Analysis of Fluorescent Proteins for Observing Single Gene Locus in a Live and Fixed Escherichia coli Cell.

Fluorescent proteins (FPs) are essential tools for advanced microscopy techniques such as super-resolution imaging, single-particle tracking, and quantitative single-molecule counting. Various FPs fused to DNA-binding proteins have been used to observe the subcellular location and movement of specific gene loci in living and fixed bacterial cells. However, quantitative assessments of the properties of FPs for gene locus measurements are still lacking. Here, we assessed various FPs to observe specific gene loci in live and fixed Escherichia coli cells using a fluorescent repressor-operator binding system (FROS), tet operator-Tet repressor proteins (TetR). Tsr-fused FPs were used to assess the intensity and photostability of various FPs (five red FPs: mCherry2, FusionRed, mRFP, mCrimson3, and dKatushka; and seven yellow FPs: SYFP2, Venus, mCitrine, YPet, mClover3, mTopaz, and EYFP) at the single-molecule level in living cells. These FPs were then used for gene locus measurements using FROS. Our results indicate that TetR-mCrimson3 (red) and TetR-EYFP (yellow) had better properties for visualizing gene loci than the other TetR-FPs. Furthermore, fixation procedures affected the clustering of diffusing TetR-FPs and altered the locations of the TetR-FP foci. Fixation with formaldehyde consistently disrupted proper DNA locus observations using TetR-FPs. Notably, the foci measured using TetR-mCrimson3 remained close to their original positions in live cells after glyoxal fixation. This in vivo study provides a cell-imaging guide for the use of FPs for gene-locus observation in E. coli and a scheme for evaluating the use of FPs for other cell-imaging purposes.

Uncovering the impact of UV radiation on mitochondria in dermal cells: a STED nanoscopy study.

Mitochondria are essential organelles in cellular energy metabolism and other cellular functions. Mitochondrial dysfunction is closely linked to cellular damage and can potentially contribute to the aging process. The purpose of this study was to investigate the subcellular structure of mitochondria and their activities in various cellular environments using super-resolution stimulated emission depletion (STED) nanoscopy. We examined the morphological dispersion of mitochondria below the diffraction limit in sub-cultured human primary skin fibroblasts and mouse skin tissues. Confocal microscopy provides only the overall morphology of the mitochondrial membrane and an indiscerptible location of nucleoids within the diffraction limit. Conversely, super-resolution STED nanoscopy allowed us to resolve the nanoscale distribution of translocase clusters on the mitochondrial outer membrane and accurately quantify the number of nucleoids per cell in each sample. Comparable results were obtained by analyzing the translocase distribution in the mouse tissues. Furthermore, we precisely and quantitatively analyzed biomolecular distribution in nucleoids, such as the mitochondrial transcription factor A (TFAM), using STED nanoscopy. Our findings highlight the efficacy of super-resolution fluorescence imaging in quantifying aging-related changes on the mitochondrial sub-structure in cells and tissues.

Single-Benzene Dual-Emitters Harness Excited-State Antiaromaticity for White Light Generation and Fluorescence Imaging.

Molecular emitters simultaneously generating light at different wavelengths have wide applications. With a small molecule, however, it is challenging to realize two independent radiative pathways. We invented the first examples of dual-emissive single-benzene fluorophores (SBFs). Two emissive tautomers are generated by synthetic modulation of the hydrogen bond acidity, which opens up pathways for excited-state proton transfer. White light is produced by a delicate balance between the energy and intensity of the emission from each tautomer. We show that the excited-state antiaromaticity of the benzene core itself dictates the proton movements driving the tautomer equilibrium. Using this simple benzene platform, a fluorinated SBF was synthesized with a record high solubility in perfluorocarbon solvents. White light-emitting devices and multicolor imaging of perfluorocarbon nanodroplets in live cells demonstrate the practical utility of these molecules.

Ultrasensitive Near-Infrared Circularly Polarized Light Detection Using 3D Perovskite Embedded with Chiral Plasmonic Nanoparticles.

