Chimeric Autofluorescent Proteins as Photophysical Model System for Multicolor Bimolecular Fluorescence Complementation.

The yellow fluorescent protein (YFP) is frequently used in a protein complementation assay called bimolecular fluorescence complementation (BiFC), and is employed to visualize protein-protein interactions. In this analysis, two different, nonfluorescent fragments of YFP are genetically attached to proteins of interest. Upon interaction of these proteins, the YFP fragments are brought into proximity close enough to reconstitute their original structure, enabling fluorescence. BiFC allows for a straightforward readout of protein-protein interactions and furthermore facilitates their functional investigation by in vivo imaging. Furthermore, it has been observed that the available color range in BiFC can be extended upon complementing fragments of different proteins that are, like YFP, derived from the Aequorea victoria green fluorescent protein, thereby allowing for a multiplexed investigation of protein-protein interactions. Some spectral characteristics of "multicolor" BiFC (mcBiFC) complexes have been reported before; however, no in-depth analysis has been performed yet. Therefore, little is known about the photophysical characteristics of these mcBiFC complexes because a proper characterization essentially relies on in vitro data. This is particularly difficult for fragments of autofluorescent proteins (AFPs) because they show a very strong tendency to form supramolecular aggregates which precipitate ex vivo. In this study, this intrinsic difficulty is overcome by directly fusing the coding DNA of different AFP fragments. Translation of the genetic sequence in Escherichia coli leads to fully functional, highly soluble fluorescent proteins with distinct properties. On the basis of their construction, they are designated chimeric AFPs, or BiFC chimeras, here. Comparison of their spectral characteristics with experimental in vivo BiFC data confirmed the utility of the chimeric proteins as a BiFC model system. In this study, nine different chimeras were thoroughly analyzed at both the ensemble and the single-molecular level. The data indicates that mutations believed to be photophysically silent significantly alter the properties of AFPs.

Light-activated phytochrome A and B interact with members of the SPA family to promote photomorphogenesis in Arabidopsis by reorganizing the COP1/SPA complex.

Phytochromes function as red/far-red photoreceptors in plants and are essential for light-regulated growth and development. Photomorphogenesis, the developmental program in light, is the default program in seed plants. In dark-grown seedlings, photomorphogenic growth is suppressed by the action of the CONSTITUTIVELY PHOTOMORPHOGENIC1 (COP1)/SUPPRESSOR OF phyA-105 (SPA) complex, which targets positive regulators of photomorphogenic growth for degradation by the proteasome. Phytochromes inhibit the COP1/SPA complex, leading to the accumulation of transcription factors promoting photomorphogenesis; yet, the mechanism by which they inactivate COP1/SPA is still unknown. Here, we show that light-activated phytochrome A (phyA) and phytochrome B (phyB) interact with SPA1 and other SPA proteins. Fluorescence resonance energy transfer-fluorescence lifetime imaging microscopy analyses show that SPAs and phytochromes colocalize and interact in nuclear bodies. Furthermore, light-activated phyA and phyB disrupt the interaction between COP1 and SPAs, resulting in reorganization of the COP1/SPA complex in planta. The light-induced stabilization of HFR1, a photomorphogenic factor targeted for degradation by COP1/SPA, correlates temporally with the accumulation of phyA in the nucleus and localization of phyA to nuclear bodies. Overall, these data provide a molecular mechanism for the inactivation of the COP1/SPA complex by phyA- and phyB-mediated light perception.

Photosynthesis in a different light: spectro-microscopy for in vivo characterization of chloroplasts.

During photosynthesis, energy conversion at the two photosystems is controlled by highly complex and dynamic adaptation processes triggered by external factors such as light quality, intensity, and duration, or internal cues such as carbon availability. These dynamics have remained largely concealed so far, because current analytical techniques are based on the investigation of isolated chloroplasts lacking full adaptation ability and are performed at non-physiologically low temperatures. Here, we use non-invasive in planta spectro-microscopic approaches to investigate living chloroplasts in their native environment at ambient temperatures. This is a valuable approach to study the complex function of these systems, because an intrinsic property-the fluorescence emission-is exploited and no additional external perturbations are introduced. Our analysis demonstrates a dynamic adjustment of not only the photosystemI/photosystemII (PSI/PSII) intensity ratio in the chloroplasts but also of the capacity of the LHCs for energy transfer in response to environmental and internal cues.

