Fluorescence Correlation Spectroscopy Measurements Research Articles

Control of Membrane Binding and Diffusion of Cholesteryl-Modified DNA Origami Nanostructures by DNA Spacers.

DNA origami nanotechnology is being increasingly used to mimic membrane-associated biophysical phenomena. Although a variety of DNA origami nanostructures has already been produced to target lipid membranes, the requirements for membrane binding have so far not been systematically assessed. Here, we used a set of elongated DNA origami structures with varying placement and number of cholesteryl-based membrane anchors to compare different strategies for their incorporation. Single and multiple cholesteryl anchors were attached to DNA nanostructures using single- and double-stranded DNA spacers of varying length. The produced DNA nanostructures were studied in terms of their membrane binding and diffusion. Our results show that the location and number of anchoring moieties play a crucial role for membrane binding of DNA nanostructures mainly if the cholesteryl anchors are in close proximity to the bulky DNA nanostructures. Moreover, the use of DNA spacers largely overcomes local steric hindrances and thus enhances membrane binding. Fluorescence correlation spectroscopy measurements demonstrate that the distinct physical properties of single- and double-stranded DNA spacers control the interaction of the amphipathic DNA nanostructures with lipid membranes. Thus, we provide a rational basis for the design of amphipathic DNA origami nanostructures to efficiently bind lipid membranes in various environments.

Residence time of singlet oxygen in membranes

Photodynamic therapy uses photosensitizers (PS) to kill cancer cells by generating reactive oxygen species – like singlet oxygen (SO) - upon illumination with visible light. PS membrane anchoring augments local SO concentration, which in turn increases photodynamic efficiency. The latter may suffer from SO’s escape into the aqueous solution or premature quenching. Here we determined the time constants of SO escape and quenching by target molecules to be in the nanosecond range, the former being threefold longer. We confined PS and dipolar target molecules either to different membrane monolayers or to the same leaflet and assessed their abundance by fluorescence correlation spectroscopy or membrane surface potential measurements. The rate at which the contribution of the dipolar target molecules to membrane dipole potential vanished, served as a measure of the photo-oxidation rate. The solution of the reaction–diffusion equations did not indicate diffusional rate limitations. Nevertheless, reducing the PS-target distance increased photodynamic efficiency by preventing other SO susceptible moieties from protecting the target. Importantly, our analytical model revealed a fourfold difference between SO generation rates per molecule of the two used PSs. Such analysis of PS quantum yield in a membrane environment may help in designing better PSs.

Diffusion Measurements of Swimming Enzymes with Fluorescence Correlation Spectroscopy.

Self-propelled chemical motors are chemically powered micro- or nanosized swimmers. The energy required for these motors' active motion derives from catalytic chemical reactions and the transformation of a fuel dissolved in the solution. While self-propulsion is now well established for larger particles, it is still unclear if enzymes, nature's nanometer-sized catalysts, are potentially also self-powered nanomotors. Because of its small size, any increase in an enzyme's diffusion due to active self-propulsion must be observed on top of the enzyme's passive Brownian motion, which dominates at this scale. Fluorescence correlation spectroscopy (FCS) is a sensitive method to quantify the diffusion properties of single fluorescently labeled molecules in solution. FCS experiments have shown a general increase in the diffusion constant of a number of enzymes when the enzyme is catalytically active. Diffusion enhancements after addition of the enzyme's substrate (and sometimes its inhibitor) of up to 80% have been reported, which is at least 1 order of magnitude higher than what theory would predict. However, many factors contribute to the FCS signal and in particular the shape of the autocorrelation function, which underlies diffusion measurements by fluorescence correlation spectroscopy. These effects need to be considered to establish if and by how much the catalytic activity changes an enzyme's diffusion. We carefully review phenomena that can play a role in FCS experiments and the determination of enzyme diffusion, including the dissociation of enzyme oligomers upon interaction with the substrate, surface binding of the enzyme to glass during the experiment, conformational changes upon binding, and quenching of the fluorophore. We show that these effects can cause changes in the FCS signal that behave similar to an increase in diffusion. However, in the case of the enzymes F1-ATPase and alkaline phosphatase, we demonstrate that there is no measurable increase in enzyme diffusion. Rather, dissociation and conformational changes account for the changes in the FCS signal in the former and fluorophore quenching in the latter. Within the experimental accuracy of our FCS measurements, we do not observe any change in diffusion due to activity for the enzymes we have investigated. We suggest useful control experiments and additional tests for future FCS experiments that should help establish if the observed diffusion enhancement is real or if it is due to an experimental or data analysis artifact. We show that fluorescence lifetime and mean intensity measurements are essential in order to identify the nature of the observed changes in the autocorrelation function. While it is clear from theory that chemically active enzymes should also act as self-propelled nanomotors, our FCS measurements show that the associated increase in diffusion is much smaller than previously reported. Further experiments are needed to quantify the contribution of the enzymes' catalytic activity to their self-propulsion. We hope that our findings help to establish a useful protocol for future FCS studies in this field and help establish by how much the diffusion of an enzyme is enhanced through catalytic activity.

