Molecular Rotors: Fluorescent Sensors for Microviscosity and Conformation of Biomolecules.

The viscosity and crowding of biological environment are considered vital for the correct cellular function, and alterations in these parameters are known to underly a number of pathologies including diabetes, malaria, cancer and neurodegenerative diseases, to name a few. Over the last decades, fluorescent molecular probes termed molecular rotors proved extremely useful for exploring viscosity, crowding, and underlying molecular interactions in biologically relevant settings. In this review, we will discuss the basic principles underpinning the functionality of these probes and will review advances in their use as sensors for lipid order, protein crowding and conformation, temperature and non-canonical nucleic acid structures in live cells and other relevant biological settings.

Cellular Visualization of G-Quadruplex RNA via Fluorescence- Lifetime Imaging Microscopy.

Over the past decade, appreciation of the roles of G-quadruplex (G4) structures in cellular regulation and maintenance has rapidly grown, making the establishment of robust methods to visualize G4s increasingly important. Fluorescent probes are commonly used for G4 detection in vitro; however, achieving sufficient selectivity to detect G4s in a dense and structurally diverse cellular environment is challenging. The use of fluorescent probes for G4 detection is further complicated by variations of probe uptake into cells, which may affect fluorescence intensity independently of G4 abundance. In this work, we report an alternative small-molecule approach to visualize G4s that does not rely on fluorescence intensity switch-on and, thus, does not require the use of molecules with exclusive G4 binding selectivity. Specifically, we have developed a novel thiazole orange derivative, TOR-G4, that exhibits a unique fluorescence lifetime when bound to G4s compared to other structures, allowing G4 binding to be sensitively distinguished from non-G4 binding, independent of the local probe concentration. Furthermore, TOR-G4 primarily colocalizes with RNA in the cytoplasm and nucleoli of cells, making it the first lifetime-based probe validated for exploring the emerging roles of RNA G4s in cellulo.

Correlation of Plasma Membrane Microviscosity and Cell Stiffness Revealed via Fluorescence-Lifetime Imaging and Atomic Force Microscopy.

The biophysical properties of cells described at the level of whole cells or their membranes have many consequences for their biological behavior. However, our understanding of the relationships between mechanical parameters at the level of cell (stiffness, viscoelasticity) and at the level of the plasma membrane (fluidity) remains quite limited, especially in the context of pathologies, such as cancer. Here, we investigated the correlations between cells' stiffness and viscoelastic parameters, mainly determined via the actin cortex, and plasma membrane microviscosity, mainly determined via its lipid profile, in cancer cells, as these are the keys to their migratory capacity. The mechanical properties of cells were assessed using atomic force microscopy (AFM). The microviscosity of membranes was visualized using fluorescence-lifetime imaging microscopy (FLIM) with the viscosity-sensitive probe BODIPY 2. Measurements were performed for five human colorectal cancer cell lines that have different migratory activity (HT29, Caco-2, HCT116, SW 837, and SW 480) and their chemoresistant counterparts. The actin cytoskeleton and the membrane lipid composition were also analyzed to verify the results. The cell stiffness (Young's modulus), measured via AFM, correlated well (Pearson r = 0.93) with membrane microviscosity, measured via FLIM, and both metrics were elevated in more motile cells. The associations between stiffness and microviscosity were preserved upon acquisition of chemoresistance to one of two chemotherapeutic drugs. These data clearly indicate that mechanical parameters, determined by two different cellular structures, are interconnected in cells and play a role in their intrinsic migratory potential.

Platinum(II)-Based Optical Probes for Imaging Quadruplex DNA Structures via Phosphorescence Lifetime Imaging Microscopy.

G-quadruplex DNA is a non-canonical structure that forms in guanine-rich regions of the genome. There is increasing evidence showing that G-quadruplexes have important biological functions, and therefore molecular tools to visualise these structures are important. Herein we report on a series of new cyclometallated platinum(II) complexes which, upon binding to G-quadruplex DNA, display an increase in their phosphorescence, acting as switch-on probes. More importantly, upon binding to G-quadruplexes they display a selective and distinct lengthening of their emission lifetime. We show that this effect can be used to selectively visualise these structures in cells using Phosphorescence Lifetime Imaging Microscopy (PLIM).

