dSTORM Imaging Research Articles

Cardiac ryanodine receptor distribution is dynamic and changed by auxiliary proteins and post-translational modification.

The effects of the immunophilins, FKBP12 and FKBP12.6, and phosphorylation on type II ryanodine receptor (RyR2) arrangement and function were examined using correlation microscopy (line scan confocal imaging of Ca2+ sparks and dual-tilt electron tomography) and dSTORM imaging of permeabilized Wistar rat ventricular myocytes. Saturating concentrations (10 µmol/L) of either FKBP12 or 12.6 significantly reduced the frequency, spread, amplitude and Ca2+ spark mass relative to control, while the tomograms revealed both proteins shifted the tetramers into a largely side-by-side configuration. Phosphorylation of immunophilin-saturated RyR2 resulted in structural and functional changes largely comparable to phosphorylation alone. dSTORM images of myocyte surfaces demonstrated that both FKBP12 and 12.6 significantly reduced RyR2 cluster sizes, while phosphorylation, even of immunophilin-saturated RyR2, increased them. We conclude that both RyR2 cluster size and the arrangement of tetramers within clusters is dynamic and respond to changes in the cellular environment. Further, these changes affect Ca2+ spark formation.

Application of an inhibitor-based probe to reveal the distribution of membrane PSMA in dSTORM imaging.

Relying on an inhibitor-based probe, we reveal the clustered distribution of membrane PSMA by dSTORM imaging and uncover its potential interaction with folate receptor. This inhibitor-based strategy realizes more accurate labeling than antibody labeling, which would make it a powerful tool in the field of dSTORM imaging.

DSTORM Imaging and Analysis of Desmosome Architecture.

Desmosomes are cell-cell junctions responsible for mechanically integrating adjacent cells. Due to the small size of the junctions, their protein architecture cannot be elucidated using conventional fluorescence microscopy. Super-resolution microscopy techniques, including dSTORM, deliver higher-resolution images which can reveal the localization or arrangement of proteins within individual desmosomes. Herein we describe an imaging and analysis method to determine the nanoscale architecture of desmosomes using super-resolution dSTORM.

Superresolution Microscopy of Drosophila Indirect Flight Muscle Sarcomeres.

Sarcomeres are extremely highly ordered macromolecular assemblies where proper structural organization is an absolute prerequisite to the functionality of these contractile units. Despite the wealth of information collected, the exact spatial arrangement of many of the H-zone and Z-disk proteins remained unknown. Recently, we developed a powerful nanoscopic approach to localize the sarcomeric protein components with a resolution well below the diffraction limit. The ease of sample preparation and the near crystalline structure of the Drosophila flight muscle sarcomeres make them ideally suitable for single molecule localization microscopy and structure averaging. Our approach allowed us to determine the position of dozens of H-zone and Z-disk proteins with a quasi-molecular, ~5-10 nm localization precision. The protocol described below provides an easy and reproducible method to prepare individual myofibrils for dSTORM imaging. In addition, it includes an in-depth description of a custom made and freely available software toolbox to process and quantitatively analyze the raw localization data.

Quantitatively Mapping the Assembly Pattern of EpCAM on Cell Membranes with Peptide Probes.

Epithelial cell adhesion molecule (EpCAM) is an important type I transmembrane protein that is overexpressed on the surfaces of most cancer cells and involved in various biological processes such as cell adhesion and cell signaling. Although it plays crucial roles in cell functions and tumorigenesis, questions concerning the detailed morphology, molecular stoichiometry, and the assembly mechanisms of EpCAM on cell membranes have not been fully elucidated. Here, we used direct stochastic optical reconstruction microscopy (dSTORM) and relied on fluorophore-conjugated peptides to quantitatively analyze the assembly pattern of EpCAM with single-molecule precision. EpCAM was found to organize heterogeneous clusters with different sizes, which contain different numbers of EpCAM molecules on MCF-7 cell membranes. Moreover, dual-color dSTORM imaging revealed a significant correlation between EpCAM and tetraspanin CD9, and part of the EpCAM clusters could be disrupted by knockdown of CD9, which indicated that EpCAM might localize in tetraspanin-enriched microdomains (TEMs) and function cooperatively with CD9 on cell membranes. In addition, the assembly of the membrane EpCAM was found to be limited by both cytoskeleton and glycosylation. Overall, our work clarified the clustered distribution of EpCAM and revealed the potential mechanisms of its clustering at the molecular level, promoting a deeper understanding of the nano-organization of membrane proteins.