Absolute Quantification of RNA Molecules Using Fluorescence Correlation Spectroscopy with Certified Reference Materials.

The accuracy and precision of quantification values of biomolecules, such as nucleic acids, are critical for the reliability of biomedical research and clinical examinations. To obtain an accurate quantitative value, it is necessary to use a measurement standard that has the same sequence and length as the target gene. The absence of an appropriate measurement standard leads to uncertain results. The development of a wide variety of different kinds of measurement standards, which have different sequences and lengths, is time-consuming and troublesome. We employed fluorescence correlation spectroscopy (FCS), which can be used to count the molecular number (absolute concentration) regardless of the molecular size and shape, without a standard curve. The confocal volume (i.e., the volume of excitation laser focus) of the FCS system was calibrated by measuring the primary standard of the fluorescent material. Furthermore, we investigated how to avoid artifacts originating from systematic aberrations or sample conditions. We validated the RNA concentration obtained from our FCS measurements using another primary standard RNA solution as a sample. Here, we describe an FCS calibration procedure with fluorescein solution standard reference material (SRM) 1932 as a primary standard and cross-validation of FCS values using RNA solutions certified reference material (CRM) 6204-a. The established method was applied to determine the concentrations of RNA samples that can be used as a laboratory working standards. The FCS method with a characterized SRM and CRM should serve as a universal method for absolute quantification of the number of biomolecules.

Photophysical Behavior of mNeonGreen, an Evolutionarily Distant Green Fluorescent Protein

Fluorescent proteins (FPs) feature complex photophysical behavior that must be considered when studying the dynamics of fusion proteins in model systems and live cells. In this work, we characterize mNeonGreen (mNG), a recently introduced FP from the bilaterian Branchiostoma lanceolatum, in comparison to the well-known hydrozoan variants enhanced green fluorescent protein (EGFP) and Aequorea coerulescens GFP by steady-state spectroscopy and fluorescence correlation spectroscopy in solutions of different pH. Blind spectral unmixing of sets of absorption spectra reveals three interconverting electronic states of mNG: a nonfluorescent protonated state, a bright state showing bell-shaped pH dependence, and a similarly bright state dominating at high pH. The gradual population of the acidic form by external protonation is reflected by increased flickering at low pH in fluorescence correlation spectroscopy measurements, albeit with much slower flicker rates and lower amplitudes as compared to Aequorea GFPs. In addition, increased flickering of mNG indicates a second deprotonation step above pH 10 leading to a slight decrease in fluorescence. Thus, mNG is distinguished from Aequorea GFPs by a two-step protonation response with opposite effects that reflects a chemically distinct chromophore environment. Despite the more complex pH dependence, mNG represents a superior FP under a broad range of conditions.

Specifically bound lambda repressor dimers promote adjacent non-specific binding.

