Plasmon-enhanced fluorescence and electrochemical aptasensor for SARS-CoV-2 Spike protein detection.

In this work, we combined plasmon-enhanced fluorescence and electrochemical (PEF-EC) transduction mechanisms to realize a highly sensitive dual-transducer aptasensor. To implement two traducers in one biosensor, a novel large-scale nanoimprint lithography process was introduced to fabricate gold nanopit arrays (AuNpA) with unique fringe structures. Light transmitting through the AuNpA samples exhibited a surface plasmon polariton peak overlapping with the excitation peak of the C7 aptamer-associated fluorophore methylene blue (MB). We observed a five and seven-times higher average fluorescence intensity over the AuNpA and fringe structure, respectively, in comparison to a plane Au film. Furthermore, the MB fluorophore was simultaneously utilized as a redox probe for electrochemical investigations and is described here as a dual transduction label for the first time. The novel dual transducer system was deployed for the detection of SARS-CoV-2 Spike protein via a C7 aptamer in combination with a strand displacement protocol. The PEF transducer exhibited a detection range from 1 fg/mL to 10 ng/mL with a detection limit of 0.07 fg/mL, while the EC traducer showed an extended dynamic range from 1 fg/mL to 100 ng/mL with a detection limit of 0.15 fg/mL. This work provides insights into an easy-to-perform, large-scale fabrication process for nanostructures enabling plasmon-enhanced fluorescence, and the development of an advanced but universal aptasensor platform.

Examining transfer of TERT to mitochondria under oxidative stress.

The primary role of telomerase is the lengthening of telomeres. Nonetheless, emerging evidence highlights additional functions of telomerase outside of the nucleus. Specifically, its catalytic subunit, TERT (Telomerase Reverse Transcriptase), is detected in the cytosol and mitochondria. Several studies have suggested an elevation in TERT concentration within mitochondria in response to oxidative stress. However, the origin of this mitochondrial TERT, whether transported from the nucleus or synthesized de novo, remains uncertain. In this study, we investigate the redistribution of TERT, labeled with a SNAP-tag, in response to oxidative stress using laser scanning fluorescence microscopy. Our findings reveal that, under our experimental conditions, there is no discernible transport of TERT from the nucleus to the mitochondria due to oxidative stress.

Monitoring GPCR conformation with GFP-inspired dyes.

Solvatochromic compounds have emerged as valuable environment-sensitive probes for biological research. Here we used thiol-reactive solvatochromic analogs of the green fluorescent protein (GFP) chromophore to track conformational changes in two proteins, recoverin and the A2A adenosine receptor (A2AAR). Two dyes showed Ca2+-induced fluorescence changes when attached to recoverin. Our best-performing dye, DyeC, exhibited agonist-induced changes in both intensity and shape of its fluorescence spectrum when attached to A2AAR; none of these effects were observed with other common environment-sensitive dyes. Molecular dynamics simulations showed that activation of the A2AAR led to a more confined and hydrophilic environment for DyeC. Additionally, an allosteric modulator of A2AAR induced distinct fluorescence changes in the DyeC spectrum, indicating a unique receptor conformation. Our study demonstrated that GFP-inspired dyes are effective for detecting structural changes in G protein-coupled receptors (GPCRs), offering advantages such as intensity-based and ratiometric tracking, redshifted fluorescence spectra, and sensitivity to allosteric modulation.

In Vitro Effect of 9,9'-Norharmane Dimer against Herpes Simplex Viruses.

