Multiparametric profiling of HER2-enriched extracellular vesicles in breast cancer using Single Extracellular VEsicle Nanoscopy.

BACKGROUND: Patients with HER2-positive breast cancer can significantly benefit from HER2-directed therapy - such as the monoclonal antibody trastuzumab. However, some patients can develop therapy resistance or change HER2 status. Thus, we urgently need new, noninvasive strategies to monitor patients frequently. Extracellular vesicles (EVs) secreted from tumor cells are emerging as potential biomarker candidates. These membrane-delimited nanoparticles harbor molecular signatures of their origin cells; report rapidly on changes to cellular status; and can be frequently sampled from accessible biofluids. RESULTS: Using Single Extracellular VEsicle Nanoscopy (SEVEN) platform that combines affinity isolation of EVs with super-resolution microscopy, here we provide multiparametric characterization of EVs with ~ 8 nm precision and molecular sensitivity. We first interrogated cell culture EVs affinity-enriched in tetraspanins CD9, CD63, and CD81; these transmembrane proteins are commonly found on EV membranes. SEVEN robustly provided critical parameters of individual, tetraspanin-enriched EVs: concentration, size, shape, molecular cargo content, and heterogeneity. Trastuzumab-resistant cells (vs. trastuzumab-sensitive) secreted more EVs. Additionally, EVs from trastuzumab-resistant cells had lower tetraspanin density and higher HER2 density. We also evaluated EVs affinity-enriched in HER2; we found that these EVs (vs. tetraspanin-enriched) were larger and more elongated. We further optimized analytical sample processing to assess a rare population of HER2-enriched EVs from patient plasma. In breast cancer patients with elevated HER2 protein expression (vs. controls), HER2-enriched EVs had distinct characteristics including typically increased number of tetraspanin molecules and larger size. Importantly, these EVs were on average 25-fold more abundant compared to no cancer controls. CONCLUSIONS: SEVEN revealed unique characteristics of HER2-enriched EVs in cultured cells and complex biological fluid. In combination with current clinical approaches, this method is well poised to support precise therapeutic decisions.

Photoactivatable BODIPYs for Live-Cell PALM.

Photoactivated localization microscopy (PALM) relies on fluorescence photoactivation and single-molecule localization to overcome optical diffraction and reconstruct images of biological samples with spatial resolution at the nanoscale. The implementation of this subdiffraction imaging method, however, requires fluorescent probes with photochemical and photophysical properties specifically engineered to enable the localization of single photoactivated molecules with nanometer precision. The synthetic versatility and outstanding photophysical properties of the borondipyrromethene (BODIPY) chromophore are ideally suited to satisfy these stringent requirements. Specifically, synthetic manipulations of the BODIPY scaffold can be invoked to install photolabile functional groups and photoactivate fluorescence under photochemical control. Additionally, targeting ligands can be incorporated in the resulting photoactivatable fluorophores (PAFs) to label selected subcellular components in live cells. Indeed, photoactivatable BODIPYs have already allowed the sub-diffraction imaging of diverse cellular substructures in live cells using PALM and can evolve into invaluable analytical probes for bioimaging applications.

Elucidation of the Clustered Nano-Architecture of Radiation-Induced DNA Damage Sites and Surrounding Chromatin in Cancer Cells: A Single Molecule Localization Microscopy Approach.