Genetic switches frequently include DNA loops secured by proteins. Recent studies of the lambda bacteriophage repressor (CI), showed that this arrangement in which the protein links two sets of three operators separated by approximately 2.3 kbp, optimizes both the stability and dynamics of DNA loops, compared to an arrangement with just two sets of two operators. Because adjacent dimers interact pairwise, we hypothesized that the odd number of operators in each set of the lambda regulatory system might have evolved to allow for semi-specific, pair-wise interactions that add stability to the loop while maintaining it dynamic. More generally, additional CI dimers may bind non-specifically to flanking DNA sequences making the genetic switch more sensitive to CI concentration. Here, we tested this hypothesis using spectroscopic and imaging approaches to study the binding of the lambda repressor (CI) dimer protein to DNA fragments. For fragments with only one operator and a short flanking sequence, fluorescence correlation spectroscopy measurements clearly indicated the presence of two distinct DNA-CI complexes; one is thought to have a non-specifically bound CI dimer on the flanking sequence. Scanning force micrographs of CI bound to DNA with all six operators revealed wild-type or mutant proteins bound at operator positions. The number of bound, wild-type proteins increased with CI concentration and was larger than expected for strictly specific binding to operators. In contrast, a mutant that fails to oligomerize beyond a dimer, D197G, only bound to operators. These data are evidence that CI cooperativity promotes oligomerization that extends from operator sites to influence the thermodynamics and kinetics of CI-mediated looping.

Lipid Membrane Interaction of Amyloidogenic Peptides

Membrane interaction appears to be a key step in the toxic pathway of many amyloidogenic proteins, such as Amyloidβ (Aβ40/42, associated with Alzheimer's disease) and hIAPP (associated with Type II diabetes). While the oligomers are generally considered to be more toxic than either the monomers or the fibrils, the exact identity of the toxic oligomer has remained a matter of debate. How much the local structure and composition of the membrane influences the binding of the oligomers is another controversial issue.Here we use a combination of single‐molecule fluorescence techniques (e.g. single molecule photobleaching (smPB) and Fluorescence Correlation Spectroscopy (FCS)) and atomic force microscopy to quantitatively address these two issues. smPB measurements of freshly prepared hIAPP in physiological solution and on a supported lipid bilayer (composed of POPC: POPG: Cholesterol 1:1:1) allow us to measure the relative affinity of each oligomeric species. Our measurements show that both monomers and small oligomers (1–5 mers) have high affinity for the membrane, and dimers appear to have the highest affinity. However, FCS measurements show that the monomers have much lower affinity than the oligomers, Though FCS is unable to resolve the contribution of individual types of oligomers. It is likely that the smPB measurements are skewed by pre‐measurement photobleaching which is a known problem with this technique. We have now developed a method to measure pre‐bleaching, and are in the process of calibrating our results.We have also addressed the influence of the local membrane structure on binding of hIAPP oligomers. This aspect may throw light on the initiation of these diseases in the human body. We use a ternary neutral lipid mixture (DOPC: Sphingomyelin: Cholesterol in 2:2:1 mole ratio) to make stable phase separated bilayers. We use an AFM to map the bilayer surface topology, and a synchronized home‐built confocal microscope to simultaneously map the distribution of the fluorescently labeled peptides on this bilayer. We find that small oligomers of hIAPP bind preferentially to the disordered (Ld) phase, compared to the ordered phase Lo, with a binding ratio of Ld:Lo = 9:1. Our studies thus quantify the membrane interaction of small n‐mers of hIAPP as a function of n, and also show that local membrane organization can play a dominant role in determining membrane interactions.Support or Funding InformationTata Institute of Fundamental ResearchThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Inhibition of amyloid beta fibril formation by monomeric human transthyretin.

