Complex tracer mobilities in polymer networks revealed by fluorescence correlation spectroscopy

Gels are elastic porous polymer networks that are accompanied by pronounced mechanical properties. Due to their biocompatibility, ‘responsive hydrogels’ (HG) have many biomedical applications ranging from biosensors and drug delivery to tissue engineering. They respond to external stimuli such as temperature and salt by changing their dimensions. Of paramount importance is the ability to engineer penetrability and diffusion of interacting molecules in the crowded HG environment, as this would enable one to optimize a specific functionality. Even though the conditions under which biomedical devices operate are rather complex, a bottom-up approach could reduce the complexity of mutually coupled parameters influencing tracer mobility. The present thesis focuses on the interaction-induced tracer diffusion in polymer solutions and their homologous gels, probed by means of Fluorescence Correlation Spectroscopy (FCS). This is a single-molecule-sensitive technique having the advantage of optimal performance under ultralow tracer concentrations, typically employed in biosensors. Two different types of hydrogels have been investigated, a conventional one with broad polydispersity in the distance between crosslink points and a so-called ‘ideal’, with uniform mesh size distribution. The former is based on a thermoresponsive polymer, exhibiting phase separation in water at temperatures close to the human body temperature. The latter represents an optimal platform to study tracer diffusion. Mobilities of different tracers have been investigated in each network, varying in size, geometry and in terms of tracer-polymer attractive strength, as perturbed by different stimuli. The thesis constitutes a systematic effort towards elucidating the role of the strength and nature of different tracer-polymer interactions, on tracer mobilities; it outlines that interactions can still be very important even in the simplified case of dilute polymer solutions; it also demonstrates that the presence of permanent crosslinks exerts distinct tracer slowdown, depending on the tracer type and the nature of the tracer-polymer interactions, expressed differently by each tracer with regard to the selected stimulus. In aqueous polymer solutions, the tracer slowdown is found to be system-dependent and no universal trend seems to hold, in contrast to predictions from scaling theory for non-interacting nanoparticle mobility and empirical relations concerning the mesh size in polymer solutions. Complex tracer dynamics in polymer networks may be distinctly expressed by FCS, depending on the specific synergy among-at least some of - the following parameters: nature of interactions, external stimuli employed, tracer size and type, crosslink density and swelling ratio.

Development of a fluorescent-based single liposome assay for studying calcium transporter LMCA1

The morphological and functional unit of all the living organisms is the cell. The transmembrane proteins, localized in the plasma membrane of cells, play a key role in the survival of the cells themselves. These proteins perform a variety of different tasks, for example the control of the homeostasis. In order to control the homeostasis, these proteins have to regulate the concentration of chemical elements, like ions, inside and outside the cell. These regulations are fundamental for the survival of the cell and to understand them we need to understand how transmembrane proteins work. Two of the most important categories of transmembrane proteins are ion channels and transporter proteins. The ion channels have been depth studied at the single molecule level since late 1970s with the development of patch-clamp technique. It is not possible to apply this technique to study the transporter proteins so a new technique is under development in order to investigate the behavior of transporter proteins at the single molecule level. This thesis describes the development of a nanoscale single liposome assay for functional studies of transporter proteins based on quantitative fluorescence microscopy in a highly-parallel manner and in real time. The transporter of interest is the prokaryotic transporter Listeria Monocytogenes Ca2+-ATPase1 (LMCA1), a structural analogue of the eukaryotic calcium pumps SERCA and PMCA. This technique will allow the characterization of LMCA1 functionality at the single molecule level. Three systematically characterized fluorescent sensors were tested at the single liposome scale in order to investigate if their properties are suitable to study the function of the transporter of interest. Further studies will be needed in order to characterize the selected calcium sensor and pH sensor both implemented together in single liposomes and in presence of the reconstituted protein LMCA1.