Chiral organic ligand-incorporated low-dimensional metal-halide perovskites have received increasing attention for next-generation photodetectors because of the direct detection capability of circularly polarized light (CPL), which overcomes the requirement for subsidiary optical components in conventional CPL photodetectors. However, most chiral perovskites have been based on low-dimensional structures that confine chiroptical responses to the ultraviolet (UV) or short-wavelength visible region and limit photocurrent due to their wide bandgap and poor charge transport. Here, chiroptical properties of 3D Cs0.05 FA0.5 MA0.45 Pb0.5 Sn0.5 I3 polycrystalline films are achieved by incorporating chiral plasmonic gold nanoparticles (AuNPs) into the mixed PbSn perovskite, without sacrificing its original optoelectronic properties. CPL detectors fabricated using chiral AuNP-embedded perovskite films can operate without external power input; they exhibit remarkable chirality in the near-infrared (NIR) region with a high anisotropy factor of responsivity (gres ) of 0.55, via giant plasmon resonance shift of chiral plasmonic AuNPs. In addition, a CPL detector array fabricated on a plastic substrate demonstrates highly sensitive self-powered NIR detection with superior flexibility and durability.

Cooperative inhibition of SNARE-mediated vesicle fusion by α-synuclein monomers and oligomers.

The primary hallmark of Parkinson's disease (PD) is the generation of Lewy bodies of which major component is α-synuclein (α-Syn). Because of increasing evidence of the fundamental roles of α-Syn oligomers in disease progression, α-Syn oligomers have become potential targets for therapeutic interventions for PD. One of the potential toxicities of α-Syn oligomers is their inhibition of SNARE-mediated vesicle fusion by specifically interacting with vesicle-SNARE protein synaptobrevin-2 (Syb2), which hampers dopamine release. Here, we show that α-Syn monomers and oligomers cooperatively inhibit neuronal SNARE-mediated vesicle fusion. α-Syn monomers at submicromolar concentrations increase the fusion inhibition by α-Syn oligomers. This cooperative pathological effect stems from the synergically enhanced vesicle clustering. Based on this cooperative inhibition mechanism, we reverse the fusion inhibitory effect of α-Syn oligomers using small peptide fragments. The small peptide fragments, derivatives of α-Syn, block the binding of α-Syn oligomers to Syb2 and dramatically reverse the toxicity of α-Syn oligomers in vesicle fusion. Our findings demonstrate a new strategy for therapeutic intervention in PD and related diseases based on this specific interaction of α-Syn.

Over-activation of a nonessential bacterial protease DegP as an antibiotic strategy.

Rising antibiotic resistance urgently begs for novel targets and strategies for antibiotic discovery. Here, we report that over-activation of the periplasmic DegP protease, a member of the highly conserved HtrA family, can be a viable strategy for antibiotic development. We demonstrate that tripodal peptidyl compounds that mimic DegP-activating lipoprotein variants allosterically activate DegP and inhibit the growth of an Escherichia coli strain with a permeable outer membrane in a DegP-dependent fashion. Interestingly, these compounds inhibit bacterial growth at a temperature at which DegP is not essential for cell viability, mainly by over-proteolysis of newly synthesized proteins. Co-crystal structures show that the peptidyl arms of the compounds bind to the substrate-binding sites of DegP. Overall, our results represent an intriguing example of killing bacteria by activating a non-essential enzyme, and thus expand the scope of antibiotic targets beyond the traditional essential proteins or pathways.

Leucine-induced localization of Leucyl-tRNA synthetase in lysosome membrane.

Leucyl-tRNA synthetase (LRS) plays major roles in providing leucine-tRNA and activating mechanistic target of rapamycin complex 1 (mTORC1) through intracellular leucine sensing. mTORC1 activated by amino acids affects the influence on physiology functions including cell proliferation, protein synthesis and autophagy in various organisms. Biochemical results demonstrating leucine sensing have been published, but visual results are lacking. Therefore, we observed the location of LRS with and without leucine using stimulated emission depletion (STED) microscopy one of the super-resolution microscopy and transmission electron microscopy (TEM). This revealed that LRS was translocated to the lysosome on addition of leucine. The translocation was inhibited by treatment with compound BC-LI-0186, disrupting the interaction between RagD and LRS. Immuno-TEM revealed a clear decrease in LRS translocation to the lysosome on addition of the inhibitor. This direct visualization of leucine sensing and LRS translocation to the lysosome was related to mTORC1 activation. To study the relationship between mTORC1 activation and LRS translocation, we monitored the change in autophagy for each condition using TEM and CLSM. The results showed a decrease in autophagy on addition of leucine, demonstrating crosstalk between leucine sensing, LRS translocation, RagD interaction, and mTORC1 activation.