Single-Molecule Blinking Fluorescence Enhancement by Surface Plasmon-Coupled Emission-Based Substrates for Single-Molecule Localization Imaging

Surface plasmon-coupled emission (SPCE) substrates to enhance the blinking fluorescence of spontaneously blinking fluorophores in single-molecule localization microscopy (SMLM) were fabricated to reduce the excitation power density requirement and reveal the distribution of fluorophore-labeled proteins on a plasma membrane with nanoscale-level resolution. The systemic investigation of the contribution of local field enhancement, modified quantum yield, and emission coupling yield through glass coverslip substrates coated with metal layers of different thicknesses revealed that the silver-layer substrate with a thickness of 44 nm produces the highest SPCE fluorescence in spontaneously blinking fluorophores, and it has a highly directional SPCE fluorescence, which helps improve the detection efficiency. Moreover, the uniform and surface-enhanced field created on the substrate surface is beneficial for fluorescence background reduction in single fluorophore detection and localization, as well as for revealing the real position of fluorophores. Consequently, compared with a glass coverslip substrate, the presented SPCE substrate demonstrated a fluorescence enhancement of 480% and an increase in blinking events from a single spontaneously blinking fluorophore; moreover, the required excitation power density for SMLM imaging was significantly reduced to 23 W cm–2 for visualizing the distribution of epidermal growth factor receptors (EGFRs) on the basal plasma membrane of A549 lung cancer cells with a localization precision of 19 ± 7 nm. Finally, the fluorophore-labeled EGFRs on the basal plasma membrane in the presence of PIKfyve-specific inhibitor treatment were explored using SPCE–SMLM imaging; the results revealed a distinct reduction in the density of localization events because of a decrease in EGFR abundance at the plasma membranes of the cells

Single-Molecule Blinking Fluorescence Enhancement by Surface Plasmon-Coupled Emission-Based Substrates for Single-Molecule Localization Imaging

Surface plasmon-coupled emission (SPCE) substrates to enhance the blinking fluorescence of spontaneously blinking fluorophores in single-molecule localization microscopy (SMLM) were fabricated to reduce the excitation power density requirement and reveal the distribution of fluorophore-labeled proteins on a plasma membrane with nanoscale-level resolution. The systemic investigation of the contribution of local field enhancement, modified quantum yield, and emission coupling yield through glass coverslip substrates coated with metal layers of different thicknesses revealed that the silver-layer substrate with a thickness of 44 nm produces the highest SPCE fluorescence in spontaneously blinking fluorophores, and it has a highly directional SPCE fluorescence, which helps improve the detection efficiency. Moreover, the uniform and surface-enhanced field created on the substrate surface is beneficial for fluorescence background reduction in single fluorophore detection and localization, as well as for revealing the real position of fluorophores. Consequently, compared with a glass coverslip substrate, the presented SPCE substrate demonstrated a fluorescence enhancement of 480% and an increase in blinking events from a single spontaneously blinking fluorophore; moreover, the required excitation power density for SMLM imaging was significantly reduced to 23 W cm–2 for visualizing the distribution of epidermal growth factor receptors (EGFRs) on the basal plasma membrane of A549 lung cancer cells with a localization precision of 19 ± 7 nm. Finally, the fluorophore-labeled EGFRs on the basal plasma membrane in the presence of PIKfyve-specific inhibitor treatment were explored using SPCE–SMLM imaging; the results revealed a distinct reduction in the density of localization events because of a decrease in EGFR abundance at the plasma membranes of the cells

Targeted Nuclear Delivery using Peptide-Coated Quantum Dots

Core/shell quantum dots (CdSe/Zns) conjugated with various nuclear localization signaling (NLS) peptides, which could facilitate the transportation of quantum dots across the plasma membrane into the nucleus, have been utilized to investigate the uptake mechanism of targeted delivery. Because of their brightness and photostability, it was possible to trace the trajectories of individual quantum dots in living cells using both confocal and total internal reflection microscopes. We found that, when the quantum dots were added to a cell culture, the peptide-coated quantum dots entered the cell nucleus while the uncoated quantum dots remained in the cytoplasm. At 8 nM, most of the peptide coated quantum dots were found in the cytoplasm due to aggregation. However, at a lower concentration (0.08 nM), approximately 25% of the NLS peptide-coated quantum dots entered the cell nucleus. We also found that some quantum dots without NLS coating could also enter the nucleus, suggesting that the size of the quantum dots may play an important role in such a process

Mouse <i>Wuho</i> (m<i>Wh</i>) is an essential gene.