Herpes simplex virus (HSV) infections are highly widespread among humans, producing symptoms ranging from ulcerative lesions to severe diseases such as blindness and life-threatening encephalitis. At present, there are no vaccines available, and some existing antiviral treatments can be ineffective or lead to adverse effects. As a result, there is a need for new anti-HSV drugs. In this report, the in vitro anti-HSV effect of 9,9'-norharmane dimer (nHo-dimer), which belongs to the ß-carboline (ßC) alkaloid family, was evaluated. The dimer exhibited no virucidal properties and did not impede either the attachment or penetration steps of viral particles. The antiviral effect was only exerted under the constant presence of the dimer in the incubation media, and the mechanism of action was found to involve later events of virus infection. Analysis of fluorescence lifetime imaging data showed that the nHo-dimer internalized well into the cells when present in the extracellular incubation medium, with a preferential accumulation into perinuclear organelles including mitochondria. After washing the host cells with fresh medium free of nHo-dimer, the signal decreased, suggesting the partial release of the compound from the cells. This agrees with the observation that the antiviral effect is solely manifested when the alkaloid is consistently present in the incubation media.

Polyelectrolyte-protein synergism: pH-responsive polyelectrolyte/insulin complexes as versatile carriers for targeted protein and drug delivery.

The co-assembly of polyelectrolytes (PE) with proteins offers a promising approach for designing complex structures with customizable morphologies, charge distribution, and stability for targeted cargo delivery. However, the complexity of protein structure limits our ability to predict the properties of the formed nanoparticles, and our goal is to identify the key triggers of the morphological transition in protein/PE complexes and evaluate their ability to encapsulate multivalent ionic drugs. A positively charged PE can assemble with a protein at pH above isoelectric point due to the electrostatic attraction and disassemble at pH below isoelectric point due to the repulsion. The additional hydrophilic block of the polymer should stabilize the particles in solution and enable them to encapsulate a negatively charged drug in the presence of PE excess. We demonstrated that diblock copolymers, poly(ethylene oxide)-block-poly(N,N-dimethylaminoethyl methacrylate) and poly(ethylene oxide)-block-poly(N,N,N-trimethylammonioethyl methacrylate), consisting of a polycation block and a neutral hydrophilic block, reversibly co-assemble with insulin in pH range between 5 and 8. Using small-angle neutron and X-ray scattering (SANS, SAXS), we showed that insulin arrangement within formed particles is controlled by intermolecular electrostatic forces between protein molecules, and can be tuned by varying ionic strength. For the first time, we observed by fluorescence that formed protein/PE complexes with excess of positive charges exhibited potential for encapsulating and controlled release of negatively charged bivalent drugs, protoporphyrin-IX and zinc(II) protoporphyrin-IX, enabling the development of nanocarriers for combination therapies with adjustable charge, stability, internal structure, and size.

Channelrhodopsin-2 Oligomerization in Cell Membrane Revealed by Photo-Activated Localization Microscopy.

Microbial rhodopsins are retinal membrane proteins that found a broad application in optogenetics. The oligomeric state of rhodopsins is important for their functionality and stability. Of particular interest is the oligomeric state in the cellular native membrane environment. Fluorescence microscopy provides powerful tools to determine the oligomeric state of membrane proteins directly in cells. Among these methods is quantitative photoactivated localization microscopy (qPALM) allowing the investigation of molecular organization at the level of single protein clusters. Here, we apply qPALM to investigate the oligomeric state of the first and most used optogenetic tool Channelrhodopsin-2 (ChR2) in the plasma membrane of eukaryotic cells. ChR2 appeared predominantly as a dimer in the cell membrane and did not form higher oligomers. The disulfide bonds between Cys34 and Cys36 of adjacent ChR2 monomers were not required for dimer formation and mutations disrupting these bonds resulted in only partial monomerization of ChR2. The monomeric fraction increased when the total concentration of mutant ChR2 in the membrane was low. The dissociation constant was estimated for this partially monomerized mutant ChR2 as 2.2±0.9 proteins/µm2 . Our findings are important for understanding the mechanistic basis of ChR2 activity as well as for improving existing and developing future optogenetic tools.

Functional GPCR Expression in Eukaryotic LEXSY System.