Dynamic control of Förster energy transfer in a photonic environment.

In this study, the effect of modified optical density of states on the rate of Förster resonant energy transfer between two closely-spaced chromophores is investigated. A model based on a system of coupled rate equations is derived to predict the influence of the environment on the molecular system. Due to the near-field character of Förster transfer, the corresponding rate constant is shown to be nearly independent of the optical mode density. An optical resonator can, however, effectively modify the donor and acceptor populations, leading to a dramatic change in the Förster transfer rate. Single-molecule measurements on the autofluorescent protein DsRed using a λ/2-microresonator are presented and compared to the theoretical model's predictions. The observed resonator-induced dequenching of the donor subunit in DsRed is accurately reproduced by the model, allowing a direct measurement of the Förster transfer rate in this otherwise inseparable multichromophoric system. With this accurate yet simple theoretical framework, new experiments can be conceived to measure normally obscured energy transfer channels in complex coupled quantum systems, e.g. in photovoltaics or light harvesting complexes.

Fluorescence microscopy.

Optical microscopy has developed as an indispensable tool for Arabidopsis cell biology. This is due to the high sensitivity, good spatial resolution, minimal invasiveness, and availability of autofluorescent proteins, which can be specifically fused to a distinct protein of interest. In this chapter, we introduce the theoretical concepts of fluorescence emission necessary to accomplish quantitative and functional cell biology using optical microscopy. The main focus lies on spectroscopic techniques, which, in addition to intensity-based studies, provide functional insight into cellular processes.

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.

Electrodynamic coupling of electric dipole emitters to a fluctuating mode density within a nanocavity.

We investigate the impact of rotational diffusion on the electrodynamic coupling of fluorescent dye molecules (oscillating electric dipoles) to a tunable planar metallic nanocavity. Fast rotational diffusion of the molecules leads to a rapidly fluctuating mode density of the electromagnetic field along the molecules' dipole axis, which significantly changes their coupling to the field as compared to the opposite limit of fixed dipole orientation. We derive a theoretical treatment of the problem and present experimental results for rhodamine 6G molecules in cavities filled with low and high viscosity liquids. The derived theory and presented experimental method is a powerful tool for determining absolute quantum yield values of fluorescence.

Degradation of Arabidopsis CRY2 is regulated by SPA proteins and phytochrome A.

The UV-A/blue light photoreceptor crytochrome2 (cry2) plays a fundamental role in the transition from the vegetative to the reproductive phase in the facultative long-day plant Arabidopsis thaliana. The cry2 protein level strongly decreases when etiolated seedlings are exposed to blue light; cry2 is first phosphorylated, polyubiquitinated, and then degraded by the 26S proteasome. COP1 is involved in cry2 degradation, but several cop1 mutants show only reduced but not abolished cry2 degradation. SUPPRESSOR OF PHYA-105 (SPA) proteins are known to work in concert with COP1, and recently direct physical interaction between cry2 and SPA1 was demonstrated. Thus, we hypothesized that SPA proteins could also play a role in cry2 degradation. To this end, we analyzed cry2 protein levels in spa mutants. In all spa mutants analyzed, cry2 degradation under continuous blue light was alleviated in a fluence rate-dependent manner. Consistent with a role of SPA proteins in phytochrome A (phyA) signaling, a phyA mutant had enhanced cry2 levels, particularly under low fluence rate blue light. Fluorescence resonance energy transfer-fluorescence lifetime imaging microscopy studies showed a robust physical interaction of cry2 with SPA1 in nuclei of living cells. Our results suggest that cry2 stability is controlled by SPA and phyA, thus providing more information on the molecular mechanisms of interaction between cryptochrome and phytochrome photoreceptors.

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.

Spectro-microscopy of living plant cells.