<p>(A) Gene targeting strategy. The gene targeting vector is a construct harboring positive (neomycin resistant gene [Neo], <i>red</i>) and negative (diphtheria toxin gene [DT], <i>blue</i>) selection markers. Through homologous recombination, the genomic region with exons 2 (E2) and 3 (E3) of m<i>Wh</i> was replaced and resulted in the introduction of one loxP site before E2, and the Neo cassette flanked by two loxP sites in Intron 3. Subsequently, E2 and E3 were deleted by Cre recombinase through recombination between two distal loxP’s. The symbols “>” and “<” represent the locations of forward and reverse primers used in PCR for genotyping. The PCR products are 3.6 kb from in wild type allele and 0.5 kb from knockout allele. (B) A representative genotyping experiment for embryos of E9.5 from heterozygous parents. PCR products can distinguish among genotypes of +/+, +/-, and -/-. (C) Knockout of m<i>Wh</i> results in DNA damage and apoptosis. The embryos genotyped in (B) were processed for western blot to examine expression of mWH, appearance of γ-H2AX for DNA damage, Cleaved PARP for apoptosis, and actin as loading controls. (D) Development of -/- embryos at E9.5 (left panel) and 10.5 (right panel) is abnormal. In E10.5 -/- embryos, they show varying morphology, potentially due to different degrees of resorption. Severe extents of resorption and are small (the left one of -/-). The other null embryo (the right one of -/-) show minor morphological defects and has brain development defects and internal bleeding. Scale bar is 1 mm.</p

DNA damage precedes apoptosis after mWH depletion in mouse JB6 cells.

<p>(A) Time course of DNA damage and apoptosis following mWH depletion. Samples were collected for analysis following mWH knockdown at 0, 24, 48, and 72 h. DNA damage (γ-H2AX signals) was detected at 48 h after siRNA treatment. Apoptotic signals including cleavage of Procaspase-9, Procaspase-3, and PARP, were detected only after 72 h of treatment. (B) Inhibitors of Caspases-9 and -3, but not for Caspase-8, block apoptosis ensuing depletion of mWH. However, DNA damage signals were unaffected and present under these conditions. Cells were co-cultured with simWH and different Caspase-specific inhibitors (final concentrations of 2 μM in the culture medium) including Z-DEVD-FMK (Caspase-3), Z-IETD-FMK (Caspase-8), and Z-LEHD-FMK (Caspase-9). After 72 h of treatment, samples were collected and processed for western blot.</p

Signaling path of cell death following depletion of hWH in human HFW cells.

<p>(A) DNA laddering reveals apoptosis in cells with hWH knockdown. (B) Activation of key signaling molecules by phosphorylation ensuing hWH knockdown. Activation of DNA damage signaling pathway was through phosphorylation of ATM (Ser1981), Chk2 (Thr68), and p53 (Ser15), but not through phosphorylation of ATR (Ser428) and Chk1 (Ser345). (C) Apoptosis was driven by an intrinsic but not extrinsic pathway. Activation of Caspases was monitored by the cleavage of Procaspase-9, Procaspase-3, and PARP, but not Procaspase-8. (D) Diagrammatic summary of DNA damage signaling and apoptotic pathway after depletion of hWH.</p

hWH knockdown results in DNA damage at the replication sites.