Viscosity-Sensitive Membrane Dyes as Tools To Estimate the Crystalline Structure of Lipid Bilayers.

Lipid membranes are crucial for cellular integrity and regulation, and tight control of their structural and mechanical properties is vital to ensure that they function properly. Fluorescent probes sensitive to the membrane's microenvironment are useful for investigating lipid membrane properties; however, there is currently a lack of quantitative correlation between the exact parameters of lipid organization and a readout from these dyes. Here, we investigate this relationship for "molecular rotors", or microviscosity sensors, by simultaneously measuring their fluorescence lifetime to determine the membrane viscosity, while using X-ray diffraction to determine the membrane's structural properties. Our results reveal a phase-dependent correlation between the membrane's structural parameters and mechanical properties measured by a BODIPY-based molecular rotor, giving excellent predictive power for the structural descriptors of the lipid bilayer. We also demonstrate that differences in membrane thickness between different lipid phases are not a prerequisite for the formation of lipid microdomains and that this requirement can be disrupted by the presence of line-active molecules. Our results underpin the use of membrane-sensitive dyes as reporters of the structure of lipid membranes.

Effects of Paclitaxel on Plasma Membrane Microviscosity and Lipid Composition in Cancer Cells.

The cell membrane is an important regulator for the cytotoxicity of chemotherapeutic agents. However, the biochemical and biophysical effects that occur in the membrane under the action of chemotherapy drugs are not fully described. In the present study, changes in the microviscosity of membranes of living HeLa-Kyoto tumor cells were studied during chemotherapy with paclitaxel, a widely used antimicrotubule agent. To visualize the microviscosity of the membranes, fluorescence lifetime imaging microscopy (FLIM) with a BODIPY 2 fluorescent molecular rotor was used. The lipid profile of the membranes was assessed using time-of-flight secondary ion mass spectrometry ToF-SIMS. A significant, steady-state decrease in the microviscosity of membranes, both in cell monolayers and in tumor spheroids, was revealed after the treatment. Mass spectrometry showed an increase in the unsaturated fatty acid content in treated cell membranes, which may explain, at least partially, their low microviscosity. These results indicate the involvement of membrane microviscosity in the response of tumor cells to paclitaxel treatment.

Dimeric Metal-Salphen Complexes Which Target Multimeric G-Quadruplex DNA.

G-Quadruplex DNA structures have attracted increasing attention due to their biological roles and potential as targets for the development of new drugs. While most guanine-rich sequences in the genome have the potential to form monomeric G-quadruplexes, certain sequences have enough guanine-tracks to give rise to multimeric quadruplexes. One of these sequences is the human telomere where tandem repeats of TTAGGG can lead to the formation of two or more adjacent G-quadruplexes. Herein we report on the modular synthesis via click chemistry of dimeric metal-salphen complexes (with NiII and PtII) bridged by either polyether or peptide linkers. We show by circular dichroism (CD) spectroscopy that they generally have higher selectivity for dimeric vs monomeric G-quadruplexes. The emissive properties of the PtII-salphen dimeric complexes have been used to study their interactions with monomeric and dimeric G-quadruplexes in vitro as well as to study their cellular uptake and localization.

Directly imaging emergence of phase separation in peroxidized lipid membranes.