In cancer therapy, the application of (fractionated) harsh radiation treatment is state of the art for many types of tumors. However, ionizing radiation is a "double-edged sword"-it can kill the tumor but can also promote the selection of radioresistant tumor cell clones or even initiate carcinogenesis in the normal irradiated tissue. Individualized radiotherapy would reduce these risks and boost the treatment, but its development requires a deep understanding of DNA damage and repair processes and the corresponding control mechanisms. DNA double strand breaks (DSBs) and their repair play a critical role in the cellular response to radiation. In previous years, it has become apparent that, beyond genetic and epigenetic determinants, the structural aspects of damaged chromatin (i.e., not only of DSBs themselves but also of the whole damage-surrounding chromatin domains) form another layer of complex DSB regulation. In the present article, we summarize the application of super-resolution single molecule localization microscopy (SMLM) for investigations of these structural aspects with emphasis on the relationship between the nano-architecture of radiation-induced repair foci (IRIFs), represented here by γH2AX foci, and their chromatin environment. Using irradiated HeLa cell cultures as an example, we show repair-dependent rearrangements of damaged chromatin and analyze the architecture of γH2AX repair clusters according to topological similarities. Although HeLa cells are known to have highly aberrant genomes, the topological similarity of γH2AX was high, indicating a functional, presumptively genome type-independent relevance of structural aspects in DSB repair. Remarkably, nano-scaled chromatin rearrangements during repair depended both on the chromatin domain type and the treatment. Based on these results, we demonstrate how the nano-architecture and topology of IRIFs and chromatin can be determined, point to the methodological relevance of SMLM, and discuss the consequences of the observed phenomena for the DSB repair network regulation or, for instance, radiation treatment outcomes.

Characterizing Binding Interactions That Are Essential for Selective Transport through the Nuclear Pore Complex.

Specific macromolecules are rapidly transported across the nuclear envelope via the nuclear pore complex (NPC). The selective transport process is facilitated when nuclear transport receptors (NTRs) weakly and transiently bind to intrinsically disordered constituents of the NPC, FG Nups. These two types of proteins help maintain the selective NPC barrier. To interrogate their binding interactions in vitro, we deployed an NPC barrier mimic. We created the stationary phase by covalently attaching fragments of a yeast FG Nup called Nsp1 to glass coverslips. We used a tunable mobile phase containing NTR, nuclear transport factor 2 (NTF2). In the stationary phase, three main factors affected binding: the number of FG repeats, the charge of fragments, and the fragment density. We also identified three main factors affecting binding in the mobile phase: the avidity of the NTF2 variant for Nsp1, the presence of nonspecific proteins, and the presence of additional NTRs. We used both experimentally determined binding parameters and molecular dynamics simulations of Nsp1FG fragments to create an agent-based model. The results suggest that NTF2 binding is negatively cooperative and dependent on the density of Nsp1FG molecules. Our results demonstrate the strengths of combining experimental and physical modeling approaches to study NPC-mediated transport.

Toward Absolute Molecular Numbers in DNA-PAINT.

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

Advances in research of DNA damage and repair in cells exposed to various types of ionizing radiation in the era of super-resolution optical microscopy.

The present work introduces new findings about the influence of different radiation types on the cells, with the concern on the micro- and nanodosimetric aspects of chromatin damage. Emphasized is the relationship between the physical parameters of the incident radiation (g-rays, protons and high-LET heavy ions), character of chromatin damage, ability of cells to repair and survive DNA damage, and risk of genetic changes. While confirming a positive correlation between the LET of ionizing radiation, complexity of induced DNA double-strand breaks (DSB), and biological effectiveness (RBE) of radiation, at the same time, we show that our understanding of this relationship is only incomplete. Our discovery that various accelerated ions with similar LET can damage DNA in different ways and kill cells with unequal efficiency, could serve as an example. In addition, many aspects of DSB repair remain to be explained, for instance, how the cell activates the particular repair pathway at sites of individual DSBs, and how it depends on the radiation used and the chromatin architecture. The discussed results may be important, above all, for newly developing hadron therapy and in the context of manned interstellar flights planning. From the methodological point of view, we point to a tremendous progress in the field of optical microscopy and its research applications. In more detail, we introduce single-molecule localization microscopy (SMLM).

Challenges and Contradictions of Metal Nano-Particle Applications for Radio-Sensitivity Enhancement in Cancer Therapy.

