A novel dual-release scaffold for fluorescent labels improves cyclic immunofluorescence.

Cyclic immunofluorescence is a powerful method to generate high-content imaging datasets for investigating cell biology and developing therapies. This method relies on fluorescent labels that determine the quality of immunofluorescence and the maximum number of staining cycles that can be performed. Here we present a novel fluorescent labelling strategy, based on antibodies conjugated to a scaffold containing two distinct sites for enzymatic cleavage of fluorophores. The scaffold is composed of a dextran decorated with short ssDNA that upon hybridization with complementary dye-modified oligos result in fluorescent molecules. The developed fluorescent labels exhibit specific staining and remarkable brightness in flow cytometry and fluorescence microscopy. We showed that the combination of DNase-mediated degradation of DNA and dextranse-mediated degradation of the dextran as two complementary enzymatic release mechanisms in one molecule, improves signal erasure from labelled epitopes. We envision that such dual-release labels with high brightness and efficient and specific erasure will advance multiplexed cyclic immunofluorescence approaches and thereby will contribute to gaining new insights in cell biology.

Fluorophore multimerization on a PEG backbone as a concept for signal amplification and lifetime modulation.

Fluorescent labels have strongly contributed to many advancements in bioanalysis, molecular biology, molecular imaging, and medical diagnostics. Despite a large toolbox of molecular and nanoscale fluorophores to choose from, there is still a need for brighter labels, e.g., for flow cytometry and fluorescence microscopy, that are preferably of molecular nature. This requires versatile concepts for fluorophore multimerization, which involves the shielding of dyes from other chromophores and possible quenchers in their neighborhood. In addition, to increase the number of readout parameters for fluorescence microscopy and eventually also flow cytometry, control and tuning of the labels' fluorescence lifetimes is desired. Searching for bright multi-chromophoric or multimeric labels, we developed PEGylated dyes bearing functional groups for their bioconjugation and explored their spectroscopic properties and photostability in comparison to those of the respective monomeric dyes for two exemplarily chosen fluorophores excitable at 488 nm. Subsequently, these dyes were conjugated with anti-CD4 and anti-CD8 immunoglobulins to obtain fluorescent conjugates suitable for the labeling of cells and beads. Finally, the suitability of these novel labels for fluorescence lifetime imaging and target discrimination based upon lifetime measurements was assessed. Based upon the results of our spectroscopic studies including measurements of fluorescence quantum yields (QY) and fluorescence decay kinetics we could demonstrate the absence of significant dye-dye interactions and self-quenching in these multimeric labels. Moreover, in a first fluorescence lifetime imaging (FLIM) study, we could show the future potential of this multimerization concept for lifetime discrimination and multiplexing.

Organosulfur/Selenium-Based Highly Fluorogenic Molecular Probes for Live-Cell Nucleolus Imaging.

We present fluorogenic cationic organo chalcogens that are highly selective to RNA. We have demonstrated that the conformational dynamics and subsequently the optical properties of these dyes can be controlled to facilitate efficient bioimaging. We report the application of organoselenium and organosulfur-based cell-permeable red-emissive probes bearing a favorable cyclic sidearm for selective and high contrast imaging of cell nucleoli. The probes exhibit high quantum yield upon interacting with RNA in an aqueous solution. An in-depth multiscale simulation study reveals that the prominent rotational freezing of the electron-donating sidearm of the probes in the microenvironment of RNA helps in attaining more planar conformation when compared to DNA. It exerts a greater extent of intramolecular charge transfer and hence leads to enhanced fluorescence emission. A systematic structure-interaction relationship study highlighted the impact of heavy-chalcogens toward the improved emissive properties of the probes.

MACSima imaging cyclic staining (MICS) technology reveals combinatorial target pairs for CAR T cell treatment of solid tumors.

Many critical advances in research utilize techniques that combine high-resolution with high-content characterization at the single cell level. We introduce the MICS (MACSima Imaging Cyclic Staining) technology, which enables the immunofluorescent imaging of hundreds of protein targets across a single specimen at subcellular resolution. MICS is based on cycles of staining, imaging, and erasure, using photobleaching of fluorescent labels of recombinant antibodies (REAfinity Antibodies), or release of antibodies (REAlease Antibodies) or their labels (REAdye_lease Antibodies). Multimarker analysis can identify potential targets for immune therapy against solid tumors. With MICS we analysed human glioblastoma, ovarian and pancreatic carcinoma, and 16 healthy tissues, identifying the pair EPCAM/THY1 as a potential target for chimeric antigen receptor (CAR) T cell therapy for ovarian carcinoma. Using an Adapter CAR T cell approach, we show selective killing of cells only if both markers are expressed. MICS represents a new high-content microscopy methodology widely applicable for personalized medicine.