Transthyretin (TTR) is a homotetrameric protein that is found in the plasma and cerebrospinal fluid. Dissociation of TTR tetramers sets off a downhill cascade of amyloid formation through polymerization of monomeric TTR. Interestingly, TTR has an additional, biologically relevant activity, which pertains to its ability to slow the progression of amyloid beta (Aβ) associated pathology in transgenic mice. In vitro, both TTR and a kinetically stable variant of monomeric TTR (M-TTR) inhibit the fibril formation of Aβ1-40/42 molecules. Published evidence suggests that tetrameric TTR binds preferentially to Aβ monomers, thus destabilizing fibril formation by depleting the pool of Aβ monomers from aggregating mixtures. Here, we investigate the effects of M-TTR on the in vitro aggregation of Aβ1-42 . Our data confirm previous observations that fibril formation of Aβ is suppressed in the presence of sub-stoichiometric amounts of M-TTR. Despite this, we find that sub-stoichiometric levels of M-TTR are not bona fide inhibitors of aggregation. Instead, they co-aggregate with Aβ to promote the formation of large, micron-scale insoluble, non-fibrillar amorphous deposits. Based on fluorescence correlation spectroscopy measurements, we find that M-TTR does not interact with monomeric Aβ. Two-color coincidence analysis of the fluorescence bursts of Aβ and M-TTR labeled with different fluorophores shows that M-TTR co-assembles with soluble Aβ aggregates and this appears to drive the co-aggregation into amorphous precipitates. Our results suggest that mimicking the co-aggregation activity with protein-based therapeutics might be a worthwhile strategy for rerouting amyloid beta peptides into inert, insoluble, and amorphous deposits.

Topology-Dependent Dynamics of Single DNA Molecules

We present the data on the conformational dynamics of circular DNA molecules with defined levels of topological complexity, characterized by the knot type K and linking number Lk. Covalently closed DNAs covalently conjugated to specific fluorophores were investigated using fluorescence correlation spectroscopy (FCS) and fluorescence cross-correlation spectroscopy (FCCS) measurements. We investigate in detail the respective effects of supercoiling and DNA knotting using Lk-topoisomer pools and specific DNA-knot types that were respectively generated by topoisomerase-I relaxation and Cre site-specific recombination. Previous Brownian-dynamics simulations by Langowski et al. (1994) suggest that increasing superhelicity leads to progressively faster dynamics on long time scales, corresponding to translational diffusion, but also on short time scales, corresponding to segmental motions within supercoiled DNA. We investigate here whether DNA knotting similarly affects translational and internal motions. The acceleration of DNA dynamics with increased supercoiling may have implications for processes in cells, such as transcriptional regulation and DNA recombination, both of which are dependent on the dynamics of juxtaposition involving multiple DNA sites.

Fluorescence Correlation Spectroscopy with Nanowire Waveguide Illumination for High Concentration Conditions

Fluorescence correlation spectroscopy (FCS), a technique based on correlating the fluorescence signal from a small sample volume, has been commonly used to study biological processes such as photophysical phenomena, binding kinetics, and intracellular dynamics. In a conventional FCS setup, the sample volume is defined by a focused laser beam for excitation and a confocal pin hole for collection resulting in a size of about one femtoliter. Evanescent waves from total internal reflection have been used to obtain a smaller sample volume, as a result of the improved confinement along the longitudinal axis for the illumination. However, applying FCS under physiological conditions where the molecule concentration is typically in the µM range requires an even smaller sample volume. Here we propose to apply FCS with nanowire waveguide illumination with nanometer-sized lateral and longitudinal confinement. Nanowire waveguides, made of a dielectric nanowire with a diameter of ∼50 nm embedded inside a metal film, allow for the efficient transmission of visible light by exploiting surface waves traveling along the dielectric-metal boundary. Such waveguides can generate a lateral illumination area of about 50 nanometers in diameter. In addition, a longitudinal confinement of around 20 nanometers is achieved with the rapidly-attenuating near fields exiting from the waveguide. The strong confinement in both lateral and longitudinal directions thus lead to a sample volume on the order of one zeptoliter, allowing for FCS measurements in the µM concentration range. By combining Brownian dynamics simulations with the illumination profile obtained from finite element method simulations of a zinc oxide nanowire waveguide in a silver metal film, we numerically calculate the correlation function to demonstrate the use of this method in the study of molecular dynamics under high concentration conditions.

Fluorescence Correlation Spectroscopy on Genomic DNA in Living Cells.

Fluorescence correlation spectroscopy (FCS) is a powerful technique used to measure diffusion, fluctuations, and other transport processes in biomolecular systems. It is, however, prone to artifacts and subject to considerable experimental difficulties when applied to living cells. In this chapter, we provide protocols to conduct quantitative FCS measurements on DNA inside living eukaryotic and prokaryotic cells. We discuss sample preparation, dye selection and characterization, FCS data acquisition, and data analysis, including a method to com pensate for photobleaching to obtain quantitatively meaningful spectra.