Lipid peroxidation is a process which is key in cell signaling and disease, it is exploited in cancer therapy in the form of photodynamic therapy. The appearance of hydrophilic moieties within the bilayer's hydrocarbon core will dramatically alter the structure and mechanical behavior of membranes. Here, we combine viscosity sensitive fluorophores, advanced microscopy, and X-ray diffraction and molecular simulations to directly and quantitatively measure the bilayer's structural and viscoelastic properties, and correlate these with atomistic molecular modelling. Our results indicate an increase in microviscosity and a decrease in the bending rigidity upon peroxidation of the membranes, contrary to the trend observed with non-oxidized lipids. Fluorescence lifetime imaging microscopy and MD simulations give evidence for the presence of membrane regions of different local order in the oxidized membranes. We hypothesize that oxidation promotes stronger lipid-lipid interactions, which lead to an increase in the lateral heterogeneity within the bilayer and the creation of lipid clusters of higher order.

Platinum(II)-Based Optical Probes for Imaging Quadruplex DNA Structures via Phosphorescence Lifetime Imaging Microscopy.

G-quadruplex DNA is a non-canonical structure that forms in guanine-rich regions of the genome. There is increasing evidence showing that G-quadruplexes have important biological functions, and therefore molecular tools to visualise these structures are important. Herein we report on a series of new cyclometallated platinum(II) complexes which, upon binding to G-quadruplex DNA, display an increase in their phosphorescence, acting as switch-on probes. More importantly, upon binding to G-quadruplexes they display a selective and distinct lengthening of their emission lifetime. We show that this effect can be used to selectively visualise these structures in cells using Phosphorescence Lifetime Imaging Microscopy (PLIM).

Emerging properties from mechanical tethering within a post-synthetically functionalised catenane scaffold.

Maintaining close spatial proximity of functional moieties within molecular systems can result in fascinating emergent properties. Whilst much work has been done on covalent tethering of functional units for myriad applications, investigations into mechanically linked systems are relatively rare. Formation of the mechanical bond is usually the final step in the synthesis of interlocked molecules, placing limits on the throughput of functionalised architectures. Herein we present the synthesis of a bis-azide [2]catenane scaffold that can be post-synthetically modified using CuAAC 'click' chemistry. In this manner we have been able to access functionalised catenanes from a common precursor and study the properties of electrochemically active, emissive and photodimerisable units within the mechanically interlocked system in comparison to non-interlocked analogues. Our data demonstrates that the greater (co-)conformational flexibility that can be obtained with mechanically interlocked systems compared to traditional covalent tethers paves the way for developing new functional molecules with exciting properties.

Development of resistance to 5-fluorouracil affects membrane viscosity and lipid composition of cancer cells.

The investigations reported here were designed to determine whether the bulk plasma membrane is involved in mechanisms of acquired resistance of colorectal cancer cells to 5-fluorouracil (5-FU). Fluorescence lifetime imaging microscopy (FLIM) of live cultured cells stained with viscosity-sensitive probe BODIPY 2 was exploited to non-invasively assess viscosity in the course of treatment and adaptation to the drug. In parallel, lipid composition of membranes was examined with the time-of-flight secondary ion mass spectrometry (ToF-SIMS). Our results showed that a single treatment with 5-FU induced only temporal changes of viscosity in 5-FU sensitive cells immediately after adding the drug. Acquisition of chemoresistance was accompanied by persistent increase of viscosity, which was preserved upon treatment without any changes. Lipidomic analysis revealed that the resistant cells had a lower level of monounsaturated fatty acids and increased sphingomyelin or decreased phosphatidylcholine in their membranes, which partly explain increase of the viscosity. Thus, we propose that a high membrane viscosity mediates the acquisition of resistance to 5-FU.

Z-α1-antitrypsin polymers impose molecular filtration in the endoplasmic reticulum after undergoing phase transition to a solid state.

Misfolding of secretory proteins in the endoplasmic reticulum (ER) features in many human diseases. In α1-antitrypsin deficiency, the pathogenic Z variant aberrantly assembles into polymers in the hepatocyte ER, leading to cirrhosis. We show that α1-antitrypsin polymers undergo a liquid:solid phase transition, forming a protein matrix that retards mobility of ER proteins by size-dependent molecular filtration. The Z-α1-antitrypsin phase transition is promoted during ER stress by an ATF6-mediated unfolded protein response. Furthermore, the ER chaperone calreticulin promotes Z-α1-antitrypsin solidification and increases protein matrix stiffness. Single-particle tracking reveals that solidification initiates in cells with normal ER morphology, previously assumed to represent a healthy pool. We show that Z-α1-antitrypsin-induced hypersensitivity to ER stress can be explained by immobilization of ER chaperones within the polymer matrix. This previously unidentified mechanism of ER dysfunction provides a template for understanding a diverse group of related proteinopathies and identifies ER chaperones as potential therapeutic targets.