Photo-isomerization of the Cyanine Dye Alexa-Fluor 647 (AF-647) in the Context of dSTORM Super-Resolution Microscopy.

Cyanine dyes, as used in super-resolution fluorescence microscopy, undergo light-induced "blinking", enabling localization of fluorophores with spatial resolution beyond the optical diffraction limit. Despite a plethora of studies, the molecular origins of this blinking are not well understood. Here, we examine the photophysical properties of a bio-conjugate cyanine dye (AF-647), used extensively in dSTORM imaging. In the absence of a potent sacrificial reductant, light-induced electron transfer and intermediates formed via the metastable, triplet excited state are considered unlikely to play a significant role in the blinking events. Instead, it is found that, under conditions appropriate to dSTORM microscopy, AF-647 undergoes reversible photo-induced isomerization to at least two long-lived dark species. These photo-isomers are characterized spectroscopically and their interconversion probed by computational means. The first-formed isomer is light sensitive and transforms to a longer-lived species in modest yield that could be involved in dSTORM related blinking. Permanent photobleaching of AF-647 occurs with very low quantum yield and is partially suppressed by the anaerobic redox buffer.

Junctophilin Proteins Tether a Cav1-RyR2-KCa3.1 Tripartite Complex to Regulate Neuronal Excitability

The excitability of CA1 hippocampal pyramidal cells is mediated by a slow AHP (sAHP) that responds to calcium increases by Cav1 calcium channels and ryanodine receptors (RyR). We used super-resolution and FRET microscopy to investigate the proximity and functional coupling among Cav1.3/Cav1.2, RyR2, and KCa3.1 potassium channels that contribute to the sAHP. dSTORM and FRET imaging shows that Cav1.3, RyR2, and KCa3.1 are organized as a triprotein complex that colocalizes with junctophilin (JPH) 3 and 4 proteins that tether the plasma membrane to the endoplasmic reticulum. JPH3 and JPH4 shRNAs dissociated a Cav1.3-RyR2-KCa3.1 complex and reduced the IsAHP. Infusing JPH3 and JPH4 antibodies into CA1 cells reduced IsAHP and spike accommodation. These data indicate that JPH3 and JPH4 proteins maintain a Cav1-RyR2-KCa3.1 complex that allows two calcium sources to act in tandem to define the activation properties of KCa3.1 channels and the IsAHP.

A stochastic view on surface inhomogeneity of nanoparticles

The interactions between and with nanostructures can only be fully understood when the functional group distribution on their surfaces can be quantified accurately. Here we apply a combination of direct stochastic optical reconstruction microscopy (dSTORM) imaging and probabilistic modelling to analyse molecular distributions on spherical nanoparticles. The properties of individual fluorophores are assessed and incorporated into a model for the dSTORM imaging process. Using this tailored model, overcounting artefacts are greatly reduced and the locations of dye labels can be accurately estimated, revealing their spatial distribution. We show that standard chemical protocols for dye attachment lead to inhomogeneous functionalization in the case of ubiquitous polystyrene nanoparticles. Moreover, we demonstrate that stochastic fluctuations result in large variability of the local group density between particles. These results cast doubt on the uniform surface coverage commonly assumed in the creation of amorphous functional nanoparticles and expose a striking difference between the average population and individual nanoparticle coverage.

Super-Resolution Microscopy Reveals the Molecular Architecture of Centriole Subdistal Appendages and Its Role in Microtubule/Golgi Anchoring