Quantitative Determination of Telomerase Activity by Combining Fluorescence Correlation Spectroscopy with Telomerase Repeat Amplification Protocol.

Telomerase is a key enzyme for maintaining the telomere length and is regarded as a versatile cancer biomarker and a potential drug target due to its important role in cancer and aging. It is necessary to develop a sensitive and reliable method for detection of telomerase activity due to its very low level in cells. In this Article, we propose an ultrasensitive and robust method for quantitative determination of telomerase activity by combining single molecule fluorescence correlation spectroscopy (FCS) with telomerase repeat amplification protocol (TRAP). The principle of this new method (FCS-TRAP) is based on measurement of the change in characteristic diffusion time and molecule number of TRAP products by FCS. The characteristic diffusion time is related to the length of TRAP products, and the molecule number represents the concentration of TRAP products. We optimized the conditions of TRAP procedure and FCS measurements. We observed that the telomerase activities are positively correlated to characteristic diffusion time and molecule number of TRAP products at optimal conditions. This method was successfully used for determination of telomerase activity of different cells, and detection of a single cell was realized. Meanwhile, this method was used to evaluate the inhibition efficiency of inhibitors, and the IC50 values obtained were in good agreement with the references. Compared to current TRAP methods, this method shows reliable quantification, ultrahigh sensitivity, and short detection time and is without separation. We believe that the FCS-TRAP method has a potential application in clinical diagnosis and screening of telomerase inhibitors.

Fluorescence-based monitoring of electronic state and ion exchange kinetics with FCS and related techniques: from T-jump measurements to fluorescence fluctuations

In this review, we give a historical view of how our research in the development and use of fluorescence correlation spectroscopy (FCS) and related techniques has its roots and how it originally evolved from the pioneering work of Manfred Eigen, his colleagues, and coworkers. Work on temperature-jump (T-jump) experiments, conducted almost 50 years ago, led on to the development of the FCS technique. The pioneering work in the 1970s, introducing and demonstrating the concept for FCS, in turn formed the basis for the breakthrough use of FCS more than 15 years later. FCS can be used for monitoring reaction kinetics, based on fluctuations at thermodynamic equilibrium, rather than on relaxation measurements following perturbations. In this review, we more specifically discuss FCS measurements on photodynamic, electronic state transitions in fluorophore molecules, and on proton exchange dynamics in solution and on biomembranes. In the latter case, FCS measurements have proven capable of casting new light on the mechanisms of proton exchange at biological membranes, of central importance to bioenergetics and signal transduction. Finally, we describe the transient-state (TRAST) spectroscopy/imaging technique, sharing features with both relaxation (T-jump) and equilibrium fluctuation (FCS) techniques. TRAST is broadly applicable for cellular and molecular studies, and we briefly outline how TRAST can provide unique information from fluorophore blinking kinetics, reflecting e.g., cellular metabolism, rare molecular encounters, and molecular stoichiometries.

Fission yeast Myo2: Molecular organization and diffusion in the cytoplasm.

Myosin-II is required for the assembly and constriction of cytokinetic contractile rings in fungi and animals. We used electron microscopy, fluorescence recovery after photobleaching (FRAP), and fluorescence correlation spectroscopy (FCS) to characterize the physical properties of Myo2 from fission yeast Schizosaccharomyces pombe. By electron microscopy, Myo2 has two heads and a coiled-coiled tail like myosin-II from other species. The first 65 nm of the tail is a stiff rod, followed by a flexible, less-ordered region up to 30 nm long. Myo2 sediments as a 7 S molecule in high salt, but aggregates rather than forming minifilaments at lower salt concentrations; this is unaffected by heavy chain phosphorylation. We used FRAP and FCS to observe the dynamics of Myo2 in live S. pombe cells and in cell extracts at different salt concentrations; both show that Myo2 with an N-terminal mEGFP tag has a diffusion coefficient of ∼ 3 µm2 s-1 in the cytoplasm of live cells during interphase and mitosis. Photon counting histogram analysis of the FCS data confirmed that Myo2 diffuses as doubled-headed molecules in the cytoplasm. FCS measurements on diluted cell extracts showed that mEGFP-Myo2 has a diffusion coefficient of ∼ 30 µm2 s-1 in 50 to 400 mM KCl concentrations.