Characterizing New Fluorescent Tools for Studying 5-HT3 Receptor Pharmacology

The pharmacological characterization of ligands depends upon the ability to accurately measure their binding properties. Fluorescence provides an alternative to more traditional approaches such as radioligand binding. Here we describe the binding and spectroscopic properties of eight fluorescent 5-HT3 receptor ligands. These were tested on purified receptors, expressed receptors on live cells, or in vivo. All compounds had nanomolar affinities with fluorescent properties extending from blue to near infra-red emission. A fluorescein-derivative had the highest affinity as measured by fluorescence polarization (FP; 1.14 nM), flow cytometry (FC; 3.23 nM) and radioligand binding (RB; 1.90 nM). Competition binding with unlabeled 5-HT3 receptor agonists (5-HT, mCPBG, quipazine) and antagonists (granisetron, palonosetron, tropisetron) yielded similar affinities in all three assays. When cysteine substitutions were introduced into the 5-HT3 receptor binding site the same changes in binding affinity were seen for both granisetron and the fluorescein-derivative, suggesting that they both adopt orientations that are consistent with co-crystal structures of granisetron with a homologous protein (5HTBP). As expected, in vivo live imaging in anaesthetized mice revealed staining in the abdominal cavity in intestines, but also in salivary glands. The unexpected presence of 5-HT3 receptors in mouse salivary glands was confirmed by Western blots. Overall, these results demonstrate the wide utility of our new high-affinity fluorescently-labeled 5-HT3 receptor probes, ranging from in vitro receptor pharmacology, including FC and FP ligand competition, to live imaging of 5-HT3 expressing tissues.

A review of fluorescent ligands for studying 5-HT3 receptors

The use of fluorescence is a valuable and increasingly accessible means of probing the pharmacology and physiology of cells and their receptors. To date, the use of fluorescence-based methods for 5-HT3 receptor research has been quite limited and, although a variety of approaches have been described, these are broadly distributed throughout the literature. In this review we condense these findings into a single, accessible source of reference with the hope of promoting the use of these valuable molecular probes.

Fluorescence correlation analysis of probe diffusion simplifies quantitative pathogen detection by PCR

A sensitive, labor-saving, and easily automatable nonradioactive procedure named APEX-FCS (amplified probe extension detected by fluorescence correlation spectroscopy) has been established to detect specific in vitro amplification of pathogen genomic sequences. As an example, Mycobacterium tuberculosis genomic DNA was subjected to PCR amplification with the Stoffel fragment of Thermus aquaticus DNA polymerase in the presence of nanomolar concentrations of a rhodamine-labeled probe (third primer), binding to the target in between the micromolar amplification primers. The probe becomes extended only when specific amplification occurs. Its low concentration avoids false-positives due to unspecific hybridization under PCR conditions. With increasing portion of extended probe molecules, the probe’s average translational diffusion properties gradually change over the course of the reaction, reflecting amplification kinetics. Following PCR, this change from a stage of high to a stage of low mobility can directly be monitored during a 30-s measurement using a fluorescence correlation spectroscopy device. Quantitation down to 10 target molecules in a background of 2.5 μg unspecific DNA without post-PCR probe manipulations could be achieved with different primer/probe combinations. The assay holds the promise to concurrently perform amplification, probe hybridization, and specific detection without opening the reaction chamber, if sealable foils are used.

Detection of HIV-1 RNA by nucleic acid sequence-based amplification combined with fluorescence correlation spectroscopy

Nucleic acid sequence-based amplification (NASBA) has proved to be an ultrasensitive method for HIV-1 diagnosis in plasma even in the primary HIV infection stage. This technique was combined with fluorescence correlation spectroscopy (FCS) which enables online detection of the HIV-1 RNA molecules amplified by NASBA. A fluorescently labeled DNA probe at nanomolar concentration was introduced into the NASBA reaction mixture and hybridizing to a distinct sequence of the amplified RNA molecule. The specific hybridization and extension of this probe during amplification reaction, resulting in an increase of its diffusion time, was monitored online by FCS. As a consequence, after having reached a critical concentration of 0.1–1 nM (threshold for unaided FCS detection), the number of amplified RNA molecules in the further course of reaction could be determined. Evaluation of the hybridization/extension kinetics allowed an estimation of the initial HIV-1 RNA concentration that was present at the beginning of amplification. The value of initial HIV-1 RNA number enables discrimination between positive and false-positive samples (caused for instance by carryover contamination)—this possibility of discrimination is an essential necessity for all diagnostic methods using amplification systems (PCR as well as NASBA). Quantitation of HIV-1 RNA in plasma by combination of NASBA with FCS may also be useful in assessing the efficacy of anti-HIV agents, especially in the early infection stage when standard ELISA antibody tests often display negative results.