<p>(A) HCT116 p53^+/+ cells were treated with either hWH or control siRNA for 24, 48, and 72 h. Afterward cells are harvested and stained with DAPI (blue), EdU (green), and γ-H2AX (red). (B) After siRNA treatment for 48h, γ-H2AX positive cells percentage in sihWH is significantly higher than that of siControl. Double asterisks indicate significant differences at <i>p</i> < 0.01, according to Student’s <i>t</i> test.</p

Localization of hWH at the site of DNA replication determined by confocal (A) and super-resolution microscopy (B).

<p>(A) HCT116 p53^+/+ cells were stained with rabbit anti-hWH antibody (red), mouse anti-FEN1 antibody (blue), goat anti-PCNA (purple), and the nascent DNA replication marker EdU (green). The merged images of hWH plus EdU, or all four signals, are in the upper-right and lower-right panels, respectively. (B) HCT116 p53^+/+ cells were stained with rabbit anti-hWH antibody (red) and EdU (green). The images were obtained by TIRF microscopy and super-resolution microscopy. The boxed areas are shown at higher magnification.</p

Approach To Deliver Two Antioxidant Enzymes with Mesoporous Silica Nanoparticles into Cells

Reactive oxygen species (ROS) are important factors in many clinical diseases. However, direct delivery of antioxidant enzymes into cells is difficult due to poor cell uptake. A proper design of delivery of enzymes by nanoparticles is very desirable for therapeutic purposes. To overcome the cell barrier problem, a designed mesoporous silica nanoparticle (MSN) system with attached TAT-fusion denatured enzyme for enhancing cell membrane penetration has been developed. Simultaneous delivery of two up–downstream antioxidant enzymes, superoxide dismutase (SOD) and glutathione peroxidase­(GPx), reveals synergistic efficiency of ROS scavenging, compared to single antioxidant enzyme delivery. TAT peptide conjugation provided a facile nonendocytosis cell uptake and escape from endosome while moving and aggregating along the cytoskeleton that would allow them to be close to each other at the same time, resulting in the cellular antioxidation cascade reaction. The two-enzyme delivery shows a significant synergistic effect for protecting cells against ROS-induced cell damage and cell cycle arrest. The nanocarrier strategy for enzyme delivery demonstrates that intracellular anti-ROS cascade reactions could be regulated by multifunctional MSNs carrying image fluorophore and relevant antioxidation enzymes

hWH physically interacts with FEN1.

<p>(A) Identification of proteins present in hWH immunoprecipitate. We induced hWH expression in cells with an ectopic expression vector of hWH with V5 and His tags. The cleared cell lysate was incubated with anti-V5 agarose beads. The bead-bound proteins were separated by SDS-PAGE and stained with Coomassie blue. Each band was sliced and identified by Mass Spectrometry. The cells transfected with empty vector served as a control. (B) Flap endonuclease 1 (FEN1), methyltransferase-like protein 1 (METTL1), and proliferating cell nuclear antigen (PCNA), three candidates identified in (A) were further confirmed with western blot. (C) Interaction between endogenous hWH and FEN1 was demonstrated by co-IP experiments using cell lysate. hWH or FEN1 from cell lysate was removed by Protein A beads by using the antibody against hWH or FEN1, followed by probing the partner protein with western blot. The bands above both hWH and FEN1 are IgG heavy chains. (D) Direct interaction between hWH and FEN1 demonstrated by pull-down assays. FEN1 tagged with C-terminal His₆, hWH tagged with C-terminal GST, and GST proteins were purified, and examined by SDS-PAGE and staining with Coomassie blue to verify their purity (left panel). hWH tagged with C-terminal GST and FEN1 tagged with C-terminal His₆ were incubated with either Glutathione beads or Ni(II)-NTA beads, followed by detecting the association of partner proteins with western blot (right panels). Negative controls were beads alone without baits. (E) Direct binding between hWH and FEN1 revealed by fluorescence anisotropy. Tetracysteine-tagged FEN1 (FEN1-CCPGCC) was labeled with ReAsH fluorophore. Fluorescence anisotropy was measured after adding varying concentrations (0–1,000 nM) of hWH to 50 nM ReAsH-labeled FEN1-CCPGCC. The data points were used to fit a binding isotherm with a dissociation constant of 130 nM.</p