From the very beginnings of radiotherapy, a crucial question persists with how to target the radiation effectiveness into the tumor while preserving surrounding tissues as undamaged as possible. One promising approach is to selectively pre-sensitize tumor cells by metallic nanoparticles. However, though the "physics" behind nanoparticle-mediated radio-interaction has been well elaborated, practical applications in medicine remain challenging and often disappointing because of limited knowledge on biological mechanisms leading to cell damage enhancement and eventually cell death. In the present study, we analyzed the influence of different nanoparticle materials (platinum (Pt), and gold (Au)), cancer cell types (HeLa, U87, and SKBr3), and doses (up to 4 Gy) of low-Linear Energy Transfer (LET) ionizing radiation (γ- and X-rays) on the extent, complexity and reparability of radiation-induced γH2AX + 53BP1 foci, the markers of double stand breaks (DSBs). Firstly, we sensitively compared the focus presence in nuclei during a long period of time post-irradiation (24 h) in spatially (three-dimensionally, 3D) fixed cells incubated and non-incubated with Pt nanoparticles by means of high-resolution immunofluorescence confocal microscopy. The data were compared with our preliminary results obtained for Au nanoparticles and recently published results for gadolinium (Gd) nanoparticles of approximately the same size (2⁻3 nm). Next, we introduced a novel super-resolution approach-single molecule localization microscopy (SMLM)-to study the internal structure of the repair foci. In these experiments, 10 nm Au nanoparticles were used that could be also visualized by SMLM. Altogether, the data show that different nanoparticles may or may not enhance radiation damage to DNA, so multi-parameter effects have to be considered to better interpret the radiosensitization. Based on these findings, we discussed on conclusions and contradictions related to the effectiveness and presumptive mechanisms of the cell radiosensitization by nanoparticles. We also demonstrate that SMLM offers new perspectives to study internal structures of repair foci with the goal to better evaluate potential differences in DNA damage patterns.

Super-resolution microscopy approaches to nuclear nanostructure imaging.

The human genome has been decoded, but we are still far from understanding the regulation of all gene activities. A largely unexplained role in these regulatory mechanisms is played by the spatial organization of the genome in the cell nucleus which has far-reaching functional consequences for gene regulation. Until recently, it appeared to be impossible to study this problem on the nanoscale by light microscopy. However, novel developments in optical imaging technology have radically surpassed the limited resolution of conventional far-field fluorescence microscopy (ca. 200nm). After a brief review of available super-resolution microscopy (SRM) methods, we focus on a specific SRM approach to study nuclear genome structure at the single cell/single molecule level, Spectral Precision Distance/Position Determination Microscopy (SPDM). SPDM, a variant of localization microscopy, makes use of conventional fluorescent proteins or single standard organic fluorophores in combination with standard (or only slightly modified) specimen preparation conditions; in its actual realization mode, the same laser frequency can be used for both photoswitching and fluorescence read out. Presently, the SPDM method allows us to image nuclear genome organization in individual cells down to few tens of nanometer (nm) of structural resolution, and to perform quantitative analyses of individual small chromatin domains; of the nanoscale distribution of histones, chromatin remodeling proteins, and transcription, splicing and repair related factors. As a biomedical research application, using dual-color SPDM, it became possible to monitor in mouse cardiomyocyte cells quantitatively the effects of ischemia conditions on the chromatin nanostructure (DNA). These novel "molecular optics" approaches open an avenue to study the nuclear landscape directly in individual cells down to the single molecule level and thus to test models of functional genome architecture at unprecedented resolution.

Recruitment of 53BP1 Proteins for DNA Repair and Persistence of Repair Clusters Differ for Cell Types as Detected by Single Molecule Localization Microscopy.

