Real-time imaging of drug-induced trapping of cellular topoisomerases and poly(ADP-ribose) polymerase 1 at the single-molecule level.

Topoisomerases (TOP1, TOP2α, and ß) are nuclear enzymes crucial for virtually all aspects of DNA metabolisms. They also are the targets of important anti-tumor chemotherapeutics that act by trapping the otherwise reversible topoisomerase-DNA covalent complex intermediates (TOPccs) that are formed during their catalytic reactions, resulting in long-lived topoisomerase DNA-protein crosslinks (TOP-DPCs) that interfere with DNA transactions. The Poly(ADP-ribose) polymerase (PARP) family protein PARP1 is activated by DNA damage to recruit DNA repair proteins, and PARP inhibitors are another class of commonly used chemotherapeutics, which bind and trap PARP molecules on DNA. To date, the trapping of TOPccs and PARP by their respective inhibitors can only be measured by immune-biochemical methods in cells. Here, we developed an imaging-based approach enabling real-time monitoring of drug-induced trapping of TOPccs and PARP1 in live cells at the single-molecule level. Capitalizing on this approach, we calculated the fraction of self-fluorescence tag-labeled topoisomerases and PARP single-molecules that are trapped by their respective inhibitors in real time. This novel technique should help elucidate the molecular processes that repair TOPcc and PARP trapping and facilitate the development of novel topoisomerase and PARP inhibitor-based therapies.

Targeting neddylation sensitizes colorectal cancer to topoisomerase I inhibitors by inactivating the DCAF13-CRL4 ubiquitin ligase complex.

Colorectal cancers (CRCs) are prevalent worldwide, yet current treatments remain inadequate. Using chemical genetic screens, we identify that co-inhibition of topoisomerase I (TOP1) and NEDD8 is synergistically cytotoxic in human CRC cells. Combination of the TOP1 inhibitor irinotecan or its bioactive metabolite SN38 with the NEDD8-activating enzyme inhibitor pevonedistat exhibits synergy in CRC patient-derived organoids and xenografts. Mechanistically, we show that pevonedistat blocks the ubiquitin/proteasome-dependent repair of TOP1 DNA-protein crosslinks (TOP1-DPCs) induced by TOP1 inhibitors and that the CUL4-RBX1 complex (CRL4) is a prominent ubiquitin ligase acting on TOP1-DPCs for proteasomal degradation upon auto-NEDD8 modification during replication. We identify DCAF13, a DDB1 and Cullin Associated Factor, as the receptor of TOP1-DPCs for CRL4. Our study not only uncovers a replication-coupled ubiquitin-proteasome pathway for the repair of TOP1-DPCs but also provides molecular and translational rationale for combining TOP1 inhibitors and pevonedistat for CRC and other types of cancers.

Targeting N-linked Glycosylation for the Therapy of Aggressive Lymphomas.

Diffuse large B-cell lymphoma (DLBCL) can be subdivided into the activated B-cell (ABC) and germinal center B cell-like (GCB) subtypes. Self-antigen engagement of B-cell receptors (BCR) in ABC tumors induces their clustering, thereby initiating chronic active signaling and activation of NF-κB and PI3 kinase. Constitutive BCR signaling is essential in some GCB tumors but primarily activates PI3 kinase. We devised genome-wide CRISPR-Cas9 screens to identify regulators of IRF4, a direct transcriptional target of NF-κB and an indicator of proximal BCR signaling in ABC DLBCL. Unexpectedly, inactivation of N-linked protein glycosylation by the oligosaccharyltransferase-B (OST-B) complex reduced IRF4 expression. OST-B inhibition of BCR glycosylation reduced BCR clustering and internalization while promoting its association with CD22, which attenuated PI3 kinase and NF-κB activation. By directly interfering with proximal BCR signaling, OST-B inactivation killed models of ABC and GCB DLBCL, supporting the development of selective OST-B inhibitors for the treatment of these aggressive cancers. SIGNIFICANCE: DLBCL depends on constitutive BCR activation and signaling. There are currently no therapeutics that target the BCR directly and attenuate its pathologic signaling. Here, we unraveled a therapeutically exploitable, OST-B-dependent glycosylation pathway that drives BCR organization and proximal BCR signaling. This article is highlighted in the In This Issue feature, p. 1749.

