Direct Stochastic Optical Reconstruction Microscopy Research Articles

Elucidating the Functional Mechanism of PTK7 in Cancer Development through Spatial Assembly Analysis Using Super Resolution Imaging.

Protein tyrosine kinase-7 (PTK7) has been reported as a vital participant in the Wnt signaling pathway, influencing tumorigenesis and metastasis. However, their specific roles in the mechanisms underlying cancer development and progression remain elusive. Here, using direct stochastic optical reconstruction microscopy (dSTORM) with aptamer-probe labeling, we first revealed that a weakening clustering distribution of PTK7 on the basal membranes happened as cellular migration increased during cancer progression. This correspondence was further supported by a diminished aggregated state of PTK7 caused by direct enhancement of cell migration. By comparing the alterations in PTK7 distribution with activation or inhibition of specific Wnt signaling pathway, we speculated that PTK7 could modulate cell migration by participating in the interplay between canonical Wnt (in MCF7 cells) and noncanonical Wnt signals (in MDA-MB-231 cells). Furthermore, we discovered that the spatial distribution morphology of PTK7 was also subject to the hydrolysis ability and activation state of the related hydrolase Matrix metallopeptidase14 (MMP14). This function-related specific assembly of PTK7 reveals a clear relationship between PTK7 and cancer. Meanwhile, potential molecular interactions predicted by the apparent assembly morphology can promote a deep understanding of the functional mechanism of PTK7 in cancer progress.

Nuclear patterns of phosphatidylinositol 4,5- and 3,4-bisphosphate revealed by super-resolution microscopy differ between the consecutive stages of RNA polymerase II transcription.

Phosphatidylinositol phosphates are powerful signaling molecules that orchestrate signaling and direct membrane trafficking in the cytosol. Interestingly, phosphatidylinositol phosphates also localize within the membrane-less compartments of the cell nucleus, where they participate in the regulation of gene expression. Nevertheless, current models of gene expression, which include condensates of proteins and nucleic acids, do not include nuclear phosphatidylinositol phosphates. This gap is partly a result of the missing detailed analysis of the subnuclear distribution of phosphatidylinositol phosphates and their relationships with gene expression. Here, we used quantitative dual-color direct stochastic optical reconstruction microscopy to analyze the nanoscale co-patterning between RNA polymerase II transcription initiation and elongation markers with respect to phosphatidylinositol 4,5- or 3,4-bisphosphate in the nucleoplasm and nuclear speckles and compared it with randomized data and cells with inhibited transcription. We found specific co-patterning of the transcription initiation marker P-S5 with phosphatidylinositol 4,5-bisphosphate in the nucleoplasm and with phosphatidylinositol 3,4-bisphosphate at the periphery of nuclear speckles. We showed the specific accumulation of the transcription elongation marker PS-2 and of nascent RNA in the proximity of phosphatidylinositol 3,4-bisphosphate associated with nuclear speckles. Taken together, this shows that the distinct spatial associations between the consecutive stages of RNA polymerase II transcription and nuclear phosphatidylinositol phosphates exhibit specificity within the gene expression compartments. Thus, in analogy to the cellular membranes, where phospholipid composition orchestrates signaling pathways and directs membrane trafficking, we propose a model in which the phospholipid identity of gene expression compartments orchestrates RNA polymerase II transcription.

Affinity fine-tuning anti-CAIX CAR-T cells mitigate on-target off-tumor side effects