DNA double stranded breaks (DSBs) are the most serious type of lesions introduced into chromatin by ionizing radiation. During DSB repair, cells recruit different proteins to the damaged sites in a manner dependent on local chromatin structure, DSB location in the nucleus, and the repair pathway entered. 53BP1 is one of the important players participating in repair pathway decision of the cell. Although many molecular biology details have been investigated, the architecture of 53BP1 repair foci and its development during the post-irradiation time, especially the period of protein recruitment, remains to be elucidated. Super-resolution light microscopy is a powerful new tool to approach such studies in 3D-conserved cell nuclei. Recently, we demonstrated the applicability of single molecule localization microscopy (SMLM) as one of these highly resolving methods for analyses of dynamic repair protein distribution and repair focus internal nano-architecture in intact cell nuclei. In the present study, we focused our investigation on 53BP1 foci in differently radio-resistant cell types, moderately radio-resistant neonatal human dermal fibroblasts (NHDF) and highly radio-resistant U87 glioblastoma cells, exposed to high-LET 15N-ion radiation. At given time points up to 24 h post irradiation with doses of 1.3 Gy and 4.0 Gy, the coordinates and spatial distribution of fluorescently tagged 53BP1 molecules was quantitatively evaluated at the resolution of 10⁻20 nm. Clusters of these tags were determined as sub-units of repair foci according to SMLM parameters. The formation and relaxation of such clusters was studied. The higher dose generated sufficient numbers of DNA breaks to compare the post-irradiation dynamics of 53BP1 during DSB processing for the cell types studied. A perpendicular (90°) irradiation scheme was used with the 4.0 Gy dose to achieve better separation of a relatively high number of particle tracks typically crossing each nucleus. For analyses along ion-tracks, the dose was reduced to 1.3 Gy and applied in combination with a sharp angle irradiation (10° relative to the cell plane). The results reveal a higher ratio of 53BP1 proteins recruited into SMLM defined clusters in fibroblasts as compared to U87 cells. Moreover, the speed of foci and thus cluster formation and relaxation also differed for the cell types. In both NHDF and U87 cells, a certain number of the detected and functionally relevant clusters remained persistent even 24 h post irradiation; however, the number of these clusters again varied for the cell types. Altogether, our findings indicate that repair cluster formation as determined by SMLM and the relaxation (i.e., the remaining 53BP1 tags no longer fulfill the cluster definition) is cell type dependent and may be functionally explained and correlated to cell specific radio-sensitivity. The present study demonstrates that SMLM is a highly appropriate method for investigations of spatiotemporal protein organization in cell nuclei and how it influences the cell decision for a particular repair pathway at a given DSB site.

Single-molecule microscopy of molecules tagged with GFP or RFP derivatives in mammalian cells using nanobody binders.

With the recent development of single-molecule localization-based superresolution microscopy, the imaging of cellular structures at a resolution below the diffraction-limit of light has become a widespread technique. While single fluorescent molecules can be resolved in the nanometer range, the delivery of these molecules to the authentic structure in the cell via traditional antibody-mediated techniques can add substantial error due to the size of the antibodies. Accurate and quantitative labeling of cellular molecules has thus become one of the bottlenecks in the race for highest resolution of target structures. Here we illustrate in detail how to use small, high affinity nanobody binders against GFP and RFP family proteins for highly generic labeling of fusion constructs with bright organic dyes. We provide detailed protocols and examples for their application in superresolution imaging and single particle tracking and demonstrate advantages over conventional labeling approaches.

High-Resolution Correlative Microscopy: Bridging the Gap between Single Molecule Localization Microscopy and Atomic Force Microscopy.

Nanoscale characterization of living samples has become essential for modern biology. Atomic force microscopy (AFM) creates topological images of fragile biological structures from biomolecules to living cells in aqueous environments. However, correlating nanoscale structure to biological function of specific proteins can be challenging. To this end we have built and characterized a correlated single molecule localization microscope (SMLM)/AFM that allows localizing specific, labeled proteins within high-resolution AFM images in a biologically relevant context. Using direct stochastic optical reconstruction microscopy (dSTORM)/AFM, we directly correlate and quantify the density of localizations with the 3D topography using both imaging modalities along (F-)actin cytoskeletal filaments. In addition, using photo activated light microscopy (PALM)/AFM, we provide correlative images of bacterial cells in aqueous conditions. Moreover, we report the first correlated AFM/PALM imaging of live mammalian cells. The complementary information provided by the two techniques opens a new dimension for structural and functional nanoscale biology.