Photophysical properties of flavonoids extracted from Syngonanthus nitens, the golden grass

Dry stems of Syngonanthus nitens, a plant of the Euriocaulaceae species, present exquisite golden shine, originated from flavonoids residing at S. nitens stem epidermis. We report here on the optical properties of flavonoids extracted, in different solvents, from dry stems of the plant. The extracts were analyzed by UV–Vis absorption and fluorescence emission spectroscopies, and data were obtained in solvents of different polarity and in aqueous media at different pHs. Time-resolved fluorescence experiments, made using single photon detection technique, fluorescence correlation spectroscopy and fluorescence anisotropy measurements were also employed to study flavonoid/solvent and flavonoid/flavonoid interactions. A consistent picture of the S. nitens flavonoid photophysical behavior and their interaction via proton transfer in aqueous suspension is obtained. The results are important to understand the photophysical process involving flavonoids in general.

Simultaneous membrane interaction of amphipathic peptide monomers, self-aggregates and cargo complexes detected by fluorescence correlation spectroscopy

Peptides able to translocate cell membranes while carrying macromolecular cargo, as cell-penetrating peptides (CPPs), can contribute to the field of drug delivery by enabling the transport of otherwise membrane impermeable molecules. Formation of non-covalent complexes between amphipathic peptides and oligonucleotides is driven by electrostatic and hydrophobic interactions. Here we investigate and quantify the coexistence of distinct molecular species in multiple equilibria, namely peptide monomer, peptide self-aggregates and peptide/oligonucleotide complexes. As a model for the complexes, we used a stearylated peptide from the PepFect family, PF14 and siRNA. PF14 has a cationic part and a lipid part, resembling some characteristics of cationic lipids. Fluorescence correlation spectroscopy (FCS) and fluorescence cross-correlation spectroscopy (FCCS) were used to detect distinct molecular entities in solution and at the plasma membrane of live cells. For that, we labeled the peptide with carboxyrhodamine 6G and the siRNA with Cyanine 5. We were able to detect fluorescent entities with diffusional properties characteristic of the peptide monomer as well as of peptide aggregates and peptide/oligonucleotide complexes. Strategies to avoid peptide adsorption to solid surfaces and self-aggregation were developed and allowed successful FCS measurements in solution and at the plasma membrane. The ratio between the detected molecular species was found to vary with pH, peptide concentration and the proximity to the plasma membrane. The present results suggest that the diverse cellular uptake mechanisms, often reported for amphipathic CPPs, might result from the synergistic effect of peptide monomers, self-aggregates and cargo complexes, distributed unevenly at the plasma membrane.

A total internal reflection-fluorescence correlation spectroscopy setup with pulsed diode laser excitation.

Fluorescence correlation spectroscopy (FCS) measures fluctuations in a (sub-)femtoliter volume to analyze the diffusive behavior of fluorescent particles. This highly sensitive method has proven to be useful for the analysis of dynamic biological systems as well as in chemistry, physics, and material sciences. It is routinely performed with commercial fluorescence microscopes, which provide a confined observation volume by the confocal technique. The evanescent wave of total internal reflectance (TIR) is used in home-built systems to permit a surface sensitive FCS analysis. We present a combined confocal and TIR-FCS setup which uses economic low-power pulsed diode lasers for excitation. Excitation and detection are coupled to time-correlated photon counting hardware. This allows simultaneous fluorescence lifetime and FCS measurements in a surface-sensitive mode. Moreover, the setup supports fluorescence lifetime correlation spectroscopy at surfaces. The excitation can be easily switched between TIR and epi-illumination to compare the surface properties with those in liquid bulk. The capabilities of the presented setup are demonstrated by measuring the diffusion coefficients of a free dye molecule, a labeled polyethylene glycol, and a fluorescent nanoparticle in confocal as well as in TIR-FCS.

