Sébastien Peter

The Arabidopsis GAGA-Binding Factor BASIC PENTACYSTEINE6 Recruits the POLYCOMB-REPRESSIVE COMPLEX1 Component LIKE HETEROCHROMATIN PROTEIN1 to GAGA DNA Motifs

A transcription factor forms a scaffold for Polycomb complex members at specific DNA motifs to control homeotic gene expression. Polycomb-repressive complexes (PRCs) play key roles in development by repressing a large number of genes involved in various functions. Much, however, remains to be discovered about PRC-silencing mechanisms as well as their targeting to specific genomic regions. Besides other mechanisms, GAGA-binding factors in animals can guide PRC members in a sequence-specific manner to Polycomb-responsive DNA elements. Here, we show that the Arabidopsis (Arabidopsis thaliana) GAGA-motif binding factor protein BASIC PENTACYSTEINE6 (BPC6) interacts with LIKE HETEROCHROMATIN PROTEIN1 (LHP1), a PRC1 component, and associates with VERNALIZATION2 (VRN2), a PRC2 component, in vivo. By using a modified DNA-protein interaction enzyme-linked immunosorbant assay, we could show that BPC6 was required and sufficient to recruit LHP1 to GAGA motif-containing DNA probes in vitro. We also found that LHP1 interacts with VRN2 and, therefore, can function as a possible scaffold between BPC6 and VRN2. The lhp1-4 bpc4 bpc6 triple mutant displayed a pleiotropic phenotype, extreme dwarfism and early flowering, which disclosed synergistic functions of LHP1 and group II plant BPC members. Transcriptome analyses supported this synergy and suggested a possible function in the concerted repression of homeotic genes, probably through histone H3 lysine-27 trimethylation. Hence, our findings suggest striking similarities between animal and plant GAGA-binding factors in the recruitment of PRC1 and PRC2 components to Polycomb-responsive DNA element-like GAGA motifs, which must have evolved through convergent evolution.

Screening for in planta protein-protein interactions combining bimolecular fluorescence complementation with flow cytometry.

Understanding protein and gene function requires identifying interaction partners using biochemical, molecular or genetic tools. In plants, searching for novel protein-protein interactions is limited to protein purification assays, heterologous in vivo systems such as the yeast-two-hybrid or mutant screens. Ideally one would be able to search for novel protein partners in living plant cells. We demonstrate that it is possible to screen for novel protein-protein interactions from a random library in protoplasted Arabidopsis plant cells and recover some of the interacting partners. Our screen is based on capturing the bi-molecular complementation of mYFP between an YN-bait fusion partner and a completely random prey YC-cDNA library with FACS. The candidate interactions were confirmed using in planta BiFC assays and in planta FRET-FLIM assays. From this work, we show that the well characterized protein Calcium Dependent Protein Kinase 3 (CPK3) interacts with APX3, HMGB5, ORP2A and a ricin B-related lectin domain containing protein At2g39050. This is one of the first randomin planta screens to be successfully employed.

Binary 2in1 Vectors Improve in Planta (Co)localization and Dynamic Protein Interaction Studies

The combination of new generation fluorescent proteins with the 2in1-cloning technique improves (co)localization and protein interaction analyses in vivo. Fluorescence-based protein-protein interaction techniques are vital tools for understanding in vivo cellular functions on a mechanistic level. However, only under the condition of highly efficient (co)transformation and accumulation can techniques such as Förster resonance energy transfer (FRET) realize their potential for providing highly accurate and quantitative interaction data. FRET as a fluorescence-based method unifies several advantages, such as measuring in an in vivo environment, real-time context, and the ability to include transient interactions as well as detecting the mere proximity of proteins. Here, we introduce a novel vector set that incorporates the benefit of the recombination-based 2in1 cloning system with the latest state-of-the-art fluorescent proteins for optimal coaccumulation and FRET output studies. We demonstrate its utility across a range of methods. Merging the 2in1 cloning system with new-generation FRET fluorophore pairs allows for enhanced detection, speeds up the preparation of clones, and enables colocalization studies and the identification of meaningful protein-protein interactions in vivo.

Determination of the in vivo redox potential by one-wavelength spectro-microscopy of roGFP

For the quantitative analysis of molecular processes in living (plant) cells, such as the perception and processing of environmental and endogenous signals, new combinatorial approaches in optical and spectroscopic technologies are required and partly already became established in many fields of the life sciences. One hallmark of the in vivo analysis of cell biological processes is the use of visible fluorescent proteins to create fluorescent fusion proteins. Recent progress has been made in generating a redox-sensitive mutant of green fluorescent proteins (roGFP), which exhibits alterations in its spectral properties in response to changes in the redox state of the surrounding medium. An established method to probe the local redox potential using roGFP is based on a ratiometric protocol. This readout modality requires two excitation wavelengths, which makes the technique less suited for in vivo studies of e.g. dynamic samples. We clarify the origin of the redox sensitivity of roGFP by ab initio calculations, which reveal a changed protonation equilibrium of the chromophore in dependence on the redox potential. Based on this finding, we test and compare different spectroscopic readout modalities with single wavelength excitation to determine the local redox potential and apply these techniques to live cell analytics.

Room temperature excitation spectroscopy of single quantum dots.

Summary We report a single molecule detection scheme to investigate excitation spectra of single emitters at room temperature. We demonstrate the potential of single emitter photoluminescence excitation spectroscopy by recording excitation spectra of single CdSe nanocrystals over a wide spectral range of 100 nm. The spectra exhibit emission intermittency, characteristic of single emitters. We observe large variations in the spectra close to the band edge, which represent the individual heterogeneity of the observed quantum dots. We also find specific excitation wavelengths for which the single quantum dots analyzed show an increased propensity for a transition to a long-lived dark state. We expect that the additional capability of recording excitation spectra at room temperature from single emitters will enable insights into the photophysics of emitters that so far have remained inaccessible.