Chemical synthesis of the precursor molecule of the Aequorea green fluorescent protein, subsequent folding, and development of fluorescence

The present paper describes the total chemical synthesis of the precursor molecule of the Aequorea green fluorescent protein (GFP). The molecule is made up of 238 amino acid residues in a single polypeptide chain and is nonfluorescent. To carry out the synthesis, a procedure, first described in 1981 for the synthesis of complex peptides, was used. The procedure is based on performing segment condensation reactions in solution while providing maximum protection to the segment. The effectiveness of the procedure has been demonstrated by the synthesis of various biologically active peptides and small proteins, such as human angiogenin, a 123-residue protein analogue of ribonuclease A, human midkine, a 121-residue protein, and pleiotrophin, a 136-residue protein analogue of midkine. The GFP precursor molecule was synthesized from 26 fully protected segments in solution, and the final 238-residue peptide was treated with anhydrous hydrogen fluoride to obtain the precursor molecule of GFP containing two Cys(acetamidomethyl) residues. After removal of the acetamidomethyl groups, the product was dissolved in 0.1 M Tris⋅HCl buffer (pH 8.0) in the presence of DTT. After several hours at room temperature, the solution began to emit a green fluorescence (λmax = 509 nm) under near-UV light. Both fluorescence excitation and fluorescence emission spectra were measured and were found to have the same shape and maxima as those reported for native GFP. The present results demonstrate the utility of the segment condensation procedure in synthesizing large protein molecules such as GFP. The result also provides evidence that the formation of the chromophore in GFP is not dependent on any external cofactor.

Quantifying single gene copy number by measuring fluorescent probe lengths on combed genomic DNA

An approach was developed for the quantification of subtle gains and losses of genomic DNA. The approach relies on a process called molecular combing. Molecular combing consists of the extension and alignment of purified molecules of genomic DNA on a glass coverslip. It has the advantage that a large number of genomes can be combed per coverslip, which allows for a statistically adequate number of measurements to be made on the combed DNA. Consequently, a high-resolution approach to mapping and quantifying genomic alterations is possible. The approach consists of applying fluorescence hybridization to the combed DNA by using probes to identify the amplified region. Measurements then are made on the linear hybridization signals to ascertain the region's exact size. The reliability of the approach first was tested for low copy number amplifications by determining the copy number of chromosome 21 in a normal and trisomy 21 cell line. It then was tested for high copy number amplifications by quantifying the copy number of an oncogene amplified in the tumor cell line GTL-16. These results demonstrate that a wide range of amplifications can be accurately and reliably quantified. The sensitivity and resolution of the approach likewise was assessed by determining the copy number of a single allele (160 kb) alteration.

Simultaneous two-photon excitation of distinct labels for dual-color fluorescence crosscorrelation analysis

Confocal fluorescence correlation spectroscopy as a time-averaging fluctuation analysis combining maximum sensitivity with high statistical confidence has proved to be a very versatile and powerful tool for detection and temporal investigation of biomolecules at ultralow concentrations on surfaces, in solutions, and in living cells. To probe the interaction of different molecular species for a detailed understanding of biologically relevant mechanisms, crosscorrelation studies on dual or multiple fluorophore assays with spectrally distinct excitation and emission are particularly promising. Despite the considerable improvement of detection specificity provided by fluorescence crosscorrelation analysis, few applications have so far been reported, presumably because of the practical challenges of properly aligning and controlling the stability of the experimental setup. In this work, we demonstrate that two-photon excitation combined with dual-color fluorescence correlation spectroscopy can be the key to simplifying simultaneous investigations of multiple fluorescent species significantly on a single-molecule scale. Two-photon excitation allows accession of common fluorophores of largely distinct emission by the same excitation wavelength, because differences in selection rules and vibronic coupling can induce considerable shifts between the one-photon and two-photon excitation spectra. The concept of dual-color two-photon fluorescence crosscorrelation analysis is introduced and experimentally demonstrated with an established assay probing the selective cleavage of dual-labeled DNA substrates by restriction endonuclease EcoRI.