Multiview confocal super-resolution microscopy.

Confocal microscopy1 remains a major workhorse in biomedical optical microscopy owing to its reliability and flexibility in imaging various samples, but suffers from substantial point spread function anisotropy, diffraction-limited resolution, depth-dependent degradation in scattering samples and volumetric bleaching2. Here we address these problems, enhancing confocal microscopy performance from the sub-micrometre to millimetre spatial scale and the millisecond to hour temporal scale, improving both lateral and axial resolution more than twofold while simultaneously reducing phototoxicity. We achieve these gains using an integrated, four-pronged approach: (1) developing compact line scanners that enable sensitive, rapid, diffraction-limited imaging over large areas; (2) combining line-scanning with multiview imaging, developing reconstruction algorithms that improve resolution isotropy and recover signal otherwise lost to scattering; (3) adapting techniques from structured illumination microscopy, achieving super-resolution imaging in densely labelled, thick samples; (4) synergizing deep learning with these advances, further improving imaging speed, resolution and duration. We demonstrate these capabilities on more than 20 distinct fixed and live samples, including protein distributions in single cells; nuclei and developing neurons in Caenorhabditis elegans embryos, larvae and adults; myoblasts in imaginal disks of Drosophila wings; and mouse renal, oesophageal, cardiac and brain tissues.

Histone hyperacetylation disrupts core gene regulatory architecture in rhabdomyosarcoma.

Core regulatory transcription factors (CR TFs) orchestrate the placement of super-enhancers (SEs) to activate transcription of cell-identity specifying gene networks, and are critical in promoting cancer. Here, we define the core regulatory circuitry of rhabdomyosarcoma and identify critical CR TF dependencies. These CR TFs build SEs that have the highest levels of histone acetylation, yet paradoxically the same SEs also harbor the greatest amounts of histone deacetylases. We find that hyperacetylation selectively halts CR TF transcription. To investigate the architectural determinants of this phenotype, we used absolute quantification of architecture (AQuA) HiChIP, which revealed erosion of native SE contacts, and aberrant spreading of contacts that involved histone acetylation. Hyperacetylation removes RNA polymerase II (RNA Pol II) from core regulatory genetic elements, and eliminates RNA Pol II but not BRD4 phase condensates. This study identifies an SE-specific requirement for balancing histone modification states to maintain SE architecture and CR TF transcription.

In situ polymerization on nanoscale metal-organic frameworks for enhanced physiological stability and stimulus-responsive intracellular drug delivery.

Metal-organic framework (MOF) nanoparticles have shown great potential as carrier platforms in theranostic applications. However, their poor physiological stability in phosphate-based media has limited their biological applications. Here, we studied the dissociation of MOF nanoparticles under physiological conditions, both in vitro and in vivo, and developed an in situ polymerization strategy on MOF nanoparticles for enhanced stability under physiological conditions and stimulus-responsive intracellular drug release. With polymer wrapped on the surface serving as a shield, the nanoscale MOFs were protected from decomposition by phosphate ions or acid and prevented the loaded cargos from leaking. An in vivo positron emission tomography (PET) study of 64Cu-labelled porphyrinic MOF indicated prolonged circulation time of the in situ polymerized MOF nanoparticles and greater tumor accumulation than unmodified MOF nanoparticles. With enhanced stability, cargos loaded into MOF nanoparticles or prodrugs conjugated on the surface can be efficiently delivered and released upon stimulus-responsive cleavage.

Gas chromatography-mass spectrometry based metabolomics profile of hippocampus and cerebellum in mice after chronic arsenic exposure.