The subdistal appendages (SDAs) are centriolar structures important to microtubule (MT) nucleation and anchoring. Recent studies suggest its role in linking submerged primary cilia to Golgi. Despite its importance, little is known about their assembly geometry and structure-function relationship. Here, we use dSTORM superresolution microscopy to resolve the organization of multiple SDA proteins and centriole-associated proteins in mammalian cells. dSTORM imaging reveals that ODF2, a core SDA protein, localizes closed to the centriole wall while Cep128 and centriolin form ring-like structure building upon it. CCDC68, Cep170 and Ninein are two layered structures with one layer in the SDA region (the SDA layer) surrounding the core SDA proteins and the other layer residing close to the centriole proximal end (the proximal layer). Dual-color dSTORM shows that these SDA proteins form a triangular structure rooted from the centriole wall, mapping to the SDA architecture resolved with electron microscopy. Interestingly, dSTORM imaging of γ-tubulin, the key MT nucleating element, is found to be enriched between the SDA and the proximal layer of Cep170 at the location where MT fibers are observed. Besides, MT fibers are also found to attach to various centriole elements including the DAs, SDAs and the proximal end of the centriole. Depletion of SDAs upon Cep128 knockout suppresses MT attachment to SDAs but not DAs. dSTORM imaging also reveals that AKAP450, a scaffold protein connecting the centrosome and Golgi, forms a cup like structure surrounding the entire centriole via its interaction with Cep170. Upon Cep170 depletion, the centrosomal AKAP450 is abolished, causing detachment of the mother centriole from Golgi. Together, using dSTORM, we systematically disclosed the structural organization of SDAs and illustrated the structural function of SDAs in MT anchoring and centriole-Golgi association.

Super-resolution imaging of cancer-associated carbohydrates using aptamer probes.

Globo H, as one of the most crucial cancer-associated carbohydrates, is exclusively overexpressed in a variety of cancers. However, the accurate localization and detailed morphology of globo H at the molecular level remain unclear. Here, we applied direct stochastic optical reconstruction microscopy (dSTORM) and relied on fluorophore-conjugated aptamers to solve the problem. The results showed that globo H organized as clusters on cell membranes with irregular shapes and different sizes from 100 to 300 nm. Significantly, globo H was found to have a higher expression level and larger clusters on various cancer cells than on non-cancer cells, which hinted that its specific distribution could be utilized for cancer diagnosis. Moreover, dual-color dSTORM imaging revealed the colocalization of globo H and other cancer-associated carbohydrates, and the clustering of globo H could be disrupted by the treatment of corresponding glycosidases, which indicated that these carbohydrates might intertwine in spatial organization and function cooperatively in cancers. Our work clarified the clustered distribution of globo H at the nanometer scale and revealed the potential interactions between cancer-associated carbohydrates, which paves the way for further understanding the relationship between the spatial structures and functions of carbohydrates in cancers.

3D dSTORM imaging reveals novel detail of ryanodine receptor localization in rat cardiac myocytes.

Key points Using 3D direct stochastic optical reconstruction microscopy (dSTORM), we developed novel approaches to quantitatively describe the nanoscale, 3D organization of ryanodine receptors (RyRs) in cardiomyocytes.Complex arrangements of RyR clusters were observed in 3D space, both at the cell surface and within the cell interior, with allocation to dyadic and non‐dyadic pools.3D imaging importantly allowed discernment of clusters overlapping in the z‐axis, for which detection was obscured by conventional 2D imaging techniques. Thus, RyR clusters were found to be significantly smaller than previous 2D estimates.Ca2+ release units (CRUs), i.e. functional groupings of neighbouring RyR clusters, were similarly observed to be smaller than earlier reports. Internal CRUs contained more RyRs in more clusters than CRUs on the cell surface, and yielded longer duration Ca2+ sparks. Cardiomyocyte contraction is dependent on Ca2+ release from ryanodine receptors (RyRs). However, the precise localization of RyRs remains unknown, due to shortcomings of imaging techniques which are diffraction limited or restricted to 2D. We aimed to determine the 3D nanoscale organization of RyRs in rat cardiomyocytes by employing direct stochastic optical reconstruction microscopy (dSTORM) with phase ramp technology. Initial observations at the cell surface showed an undulating organization of RyR clusters, resulting in their frequent overlap in the z‐axis and obscured detection by 2D techniques. Non‐overlapping clusters were imaged to create a calibration curve for estimating RyR number based on recorded fluorescence blinks. Employing this method at the cell surface and interior revealed smaller RyR clusters than 2D estimates, as erroneous merging of axially aligned RyRs was circumvented. Functional groupings of RyR clusters (Ca2+ release units, CRUs), contained an average of 18 and 23 RyRs at the surface and interior, respectively, although half of all CRUs contained only a single ‘rogue’ RyR. Internal CRUs were more tightly packed along z‐lines than surface CRUs, contained larger and more numerous RyR clusters, and constituted ∼75% of the roughly 1 million RyRs present in an average cardiomyocyte. This complex internal 3D geometry was underscored by correlative imaging of RyRs and t‐tubules, which enabled quantification of dyadic and non‐dyadic RyR populations. Mirroring differences in CRU size and complexity, Ca2+ sparks originating from internal CRUs were of longer duration than those at the surface. These data provide novel, nanoscale insight into RyR organization and function across cardiomyocytes.