Cationic helicenes as selective G4 DNA binders and optical probes for cellular imaging.

The important role that G-quadruplex DNA (G4 DNA) structures play in regulating biological processes is becoming widely recognised. These structures have also been proposed to be attractive drug targets. Therefore, there has been significant interest in developing small molecules that can selectively bind to G4 DNA over other topologies. In this paper we investigate the interaction between DNA and helical compounds (helicenes) based on a central carbocation trisubstituted with aromatic rings. We show that the non-planar structure of these helicenes results in a significantly reduced affinity for dsDNA when compared to their planar analogues, whilst maintaining a high affinity for G4 DNA. Additionally, the right- and left-handed enantiomers of one of these helicenes recognise the chiral DNA environments of G4 and dsDNA differently. We show that upon DNA binding the helicenes display a fluorescence switch-on effect, which we have successfully used for cellular imaging in live and fixed U2OS cells, staining mitochondria and the nucleus, respectively.

The Role of Plasma Membrane Viscosity in the Response and Resistance of Cancer Cells to Oxaliplatin.

Maintenance of the biophysical properties of membranes is essential for cell survival upon external perturbations. However, the links between a fluid membrane state and the drug resistance of cancer cells remain elusive. Here, we investigated the role of membrane viscosity and lipid composition in the responses of cancer cells to oxaliplatin and the development of chemoresistance. Plasma membrane viscosity was monitored in live colorectal cancer cells and tumor xenografts using two-photon excited fluorescence lifetime imaging microscopy (FLIM) using the fluorescent molecular rotor BODIPY 2. The lipid profile was analyzed using time-of-flight secondary ion mass spectrometry (ToF-SIMS). It was found that the plasma membrane viscosity increased upon oxaliplatin treatment, both in vitro and in vivo, and that this correlated with lower phosphatidylcholine and higher cholesterol content. The emergence of resistance to oxaliplatin was accompanied by homeostatic adaptation of the membrane lipidome, and the recovery of lower viscosity. These results suggest that maintaining a constant plasma membrane viscosity via remodeling of the lipid profile is crucial for drug resistance in cancer.

Visualising UV-A light-induced damage to plasma membranes of eye lens.

An eye lens is constantly exposed to the solar UV radiation, which is considered the most important external source of age-related changes to eye lens constituents. The accumulation of modifications of proteins and lipids with age can eventually lead to the development of progressive lens opacifications, such as cataracts. Though the impact of solar UV radiation on the structure and function of proteins is actively studied, little is known about the effect of photodamage on plasma membranes of lens cells. In this work we exploit Fluorescence Lifetime Imaging Microscopy (FLIM), together with viscosity-sensitive fluorophores termed molecular rotors, to study the changes in viscosity of plasma membranes of porcine eye lens resulting from two different types of photodamage: Type I (electron transfer) and Type II (singlet oxygen) reactions. We demonstrate that these two types of photodamage result in clearly distinct changes in viscosity - a decrease in the case of Type I damage and an increase in the case of Type II processes. Finally, to simulate age-related changes that occur in vivo, we expose an intact eye lens to UV-A light under anaerobic conditions. The observed decrease in viscosity within plasma membranes is consistent with the ability of eye lens constituents to sensitize Type I photodamage under natural irradiation conditions. These changes are likely to alter the transport of metabolites and predispose the whole tissue to the development of pathological processes such as cataracts.

Molecular rotors as reporters for viscosity of solutions of collagen like peptides.