Spectro-microscopy, a combination of fluorescence microscopy with spatially resolved spectroscopic techniques, provides new and exciting tools for functional cell biology in living organisms. This review focuses on recent developments in spectro-microscopic applications for the investigation of living plant cells in their native tissue context. The application of spectro-microscopic methods led to the recent discovery of a fast signal response pathway for the brassinosteroide receptor BRI1 in the plasma membrane of living plant cells. Moreover, the competence of different plant cell types to respond to environmental or endogenous stimuli was determined in vivo by correlation analysis of different optical and spectroscopic readouts such as fluorescence lifetime (FLT). Furthermore, a new spectro-microscopic technique, fluorescence intensity decay shape analysis microscopy (FIDSAM), has been developed. FIDSAM is capable of imaging low-expressed fluorophore-tagged proteins at high spatial resolution and precludes the misinterpretation of autofluorescence artifacts. In addition, FIDSAM provides a very effective and sensitive tool on the basis of Förster resonance energy transfer (FRET) for the qualitative and quantitative determination of protein-protein interaction. Finally, we report on the quantitative analysis of the photosystem I and II (PSI/PSII) ratio in the chloroplasts of living Arabidopsis plants at room temperature, using high-resolution, spatially resolved fluorescence spectroscopy. With this technique, it was not only possible to measure PSI/PSII ratios, but also to demonstrate the differential competence of wild-type and carbohydrate-deficient plants to adapt the PSI/PSII ratio to different light conditions. In summary, the information content of standard microscopic images is extended by several dimensions by the use of spectro-microscopic approaches. Therefore, novel cell physiological and molecular topics can be addressed and valuable insights into molecular and subcellular processes can be obtained in living plants.

Room temperature excitation spectroscopy of single quantum dots.

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.

The activation of the Arabidopsis P-ATPase 1 by the brassinosteroid receptor BRI1 is independent of threonine 948 phosphorylation.

The plasma membrane-spanning receptor brassinosteroid insenstive 1 (BRI1) rapidly induces plant cell wall expansion in response to brassinosteroids such as brassinolide (BL). Wall expansion is accompanied by a rapid hyperpolarisation of the plasma membrane which is recordable by measuring the fluorescence lifetime (FLT) of the green fluorescent protein (GFP) fused to BRI1. For the BL induction of hyperpolarisation and wall expansion, the activation of the plasma membrane P-type H+-ATPase is necessary. Furthermore, the activation of the P-ATPase requires BRI1 kinase activity and appears to be mediated by a BL-modulated association of BRI1 with the proton pump. Here, we show that BRI1 also associates with a mutant version of the Arabidopsis P-ATPase 1 (AHA1) characterized by an exchange of a well-known regulatory threonine for a non-phosphorylatable residue in the auto-inhibitory C-terminal domain. Even more important, BRI1 is still able to activate this AHA1 mutant in response to BL. This suggests a novel mechanism for the enzymatic activation of the P-ATPase by BRI1 in the plasma membrane. Furthermore, we demonstrate that the FLT of BRI1-GFP can be used as a non-invasive probe to analyse long-distance BL signaling in Arabidopsis seedlings.

Alanine zipper-like coiled-coil domains are necessary for homotypic dimerization of plant GAGA-factors in the nucleus and nucleolus.

GAGA-motif binding proteins control transcriptional activation or repression of homeotic genes. Interestingly, there are no sequence similarities between animal and plant proteins. Plant BBR/BPC-proteins can be classified into two distinct groups: Previous studies have elaborated on group I members only and so little is known about group II proteins. Here, we focused on the initial characterization of AtBPC6, a group II protein from Arabidopsis thaliana. Comparison of orthologous BBR/BPC sequences disclosed two conserved signatures besides the DNA binding domain. A first peptide signature is essential and sufficient to target AtBPC6-GFP to the nucleus and nucleolus. A second domain is predicted to form a zipper-like coiled-coil structure. This novel type of domain is similar to Leucine zippers, but contains invariant alanine residues with a heptad spacing of 7 amino acids. By yeast-2-hybrid and BiFC-assays we could show that this Alanine zipper domain is essential for homotypic dimerization of group II proteins in vivo. Interhelical salt bridges and charge-stabilized hydrogen bonds between acidic and basic residues of the two monomers are predicted to form an interaction domain, which does not follow the classical knobs-into-holes zipper model. FRET-FLIM analysis of GFP/RFP-hybrid fusion proteins validates the formation of parallel dimers in planta. Sequence comparison uncovered that this type of domain is not restricted to BBR/BPC proteins, but is found in all kingdoms.