One of the major hurdles that has hindered the success of chimeric antigen receptor (CAR) T cell therapies against solid tumors is on-target off-tumor (OTOT) toxicity due to sharing of the same epitopes on normal tissues. To elevate the safety profile of CAR-T cells, an affinity/avidity fine-tuned CAR was designed enabling CAR-T cell activation only in the presence of a highly expressed tumor associated antigen (TAA) but not when recognizing the same antigen at a physiological level on healthy cells. Using direct stochastic optical reconstruction microscopy (dSTORM) which provides single-molecule resolution, and flow cytometry, we identified high carbonic anhydrase IX (CAIX) density on clear cell renal cell carcinoma (ccRCC) patient samples and low-density expression on healthy bile duct tissues. A Tet-On doxycycline-inducible CAIX expressing cell line was established to mimic various CAIX densities, providing coverage from CAIX-high skrc-59 tumor cells to CAIX-low MMNK-1 cholangiocytes. Assessing the killing of CAR-T cells, we demonstrated that low-affinity/high-avidity fine-tuned G9 CAR-T has a wider therapeutic window compared to high-affinity/high-avidity G250 that was used in the first anti-CAIX CAR-T clinical trial but displayed serious OTOT effects. To assess the therapeutic effect of G9 on patient samples, we generated ccRCC patient derived organotypic tumor spheroid (PDOTS) ex vivo cultures and demonstrated that G9 CAR-T cells exhibited superior efficacy, migration and cytokine release in these miniature tumors. Moreover, in an RCC orthotopic mouse model, G9 CAR-T cells showed enhanced tumor control compared to G250. In summary, G9 has successfully mitigated OTOT side effects and in doing so has made CAIX a druggable immunotherapeutic target.

Increased vesicular dynamics and nanoscale clustering of IL-2 after T cell activation

T cells coordinate intercellular communication through the meticulous regulation of cytokine secretion. Direct visualization of vesicular transport and intracellular distribution of cytokines provides valuable insights into the temporal and spatial mechanisms involved in regulation. Employing Jurkat E6-1 T cells and interleukin-2 (IL-2) as a model system, we investigated vesicular dynamics using single-particle tracking and the nanoscale distribution of intracellular IL-2 in fixed T cells using superresolution microscopy. Live-cell imaging revealed that in vitro activation resulted in increased vesicular dynamics. Direct stochastic optical reconstruction microscopy and 3D structured illumination microscopy revealed nanoscale clustering of IL-2. In vitro activation correlated with spatial accumulation of IL-2 nanoclusters into more pronounced and elongated clusters. These observations provide visual evidence that accelerated vesicular transport and spatial concatenation of IL-2 clusters at the nanoscale may constitute a potential mechanism for modulating cytokine release by Jurkat T cells.

Versatile Endogenous Editing of GluRIIA in Drosophila melanogaster.

Glutamate receptors at the postsynaptic side translate neurotransmitter release from presynapses into postsynaptic excitation. They play a role in many forms of synaptic plasticity, e.g., homeostatic scaling of the receptor field, activity-dependent synaptic plasticity and the induction of presynaptic homeostatic potentiation (PHP). The latter process has been extensively studied at Drosophila melanogaster neuromuscular junctions (NMJs). The genetic removal of the glutamate receptor subunit IIA (GluRIIA) leads to an induction of PHP at the synapse. So far, mostly imprecise knockouts of the GluRIIA gene have been utilized. Furthermore, mutated and tagged versions of GluRIIA have been examined in the past, but most of these constructs were not expressed under endogenous regulatory control or involved the mentioned imprecise GluRIIA knockouts. We performed CRISPR/Cas9-assisted gene editing at the endogenous locus of GluRIIA. This enabled the investigation of the endogenous expression pattern of GluRIIA using tagged constructs with an EGFP and an ALFA tag for super-resolution immunofluorescence imaging, including structured illumination microscopy (SIM) and direct stochastic optical reconstruction microscopy (dSTORM). All GluRIIA constructs exhibited full functionality and PHP could be induced by philanthotoxin at control levels. By applying hierarchical clustering algorithms to analyze the dSTORM data, we detected postsynaptic receptor cluster areas of ~0.15 µm2. Consequently, our constructs are suitable for ultrastructural analyses of GluRIIA.

Super-Resolution Imaging of Tau Proteins in Isolated and Immobilized Brain Synaptosomes.