Intake of arsenic (As) via drinking water has been a serious threat to global public health. Though there are numerous reports of As neurotoxicity, its pathogenesis mechanisms remain vague especially its chronic effects on metabolic network. Hippocampus is a renowned area in relation to learning and memory, whilst recently, cerebellum is argued to be involved with process of cognition. Therefore, the study aimed to explore metabolomics alternations in these two areas after chronic As exposure, with the purpose of further illustrating details of As neurotoxicity. Twelve 3-week-old male C57BL/6J mice were divided into two groups, receiving deionized drinking water (control group) or 50 mg/L of sodium arsenite (via drinking water) for 24 weeks. Learning and memory abilities were tested by Morris water maze (MWM) test. Pathological and morphological changes of hippocampus and cerebellum were captured via transmission electron microscopy (TEM). Metabolic alterations were analyzed by gas chromatography-mass spectrometry (GC-MS). MWM test confirmed impairments of learning and memory abilities of mice after chronic As exposure. Metabolomics identifications indicated that tyrosine increased and aspartic acid (Asp) decreased simultaneously in both hippocampus and cerebellum. Intermediates (succinic acid) and indirect involved components of tricarboxylic acid cycle (proline, cysteine, and alanine) were found declined in cerebellum, indicating disordered energy metabolism. Our findings suggest that these metabolite alterations are related to As-induced disorders of amino acids and energy metabolism, which might therefore, play an important part in mechanisms of As neurotoxicity.

Single-shot super-resolution total internal reflection fluorescence microscopy.

We combined instant structured illumination microscopy (iSIM) with total internal reflection fluorescence microscopy (TIRFM) in an approach referred to as instant TIRF-SIM, thereby improving the lateral spatial resolution of TIRFM to 115 ± 13 nm without compromising speed, and enabling imaging frame rates up to 100 Hz over hundreds of time points. We applied instant TIRF-SIM to multiple live samples and achieved rapid, high-contrast super-resolution imaging close to the coverslip surface.

A RAS-CaMKKß-AMPKα2 pathway promotes senescence by licensing post-translational activation of C/EBPß through a novel 3'UTR mechanism.

Oncogene-induced senescence (OIS) is an intrinsic tumor suppression mechanism that requires the p53 and RB pathways and post-translational activation of C/EBPß through the RAS-ERK cascade. We previously reported that in transformed/proliferating cells, C/EBPß activation is inhibited by G/U-rich elements (GREs) in its 3'UTR. This mechanism, termed "3'UTR regulation of protein activity" (UPA), maintains C/EBPß in a low-activity state in tumor cells and thus facilitates senescence bypass. Here we show that C/EBPß UPA is overridden by AMPK signaling. AMPK activators decrease cytoplasmic levels of the GRE binding protein HuR, which is a key UPA component. Reduced cytoplasmic HuR disrupts 3'UTR-mediated trafficking of Cebpb transcripts to the peripheral cytoplasm-a fundamental feature of UPA-thereby stimulating C/EBPß activation and growth arrest. In primary cells, oncogenic RAS triggers a Ca++-CaMKKß-AMPKα2-HuR pathway, independent of AMPKα1, that is essential for C/EBPß activation and OIS. This axis is disrupted in cancer cells through down-regulation of AMPKα2 and CaMKKß. Thus, CaMKKß-AMPKα2 signaling constitutes a key tumor suppressor pathway that activates a novel UPA-cancelling mechanism to unmask the cytostatic and pro-senescence functions of C/EBPß.

Reflective imaging improves spatiotemporal resolution and collection efficiency in light sheet microscopy.

Light-sheet fluorescence microscopy (LSFM) enables high-speed, high-resolution, and gentle imaging of live specimens over extended periods. Here we describe a technique that improves the spatiotemporal resolution and collection efficiency of LSFM without modifying the underlying microscope. By imaging samples on reflective coverslips, we enable simultaneous collection of four complementary views in 250 ms, doubling speed and improving information content relative to symmetric dual-view LSFM. We also report a modified deconvolution algorithm that removes associated epifluorescence contamination and fuses all views for resolution recovery. Furthermore, we enhance spatial resolution (to <300 nm in all three dimensions) by applying our method to single-view LSFM, permitting simultaneous acquisition of two high-resolution views otherwise difficult to obtain due to steric constraints at high numerical aperture. We demonstrate the broad applicability of our method in a variety of samples, studying mitochondrial, membrane, Golgi, and microtubule dynamics in cells and calcium activity in nematode embryos.

Probing the target search of DNA-binding proteins in mammalian cells using TetR as model searcher.