Rapid and accurate analysis of stem cell-derived extracellular vesicles with super resolution microscopy and live imaging

Extracellular vesicles (EVs) have prevalent roles in cancer biology and regenerative medicine. Conventional techniques for characterising EVs including electron microscopy (EM), nanoparticle tracking analysis (NTA) and tuneable resistive pulse sensing (TRPS), have been reported to produce high variability in particle count (EM) and poor sensitivity in detecting EVs below 50 nm in size (NTA and TRPS), making accurate and unbiased EV analysis technically challenging. This study introduces direct stochastic optical reconstruction microscopy (d-STORM) as an efficient and reliable characterisation approach for stem cell-derived EVs. Using a photo-switchable lipid dye, d-STORM imaging enabled rapid detection of EVs down to 20–30 nm in size with higher sensitivity and lower variability compared to EM, NTA and TRPS techniques. Imaging of EV uptake by live stem cells in culture further confirmed the potential of this approach for downstream cell biology applications and for the analysis of vesicle-based cell-cell communication.

PO-132 Alpha5 beta1 integrin provides glioma cell resistance to EGFR tyrosine kinase inhibitors. role of membrane trafficking

IntroductionOverexpression of epidermal growth factor (EGFR) drives glioblastoma (GBM) cell invasion and tumour progression. EGFR has been a major therapeutic target in GBM. However, clinical trials were disappointing, and we are still missing molecular basis to explain these poor results. In other solid tumours, extracellular matrix proteins found in the tumour microenvironment and their heterodimeric integrin receptors trigger resistance to EGFR targeted therapy. We recently established that the fibronectin receptor, α5β1 integrin is a pertinent therapeutic target in GBM. Therefore, we seek to evaluate whether α5β1 integrin is involved in therapy targeting EGFR in GBM.Material and methodsWe used GBM cell line (U87) overexpressing or down expressing α5 integrin subunit. EGFR was inhibited by clinically approved tyrosine kinase inhibitors (TKIs) (gefitinib, erlotinib, lapatinib). For cell motility assays, we performed spheroid dissemination assays, in 2D and 3D environment and chemotaxis assays through Boyden chambers. We also evaluated the impact of integrin expression on cell clonogenicity and cell grow th (2D, spheroids) in presence of EGFR inhibitors. Confocal microscopy confirmed integrin, EGFR and endosomes markers localization. Protein colocalization in early-endosomes was confirmed by confocal image analysis and dSTORM super-resolution.Results and discussionsWe showed that loss of integrin α5 expression increased U87 cell sensitivity to TKIs in spheroid cell evasion assays but did not affect TKIs efficiency on chemotaxis, cell growth (2D or 3D) or cell clonogenicity. EGFR is thus critical for cell dissemination from GBM tumour spheroïds and overexpression of α5 integrin can circumvent EGFR inhibition by TKIs. Using confocal imaging, we showed that TKIs provoked a massive relocalization of integrin and EGFR in Rab5, EEA1 or LRP1 positive endosomes. Dynamic studies confirmed that TKIs increased EGFR receptor endocytosis. By colocalization studies and super-resolution dSTORM imaging, we clearly showed that in intracellular vesicules integrin and EGFR are in close proximity, suggesting a potential interaction.ConclusionWe showed in GBM cells that α5β1 integrin expression triggers resistance to EGFR-targeting TKIs and that TKIs alter EGFR and integrin membrane trafficking. These data point out the potential importance of integrin and EGFR endocytosis and trafficking in GBM resistance to TKIs.

Super-resolution Imaging of Individual Human Subchromosomal Regions in Situ Reveals Nanoscopic Building Blocks of Higher-Order Structure.