A fast brassinolide-regulated response pathway in the plasma membrane of Arabidopsis thaliana.

To understand molecular processes in living plant cells, quantitative spectro-microscopic technologies are required. By combining fluorescence lifetime spectroscopy with confocal microscopy, we studied the subcellular properties and function of a GFP-tagged variant of the plasma membrane-bound brassinosteroid receptor BRI1 (BRI1-GFP) in living cells of Arabidopsis seedlings. Shortly after adding brassinolide, we observed BRI1-dependent cell-wall expansion, preceding cell elongation. In parallel, the fluorescence lifetime of BRI1-GFP decreased, indicating an alteration in the receptor's physico-chemical environment. The parameter modulating the fluorescence lifetime of BRI1-GFP was found to be BL-induced hyperpolarization of the plasma membrane. Furthermore, for induction of hyperpolarization and cell-wall expansion, activation of the plasma membrane P-ATPase was necessary. This activation required BRI1 kinase activity, and was mediated by BL-modulated interaction of BRI1 with the P-ATPase. Our results were used to develop a model suggesting that there is a fast BL-regulated signal response pathway within the plasma membrane that links BRI1 with P-ATPase for the regulation of cell-wall expansion.

Application of FLIM-FIDSAM for the in vivo analysis of hormone competence of different cell types.

Background fluorescence derived from subcellular compartments is a major drawback in high-resolution live imaging, especially of plant cells. A novel technique for contrast enhancement of fluorescence images of living cells expressing fluorescent fusion proteins termed fluorescence intensity decay shape analysis microscopy (FIDSAM) has been recently published and is applied here to plant cells expressing wild-type levels of a low-abundant membrane protein (BRI1-EGFP), demonstrating the applicability of FIDSAM to samples exhibiting about 80% autofluorescence. Furthermore, the combination of FIDSAM and fluorescence lifetime imaging microscopy enables the simultaneous determination and quantification of different ligand-specific responses in living cells with high spatial and temporal resolution even in samples with high autofluorescence background. Correlation of different responses can be used to determine the hormone ligand competence of different cell types as demonstrated here in BRI1-EGFP-expressing root and hypocotyl cells.

Analysis of Arabidopsis with highly reduced levels of malate and fumarate sheds light on the role of these organic acids as storage carbon molecules.

While malate and fumarate participate in a multiplicity of pathways in plant metabolism, the function of these organic acids as carbon stores in C(3) plants has not been deeply addressed. Here, Arabidopsis (Arabidopsis thaliana) plants overexpressing a maize (Zea mays) plastidic NADP-malic enzyme (MEm plants) were used to analyze the consequences of sustained low malate and fumarate levels on the physiology of this C(3) plant. When grown in short days (sd), MEm plants developed a pale-green phenotype with decreased biomass and increased specific leaf area, with thin leaves having lower photosynthetic performance. These features were absent in plants growing in long days. The analysis of metabolite levels of rosettes from transgenic plants indicated similar disturbances in both sd and long days, with very low levels of malate and fumarate. Determinations of the respiratory quotient by the end of the night indicated a shift from carbohydrates to organic acids as the main substrates for respiration in the wild type, while MEm plants use more reduced compounds, like fatty acids and proteins, to fuel respiration. It is concluded that the alterations observed in sd MEm plants are a consequence of impairment in the supply of carbon skeletons during a long dark period. This carbon starvation phenotype observed at the end of the night demonstrates a physiological role of the C(4) acids, which may be a constitutive function in plants.

Fluorescence intensity decay shape analysis microscopy (FIDSAM) for quantitative and sensitive live-cell imaging: a novel technique for fluorescence microscopy of endogenously expressed fusion-proteins.