Many cellular functions rely on DNA-binding proteins finding and associating to specific sites in the genome. Yet the mechanisms underlying the target search remain poorly understood, especially in the case of the highly organized mammalian cell nucleus. Using as a model Tet repressors (TetRs) searching for a multi-array locus, we quantitatively analyse the search process in human cells with single-molecule tracking and single-cell protein-DNA association measurements. We find that TetRs explore the nucleus and reach their target by 3D diffusion interspersed with transient interactions with non-cognate sites, consistent with the facilitated diffusion model. Remarkably, nonspecific binding times are broadly distributed, underlining a lack of clear delimitation between specific and nonspecific interactions. However, the search kinetics is not determined by diffusive transport but by the low association rate to nonspecific sites. Altogether, our results provide a comprehensive view of the recruitment dynamics of proteins at specific loci in mammalian cells.

Fast multicolor 3D imaging using aberration-corrected multifocus microscopy.

Conventional acquisition of three-dimensional (3D) microscopy data requires sequential z scanning and is often too slow to capture biological events. We report an aberration-corrected multifocus microscopy method capable of producing an instant focal stack of nine 2D images. Appended to an epifluorescence microscope, the multifocus system enables high-resolution 3D imaging in multiple colors with single-molecule sensitivity, at speeds limited by the camera readout time of a single image.

Nuclear targeting dynamics of gold nanoclusters for enhanced therapy of HER2+ breast cancer.

Recent advances in fluorescent metal nanoclusters have spurred tremendous interest in nanomedicine due to the ease of fabrication, excellent biocompatibility, and, more importantly, excellent wavelength-dependent tunability. Herein, we report our findings on fluorescent BSA-protected gold nanoclusters (AuNCs), ∼2 nm in size conjugated with Herceptin (AuNCs-Her), for specific targeting and nuclear localization in ErbB2 over-expressing breast cancer cells and tumor tissue as a novel fluorescent agent for simultaneous imaging and cancer therapy. More interestingly, we found that AuNCs-Her could escape the endolysosomal pathway and enter the nucleus of cancer cells to enhance the therapeutic efficacy of Herceptin. We elucidate the diffusion characteristics (diffusion time and number of diffusers) and concentration of the fluorescing clusters in the nucleus of live cells. Our findings also suggest that the nuclear localization effect of AuNCs-Her enhances the anticancer therapeutic efficacy of Herceptin as evidenced by the induction of DNA damage. This study not only discusses a new nanomaterial platform for nuclear delivery of drugs but also provides important insights on nuclear targeting for enhanced therapy.

Measurement of the attachment and assembly of small amyloid-ß oligomers on live cell membranes at physiological concentrations using single-molecule tools.

It is thought that the pathological cascade in Alzheimer's disease is initiated by the formation of amyloid-ß (Aß) peptide complexes on cell membranes. However, there is considerable debate about the nature of these complexes and the type of solution-phase Aß aggregates that may contribute to their formation. Also, it is yet to be shown that Aß attaches strongly to living cell membranes, and that this can happen at low, physiologically relevant Aß concentrations. Here, we simultaneously measure the aggregate size and fluorescence lifetime of fluorescently labeled Aß(1-40) on and above the membrane of cultured PC12 cells at near-physiological concentrations. We find that at 350 nM Aß concentration, large (>>10 nm average hydrodynamic radius) assemblies of codiffusing, membrane-attached Aß molecules appear on the cell membrane together with a near-monomeric species. When the extracellular concentration is 150 nM, the membrane contains only the smaller species, but with a similar degree of attachment. At both concentrations, the extracellular solution contains only small (∼2.3 nm average hydrodynamic radius) Aß oligomers or monomers. We conclude that at near-physiological concentrations only the small oligomeric Aß species are relevant, they are capable of attaching to the cell membrane, and they assemble in situ to form much larger complexes.

Fluorescence lifetime cross correlation spectroscopy resolves EGFR and antagonist interaction in live cells.