Tau protein has important physiological functions at both presynaptic and postsynaptic terminals. Pathological tau species are also associated with synaptic dysfunctions in several neurodegenerative disorders, especially Alzheimer's disease. To understand tau distribution inside synaptic compartments, super-resolution imaging is required. Here, we describe a facile protocol to immobilize and image brain synaptosomes without aggregation artefacts, by substituting the standard fixative paraformaldehyde with ethylene glycol bis(succinimidyl succinate) (EGS). Super-resolution imaging of tau proteins is achieved through three-color direct stochastic optical reconstruction microscopy (dSTORM). Tau protein is found to colocalize with synaptic vesicles as well as postsynaptic densities.

A Photoactive Supramolecular Complex Targeting PD-L1 Reveals a Weak Correlation between Photoactivation Efficiency and Receptor Expression Levels in Non-Small-Cell Lung Cancer Tumor Models.

Photo-immunotherapy uses antibodies conjugated to photosensitizers to produce nanostructured constructs endowed with targeting properties and photo-inactivation capabilities towards tumor cells. The superficial receptor density on cancer cells is considered a determining factor for the efficacy of the photodynamic treatment. In this work, we propose the use of a photoactive conjugate that consists of the clinical grade PD-L1-binding monoclonal antibody Atezolizumab, covalently linked to either the well-known photosensitizer eosin or the fluorescent probe Alexa647. Using single-molecule localization microscopy (direct stochastic optical reconstruction microscopy, dSTORM), and an anti-PD-L1 monoclonal antibody labelled with Alexa647, we quantified the density of PD-L1 receptors exposed on the cell surface in two human non-small-cell lung cancer lines (H322 and A549) expressing PD-L1 to a different level. We then investigated if this value correlates with the effectiveness of the photodynamic treatment. The photodynamic treatment of H322 and A549 with the photo-immunoconjugate demonstrated its potential for PDT treatments, but the efficacy did not correlate with the PD-L1 expression levels. Our results provide additional evidence that receptor density does not determine a priori the level of photo-induced cell death.

Differential CD38 Expression on Daratumumab-Naïve Vs. Resistant Multiple Myeloma - a Direct Stochastic Optical Reconstruction Microscopy ( dSTORM) Analysis in 53 Patients

Background: The anti-CD38 antibody Daratumumab (Dara) has become the new standard of care for the treatment of newly-diagnosed and relapsed/ refractory multiple myeloma (MM). At the same time, Dara resistance remains poorly understood. Potential resistance mechanisms have been proposed and range from intrinsic downregulation in CD38 dim MM cells to genetic alterations and splicing variants which may affect CD38 epitope expression (Adamia, ASH 2022). Here, we used super-resolution microscopy for high-precision quantification of CD38 in MM and piloted this approach in a large cohort of 53 MM patients with vs. without Dara resistance. Methods: Direct stochastic optical reconstruction microscopy ( dSTORM) is a novel imaging modality based on photoswitching of certain fluorophores coupled to antibodies for molecular representation of surface molecules at unprecedented levels of resolution (<20 nM). To correlate CD38 expression data with clinical outcome, we divided our cohort based on treatment status into two groups: Dara-naïve (n = 31) vs. Dara-resistant (n = 22) patients. Imaging data was complemented by phenotypic assessment using a well-established multi-epitope anti-CD38 antibody (Cytognos), as per standard-of-care. Results: Mean CD38 receptor density as measured with Dara via dSTORM was significantly higher in Dara-naïve (of 48.81 ± 29.65 receptors/µm²) vs. Dara-resistant patients (15.52 ± 3.9 receptors/µm², p = 0.016). This finding was corroborated by multi-epitope phenotypic analysis which revealed mean CD38 receptor expression levels of 32.09 ± 13.14 receptors/µm² in Dara-naïve vs. 21.65 ± 17.73 receptors/µm² in Dara-resistant patients ( p = 0.05). Lower CD38 expression via dSTORM vs. multi-epitope anti-CD38 staining was consistently observed in our cohort and may reflect ongoing blockage of the receptor binding site by Dara in cases with shorter time to last Dara application (median 53 days, range 1-189 days). As there was no correlation with the interval between Dara application and sampling however, splice-site mutations affecting CD38 receptor accessibility as proposed by Adamia et al. may depict a more relevant mechanism of resistance in our Dara-resistant cohort. In a next step, we therefore investigated alternative epitope binding in Dara-resistant patients. To this end, we investigated antibody binding by dSTORM imaging for isatuximab (Isa), an alternative CD38-directed monoclonal antibody that binds to a location in the C-terminal portion of the CD38 extracellular domain which is entirely different from the Dara epitope. Interestingly, there was a trend for increased detection rate with Isa for mean CD38 receptor expression in Dara-resistant patients (22.2 ± 4.6 receptors/µm²) vs. Dara (15.5 ± 3.9 receptors/µm², p = 0.1) and on par with CD38 receptor expression. No data was available to assess Dara binding in Isa pre-exposed patients. Conclusion: Innovative microscopy using dSTORM allows for high-resolution epitope quantification at unprecedented granularity. While this finding enables visual proof of differential epitope binding of Dara vs. Isa on CD38 in a clinically feasible setting, correlation studies to determine the clinical relevance of this observation remain ongoing.