Fluorescent studies of living plant cells such as confocal microscopy and fluorescence lifetime imaging often suffer from a strong autofluorescent background contribution that significantly reduces the dynamic image contrast and the quantitative access to sub-cellular processes at high spatial resolution. Here, we present a novel technique--fluorescence intensity decay shape analysis microscopy (FIDSAM)--to enhance the dynamic contrast of a fluorescence image of at least one order of magnitude. The method is based on the analysis of the shape of the fluorescence intensity decay (fluorescence lifetime curve) and benefits from the fact that the decay patterns of typical fluorescence label dyes strongly differ from emission decay curves of autofluorescent sample areas. Using FIDSAM, we investigated Arabidopsis thaliana hypocotyl cells in their tissue environment, which accumulate an eGFP fusion of the plasma membrane marker protein LTI6b (LTI6b-eGFP) to low level. Whereas in conventional confocal fluorescence images, the membranes of neighboring cells can hardly be optically resolved due to the strong autofluorescence of the cell wall, FIDSAM allows for imaging of single, isolated membranes at high spatial resolution. Thus, FIDSAM will enable the sub-cellular analysis of even low-expressed fluorophore-tagged proteins in living plant cells. Furthermore, the combination of FIDSAM with fluorescence lifetime imaging provides the basis to study the local physico-chemical environment of fluorophore-tagged biomolecules in living plant cells.

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.

Microcavities: tailoring the optical properties of single quantum emitters.

We present a general review of different microresonator structures and how they can be used in future device applications in modern analytical methods by tailoring the optical properties of single quantum emitters. The main emphasis is on the tunable lambda/2-Fabry-Perot-type microresonator which we used to obtain the results presented in this article. By varying the mirror distance the local mode structure of the electromagnetic field is altered and thus the radiative coupling of fluorescent single quantum emitters embedded inside the resonator to that field is changed, too. As a result a modification of the optical properties of these quantum emitters can be observed. We present experimental as well as theoretical results illustrating this effect. Furthermore, the developed resonator can be used to determine the longitudinal position of embedded emitters with an accuracy of lambda/60 by analyzing the excitation patterns of nano-sized fluorescent polymer spheres after excitation with a radially polarized doughnut mode laser beam. Finally, we will apply this resonator to a biological system and demonstrate the modification of Förster resonant energy transfer (FRET) efficiency by inhibiting the excited state energy transfer from the donor to the acceptor chromophore of a single DsRed protein.

Novel application of fluorescence lifetime and fluorescence microscopy enables quantitative access to subcellular dynamics in plant cells.

BACKGROUND: Optical and spectroscopic technologies working at subcellular resolution with quantitative output are required for a deeper understanding of molecular processes and mechanisms in living cells. Such technologies are prerequisite for the realisation of predictive biology at cellular and subcellular level. However, although established in the physical sciences, these techniques are rarely applied to cell biology in the plant sciences. PRINCIPAL FINDINGS: Here, we present a combined application of one-chromophore fluorescence lifetime microscopy and wavelength-selective fluorescence microscopy to analyse the function of a GFP fusion of the Brassinosteroid Insensitive 1 Receptor (BRI1-GFP) with high spatial and temporal resolution in living Arabidopsis cells in their tissue environment. We show a rapid, brassinolide-induced cell wall expansion and a fast BR-regulated change in the BRI1-GFP fluorescence lifetime in the plasmamembrane in vivo. Both cell wall expansion and changes in fluorescence lifetime reflect early BR-induced and BRI1-dependent physiological or signalling processes. Our experiments also show the potential of one-chromophore fluorescence lifetime microscopy for the in vivo monitoring of the biochemical and biophysical subcellular environment using GFP fusion proteins as probes. SIGNIFICANCE: One-chromophore fluorescence lifetime microscopy, combined with wavelength-specific fluorescence microscopy, opens up new frontiers for in vivo dynamic and quantitative analysis of cellular processes at high resolution which are not addressable by pure imaging technologies or transmission electron microscopy.