FRET-based assay for intracellular evaluation of α-synuclein aggregation inhibitors.

Aggregation of small neuronal protein α-synuclein (αSyn) in amyloid fibrils is considered to be one of the main causes of Parkinson's disease. Inhibition of this aggregation is a promising approach for disease treatment. Dozens of compounds able to inhibit αSyn fibrillization in solution were developed during the last decade. However, the applicability of most of them in the cellular environment was not established because of the absence of a suitable cell-based assay. In this work, we developed an assay for testing αSyn aggregation inhibitors in cells that is based on fluorescence resonance energy transfer (FRET) between labeled αSyn molecules in fibrils. The assay directly reports the amount of fibrillized αSyn and is more reliable than the assays based on cell viability. Moreover, we showed that cell viability decline does not always correlate with the amount of misfolded αSyn. The developed FRET-based assay does not interfere with the aggregation process and is suitable for high-throughput testing of αSyn aggregation inhibitors. Its application can sort out non-specific inhibitors and thus significantly facilitate the development of drugs for Parkinson`s disease.

Rationally Designed Protein-Based Inhibitor of α-Synuclein Fibrillization in Cells.

Misfolding of the neuronal protein α-synuclein (αSyn) into amyloid fibrils is involved in the development of Parkinson's disease (PD), and inhibition of this process is considered to be a promising therapeutic approach. In this work, we engineered protein inhibitors that bind to fibrils with higher affinity than the monomeric αSyn. They were developed based on the recent structural data of the αSyn fibrils and were shown to prevent fibril elongation upon binding to fibril ends. These inhibitors are highly selective to the misfolded αSyn, nontoxic, and active in cytosol in small concentrations. The best-performing inhibitor shows IC50 ∼10 nM in a cell-based assay, which corresponds to the ∼1:60 molar ratio to αSyn. It can suppress the formation of αSyn aggregates in cells that can be potentially used to slow down the spreading of the pathological aggregates from cell to cell during the course of the PD.

Fluorescent Probe for Selective Imaging of α-Synuclein Fibrils in Living Cells.

Plaques of amyloid fibrils composed of neuronal protein α-synuclein are one of the hallmarks of Parkinson's disease, and their selective imaging is crucial to study the mechanism of its pathogenesis. However, the existing fluorescent probes for amyloids are efficient only in solution and tissue systems, and they are not selective enough for the visualization of amyloid fibrils in living cells. In this study, we present two molecular rotor-based probes RB1 and RB2. These thiazolium probes show affinity to α-synuclein fibrils and turn-on fluorescence response upon interactions. Because of its extended π-conjugation and high rotational degree of freedom, RB1 exhibits a 76 nm red-shift of absorption maxima and 112-fold fluorescence enhancement upon binding to amyloid fibrils. Owing to its strong binding affinity to α-synuclein fibrils, RB1 can selectively stain them in the cytoplasm of living HeLa and SH-SY5Y cells with high optical contrast. RB1 is a cell-permeable and noncytotoxic probe. Taken together, we have demonstrated that RB1 is an amyloid probe with an outstanding absorption red-shift that can be used for intracellular imaging of α-synuclein fibrils.

Influence of Lipid Membranes on α-Synuclein Aggregation.

α-Synuclein is a neuronal protein involved in synaptic vesicle trafficking. During the course of Parkinson's disease, it aggregates, forming amyloid fibrils that accumulate in the midbrain. This pathological fibrillization process is strongly modulated by physiological interactions of α-synuclein with lipid membranes. However, the detailed mechanism of this effect remains unclear. In this work, we used environment-sensitive fluorescent dyes to study the influence of model lipid membranes on the kinetics of α-synuclein fibrillization. We observed that formation of the fibrils from α-synuclein monomers is strongly delayed even by small amounts of lipids. Furthermore, we found that membrane-bound α-synuclein monomers are not involved in fibril elongation. Hence, presence of lipids slows down fibril growth proportionally to the fraction of membrane-bound protein.

Di(benzothienyl)cyclobutenes: Toward Strained Photoswitchable Fluorophores.