Live-cell super-resolution imaging of actin using LifeAct-14 with a PAINT-based approach.

We present direct-LIVE-PAINT, an easy-to-implement approach for the nanoscopic imaging of protein structures in live cells using labeled binding peptides. We demonstrate the feasibility of direct-LIVE-PAINT with an actin-binding peptide fused to EGFP, the location of which can be accurately determined as it transiently binds to actin filaments. We show that direct-LIVE-PAINT can be used to image actin structures below the diffraction-limit of light and have used it to observe the dynamic nature of actin in live cells. We envisage a similar approach could be applied to imaging other proteins within live mammalian cells.

Photoactivatable Fluorophores for Bioimaging Applications.

Photoactivatable fluorophores provide the opportunity to switch fluorescence on exclusively in a selected area within a sample of interest at a precise interval of time. Such a level of spatiotemporal fluorescence control enables the implementation of imaging schemes to monitor dynamic events in real time and visualize structural features with nanometer resolution. These transformative imaging methods are contributing fundamental insights on diverse cellular processes with profound implications in biology and medicine. Current photoactivatable fluorophores, however, become emissive only after the activation event, preventing the acquisition of fluorescence images and, hence, the visualization of the sample prior to activation. We developed a family of photoactivatable fluorophores capable of interconverting between emissive states with spectrally resolved fluorescence, instead of switching from a nonemissive state to an emissive one. We demonstrated that our compounds allow the real-time monitoring of molecules diffusing across the cellular blastoderm of developing embryos as well as of polymer beads translocating along the intestinal tract of live nematodes. Additionally, they also permit the tracking of single molecules in the lysosomal compartments of live cells and the visualization of these organelles with nanometer resolution. Indeed, our photoactivatable fluorophores may evolve into invaluable analytical tools for the investigation of the fundamental factors regulating the functions and structures of cells at the molecular level.

Nano-Architecture of Persistent Focal DNA Damage Regions in the Minipig Epidermis Weeks after Acute γ-Irradiation.

Exposure to high acute doses of ionizing radiation (IR) can induce cutaneous radiation syndrome. Weeks after such radiation insults, keratinocyte nuclei of the epidermis exhibit persisting genomic lesions that present as focal accumulations of DNA double-strand break (DSB) damage marker proteins. Knowledge about the nanostructure of these genomic lesions is scarce. Here, we compared the chromatin nano-architecture with respect to DNA damage response (DDR) factors in persistent genomic DNA damage regions and healthy chromatin in epidermis sections of two minipigs 28 days after lumbar irradiation with ~50 Gy γ-rays, using single-molecule localization microscopy (SMLM) combined with geometric and topological mathematical analyses. SMLM analysis of fluorochrome-stained paraffin sections revealed, within keratinocyte nuclei with perisitent DNA damage, the nano-arrangements of pATM, 53BP1 and Mre11 DDR proteins in γ-H2AX-positive focal chromatin areas (termed macro-foci). It was found that persistent macro-foci contained on average ~70% of 53BP1, ~23% of MRE11 and ~25% of pATM single molecule signals of a nucleus. MRE11 and pATM fluorescent tags were organized in focal nanoclusters peaking at about 40 nm diameter, while 53BP1 tags formed nanoclusters that made up super-foci of about 300 nm in size. Relative to undamaged nuclear chromatin, the enrichment of DDR protein signal tags in γ-H2AX macro-foci was on average 8.7-fold (±3) for 53BP1, 3.4-fold (±1.3) for MRE11 and 3.6-fold (±1.8) for pATM. The persistent macro-foci of minipig epidermis displayed a ~2-fold enrichment of DDR proteins, relative to DSB foci of lymphoblastoid control cells 30 min after 0.5 Gy X-ray exposure. A lasting accumulation of damage signaling and sensing molecules such as pATM and 53BP1, as well as the DSB end-processing protein MRE11 in the persistent macro-foci suggests the presence of diverse DNA damages which pose an insurmountable problem for DSB repair.