Secondary Genetic Events Impact the Expression of Key Immunotargets on the Surface of Multiple Myeloma Cells

Background: Multiple myeloma (MM) is characterized by complex genomic aberrations including primary translocations of the immunoglobin heavy chain (IGH) locus and secondary mutations involving key oncogenic drivers. This temporal complexity represents a constant challenge for the clinical management of MM, but at the same time may provide new avenues for immunotherapy-based approaches including CAR-T cells and T-cell engaging antibodies. In this study, we investigated the impact of secondary genetic events on the surface expression of common targets for immunotherapy in MM. Methods: Cell models harboring two hotspot activating mutations in KRAS (G12A, A146T) and TP53 (R282W) were created using the MM cell lines OPM-2 and AMO1 via CRISPR-Cas9 technology. Direct stochastic optical reconstruction microscopy ( dSTORM) was used for high-resolution receptor density estimation and epitope quantification of common immunotherapeutic targets such as CD38, BCMA and SLAMF7. Results: dSTORM imaging of cell line models carrying patient-derived secondary genetic alterations revealed a distinct pattern linked to each mutation respectively. In KRAS G12A, we observed a reduction in BCMA density by 2-fold as compared to wild type cells (5.9 ± 0.3 vs. 10.8 ± 1.2 localization clusters/µm 2, p=0005). This reduction in BCMA surface expression was accompanied by a decrease in other key immunotargets, including SLAMF7 (6.0 ± 0.8 vs 11.8 ± 0.8 localization clusters/µm 2, p <0,0001) and CD38 (12.2 ± 1.5 vs 17.9 ± 1.6 localization clusters/µm 2, p=0,012). In KRAS A146, CD38 density increased, whereas mean BCMA and SLAMF7 localizations were consistently reduced as compared to wild type control. To ensure that BCMA downregulation is a consistent feature in genomic high-risk disease, we next simulated a TP53 double-hit situation by introducing an inactivating hotspot mutation R282W in a TP53 deletion background. Surfaceome profiling by dSTORM revealed a minor increase in CD38 levels for TP53R282W/Del as compared to wild type control (51.5 ± 5.7 vs. 36.4 ± 4.2 localization clusters/µm 2, p=0.06). Interestingly, the expression level of BCMA, known to be comparably low on WT tumor cells, further decreased in TP53 double-hit cells (0.6 ± 0.1 vs 1.1 ± 0.1 localization clusters/µm 2, p=0.002), whereas no major differences in the cluster density of SLAMF7 were observed. This leaves room for speculation whether these effects on biallelic TP53 inactivated tumor cells may underlie reduced susceptibility towards BCMA-targeting therapy. Of note, we also observed altered cell morphology in the TP53 double-hit model, characterized by elongated stress fibers enriched with CD138, potentially interfering with receptor accessibility of surface receptors (Figure 1). Recently, secondary genetic events have been reported in BCMA and GPRC5D following targeted immunotherapy inducing acquired drug resistance. We created cell models representing homo- and heterozygous genetic alterations in BCMA and GPRC5D, studies to assess their effects on the surfaceome are ongoing and will be presented at the meeting. Summary: Our data suggest that surface epitope expression in MM cells seems to be less agnostic to secondary genomic events than expected. These findings may provide a direct link between genetic high-risk disease and inferior efficacy of anti-BCMA immunotherapies in the context of e.g. TP53 double-hit MM.