G protein-coupled receptors (GPCRs) form the largest superfamily of membrane proteins in the human genome, and represent one of the most important classes of drug targets. Their structural studies facilitate rational drug discovery. However, atomic structures of only about 20% of human GPCRs have been solved to date. Recombinant production of GPCRs for structural studies at a large scale is challenging due to their low expression levels and stability. Therefore, in this study, we explored the efficacy of the eukaryotic system LEXSY (Leishmania tarentolae) for GPCR production. We selected the human A2A adenosine receptor (A2AAR), as a model protein, expressed it in LEXSY, purified it, and compared with the same receptor produced in insect cells, which is the most popular expression system for structural studies of GPCRs. The A2AAR purified from both expression systems showed similar purity, stability, ligand-induced conformational changes and structural dynamics, with a remarkably higher protein yield in the case of LEXSY expression. Overall, our results suggest that LEXSY is a promising platform for large-scale production of GPCRs for structural studies.

Membrane manipulation by free fatty acids improves microbial plant polyphenol synthesis.

Microbial synthesis of nutraceutically and pharmaceutically interesting plant polyphenols represents a more environmentally friendly alternative to chemical synthesis or plant extraction. However, most polyphenols are cytotoxic for microorganisms as they are believed to negatively affect cell integrity and transport processes. To increase the production performance of engineered cell factories, strategies have to be developed to mitigate these detrimental effects. Here, we examine the accumulation of the stilbenoid resveratrol in the cell membrane and cell wall during its production using Corynebacterium glutamicum and uncover the membrane rigidifying effect of this stilbenoid experimentally and with molecular dynamics simulations. A screen of free fatty acid supplements identifies palmitelaidic acid and linoleic acid as suitable additives to attenuate resveratrol's cytotoxic effects resulting in a three-fold higher product titer. This cost-effective approach to counteract membrane-damaging effects of product accumulation is transferable to the microbial production of other polyphenols and may represent an engineering target for other membrane-active bioproducts.

Growth-rate dependency of ribosome abundance and translation elongation rate in Corynebacterium glutamicum differs from that in Escherichia coli.

Bacterial growth rate (µ) depends on the protein synthesis capacity of the cell and thus on the number of active ribosomes and their translation elongation rate. The relationship between these fundamental growth parameters have only been described for few bacterial species, in particular Escherichia coli. Here, we analyse the growth-rate dependency of ribosome abundance and translation elongation rate for Corynebacterium glutamicum, a gram-positive model species differing from E. coli by a lower growth temperature optimum and a lower maximal growth rate. We show that, unlike in E. coli, there is little change in ribosome abundance for µ <0.4 h-1 in C. glutamicum and the fraction of active ribosomes is kept above 70% while the translation elongation rate declines 5-fold. Mathematical modelling indicates that the decrease in the translation elongation rate can be explained by a depletion of translation precursors.

KCC2 reverse mode helps to clear postsynaptically released potassium at glutamatergic synapses.

Extracellular potassium [K+]o elevation during synaptic activity retrogradely modifies presynaptic release and astrocytic uptake of glutamate. Hence, local K+ clearance and replenishment mechanisms are crucial regulators of glutamatergic transmission and plasticity. Based on recordings of astrocytic inward rectifier potassium current IKir and K+-sensitive electrodes as sensors of [K+]o as well as on in silico modeling, we demonstrate that the neuronal K+-Cl- co-transporter KCC2 clears local perisynaptic [K+]o during synaptic excitation by operating in an activity-dependent reversed mode. In reverse mode, KCC2 replenishes K+ in dendritic spines and complements clearance of [K+]o, therewith attenuating presynaptic glutamate release and shortening LTP. We thus demonstrate a physiological role of KCC2 in neuron-glial interactions and regulation of synaptic signaling and plasticity through the uptake of postsynaptically released K+.

Sub-millisecond conformational dynamics of the A2A adenosine receptor revealed by single-molecule FRET.

