ growth by protein incorporation was proposed as a core mechanism in presynaptic homeostatic potentiation (PHP). Localization microscopy of an abundant AZ scaffold protein uncovered that instead of growing, AZs are compacted in acute and chronic PHP. At lower imaging resolution, however, AZs appear larger and brighter although protein numbers are not increased. In summary, our findings suggest AZ compaction as new and effective mechanism to raise presynaptic protein density and transmitter release in PHP.

neuroscience↗

bioRxiv2018

Bioorthogonal labeling with tetrazine-dyes forsuper-resolution microscopy

Genetic code expansion (GCE) technology allows the specific incorporation of functionalized noncanonical amino acids (ncAAs) into proteins. Here, we investigated the Diels-Alder reaction between trans-cyclooct-2-ene (TCO)-modified ncAAs, and 22 known and novel 1,2,4,5-tetrazine-dye conjugates spanning the entire visible wavelength range. A hallmark of this reaction is its fluorogenicity - the tetrazine moiety can elicit substantial quenching of the dye. We discovered that photoinduced electron transfer (PET) from the excited dye to tetrazine as the main quenching mechanism in red-absorbing oxazine and rhodamine derivatives. Upon reaction with dienophiles quenching interactions are reduced resulting in a considerable increase in fluorescence intensity. Efficient and specific labeling of all tetrazine-dyes investigated permits super-resolution microscopy with high signal-to-noise ratio even at the single-molecule level. The different cell permeability of tetrazine-dyes can be used advantageously for specific intra- and extracellular labeling of proteins and highly sensitive fluorescence imaging experiments in fixed and living cells.

biochemistry↗