We have studied the suitability of using a molecular rotor-based steady-state fluorometric assay for evaluating changes in both the conformation and the viscosity of collagen-like peptide solutions. Our results indicate that a positive charge incorporated on the hydrophobic tail of the BODIPY molecular rotor favours the dye specificity as a reporter for viscosity of these solutions.

Probing Metabolism and Viscosity of Cancer Cells using Fluorescence Lifetime Imaging Microscopy.

Viscosity is an important physical property of a biological membrane, as it is one of the key parameters for the regulation of morphological and physiological state of living cells. Plasma membranes of tumor cells are known to have significant alterations in their composition, structure, and functional characteristics. Along with dysregulated metabolism of glucose and lipids, these specific membrane properties help tumor cells to adapt to the hostile microenvironment and develop resistance to drug therapies. Here, we demonstrate the use of fluorescence lifetime imaging microscopy (FLIM) to sequentially image cellular metabolism and plasma membrane viscosity in live cancer cell culture. Metabolic assessments are performed by detecting fluorescence of endogenous metabolic cofactors, such as reduced nicotinamide adenine dinucleotide NAD(P)H and oxidized flavins. Viscosity is measured using a fluorescent molecular rotor, a synthetic viscosity-sensitive dye, with a strong fluorescence lifetime dependence on the viscosity of the immediate environment. In combination, these techniques enable us to better understand the links between membrane state and metabolic profile of cancer cells and to visualize the changes induced by chemotherapy.

Selective Detection of Cu+ Ions in Live Cells via Fluorescence Lifetime Imaging Microscopy.

Copper is an essential trace element in living organisms with its levels and localisation being carefully managed by the cellular machinery. However, if misregulated, deficiency or excess of copper ions can lead to several diseases. Therefore, it is important to have reliable methods to detect, monitor and visualise this metal in cells. Herein we report a new optical probe based on BODIPY, which shows a switch-on in its fluorescence intensity upon binding to copper(I), but not in the presence of high concentration of other physiologically relevant metal ions. More interestingly, binding to copper(I) leads to significant changes in the fluorescence lifetime of the new probe, which can be used to visualize copper(I) pools in lysosomes of live cells via fluorescence lifetime imaging microscopy (FLIM).

Cyclopropyl Substituents Transform the Viscosity-Sensitive BODIPY Molecular Rotor into a Temperature Sensor.

A quantitative fluorescent probe that responds to changes in temperature is highly desirable for studies of biological environments, particularly in cellulo. Here, we report new cell-permeable fluorescence probes based on the BODIPY moiety that respond to environmental temperature. The new probes were developed on the basis of a well-established BODIPY-based viscosity probe by functionalization with cyclopropyl substituents at α and ß positions of the BODIPY core. In contrast to the parent BODIPY fluorophore, α-cyclopropyl-substituted fluorophore displays temperature-dependent time-resolved fluorescence decays showing greatly diminished viscosity dependence, making it an attractive sensor to be used with fluorescence lifetime imaging microscopy (FLIM). We performed theoretical calculations that help rationalize the effect of the cyclopropyl substituents on the photophysical behavior of the new BODIPYs. In summary, we designed an attractive new quantitative FLIM-based temperature probe that can be used for temperature sensing in live cells.

Metal complexes as optical probes for DNA sensing and imaging.

Transition and lanthanide metal complexes have rich photophysical properties that can be used for cellular imaging, biosensing and phototherapy. One of the applications of such luminescent compounds is the detection and visualisation of nucleic acids. In this brief review, we survey the recent literature on the use of luminescent metal complexes (including ReI, RuII, OsII, IrIII, PtII, EuIII and TbIII) as DNA optical probes, including examples of compounds that bind selectively to non-duplex DNA topologies such as quadruplex, i-motif and DNA mismatches. We discuss the applications of metal-based luminescent complexes in cellular imaging, including time-resolved microscopy and super-resolution techniques. Their applications in biosensing and phototherapy are briefly mentioned in the relevant sections.