Bis(benzothienyl)ethene sulfones are very interesting molecules for super-resolution microscopy due to their photoswitching properties. However, functionalization of the 'classical' bis(benzothienyl)ethene sulfones with a five-membered central ring leads to significant decrease of quantum yields of photoconversion of the fluorescent closed form of the dye to the non-fluorescent open form that limits their application in microscopy. Here, we designed and synthesized diarylethenes with a fluorinated four-membered central ring that adds extra strain to the closed form of the dye. The reaction mechanism of their formation was studied, and byproducts formed upon structural rearrangement of the benzothiophene fragment were characterized. The photochromic properties of the new molecules were investigated by NMR and absorption spectroscopy. Some of these compounds show enhanced tendency to ring opening and have quantum yields of the ring-opening reaction in the range of 0.2-0.5.

Reversible spatial and temporal control of lipid signaling.

Herein, we introduce versatile molecular tools that enable specific delivery and visualization of photoswitchable lipids at cellular membranes, namely at the plasma membrane and internal membranes. These molecules were prepared by tethering ortho-nitrobenzyl-based fluorescent cages with a signaling lipid bearing an azobenzene photoswitch. They permit two sequential photocontrolled reactions, which are uncaging of a lipid analogue and then its repeated activation and deactivation. We used these molecules to activate GPR40 receptor transiently expressed in HeLa cells and demonstrated downstream modulation of intracellular Ca2+ levels.

Structural Optimization of Inhibitors of α-Synuclein Fibril Growth: Affinity to the Fibril End as a Crucial Factor.

BACKGROUND: Misfolding of the neuronal protein α-synuclein into amyloid fibrils is a pathological hallmark of Parkinson's disease, a neurodegenerative disorder that has no cure. Inhibition of the fibril growth is considered a promising therapeutic approach. However, the majority of the existing inhibitors are either unspecific or work at high micromolar concentrations. Earlier, we created a protein-based inhibitor of α-synuclein fibril growth that consists of an α-synuclein moiety and a bulky group. It specifically binds to α-synuclein fibril ends and blocks them by creating steric hindrance to subsequent monomer binding. RESULTS: In this work, we prepared a series of inhibitors with modified α-synuclein moieties and bulky groups of different structure, size, and position. We studied the structure-activity relationship of these inhibitors and optimized them by improving affinity to the fibril end and blocking efficiency. The inhibitors were tested in a Thioflavin T-based kinetic assay, and their affinity to the fibril ends was measured by fluorescence anisotropy. We showed that decrease in electrostatic repulsion between inhibitor and fibril end improved the inhibitor efficiency. Inhibitors with rigid ß-sheet-rich bulky groups bind to fibril ends stronger than monomeric α-synuclein and therefore have a high inhibition efficiency, showing a linear correlation between Kd and IC50. SIGNIFICANCE: We determined which properties of inhibitor molecules are the most important for good performance and found that the inhibitor affinity to the fibril end is a key feature that determines its inhibition efficiency. Applying this knowledge, we improved existing inhibitors and reached IC50 value of 300 nM.

α-Synuclein Dimers as Potent Inhibitors of Fibrillization.

Aggregation of the neuronal protein α-synuclein into amyloid fibrils plays a central role in the development of Parkinson's disease. Growth of fibrils can be suppressed by blocking fibril ends from their interaction with monomeric proteins. In this work, we constructed inhibitors that bind to the ends of α-synuclein amyloid fibrils with very high affinity. They are based on synthetic α-synuclein dimers and interact with fibrils via two monomeric subunits adopting conformation that efficiently blocks fibril elongation. By tuning the charge of dimers, we further enhanced the binding affinity and prepared a construct that inhibits fibril elongation at nanomolar concentration (IC50 ≈ 20 nM). To the best of our knowledge, it is the most efficient inhibitor of α-synuclein fibrillization.

Nitrobenzyl-based fluorescent photocages for spatial and temporal control of signalling lipids in cells.

Here we present a set of fluorescent cages prepared by tethering fluorescent dyes to a photolabile group. The developed molecules enable caging of signalling lipids, their delivery to specific cellular membranes, with further imaging, quantification, and controlled photorelease of active lipids in living cells.

α-Synuclein aggregation at low concentrations.