Abstract 1404: Regulation of 53BP1 by the structural nuclear protein NuMA

Abstract Cells repair DNA double-strand breaks (DSB) through two major pathways: homologous recombination (HR) and non-homologous end joining (NHEJ). The choice between these two pathways is critical to cell survival and is altered in cancers. 53BP1 is a protein with an important role in this choice: it promotes NHEJ by blocking CtIP-dependent DNA resection. Here, we present a new negative regulation mechanism of 53BP1 that relies on the structural protein NuMA. This protein participates in mitotic spindle assembly, influences chromatin organization during the interphase and modulates the chromatin response to DNA damage. We identified NuMA in a proteomics analysis of a 53BP1-interacting proteins. The interaction was confirmed by reciprocal immunoprecipitation and with a FRET assay. In response to DSB, we measured greater than 50% decreased 53BP1-NuMA binding, which suggests that NuMA may restrain 53BP1 diffusion in the absence of DNA damage. Fluorescence correlation spectroscopy (FCS) measurements in cells expressing GFP-tagged 53BP1 support this hypothesis. Moreover, NuMA overexpression inhibited 53BP1 recruitment at DNA damage sites in laser microirradiation assays, whereas NuMA silencing had the opposite effect. 53BP1 plays an essential role in B cell class switch recombination and mediates PARP inhibitor sensitivity in BRCA1-null cells. NuMA overexpression prevented immunoglobulin switching, and overwrote cancer cell sensitivity to PARP inhibitors to the same extent as 53BP1 loss of function. Our results help understand how 53BP1 is controlled, and shed light on the mechanisms regulating PARPi sensitivity. Citation Format: Naike Salvador Moreno. Regulation of 53BP1 by the structural nuclear protein NuMA [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1404. doi:10.1158/1538-7445.AM2017-1404

Quantifying transcription factor-DNA binding in single cells in vivo with photoactivatable fluorescence correlation spectroscopy.

Probing transcription factor (TF)-DNA interactions remains challenging in complex in vivo systems such as mammalian embryos, especially when TF copy numbers and fluorescence background are high. To address this difficulty, fluorescence correlation spectroscopy (FCS) can be combined with the use of photoactivatable fluorescent proteins to achieve selective photoactivation of a subset of tagged TF molecules. This approach, termed paFCS, enables FCS measurements within single cell nuclei inside live embryos, and obtains autocorrelation data of a quality previously only attainable in simpler in vitro cell culture systems. Here, we present a protocol demonstrating the applicability of paFCS in developing mouse embryos by outlining its implementation on a commercial laser-scanning microscope. We also provide procedures for optimizing the photoactivation and acquisition parameters and determining key parameters describing TF-DNA binding. The entire procedure can be performed within ∼2 d (excluding embryo culture time), although the acquisition of each paFCS data set takes only ∼10 min. This protocol can be used to noninvasively reveal cell-to-cell variation in TF dynamics, as well as critical, fate-predicting changes over the course of early embryonic development.

Cholesterol Based Surface Active Ionic Liquid That Can Form Microemulsions and Spontaneous Vesicles.

In this article, we have reported the synthesis and physicochemical characterization of a novel l-glycine amino acid derived cholesterol based surface active ionic liquid (SAIL). This SAIL has been explored for the preparation of ionic liquid (IL)-in-oil microemulsions and vesicles. The formation of IL-in-oil microemulsion is characterized by construction of a ternary phase diagram, dynamic light scattering (DLS) measurement, proton nuclear magnetic resonance (1H NMR) study, fluorescence measurement using coumarin 480 (C-480) as a molecular probe, and also by recording the diffusion behavior of the molecular probe rhodamine 6G (R6G) in microemulsion droplets through the fluorescence correlation spectroscopy (FCS) technique. Similarly, the spontaneous vesicle formation from the SAIL in water has been established using DLS, transmission electron microscopy (TEM), cryogenic-transmission electron microscopy (cryo-TEM), field emission scanning electron microscopy (FESEM), atomic force microscopy (AFM), FCS, and fluorescence lifetime imaging microscopy (FLIM) measurements. These aggregates may potentially serve as good biomimicking models and possible drug carriers.