Advanced image-free analysis of the nano-organization of chromatin and other biomolecules by Single Molecule Localization Microscopy (SMLM).

The cell as a system of many components, governed by the laws of physics and chemistry drives molecular functions having an impact on the spatial organization of these systems and vice versa. Since the relationship between structure and function is an almost universal rule not only in biology, appropriate methods are required to parameterize the relationship between the structure and function of biomolecules and their networks, the mechanisms of the processes in which they are involved, and the mechanisms of regulation of these processes. Single molecule localization microscopy (SMLM), which we focus on here, offers a significant advantage for the quantitative parametrization of molecular organization: it provides matrices of coordinates of fluorescently labeled biomolecules that can be directly subjected to advanced mathematical analytical procedures without the need for laborious and sometimes misleading image processing. Here, we propose mathematical tools for comprehensive quantitative computer data analysis of SMLM point patterns that include Ripley distance frequency analysis, persistent homology analysis, persistent 'imaging', principal component analysis and co-localization analysis. The application of these methods is explained using artificial datasets simulating different, potentially possible and interpretatively important situations. Illustrative analyses of real complex biological SMLM data are presented to emphasize the applicability of the proposed algorithms. This manuscript demonstrated the extraction of features and parameters quantifying the influence of chromatin (re)organization on genome function, offering a novel approach to study chromatin architecture at the nanoscale. However, the ability to adapt the proposed algorithms to analyze essentially any molecular organizations, e.g., membrane receptors or protein trafficking in the cytosol, offers broad flexibility of use.

Super-resolution imaging and quantitative analysis of microtubule arrays in model neurons show that epothilone D increases the density but decreases the length and straightness of microtubules in axon-like processes.

Microtubules are essential for the development of neurons and the regulation of their structural plasticity. Microtubules also provide the structural basis for the long-distance transport of cargo. Various factors influence the organization and dynamics of neuronal microtubules, and disturbance of microtubule regulation is thought to play a central role in neurodegenerative diseases. However, imaging and quantitative assessment of the microtubule organization in the densely packed neuronal processes is challenging. The development of super-resolution techniques combined with the use of nanobodies offers new possibilities to visualize microtubules in neurites in high resolution. In combination with recently developed computational analysis tools, this allows automated quantification of neuronal microtubule organization with high precision. Here we have implemented three-dimensional DNA-PAINT (Point Accumulation in Nanoscale Topography), a single-molecule localization microscopy (SMLM) technique, which allows us to acquire 3D arrays of the microtubule lattice in axons of model neurons (neuronally differentiated PC12 cells) and dendrites of primary neurons. For the quantitative analysis of the microtubule organization, we used the open-source software package SMLM image filament extractor (SIFNE). We found that treatment with nanomolar concentrations of the microtubule-targeting drug epothilone D (EpoD) increased microtubule density in axon-like processes of model neurons and shifted the microtubule length distribution to shorter ones, with a mean microtubule length of 2.39 µm (without EpoD) and 1.98 µm (with EpoD). We also observed a significant decrease in microtubule straightness after EpoD treatment. The changes in microtubule density were consistent with live-cell imaging measurements of ensemble microtubule dynamics using a previously established Fluorescence Decay After Photoactivation (FDAP) assay. For comparison, we determined the organization of the microtubule array in dendrites of primary hippocampal neurons. We observed that dendritic microtubules have a very similar length distribution and straightness compared to microtubules in axon-like processes of a neuronal cell line. Our data show that super-resolution imaging of microtubules followed by algorithm-based image analysis represents a powerful tool to quantitatively assess changes in microtubule organization in neuronal processes, useful to determine the effect of microtubule-modulating conditions. We also provide evidence that the approach is robust and can be applied to neuronal cell lines or primary neurons, both after incorporation of labeled tubulin and by anti-tubulin antibody staining.