BACKGROUND: Aggregation of the neuronal protein α-synuclein into amyloid fibrils is a hallmark of Parkinson's disease. The propensity of α-synuclein to aggregate increases with the protein concentration. For the development of efficient inhibitors of α-synuclein aggregation, it is important to know the critical concentration of aggregation (the concentration of monomeric protein, below which the protein does not aggregate). METHODS: We performed in vitro aggregation studies of α-synuclein at low concentrations (0.11-20 µM). Aggregation kinetics was measured by ThT fluorescence. Obtained aggregates were characterized using CD-spectroscopy, fluorescent spectroscopy, dynamic light scattering and AFM imaging. RESULTS: Monomeric α-synuclein at concentrations 0.45 µM and above was able to bind to fibril ends resulting in fibril growth. At the protein concentrations below 0.4 µM, monomers did not fibrillize, and fibrils disaggregated. In the absence of seeds, fibrils were formed only at monomer concentrations higher than 10 µM. At low micromolar concentrations, we observed formation of prefibrillar amyloid aggregates, which are able to induce fibril formation in α-synuclein solutions of high concentrations. CONCLUSIONS: The critical concentration of α-synuclein fibril growth is ~0.4 µM. Prefibrillar amyloid aggregates appear at concentrations between 0.45 and 3 µM and are an intermediate state between monomers and fibrils. Although morphologically different from fibrils, prefibrillar aggregates have similar properties to those of fibrils. GENERAL SIGNIFICANCE: We determined the critical concentration of α-synuclein fibril growth. We showed that fibrils can grow at much lower monomer concentrations than that required for de novo fibril formation. We characterized a prefibrillar intermediate species formed upon aggregation of α-synuclein at low micromolar concentration.

Optical tools for understanding the complexity of ß-cell signalling and insulin release.

Following stimulation, pancreatic ß-cells must orchestrate a plethora of signalling events to ensure the appropriate release of insulin and maintenance of normal glucose homeostasis. Failure at any point in this cascade leads to impaired insulin secretion, elevated blood levels of glucose and eventually type 2 diabetes mellitus. Likewise, ß-cell replacement or regeneration strategies for the treatment of both type 1 and type 2 diabetes mellitus might fail if the correct cell signalling phenotype cannot be faithfully recreated. However, current understanding of ß-cell function is complicated because of the highly dynamic nature of their intracellular and intercellular signalling as well as insulin release itself. ß-Cells must precisely integrate multiple signals stemming from multiple cues, often with differing intensities, frequencies and cellular and subcellular localizations, before converging these signals onto insulin exocytosis. In this respect, optical approaches with high resolution in space and time are extremely useful for properly deciphering the complexity of ß-cell signalling. An increased understanding of ß-cell signalling might identify new mechanisms underlying insulin release, with relevance for future drug therapy and de novo stem cell engineering of functional islets.

Endogenous Fatty Acids Are Essential Signaling Factors of Pancreatic ß-Cells and Insulin Secretion.

The secretion of insulin from ß-cells depends on extracellular factors, in particular glucose and other small molecules, some of which act on G-protein-coupled receptors. Fatty acids (FAs) have been discussed as exogenous secretagogues of insulin for decades, especially after the FA receptor GPR40 (G-protein-coupled receptor 40) was discovered. However, the role of FAs as endogenous signaling factors has not been investigated until now. In the present work, we demonstrate that lowering endogenous FA levels in ß-cell medium by stringent washing or by the application of FA-free (FAF) BSA immediately reduced glucose-induced oscillations of cytosolic Ca2+ ([Ca2+]i oscillations) in MIN6 cells and mouse primary ß-cells, as well as insulin secretion. Mass spectrometry confirmed BSA-mediated removal of FAs, with palmitic, stearic, oleic, and elaidic acid being the most abundant species. [Ca2+]i oscillations in MIN6 cells recovered when BSA was replaced by buffer or as FA levels in the supernatant were restored. This was achieved by recombinant lipase-mediated FA liberation from membrane lipids, by the addition of FA-preloaded FAF-BSA, or by the photolysis of cell-impermeant caged FAs. Our combined data support the hypothesis of FAs as essential endogenous signaling factors for ß-cell activity and insulin secretion.

Inhibition of α-Synuclein Amyloid Fibril Elongation by Blocking Fibril Ends.

Misfolding of the protein α-synuclein (αSyn) into amyloid fibrils plays a central role in the development of Parkinson's disease. Most approaches for the inhibition of αSyn fibril formation are based on stabilizing the native monomeric form of the protein or destabilizing the fibrillized misfolded form. They require high concentrations of inhibitor and therefore cannot be easily used for therapies. In this work, we designed an inhibitor (Inh-ß) that selectively binds the growing ends of αSyn fibrils and creates steric hindrance for the binding of monomeric αSyn. This approach permits the inhibition of fibril formation at Inh-ß concentrations (IC50 =850 nm) much lower than the concentration of monomeric αSyn. We studied its kinetic mechanism in vitro and identified the reactions that limit inhibition efficiency. It is shown that blocking of αSyn fibril ends is an effective approach to inhibiting fibril growth and provides insights for the development of effective inhibitors of αSyn aggregation.