Optical microresonator modifies the efficiency of the fluorescence resonance energy transfer in the autofluorescent protein DsRed

We investigate experimentally the modifications of the fluorescence properties of the bichromophoric fluorescent resonance energy transfer (FRET) system DsRed imposed by optical confinement. The confinement-condition is realized by a novel λ/2-microresonator that modifies the local photonic mode density in the vicinity of the proteins while maintaining a physiological environment for the embedded biological molecules. The experimental ratio of the fluorescence intensities and lifetimes, respectively, of donor and acceptor chromophores varies by up to a one order of magnitude as we vary the mirror spacing of the microresonator with nanometer-precision. Since these ratios determine the FRET efficiency, we modify the yield of the excited state energy transfer in rigidly coupled FRET pairs without chemically or physically perturbating the chromophoric subunits. We show that the microresonator-controlled inhibition of the acceptor fluorescence results in a loss of transfer efficiency of excited state energy from donor to acceptor, an effect that enables the spectral isolation and efficient observation of donor chromophores both in DsRed ensembles and on the single protein level. This constitutes an important application of microcavity-enhanced single molecule spectroscopy of biological systems and shows the potential of optical confinement for applications in nano-biophotonics.

Detecting the same individual protein and its photoproducts via fluorescence and surface-enhanced Raman spectroscopic imaging.

We present a novel multiparameter microscopy approach allowing for both fluorescence and Raman imaging and spectroscopy of the same individual autofluorescent protein and its photoproduct by colocalization of the same species in the respective spectroscopic images. For the investigated bichromophoric autofluorescent protein DsRed_N42H we are able to assign different Raman spectra to the photoproducts of the distinct chromophores. Furthermore, a careful analysis of Raman spectra taken from native proteins in comparison to Raman spectra from photobleached species allows for a feasible estimation of the underlying photodegeneration processes of the individual spectral forms.

Fluorescence intensity decay shape analysis microscopy (FIDSAM) for quantitative and sensitive live-cell imaging

Fluorescence microscopy became an invaluable tool in cell biology in the past 20 years. However, the information that lies in these studies is often corrupted by a cellular fluorescence background known as autofluorescence. Since the unspecific background often overlaps with most commonly used labels in terms of fluorescence spectra and fluorescence lifetime, the use of spectral filters in the emission beampath or timegating in fluorescence lifetime imaging (FLIM) is often no appropriate means for distinction between signal and background. Despite the prevalence of fluorescence techniques only little progress has been reported in techniques that specifically suppress autofluorescence or that clearly discriminate autofluorescence from label fluorescence. Fluorescence intensity decay shape analysis microscopy (FIDSAM) is a novel technique which is based on the image acquisition protocol of FLIM. Whereas FLIM spatially resolved maps the average fluorescence lifetime distribution in a heterogeneous sample such as a cell, FIDSAM enhances the dynamic image contrast by determination of the autofluorescence contribution by comparing the fluorescence decay shape to a reference function. The technique therefore makes use of the key difference between label and autofluorescence, i.e. that for label fluorescence only one emitting species contributes to fluorescence intensity decay curves whereas many different species of minor intensity contribute to autofluorescence. That way, we were able to suppress autofluorescence contributions from chloroplasts in Arabidopsis stoma cells and from cell walls in Arabidopsis hypocotyl cells to background level. Furthermore, we could extend the method to more challenging labels such as the cyan fluorescent protein CFP in human fibroblasts.

Multiparameter Single Molecule Spectroscopy gives insight into the complex Photophysics of Fluorescence Energy Transfer (FRET) coupled Biosystems

Since the discovery of the technique in the early 1990s, single molecule spectroscopy has been used as a powerful tool to investigate and characterize fluorescent molecules, revealing insights into molecular behavior far beyond the information content that can be obtained by conventional ensemble studies. Several spectroscopic techniques have been established at the single molecule level, including spectrally resolved fluorescence, fluorescence lifetime investigations, or single molecule Raman measurements. However, the combination of two or more of these spectroscopies applied to the same individual molecule in multiparameter approaches yields a deeper understanding of molecular systems. In this contribution, we present our results of combined spectrally- and time-resolved fluorescence microscopy of the intrinsic fluorescence energy transfer (FRET) system of the red fluorescent protein DsRed. Correlating the results obtained from the two spectroscopic techniques, we are able to determine all relevant parameters to describe the energy transfer processes within the DsRed system without any further assumptions. We further discuss fluorescence and surface enhanced Raman scattering (SERS) spectroscopy of the same individual DsRed unit, which can help to propose mechanisms for photodegeneration of the distinct chromophores involved.

Bimodal single molecule microscopy: multiparameter spectroscopy gives insight into photodegradation processes

The triumphal course of optical single molecule studies mainly focuses on fluorescence based techniques. However, the structural insight, which can be gained by these methods, is often rather limited due to the broad and unstructured fluorescence spectra. This restriction may be overcome by surface enhanced Raman spectroscopy (SERS), which provides a chemical fingerprint of the investigated species. Nevertheless, for complex systems such as e. g. proteins, the interpretation of the obtained spectra is often too intricate. We present a novel bimodal microscopy approach to correlate the information content of the two spectroscopic modes on the single molecule level. By performing both, fluorescence and SERS spectroscopy on the same individual bichromophoric autofluorescent protein, we are able to assign distinct Raman bands to isolated fluorescence forms. On basis of these data we compare Raman spectra of native and photobleached proteins independently for the two distinct spectral forms. This in turn enables us to study the individual photodegradation processes and to open the field of elucidating the chemical structure of these compounds by spectroscopic methods in the single molecule level.