Fluorescence correlation or cross-correlation spectroscopy (FCS or FCCS), a single molecule technique, has the ability to provide highly sensitive information on interaction and dynamics of biomolecules both in vitro and in vivo. However, the inherent drawback of FCS is that species with similar molecular weight could not be differentiated. Although FCCS could resolve this through cross-correlation, it suffers from nonideal confocal volume overlap and spectral cross-talk which limits its application. In this work, we demonstrate for the first time the applicability of fluorescence lifetime correlation spectroscopy (FLCS) to monitor the interaction of an antagonist antibody with the epidermal growth factor receptor (EGFR) in live cells. As a proof of concept, we demonstrate the interaction of Cy5 labeled IgG and Alexa633 labeled anti-IgG using a single laser source (636 nm excitation) in vitro. The autocorrelation functions were separated based on their respective lifetime with a single detector and their K(d) value was determined to be 11 +/- 3 nM. An in vivo application constituting the interaction of EGFR neutralizing antibody labeled with Alexa488 and EGFR-GFP in live HEK293 cells was successfully demonstrated. The binding specificity of EGFR neutralizing antibody was confirmed by fluorescence lifetime cross-correlation measurements and fluorescence lifetime imaging (FLIM). The dissociation constant of this complex was found to be 9.2 +/- 2.7 nM. A quantitative assessment of receptor density calculations show that the density of EGFR significantly decreased, from 540 +/- 64 receptors/microm(2) to 38 +/- 7 receptors/microm(2) upon addition of the neutralizing EGFR antibody, indicating that the antagonist could induce receptor internalization. The demonstrated work not only opens up new opportunities in studying protein-protein interactions in solutions and in live cells but also provide new insights in biology to understand how the antagonists influence EGFR through live cell quantification and imaging.

Quantitative investigation of compartmentalized dynamics of ErbB2 targeting gold nanorods in live cells by single molecule spectroscopy.

Understanding the diffusion dynamics and receptor uptake mechanism of nanoparticles in cancer cells is crucial to the rational design of multifunctional nanoprobes for targeting and delivery. In this report, for the first time, we quantify the localization and evaluate the diffusion times of Herceptin-conjugated gold nanorods (H-GNRs) in different cell organelles by fluorescence correlation spectroscopy (FCS) and examine the endocytic diffusion of H-GNRs in live ErbB2 overexpressing SK-BR-3 cells. First, by colocalizing H-GNRs in different cellular organelles depicted by the respective markers, we demonstrate that H-GNRs colocalize with the endosome and lysosome but not with the Golgi apparatus. Our study shows that Herceptin-conjugated GNRs have similar intracellular localization characteristics as Herceptin-ErbB2 complex, with a higher concentration found in the endosome (72 +/- 20.6 nM) than lysosome (9.4 +/- 4.2 nM) after internalization. The demonstrated approach and findings not only lay the foundations for a quantitative understanding of the fate of nanoparticle-based targeting but also provide new insights into the rational design of nanoparticle delivery systems for effective treatment.

Gold nanorod/Fe3O4 nanoparticle "nano-pearl-necklaces" for simultaneous targeting, dual-mode imaging, and photothermal ablation of cancer cells.

Gold and pearls: Multifunctional nanoparticles, each composed of a single, amine-modified gold nanorod, decorated with multiple "pearls" of Fe(3)O(4) nanoparticles capped with carboxy groups, are prepared. Their effectiveness in simultaneous targeting, dual-mode imaging, and photothermal ablation of breast cancer cells is demonstrated.

Meso-tetra (carboxyphenyl) porphyrin (TCPP) nanoparticles were internalized by SW480 cells by a clathrin-mediated endocytosis pathway to induce high photocytotoxicity.