It is widely recognized that the higher-order spatial organization of the genome, beyond the nucleosome, plays an important role in many biological processes. However, to date, direct information on even such fundamental structural details as the typical sizes and DNA content of these higher-order structures in situ is poorly characterized. Here, we examine the nanoscopic DNA organization within human nuclei using super-resolution direct stochastic optical reconstruction microscopy (dSTORM) imaging and 5-ethynyl-2'-deoxyuridine click chemistry, studying single fully labeled chromosomes within an otherwise unlabeled nuclei to improve the attainable resolution. We find that, regardless of nuclear position, individual subchromosomal regions consist of three different levels of DNA compaction: (i) dispersed chromatin; (ii) nanodomains of sizes ranging tens of nanometers containing a few kilobases (kb) of DNA; and (iii) clusters of nanodomains. Interestingly, the sizes and DNA content of the nanodomains are approximately the same at the nuclear periphery, nucleolar proximity, and nuclear interior, suggesting that these nanodomains share a roughly common higher-order architecture. Overall, these results suggest that DNA compaction within the eukaryote nucleus occurs via the condensation of DNA into few-kb nanodomains of approximately similar structure, with further compaction occurring via the clustering of nanodomains.

Enhanced labeling density and whole-cell 3D dSTORM imaging by repetitive labeling of target proteins

With continuing advances in the resolving power of super-resolution microscopy, the inefficient labeling of proteins with suitable fluorophores becomes a limiting factor. For example, the low labeling density achieved with antibodies or small molecule tags limits attempts to reveal local protein nano-architecture of cellular compartments. On the other hand, high laser intensities cause photobleaching within and nearby an imaged region, thereby further reducing labeling density and impairing multi-plane whole-cell 3D super-resolution imaging. Here, we show that both labeling density and photobleaching can be addressed by repetitive application of trisNTA-fluorophore conjugates reversibly binding to a histidine-tagged protein by a novel approach called single-epitope repetitive imaging (SERI). For single-plane super-resolution microscopy, we demonstrate that, after multiple rounds of labeling and imaging, the signal density is increased. Using the same approach of repetitive imaging, washing and re-labeling, we demonstrate whole-cell 3D super-resolution imaging compensated for photobleaching above or below the imaging plane. This proof-of-principle study demonstrates that repetitive labeling of histidine-tagged proteins provides a versatile solution to break the ‘labeling barrier’ and to bypass photobleaching in multi-plane, whole-cell 3D experiments.

A peptide tag-specific nanobody enables high-quality labeling for dSTORM imaging

Dense fluorophore labeling without compromising the biological target is crucial for genuine super-resolution microscopy. Here we introduce a broadly applicable labeling strategy for fixed and living cells utilizing a short peptide tag-specific nanobody (BC2-tag/bivBC2-Nb). BC2-tagging of ectopically introduced or endogenous proteins does not interfere with the examined structures and bivBC2-Nb staining results in a close-grained fluorophore labeling with minimal linkage errors. This allowed us to perform high-quality dSTORM imaging of various targets in mammalian and yeast cells. We expect that this versatile strategy will render many more demanding cellular targets amenable to dSTORM imaging.

Super-Resolution (dSTORM) Imaging of Calcium Handling Proteins in Cardiomyocytes

Super-Resolution (dSTORM) Imaging of Calcium Handling Proteins in Cardiomyocytes

3D dSTORM Imaging Reveals Disassembly of Ryanodine Receptor Clusters in Failing Cardiomyocytes

3D dSTORM Imaging Reveals Disassembly of Ryanodine Receptor Clusters in Failing Cardiomyocytes

Multi-Structure Super-Resolution Imaging using DNA Strand Displacement

We describe a simple new method for super-resolution of imaging of many different structures/proteins in the same cell using a sequential imaging strategy where one of the best dSTORM dyes (Alex647), is used to image all structures. The simple optical setup needs no spectrometers, beam splitters or channel registration. Briefly, antibodies are pre-conjugated to a ‘protector’ strand of ssDNA. After cellular labeling and before imaging, a complementary ‘template’ strand, which is labeled with the dye, is added to the sample to bind and label the protector. After imaging, a third ‘invader’ ssDNA is introduced that gains a toe-hold on the template, and competes it off the antibody-protector complex. Orthogonal sets of protector\template\invader are used to label different cellular structures. All antibody labeling is done at once, before imaging. Both the template binding and invader action only require about ∼ 1 minute, allowing quick progression through the different structures. We demonstrate the concept by performing dSTORM imaging on several different proteins in a single cell. The concept is easily extended to >10 proteins and could be used for other SR techniques such as STED and SIM.

Protocol for the Isolation and Super-resolution dSTORM Imaging of RyR2 in Cardiac Myocytes

Protocol for the Isolation and Super-resolution dSTORM Imaging of RyR2 in Cardiac Myocytes