Intranuclear diffusion and hybridization state of oligonucleotides measured by fluorescence correlation spectroscopy in living cells

Fluorescein-labeled oligodeoxynucleotides (oligos) were introduced into cultured rat myoblasts, and their molecular movements inside the nucleus were studied by fluorescence correlation spectroscopy (FCS) and fluorescence recovery after photobleaching (FRAP). FCS revealed that a large fraction of both intranuclear oligo(dT) (43%) and oligo(dA) (77%) moves rapidly with a diffusion coefficient of 4 × 10−7 cm2/s. Interestingly, this rate of intranuclear oligo movement is similar to their diffusion rates measured in aqueous solution. In addition, we detected a large fraction (45%) of the intranuclear oligo(dT), but not oligo(dA), diffusing at slower rates (≤1 × 10−7 cm2/s). The amount of this slower-moving oligo(dT) was greatly reduced if the oligo(dT) was prehybridized in solution with (unlabeled) oligo(dA) prior to introduction to cells, presumably because the oligo(dT) was then unavailable for subsequent hybridization to endogenous poly(A) RNA. The FCS-measured diffusion rate for much of the slower oligo(dT) population approximated the diffusion rate in aqueous solution of oligo(dT) hybridized to a large polyadenylated RNA (1.0 × 10−7 cm2/s). Moreover, this intranuclear movement rate falls within the range of calculated diffusion rates for an average-sized heterogeneous nuclear ribonucleoprotein particle in aqueous solution. A subfraction of oligo(dT) (15%) moved over 10-fold more slowly, suggesting it was bound to very large macromolecular complexes. Average diffusion coefficients obtained from FRAP experiments were in agreement with the FCS data. These results demonstrate that oligos can move about within the nucleus at rates comparable to those in aqueous solution and further suggest that this is true for large ribonucleoprotein complexes as well.

Transverse relaxation-optimized NMR spectroscopy with the outer membrane protein OmpX in dihexanoyl phosphatidylcholine micelles

The 2H,13C,15N-labeled, 148-residue integral membrane protein OmpX from Escherichia coli was reconstituted with dihexanoyl phosphatidylcholine (DHPC) in mixed micelles of molecular mass of about 60 kDa. Transverse relaxation-optimized spectroscopy (TROSY)-type triple resonance NMR experiments and TROSY-type nuclear Overhauser enhancement spectra were recorded in 2 mM aqueous solutions of these mixed micelles at pH 6.8 and 30°C. Complete sequence-specific NMR assignments for the polypeptide backbone thus have been obtained. The 13C chemical shifts and the nuclear Overhauser effect data then resulted in the identification of the regular secondary structure elements of OmpX/DHPC in solution and in the collection of an input of conformational constraints for the computation of the global fold of the protein. The same type of polypeptide backbone fold is observed in the presently determined solution structure and the previously reported crystal structure of OmpX determined in the presence of the detergent n-octyltetraoxyethylene. Further structure refinement will have to rely on the additional resonance assignment of partially or fully protonated amino acid side chains, but the present data already demonstrate that relaxation-optimized NMR techniques open novel avenues for studies of structure and function of integral membrane proteins.