Quantitatively Lighting up the Spatial Organization of CD47/SIRPα Immune Checkpoints on the Cellular Membrane with Single-Molecule Localization Microscopy.

Immunotherapy including immune checkpoint inhibition has reinvigorated the current cancer treatment field. The development of efficient cancer immunotherapies depends on a thorough understanding of the status of immune checkpoints and how they interact. However, the distribution and spatial organization changes of immune checkpoints during their interactions at the single-molecule level remain difficult to directly visualize due to the lack of in situ imaging techniques with appropriate spatial and stoichiometric resolution. Herein, we report the direct visualization and quantification of the spatial distribution and organization of CD47 on the bladder tumor cell membrane and SIRPα on the macrophage membrane by using a single-molecule localization imaging technique called quantitative direct stochastic optical reconstruction microscopy (QdSTORM). Results showed that a portion of CD47 and SIRPα was present on cell membranes as heterogeneous clusters of varying sizes and densities prior to activation. Quantitative analyses of the reconstructed super-resolution images and theoretical simulation revealed that CD47 and SIRPα were reorganized into larger clusters upon binding to each other. Furthermore, we found that blocking the immune checkpoint interaction with small-molecule inhibitors or antibodies significantly impacted the spatial clustering behavior of CD47 on bladder tumor cells, demonstrating the promise of our QdSTORM strategy in elucidating the molecular mechanisms underlying immunotherapy. This work offers a promising strategy to advance our understanding of immune checkpoint state and interactions while also contributing to the fields including signal regulation and cancer therapy.

Super-Resolution-Chip: an in-vitro platform that enables super-resolution microscopy of co-cultures and 3D systems.

The development of organs-on-a-chip platforms has revolutionized in-vitro cellular culture by allowing cells to be grown in an environment that better mimics human physiology. However, there is still a challenge in integrating those platforms with advanced imaging technology. This is extremely important when we want to study molecular changes and subcellular processes on the level of a single molecule using super-resolution microscopy (SRM), which has a resolution beyond the diffraction limit of light. Currently, existing platforms that include SRM have certain limitations, either as they only support 2D monocultures, without flow or as they demand a lot of production and handling. In this study, we developed a Super-Res-Chip platform, consisting of a 3D-printed chip and a porous membrane, that could be used to co-culture cells in close proximity either in 2D or in 3D while allowing SRM on both sides of the membrane. To demonstrate the functionality of the device, we co-cultured in endothelial and epithelial cells and used direct stochastic optical reconstruction microscopy (dSTORM) to investigate how glioblastoma cells affect the expression of the gap-junction protein Connexin43 in endothelial cells grown in 2D and in 3D. Cluster analysis of Connexin43 distribution revealed no difference in the number of clusters, their size, or radii, but did identify differences in their density. Furthermore, the spatial resolution was high also when the cells were imaged through the membrane (20-30 nm for x-y) and 10-20 nm when imaged directly both for 2D and 3D conditions. Overall, this chip allows to characterize of complex cellular processes on a molecular scale in an easy manner and improved the capacity for imaging in a single molecule resolution complex cellular organization.