Modification of C Terminus Provides New Insights into the Mechanism of α-Synuclein Aggregation.

Aggregation of neuronal protein α-synuclein leads to the formation of amyloid fibrils, which are associated with the development of Parkinson's disease. The mechanism of α-synuclein pathology is not fully understood and is a subject of active research in the field. To tackle this problem, the fusions of fluorescent proteins to α-synuclein C-terminus are often used in cellular and animal studies. The effects induced by such α-synuclein sequence extension on α-synuclein aggregation propensity are, however, not systematically examined despite the evidence that the negatively charged C-terminus plays a critical role in the regulation of α-synuclein aggregation. In this work, we investigated how the charge and length variations of the C-terminus affect the aggregation propensity of α-synuclein. To address these questions, we prepared mutants of α-synuclein carrying additional moieties of different charge and length at the protein C-terminus. We determined the rates of two different aggregation stages (primary nucleation and elongation) based on a thioflavin T kinetic assay. We observed that all mutants bearing neutrally charged moieties of different length fibrilized slower than wild-type α-synuclein. The primary nucleation and elongation rates strongly decreased with increase of the C-terminal extension length. Meanwhile, charge variation of the C-terminus significantly changed the rate of α-synuclein nucleation, but did not markedly affect the rate of fibril elongation. Our data demonstrate that both the charge and length of the C-terminus play an important role at the stage of initial fibril formation, but the stage of fibril elongation is affected mainly by the length of C-terminal extension. In addition, our results suggest that there are at least two steps of incorporation of α-synuclein monomers into the amyloid fibril: namely, the initial monomer binding to the fibril end (charge-dependent, relatively fast), and the subsequent conformational change of the protein (charge-independent, relatively slow, and thus the rate-limiting step).

A Ratiometric Sensor for Imaging Insulin Secretion in Single ß Cells.

Despite the urgent need for assays to visualize insulin secretion there is to date no reliable method available for measuring insulin release from single cells. To address this need, we developed a genetically encoded reporter termed RINS1 based on proinsulin superfolder GFP (sfGFP) and mCherry fusions for monitoring insulin secretion. RINS1 expression in MIN6 ß cells resulted in proper processing yielding single-labeled insulin species. Unexpectedly, glucose or drug stimulation of insulin secretion in ß cells led to the preferential release of the insulin-sfGFP construct, while the mCherry-fused C-peptide remained trapped in exocytic granules. This physical separation was used to monitor glucose-stimulated insulin secretion ratiometrically by total internal reflection fluorescence microscopy in single MIN6 and primary mouse ß cells. Further, RINS1 enabled parallel monitoring of pulsatile insulin release in tolbutamide-treated ß cells, demonstrating the potential of RINS1 for investigations of antidiabetic drug candidates at the single-cell level.

Environmentally sensitive probes for monitoring protein-membrane interactions at nanomolar concentrations.

Solvatochromic probes are suitable tools for quantitative characterization of protein-membrane interactions. Based on diverse fluorophores these probes have different fluorescent properties and therefore demonstrate different responses when applied for sensing the interactions of biomolecules. Surprisingly, to the best of our knowledge, no systematic comparison of the sensitivities of solvatochromic dyes for monitoring protein-membrane interactions was described. Hence, a rational choice of an optimal environmentally sensitive probe for such experiments is usually not a straightforward task. In this work we developed a series of thiol-reactive fluorescent probes based on the fluorophores with high sensitivity to their environment and compared them with two widely used DNS and DMN probes. We investigated the responses of these probes to the interaction of probe-labeled presynaptic protein α-synuclein with lipid membranes. We observed that newly synthesized probes based on fluorene and chromone dyes, which combine the strongest brightness and significant changes of fluorescence intensity, demonstrated the highest sensitivity to interaction of α-synuclein with lipid membranes. They are especially beneficial for sensing in scattering media such as solutions of lipid vesicles. We show that the described probes permit quantitative measurements of α-synuclein binding to lipid membranes at low nanomolar concentrations. We developed a detailed protocol for measuring Kd and binding stoichiometry for interaction of soluble peripheral proteins with membranes based on the response of the environmentally sensitive fluorescent probes. We applied this protocol for quantification of the affinity of α-synuclein to anionic membranes and found that it is substantially higher than it was earlier reported.