Chaotropic Perturbation of Noncovalent Interactions of the Hemagglutinin Tag Monoclonal Antibody Fragment Enables Superresolution Molecular Census.

Antibody-antigen interactions represent one of the most exploited biomolecular interactions in experimental biology. While numerous techniques harnessed immobilized antibodies for nanoscale fluorescence imaging, few utilized their reversible binding kinetics. Here, we investigated noncovalent interactions of the monoclonal hemagglutinin (HA) epitope tag antibody, 12CA5, in the fixed cellular environment. We observed that the use of a chaotropic agent, potassium thiocyanate (KSCN), promoted the dissociation of the 12CA5 antibody fragment (Fab), which already displayed faster dissociation compared to its immunoglobulin G (IgG) counterpart. Molecular dynamic simulations revealed notable root-mean-square deviations and destabilizations in the presence of KSCN, while the hydrogen-bonding network remained primarily unaffected at the antigen-binding site. The reversible interactions enabled us to achieve a superresolution molecular census of local populations of 3xHA tagged microtubule fibers with improved molecular quantification consistency compared to single-molecule localization microscopy (SMLM) techniques utilizing standard immunofluorescence staining for sample labeling. Our technique, termed superresolution census of molecular epitope tags (SR-COMET), highlights the utilization of reversible antibody-antigen interactions for SMLM-based quantitative superresolution imaging.

Incorporation of Low Concentrations of Gold Nanoparticles: Complex Effects on Radiation Response and Fate of Cancer Cells.

(1) Background: In oncology research, a long-standing discussion exists about pros and cons of metal nanoparticle-enhanced radiotherapy and real mechanisms behind the tumor cell response to irradiation (IR) in presence of gold nanoparticles (GNPs). A better understanding of this response is, however, necessary to develop more efficient and safety nanoparticle (NP) types designed to disturb specific processes in tumor cells. (2) Aims and Methods: We combined 3D confocal microscopy and super-resolution single molecule localization microscopy (SMLM) to analyze, at the multiscale, the early and late effects of 10 nm-GNPs on DNA double strand break (DSB) induction and repair in tumor cells exposed to different doses of photonic low-LET (linear energy transfer) radiation. The results were correlated to different aspects of short and long-term cell viability. SkBr3 breast cancer cells (selected for the highest incidence of this cancer type among all cancers in women, and because most breast tumors are treated with IR) were incubated with low concentrations of GNPs and irradiated with 60Co γ-rays or 6 MV X-rays. In numerous post-irradiation (PI) times, ranging from 0.5 to 24 h PI, the cells were spatially (3D) fixed and labeled with specific antibodies against γH2AX, 53BP1 and H3K9me3. The extent of DSB induction, multi-parametric micro- and nano-morphology of γH2AX and 53BP1 repair foci, DSB repair kinetics, persistence of unrepaired DSBs, nanoscale clustering of γH2AX and nanoscale (hetero)chromatin re-organization were measured by means of the mentioned microscopy techniques in dependence of radiation dose and GNP concentration. (3) Results: The number of γH2AX/53BP1 signals increased after IR and an additional increase was observed in GNP-treated (GNP(+)) cells compared to untreated controls. However, this phenomenon reflected slight expansion of the G2-phase cell subpopulation in irradiated GNP(+) specimens instead of enhanced DNA damage induction by GNPs. This statement is further supported by some micro- and nano-morphological parameters of γH2AX/53BP1 foci, which slightly differed for cells irradiated in absence or presence of GNPs. At the nanoscale, Ripley's distance frequency analysis of SMLM signal coordinate matrices also revealed relaxation of heterochromatin (H3K9me3) clusters upon IR. These changes were more prominent in presence of GNPs. The slight expansion of radiosensitive G2 cells correlated with mostly insignificant but systematic decrease in post-irradiation survival of GNP(+) cells. Interestingly, low GNP concentrations accelerated DSB repair kinetics; however, the numbers of persistent γH2AX/53BP1 repair foci were slightly increased in GNP(+) cells. (4) Conclusions: Low concentrations of 10-nm GNPs enhanced the G2/M cell cycle arrest and the proportion of radiosensitive G2 cells, but not the extent of DNA damage induction. GNPs also accelerated DSB repair kinetics and slightly increased presence of unrepaired γH2AX/53BP1 foci at 24 h PI. GNP-mediated cell effects correlated with slight radiosensitization of GNP(+) specimens, significant only for the highest radiation dose tested (4 Gy).