Spatial mapping and quantitative evaluation of protein corona on PEGylated mesoporous silica particles by super-resolution fluorescence microscopy

Nanoparticles (NPs) adsorb serum proteins when exposed to biological fluids, forming a dynamic protein corona that has a profound impact on their overall biological profile and fate. Polyethylene glycol (PEG) modification is the most widely used strategy to mitigate and inhibit protein corona formation. Nevertheless, the accurate mapping and quantification of PEG inhibition effects on protein corona formation have scarcely been reported. Herein, we demonstrate the direct observation and quantification of protein corona adsorbed onto PEGylated mesoporous silica particles by direct stochastic optical reconstruction microscopy (dSTORM). The variation tendency of protein penetration depth in terms of PEG molecular weights and incubated time is investigated for the first time. The maximum penetration depths present slight increase with the prolonged incubation time, while they tend to remarkably decrease with increased chain length of modified PEG. Moreover, the co-localization of preformed protein corona with lysosomes and the destination of adsorbed protein are demonstrated. Our method provides important technical characterization information and in-depth understanding of protein corona adsorbed onto PEGylated mesoporous silica particles. This shines new light on the behaviors of silica materials in cells and may promote their practical applications in biomedicine.

Heterogeneity of Signaling Complex Nanostructure in T Cells Activated Via the T Cell Antigen Receptor.

Activation of the T cell antigen receptor (TCR) is a key step in initiating the adaptive immune response. Single-molecule localization techniques have been used to investigate the arrangement of proteins within the signaling complexes formed around activated TCRs, but a clear picture of nanoscale organization in stimulated T cells has not emerged. Here, we have improved the examination of T cell nanostructure by visualizing individual molecules of six different proteins in a single sample of activated Jurkat T cells using the multiplexed antibody-size limited direct stochastic optical reconstruction microscopy (madSTORM) technique. We formally define irregularly shaped regions of interest, compare areas where signaling complexes are concentrated with other areas, and improve the statistical analyses of the locations of molecules. We show that nanoscale organization of proteins is mainly confined to the areas with dense concentrations of TCR-based signaling complexes. However, randomly distributed molecules are also found in some areas containing concentrated signaling complexes. These results are consistent with the view that the proteins within signaling complexes are connected by numerous weak interactions, leading to flexible, dynamic, and mutable structures which produce large variations in the nanostructure found in activated T cells.

Direct observation of secondary nucleation along the fibril surface of the amyloid β 42 peptide

Alzheimer's disease is a neurodegenerative condition which involves heavy neuronal cell death linked to oligomers formed during the aggregation process of the amyloid β peptide 42 (Aβ42). The aggregation of Aβ42 involves both primary and secondary nucleation. Secondary nucleation dominates the generation of oligomers and involves the formation of new aggregates from monomers on catalytic fibril surfaces. Understanding the molecular mechanism of secondary nucleation may be crucial in developing a targeted cure. Here, the self-seeded aggregation of WT Aβ42 is studied using direct stochastic optical reconstruction microscopy (dSTORM) with separate fluorophores in seed fibrils and monomers. Seeded aggregation proceeds faster than nonseeded reactions because the fibrils act as catalysts. The dSTORM experiments show that monomers grow into relatively large aggregates on fibril surfaces along the length of fibrils before detaching, thus providing a direct observation of secondary nucleation and growth along the sides of fibrils. The experiments were repeated for cross-seeded reactions of the WT Aβ42 monomer with mutant Aβ42 fibrils that do not catalyze the nucleation of WT monomers. While the monomers are observed by dSTORM to interact with noncognate fibril surfaces, we fail to notice any growth along such fibril surfaces. This implies that the failure to nucleate on the cognate seeds is not a lack of monomer association but more likely a lack of structural conversion. Our findings support a templating role for secondary nucleation, which can only take place if the monomers can copy the underlying parent structure without steric clashes or other repulsive interactions between nucleating monomers.

Subunit-Dependent Surface Mobility and Localization of NMDA Receptors in Hippocampal Neurons Measured Using Nanobody Probes.