We studied the uptake of meso-tetra (carboxyphenyl) porphyrin (TCPP) nanoparticles by SW480 cells and carried out a systematic investigation of the cellular internalization mechanism of TCPP nanoparticles, also studied the photocytotoxicity of TCPP nanoparticles. At first, meso-tetra (carboxyphenyl) porphyrin (TCPP) nanoparticles were prepared by the method of mixing solvent techniques. SW480 cellular uptakes of photosensitizers (TCPP nanoparticles, TCPP-loaded poly (lactic-co-glycolic acid) (PLGA) nanoparticles and free TCPP) were analyzed by the method of fluorospectrophotometry. Endocytosis mechanism investigation was carried out by preincubating SW480 cells at 4 degrees C, and preincubating SW480 cells with sucrose, K+-free buffer solution and filipin. Clathrin HC expression after incubating SW480 cells with these three photosensitizers was analyzed by methods of Western blot and RT-PCR. At last, we analyzed the photo-cytotoxicity after incubating SW480 cells with photosensitizers and receiving irradiation. SW480 cells showed rapid uptake (0.0083fmoles TCPP/cell) of TCPP nanoparticles after 1h incubation. We also demonstrated that the uptake of TCPP nanoparticles by SW480 cells was a clathrin-mediated endocytosis pathway. As a result of rapid internalization of TCPP nanoparticles by SW480 cells, this special photosensitizer showed very high photocytotoxic effect on SW480 cells in vitro. The nano-sized photosensitizer with no matrix cover: TCPP nanoparticles, can produce higher photocytotoxicity than other photosensitizers (TCPP-loaded PLGA nanoparticles and free TCPP). The in vivo tumor growth inhibition experiment indicated that TCPP nanoparticles plus PDT treatment induced the most dramatic tumor-inhibiting efficacy in all TCPP treated groups. The results of this study suggest that TCPP nanoparticles represent a potential and powerful photodynamic therapy agent.

Adenosine A(2A) receptors assemble into higher-order oligomers at the plasma membrane.

Oligomerization of G protein-coupled receptors (GPCRs) is known to play important roles in regulating receptor pharmacology and function. Whereas many bivalent GPCR interactions have been described, the stoichiometry and localization of GPCR oligomers are largely unknown. We have used bimolecular fluorescence complementation (BiFC) to study adenosine A(2A) receptor (A(2A)R) oligomerization. The data suggest specificity of the A(2A)R/A(2A)R interaction monitored by BiFC and proper sub-cellular localization of tagged receptors. Moreover, using a novel approach combining fluorescence resonance energy transfer and BiFC, we found that at least three A(2A) receptors assemble into higher-order oligomers at the plasma membrane in Cath.A differentiated neuronal cells.

Arg-Gly-Asp (RGD) peptide conjugated poly(lactic acid)-poly(ethylene oxide) micelle for targeted drug delivery.

In this study, a new poly(lactic acid)-poly (ethylene oxide)-Arg-Gly-Asp (PLA-PEO-RGD) derivative was synthesized, and paclitaxel-loaded PLA-PEO-RGD micelles were prepared by this derivative. The solubility assay showed that micelles mixed with Pluronic F-68 as surfactant could increase the solubility of this hydrophobic paclitaxel in aqueous solution. The cell-binding assay showed that PLA-PEO-RGD micelle (IC(50) = 11.13 +/- 1.38 nmol/L) had about 3.6-fold higher integrin avidity than PLA-PEO-RGD conjugates (IC(50) = 40.33 +/- 3.12 nmol/L). The avidity of micelle was also higher than RGD4C peptide (IC(50) = 24.44 +/- 1.21 nmol/L). The in vitro drug release profile of drug-loaded PLA-PEO-RGD micelles exhibited initial burst release to 37% +/- 2% (w/w) during the first 12 h, and then the release rate became steady in a controlled release manner. Furthermore, treatment of the MDA-MB-435 breast cancer cell line with paclitaxel-loaded PLA-PEO-RGD micelles yielded cytotoxicities, with EC(50) values of approximately 30 mumol/L. The paclitaxel-loaded PLA-PEO-RGD micelles treated group showed the most dramatic tumor reduction in MDA-MB-435 tumor-bearing nude mice, and the final mean tumor load was 31 +/- 16 mm(3) (mean +/- SD; n = 8). (125)I-labeled micelles administration resulted in significant (p < 0.001) higher tumor uptake (2.68% +/- 0.14%, ID/g) of PLA-PEO-RGD micelles compared to PLA-PEO micelles (0.84% +/- 0.09%, ID/g) after 2.5 h postinjection. Biodistribution study showed the best blood clearance of PLA-PEO-RGD micelles after 4.5 h postinjection. The results of this study suggest that paclitaxel-loaded PLA-PEO-RGD micelles based on the specific recognition of alpha(V)beta(3) integrin represent a potential and powerful target delivery technology.