Probing Chromatin Compaction and Its Epigenetic States in situ With Single-Molecule Localization-Based Super-Resolution Microscopy.

Chromatin organization play a vital role in gene regulation and genome maintenance in normal biological processes and in response to environmental insults. Disruption of chromatin organization imposes a significant effect on many cellular processes and is often associated with a range of pathological processes such as aging and cancer. Extensive attention has been attracted to understand the structural and functional studies of chromatin architecture. Biochemical assays coupled with the state-of-the-art genomic technologies have been traditionally used to probe chromatin architecture. Recent advances in single molecule localization microscopy (SMLM) open up new opportunities to directly visualize higher-order chromatin architecture, its compaction status and its functional states at nanometer resolution in the intact cells or tissue. In this review, we will first discuss the recent technical advantages and challenges of using SMLM to image chromatin architecture. Next, we will focus on the recent applications of SMLM for structural and functional studies to probe chromatin architecture in key cellular processes. Finally, we will provide our perspectives on the recent development and potential applications of super-resolution imaging of chromatin architecture in improving our understanding in diseases.

Quantitative single-molecule imaging of TNFR1 reveals zafirlukast as antagonist of TNFR1 clustering and TNFα-induced NF-ĸB signaling.

TNFR1 is a crucial regulator of NF-ĸB-mediated proinflammatory cell survival responses and programmed cell death (PCD). Deregulation of TNFα- and TNFR1-controlled NF-ĸB signaling underlies major diseases, like cancer, inflammation, and autoimmune diseases. Therefore, although being routinely used, antagonists of TNFα might also affect TNFR2-mediated processes, so that alternative approaches to directly antagonize TNFR1 are beneficial. Here, we apply quantitative single-molecule localization microscopy (SMLM) of TNFR1 in physiologic cellular settings to validate and characterize TNFR1 inhibitory substances, exemplified by the recently described TNFR1 antagonist zafirlukast. Treatment of TNFR1-mEos2 reconstituted TNFR1/2 knockout mouse embryonic fibroblasts (MEFs) with zafirlukast inhibited both ligand-independent preligand assembly domain (PLAD)-mediated TNFR1 dimerization as well as TNFα-induced TNFR1 oligomerization. In addition, zafirlukast-mediated inhibition of TNFR1 clustering was accompanied by deregulation of acute and prolonged NF-ĸB signaling in reconstituted TNFR1-mEos2 MEFs and human cervical carcinoma cells. These findings reveal the necessity of PLAD-mediated, ligand-independent TNFR1 dimerization for NF-ĸB activation, highlight the PLAD as central regulator of TNFα-induced TNFR1 oligomerization, and demonstrate that TNFR1-mEos2 MEFs can be used to investigate TNFR1-antagonizing compounds employing single-molecule quantification and functional NF-ĸB assays at physiologic conditions.