The complex pharmacology of G-protein-coupled receptors (GPCRs) is defined by their multi-state conformational dynamics. Single-molecule Förster Resonance Energy Transfer (smFRET) is well suited to quantify dynamics for individual protein molecules; however, its application to GPCRs is challenging. Therefore, smFRET has been limited to studies of inter-receptor interactions in cellular membranes and receptors in detergent environments. Here, we performed smFRET experiments on functionally active human A2A adenosine receptor (A2AAR) molecules embedded in freely diffusing lipid nanodiscs to study their intramolecular conformational dynamics. We propose a dynamic model of A2AAR activation that involves a slow (>2 ms) exchange between the active-like and inactive-like conformations in both apo and antagonist-bound A2AAR, explaining the receptor's constitutive activity. For the agonist-bound A2AAR, we detected faster (390 ± 80 µs) ligand efficacy-dependent dynamics. Our work establishes a general smFRET platform for GPCR investigations that can potentially be used for drug screening and/or mechanism-of-action studies.

Development and Characterization of Flavin-Binding Fluorescent Proteins, Part II: Advanced Characterization.

Flavin-based fluorescent proteins (FbFPs), a class of small fluorescent proteins derived from light-oxygen-voltage (LOV) domains, bind ubiquitous endogenous flavins as chromophores. Due to their unique properties, they can be used as versatile in vivo reporter proteins under aerobic and anaerobic conditions. This chapter presents methodologies for in-depth characterization of the biochemical, spectroscopic, photophysical, and photochemical properties of FbFPs.

Nitric Oxide Sensing by a Blue Fluorescent Protein.

S-Nitrosylation of cysteine residues is an important molecular mechanism for dynamic, post-translational regulation of several proteins, providing a ubiquitous redox regulation. Cys residues are present in several fluorescent proteins (FP), including members of the family of Aequorea victoria Green Fluorescent Protein (GFP)-derived FPs, where two highly conserved cysteine residues contribute to a favorable environment for the autocatalytic chromophore formation reaction. The effect of nitric oxide on the fluorescence properties of FPs has not been investigated thus far, despite the tremendous role FPs have played for 25 years as tools in cell biology. We have examined the response to nitric oxide of fluorescence emission by the blue-emitting fluorescent protein mTagBFP2. To our surprise, upon exposure to micromolar concentrations of nitric oxide, we observed a roughly 30% reduction in fluorescence quantum yield and lifetime. Recovery of fluorescence emission is observed after treatment with Na-dithionite. Experiments on related fluorescent proteins from different families show similar nitric oxide sensitivity of their fluorescence. We correlate the effect with S-nitrosylation of Cys residues. Mutation of Cys residues in mTagBFP2 removes its nitric oxide sensitivity. Similarly, fluorescent proteins devoid of Cys residues are insensitive to nitric oxide. We finally show that mTagBFP2 can sense exogenously generated nitric oxide when expressed in a living mammalian cell. We propose mTagBFP2 as the starting point for a new class of genetically encoded nitric oxide sensors based on fluorescence lifetime imaging.

The Functional Characterization of GCaMP3.0 Variants Specifically Targeted to Subcellular Domains.

Calcium (Ca2+) ions play a pivotal role in physiology and cellular signaling. The intracellular Ca2+ concentration ([Ca2+]i) is about three orders of magnitude lower than the extracellular concentration, resulting in a steep transmembrane concentration gradient. Thus, the spatial and the temporal dynamics of [Ca2+]i are ideally suited to modulate Ca2+-mediated cellular responses to external signals. A variety of highly sophisticated methods have been developed to gain insight into cellular Ca2+ dynamics. In addition to electrophysiological measurements and the application of synthetic dyes that change their fluorescent properties upon interaction with Ca2+, the introduction and the ongoing development of genetically encoded Ca2+ indicators (GECI) opened a new era to study Ca2+-driven processes in living cells and organisms. Here, we have focused on one well-established GECI, i.e., GCaMP3.0. We have systematically modified the protein with sequence motifs, allowing localization of the sensor in the nucleus, in the mitochondrial matrix, at the mitochondrial outer membrane, and at the plasma membrane. The individual variants and a cytosolic version of GCaMP3.0 were overexpressed and purified from E. coli cells to study their biophysical properties in solution. All versions were examined to monitor Ca2+ signaling in stably transfected cell lines and in primary cortical neurons transduced with recombinant Adeno-associated viruses (rAAV). In this comparative study, we provide evidence for a robust approach to reliably trace Ca2+ signals at the (sub)-cellular level with pronounced temporal resolution.