Establishment of medaka (Oryzias latipes) transgenic lines with the expression of green fluorescent protein fluorescence exclusively in germ cells: A useful model to monitor germ cells in a live vertebrate

We have generated transgenic medaka (teleost, Oryzias latipes), which allow us to monitor germ cells by green fluorescent protein (GFP) fluorescence in live specimens. Two medaka strains, himedaka (orange–red variety) and inbred QurtE, were used. The transgenic lines were achieved by microinjection of a construct containing the putative promoter region and 3′ region of the medaka vasa gene (olvas). The intensity of GFP fluorescence increases dramatically in primordial germ cells (PGCs) located in the ventrolateral region of the posterior intestine around stage 25 (the onset of blood circulation). Whole-mount in situ hybridization and monitoring of ectopically located cells by GFP fluorescence suggested that (i) the increase in zygotic olvas expression occurs after PGC specification and (ii) PGCs can maintain their cell characteristics ectopically after stages 20–25. Around the day of hatching, the QurtE strain clearly exhibits sexual dimorphisms in the number of GFP fluorescent germ cells, a finding consistent with the appearance of leucophores, a sex-specific marker of QurtE. The GFP expression persists throughout the later stages in the mature ovary and testis. Thus, these transgenic medaka represent a live vertebrate model to investigate how germ cells migrate to form sexually dimorphic gonads, as well as a potential assay system for environmental substances that may affect gonad development. The use of a transgenic construct as a selective marker to efficiently isolate germ-line-transmitting founders during embryogenesis is also discussed.

Fluorescence correlation spectroscopy: Diagnostics for sparse molecules

The robust glow of molecular fluorescence renders even sparse molecules detectable and susceptible to analysis for concentration, mobility, chemistry, and photophysics. Correlation spectroscopy, a statistical-physics-based tool, gleans quantitative information from the spontaneously fluctuating fluorescence signals obtained from small molecular ensembles. This analytical power is available for studying molecules present at minuscule concentrations in liquid solutions (less than one nanomolar), or even on the surfaces of living cells at less than one macromolecule per square micrometer. Indeed, routines are becoming common to detect, locate, and examine individual molecules under favorable conditions.

Ultra-fast excited state dynamics in green fluorescent protein: multiple states and proton transfer.

The green fluorescent protein (GFP) of the jellyfish Aequorea Victoria has attracted widespread interest since the discovery that its chromophore is generated by the autocatalytic, posttranslational cyclization and oxidation of a hexapeptide unit. This permits fusion of the DNA sequence of GFP with that of any protein whose expression or transport can then be readily monitored by sensitive fluorescence methods without the need to add exogenous fluorescent dyes. The excited state dynamics of GFP were studied following photo-excitation of each of its two strong absorption bands in the visible using fluorescence upconversion spectroscopy (about 100 fs time resolution). It is shown that excitation of the higher energy feature leads very rapidly to a form of the lower energy species, and that the excited state interconversion rate can be markedly slowed by replacing exchangeable protons with deuterons. This observation and others lead to a model in which the two visible absorption bands correspond to GFP in two ground-state conformations. These conformations can be slowly interconverted in the ground state, but the process is much faster in the excited state. The observed isotope effect suggests that the initial excited state process involves a proton transfer reaction that is followed by additional structural changes. These observations may help to rationalize and motivate mutations that alter the absorption properties and improve the photo stability of GFP.

Simultaneous fluorescence-activated cell sorter analysis of two distinct transcriptional elements within a single cell using engineered green fluorescent proteins.

Green fluorescent protein (GFP) is widely used as a reporter gene in both prokaryotes and eukaryotes. However, the fluorescence levels of wild-type GFP (wtGFP) are not bright enough for fluorescence-activated cell sorting or flow cytometry. Several GFP variants were generated that are brighter or have altered excitation spectra when expressed in prokaryotic cells. We engineered two GFP genes with different combinations of these mutations, GFP(S65T,V163A) termed GFP-Bex1, and GFP(S202F,T203I,V163A) termed GFP-Vex1. Both show enhanced brightness and improved signal-to-noise ratios when expressed in mammalian cells and appropriately excited, compared with wtGFP. Each mutant retains only one of the two excitation peaks of the wild-type protein. GFP-Bex1 excites at 488 nm (blue) and GFP-Vex1 excites at 406 nm (violet), both of which are available laser lines. Excitation at these wavelengths allows for the independent analyses of these mutants by fluorescence-activated cell sorting, permitting simultaneous, quantitative detection of expression from two different genes within single mammalian cells.