NMDA receptors (NMDARs) are ionotropic glutamate receptors that play a key role in excitatory neurotransmission. The number and subtype of surface NMDARs are regulated at several levels, including their externalization, internalization, and lateral diffusion between the synaptic and extrasynaptic regions. Here, we used novel anti-GFP (green fluorescent protein) nanobodies conjugated to either the smallest commercially available quantum dot 525 (QD525) or the several nanometer larger (and thus brighter) QD605 (referred to as nanoGFP-QD525 and nanoGFP-QD605, respectively). Targeting the yellow fluorescent protein-tagged GluN1 subunit in rat hippocampal neurons, we compared these two probes to a previously established larger probe, a rabbit anti-GFP IgG together with a secondary IgG conjugated to QD605 (referred to as antiGFP-QD605). The nanoGFP-based probes allowed faster lateral diffusion of the NMDARs, with several-fold increased median values of the diffusion coefficient (D). Using thresholded tdTomato-Homer1c signals to mark synaptic regions, we found that the nanoprobe-based D values sharply increased at distances over 100 nm from the synaptic edge, while D values for antiGFP-QD605 probe remained unchanged up to a 400 nm distance. Using the nanoGFP-QD605 probe in hippocampal neurons expressing the GFP-GluN2A, GFP-GluN2B, or GFP-GluN3A subunits, we detected subunit-dependent differences in the synaptic localization of NMDARs, D value, synaptic residence time, and synaptic-extrasynaptic exchange rate. Finally, we confirmed the applicability of the nanoGFP-QD605 probe to study differences in the distribution of synaptic NMDARs by comparing to data obtained with nanoGFPs conjugated to organic fluorophores, using universal point accumulation imaging in nanoscale topography and direct stochastic optical reconstruction microscopy.SIGNIFICANCE STATEMENT Our study systematically compared the localization and mobility of surface NMDARs containing GFP-GluN2A, GFP-GluN2B, or GFP-GluN3A subunits expressed in rodent hippocampal neurons, using anti-green fluorescent protein (GFP) nanobodies conjugated to the quantum dot 605 (nanoGFP-QD605), as well as nanoGFP probes conjugated with small organic fluorophores. Our comprehensive analysis showed that the method used to delineate the synaptic region plays an important role in the study of synaptic and extrasynaptic pools of NMDARs. In addition, we showed that the nanoGFP-QD605 probe has optimal parameters for studying the mobility of NMDARs because of its high localization accuracy comparable to direct stochastic optical reconstruction microscopy and longer scan time compared with universal point accumulation imaging in nanoscale topography. The developed approaches are readily applicable to the study of any GFP-labeled membrane receptors expressed in mammalian neurons.

Coronaviruses Use ACE2 Monomers as Entry‐Receptors

AbstractThe 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 surface. It has been suggested that trimeric S proteins preferably bind to plasma membrane areas with high concentrations of possibly multimeric ACE2 receptors to achieve a higher binding and infection efficiency. 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 μm−2. In addition, binding of trimeric S proteins does not induce the formation of ACE2 oligomers 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 provides SARS‐CoV‐2 a high infectivity.

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 surface. It has been suggested that trimeric S proteins preferably bind to plasma membrane areas with high concentrations of possibly multimeric ACE2 receptors to achieve a higher binding and infection efficiency. 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 μm-2 . In addition, binding of trimeric S proteins does not induce the formation of ACE2 oligomers 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 provides SARS-CoV-2 a high infectivity.

Assembly Characterization of Human Equilibrium Nucleoside Transporter 1 (hENT1) by Inhibitor Probe-Based dSTORM Imaging.

Nucleoside transporters (NTs) play an important role in the metabolism of nucleoside substances and the efficacy of nucleoside drugs. Its spatial information related to biofunctions at the single-molecule level remains unclear, owing to the limitation of the existing labeling methods and traditional imaging methods. Therefore, we synthesize the inhibitor-based fluorescent probe SAENTA-Cy5 and apply direct stochastic optical reconstruction microscopy (dSTORM) to conduct refined observation of human equilibrative nucleoside transporter 1 (hENT1), the most important and famous member of NTs. We first demonstrate the labeling specificity and superiority of SAENTA-Cy5 to the antibody probe. Then, we found different assembly patterns of hENT1 on the apical and basal membranes, which are further investigated to be caused by varying associations of membrane carbohydrates, membrane classical functional domains (lipid rafts), and associated membrane proteins (EpCAM). Our work provides an efficient method for labeling hENT1, which contributes to realize fine observation of NTs. The findings on the assembly features and potential assembly mechanism of hENT1 promote a better understanding of its biofunction, which facilitates further investigations on how NTs work in the metabolism of nucleoside and nucleoside analogues.