Role of Mitochondrial Protein Import in Age-Related Neurodegenerative and Cardiovascular Diseases.

Mitochondria play a critical role in providing energy, maintaining cellular metabolism, and regulating cell survival and death. To carry out these crucial functions, mitochondria employ more than 1500 proteins, distributed between two membranes and two aqueous compartments. An extensive network of dedicated proteins is engaged in importing and sorting these nuclear-encoded proteins into their designated mitochondrial compartments. Defects in this fundamental system are related to a variety of pathologies, particularly engaging the most energy-demanding tissues. In this review, we summarize the state-of-the-art knowledge about the mitochondrial protein import machinery and describe the known interrelation of its failure with age-related neurodegenerative and cardiovascular diseases.

Glial Chloride Homeostasis Under Transient Ischemic Stress.

High water permeabilities permit rapid adjustments of glial volume upon changes in external and internal osmolarity, and pathologically altered intracellular chloride concentrations ([Cl-]int) and glial cell swelling are often assumed to represent early events in ischemia, infections, or traumatic brain injury. Experimental data for glial [Cl-]int are lacking for most brain regions, under normal as well as under pathological conditions. We measured [Cl-]int in hippocampal and neocortical astrocytes and in hippocampal radial glia-like (RGL) cells in acute murine brain slices using fluorescence lifetime imaging microscopy with the chloride-sensitive dye MQAE at room temperature. We observed substantial heterogeneity in baseline [Cl-]int, ranging from 14.0 ± 2.0 mM in neocortical astrocytes to 28.4 ± 3.0 mM in dentate gyrus astrocytes. Chloride accumulation by the Na+-K+-2Cl- cotransporter (NKCC1) and chloride outward transport (efflux) through K+-Cl- cotransporters (KCC1 and KCC3) or excitatory amino acid transporter (EAAT) anion channels control [Cl-]int to variable extent in distinct brain regions. In hippocampal astrocytes, blocking NKCC1 decreased [Cl-]int, whereas KCC or EAAT anion channel inhibition had little effect. In contrast, neocortical astrocytic or RGL [Cl-]int was very sensitive to block of chloride outward transport, but not to NKCC1 inhibition. Mathematical modeling demonstrated that higher numbers of NKCC1 and KCC transporters can account for lower [Cl-]int in neocortical than in hippocampal astrocytes. Energy depletion mimicking ischemia for up to 10 min did not result in pronounced changes in [Cl-]int in any of the tested glial cell types. However, [Cl-]int changes occurred under ischemic conditions after blocking selected anion transporters. We conclude that stimulated chloride accumulation and chloride efflux compensate for each other and prevent glial swelling under transient energy deprivation.

Accessing Mitochondrial Protein Import in Living Cells by Protein Microinjection.

Mitochondrial protein biogenesis relies almost exclusively on the expression of nuclear-encoded polypeptides. The current model postulates that most of these proteins have to be delivered to their final mitochondrial destination after their synthesis in the cytoplasm. However, the knowledge of this process remains limited due to the absence of proper experimental real-time approaches to study mitochondria in their native cellular environment. We developed a gentle microinjection procedure for fluorescent reporter proteins allowing a direct non-invasive study of protein transport in living cells. As a proof of principle, we visualized potential-dependent protein import into mitochondria inside intact cells in real-time. We validated that our approach does not distort mitochondrial morphology and preserves the endogenous expression system as well as mitochondrial protein translocation machinery. We observed that a release of nascent polypeptides chains from actively translating cellular ribosomes by puromycin strongly increased the import rate of the microinjected pre-protein. This suggests that a substantial amount of mitochondrial translocase complexes was involved in co-translational protein import of endogenously expressed pre-proteins. Our protein microinjection method opens new possibilities to study the role of mitochondrial protein import in cell models of various pathological conditions as well as aging processes.