Super-resolution imaging pinpoints the periodic ultrastructure at the human node of Ranvier and its disruption in patients with polyneuropathy

The node of Ranvier is the key element in saltatory conduction along myelinated axons, but its specific protein organization remains elusive in the human species. To shed light on nanoscale anatomy of the human node of Ranvier in health and disease, we assessed human nerve biopsies of patients with polyneuropathy by super-resolution fluorescence microscopy. We applied direct stochastic optical reconstruction microscopy (dSTORM) and supported our data by high-content confocal imaging combined with deep learning-based analysis. As a result, we revealed a ∼ 190 nm periodic protein arrangement of cytoskeletal proteins and axoglial cell adhesion molecules in human peripheral nerves. In patients with polyneuropathy, periodic distances increased at the paranodal region of the node of Ranvier, both at the axonal cytoskeleton and at the axoglial junction. In-depth image analysis revealed a partial loss of proteins of the axoglial complex (Caspr-1, neurofascin-155) in combination with detachment from the cytoskeletal anchor protein ß2-spectrin. High content analysis showed that such paranodal disorganization occurred especially in acute and severe axonal neuropathy with ongoing Wallerian degeneration and related cytoskeletal damage. We provide nanoscale and protein-specific evidence for the prominent, but vulnerable role of the node of Ranvier for axonal integrity. Furthermore, we show that super-resolution imaging can identify, quantify and map elongated periodic protein distances and protein interaction in histopathological tissue samples. We thus introduce a promising tool for further translational applications of super resolution microscopy.

Possible frequent multiple mitochondrial DNA copies in a single nucleoid in HeLa cells

Previously, a number of ~ 1.4 of mitochondrial DNA (mtDNA) molecules in a single nucleoid was reported, which would reflect a minimum nucleoid division. We applied 3D-double-color direct stochastic optical reconstruction microscopy (dSTORM), i.e. nanoscopy with ~ 25–40 nm x,y-resolution, together with our novel method of Delaunay segmentation of 3D data to identify unbiased 3D-overlaps. Noncoding D-loops were recognized in HeLa cells by mtDNA fluorescence in situ hybridization (mtFISH) 7S-DNA 250-bp probe, containing biotin, visualized by anti-biotin/Cy3B-conjugated antibodies. Other mtFISH probes with biotin or Alexa Fluor 647 (A647) against ATP6-COX3 gene overlaps (1,100 bp) were also used. Nucleoids were imaged by anti-DNA/(A647-)-Cy3B-conjugated antibodies. Resulting histograms counting mtFISH-loci/nucleoid overlaps demonstrated that 45% to 70% of visualized nucleoids contained two or more D-loops or ATP6-COX3-loci, indicating two or more mtDNA molecules per nucleoid. With increasing number of mtDNA per nucleoid, diameters were larger and their distribution histograms peaked at ~ 300 nm. A wide nucleoid diameter distribution was obtained also using 2D-STED for their imaging by anti-DNA/A647. At unchanged mtDNA copy number in osteosarcoma 143B cells, TFAM expression increased nucleoid spatial density 1.67-fold, indicating expansion of existing mtDNA and its redistribution into more nucleoids upon the higher TFAM/mtDNA stoichiometry. Validation of nucleoid imaging was also done with two TFAM mutants unable to bend or dimerize, respectively, which reduced both copy number and nucleoid spatial density by 80%. We conclude that frequently more than one mtDNA molecule exists within a single nucleoid in HeLa cells and that mitochondrial nucleoids do exist in a non-uniform size range.