The molecular basis of spectral tuning in blue- and red-shifted flavin-binding fluorescent proteins.

Photoactive biological systems modify the optical properties of their chromophores, known as spectral tuning. Determining the molecular origin of spectral tuning is instrumental for understanding the function and developing applications of these biomolecules. Spectral tuning in flavin-binding fluorescent proteins (FbFPs), an emerging class of fluorescent reporters, is limited by their dependency on protein-bound flavins, whose structure and hence electronic properties cannot be altered by mutation. A blue-shifted variant of the plant-derived improved light, oxygen, voltage FbFP has been created by introducing a lysine within the flavin-binding pocket, but the molecular basis of this shift remains unconfirmed. We here structurally characterize the blue-shifted improved light, oxygen, voltage variant and construct a new blue-shifted CagFbFP protein by introducing an analogous mutation. X-ray structures of both proteins reveal displacement of the lysine away from the chromophore and opening up of the structure as instrumental for the blue shift. Site saturation mutagenesis and high-throughput screening yielded a red-shifted variant, and structural analysis revealed that the lysine side chain of the blue-shifted variant is stabilized close to the flavin by a secondary mutation, accounting for the red shift. Thus, a single additional mutation in a blue-shifted variant is sufficient to generate a red-shifted FbFP. Using spectroscopy, X-ray crystallography, and quantum mechanics molecular mechanics calculations, we provide a firm structural and functional understanding of spectral tuning in FbFPs. We also show that the identified blue- and red-shifted variants allow for two-color microscopy based on spectral separation. In summary, the generated blue- and red-shifted variants represent promising new tools for application in life sciences.

Photothermal therapy with silver nanoplates in HeLa cells studied by in situ fluorescence microscopy.

Photothermal therapy (PTT) is a noninvasive treatment for cancer relying on the incorporation of NIR-light absorbing nanomaterials into cells, which upon illumination release heat causing thermally induced cell death. We prove that irradiation of aqueous suspensions of poly(vinylpyrrolidone)-coated silver nanoplates (PVPAgNP) or PVPAgNP in HeLa cells with red or NIR lasers causes a sizeable photothermal effect, which in cells can be visualized with the temperature sensing fluorophore Rhodamine B (RhB) using spinning disk confocal fluorescence microscopy or fluorescence lifetime imaging. Upon red-light irradiation of cells that were incubated with both, RhB and PVPAgNP at concentrations with no adverse effects on cell viability, a substantial heat release is detected. Initiation of cell death by photothermal effect is observed by positive signals of fluorescent markers for early and late apoptosis. Surprisingly, a new nanomaterial-assisted cell killing mode is operating when PVPAgNP-loaded HeLa cells are excited with moderate powers of fs-pulsed NIR light. Small roundish areas are generated with bright and fast (<1 ns) decaying emission, which expand fast and destroy the whole cell in seconds. This characteristic emission is assigned to efficient optical breakdown initiation around the strongly absorbing PVPAgNP leading to plasma formation that spreads fast through the cell.

Three dimensional drift control at nano-scale in single molecule localization microscopy.

Super-resolution imaging based on single molecule localization of cellular structures on nanometer scale requires to record a series of wide-field or TIRF images resulting in a considerable recording time (typically of minutes). Therefore, sample drift becomes a critical problem and will lower the imaging precision. Herein we utilized morphological features of the specimen (mammalian cells) itself as reference markers replacing the traditionally used markers (e.g., artificial fiduciary markers, fluorescent beads, or metal nanoparticles) for sample drift compensation. We achieved sub-nanometer localization precision <1.0 nm in lateral direction and <6.0 nm in axial direction, which is well comparable with the precision achieved with the established methods using artificial position markers added to the specimen. Our method does not require complex hardware setup, extra labelling or markers, and has the additional advantage of the absence of photobleaching, which caused precision decrease during the course of super-resolution measurement. The achieved improvement of quality and resolution in reconstructed super-resolution images by application of our drift-correction method is demonstrated by single molecule localization-based super-resolution imaging of F-actin in fixed A549 cells.