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The sequestration of enzymes and associated processes into sub-cellular domains, called organelles, is considered a defining feature of eukaryotic cells. However, what leads to specific outcomes and allows a eukaryotic cell to function singularly is the interactivity and exchanges between discrete organelles. Our ability to observe and assess sub-cellular interactions in living plant cells has expanded greatly following the creation of fluorescent fusion proteins targeted to different organelles. Notably, organelle interactivity changes quickly in response to stress and reverts to a normal less interactive state as homeostasis is re-established. Using key observations of some of the organelles present in a plant cell, this chapter provides a brief overview of our present understanding of organelle interactions in plant cells.

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Macrophages are a type of white blood cells that play key roles in innate immune responses as a part of cellular immunity for host defence and tissue homeostasis. To perform diverse functions, macrophages show high plasticity by transforming to polarized states. They are mainly identified as unpolarized, pro-inflammatory and antiinflammatory states and termed as M0, M1 and M2 macrophages respectively. Discriminating polarized states is important due to strict implication with inflammatory conditions resulting in many diseases as chronic inflammation, neurodegeneration, and cancer etc. Many polarization protein markers have been identified and applied to investigate expression profiles through PCR and other techniques with antibodies. However, they are time and cost consuming and sometimes show insufficient performances. We focused on the mannose receptor (CD206) as representative marker of M2 macrophage recognising terminal mannose. We developed dose dependent mannosylated fluorescent proteins (FPs) by conjugations with mannose derivative for around 20 modifiable sites on FPs surfaces. Maximum modifications did not spoil various features of FPs. We found further sensitive and specific discriminations among M2, M1 and M0 macrophages after treating polarized macrophages with adequately conditioned FPs compared to already established approaches using anti CD206 antibody through flow cytometric analysis. These results might be derived from direct ligand utilizations and increased avidity due to multivalent bindings with abundantly modified multimeric FPs. Our strategy is simple but addresses disadvantages of preceding methods. Moreover, this strategy is applicable to detect other cell surface receptors as FPs can be modified with ligands or recognizable aptamer like molecules.

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Epigenetic modifications (methylation, acetylation, etc.) of core histones play a key role in regulation of gene expression. Thus, the epigenome changes strongly during various biological processes such as cell differentiation and dedifferentiation. Classical methods of analysis of epigenetic modifications such as mass-spectrometry and chromatin immuno-precipitation, work with fixed cells only. Here we present a genetically encoded fluorescent probe, MPP8-Green, for detecting H3K9me3, a histone modification associated with inactive chromatin. This probe, based on the chromodomain of MPP8, allows for visualization of H3K9me3 epigenetic landscapes in single living cells. We used this probe to track changes in H3K9me3 landscapes during the differentiation of induced pluripotent stem cells (iPSCs) into induced neurons. Our findings revealed two major waves of global H3K9me3 reorganization during 4-day differentiation, namely on the first and third days, whereas nearly no changes occurred on the second and fourth days. The proposed method LiveMIEL (Live-cell Microscopic Imaging of Epigenetic Landscapes), which combines genetically encoded epigenetic probes and machine learning approaches, enables classification of multiparametric epigenetic signatures of single cells during stem cell differentiation and potentially in other biological models.

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Crystal polymorphism serves as a strategy to study the conformational flexibility of proteins. However, the relationship between protein crystal packing and protein conformation often remains elusive. In this study, two distinct crystal forms of a green fluorescent protein variant, NowGFP, are compared: a previously identified monoclinic form (space group C2) and a newly discovered orthorhombic form (space group P212121). Comparative analysis reveals that both crystal forms exhibit nearly identical linear assemblies of NowGFP molecules interconnected through similar crystal contacts. However, a notable difference lies in the stacking of these assemblies: parallel in the monoclinic form and perpendicular in the orthorhombic form. This distinct mode of stacking leads to different crystal contacts and induces structural alteration in one of the two molecules within the asymmetric unit of the orthorhombic crystal form. This new conformational state captured by orthorhombic crystal packing exhibits two unique features: a conformational shift of the ß-barrel scaffold and a restriction of pH-dependent shifts of the key residue Lys61, which is crucial for the pH-dependent spectral shift of this protein. These findings demonstrate a clear connection between crystal packing and alternative conformational states of proteins, providing insights into how structural variations influence the function of fluorescent proteins.

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Far-red fluorescent proteins (FPs) have emerged as indispensable tools in in vivo imaging, playing a pivotal role in elucidating fundamental mechanisms and addressing application issues in biotechnology and biomedical fields. Their ability for deep penetration, coupled with reduced light scattering and absorption, robust resistance to autofluorescence, and diminished phototoxicity, has positioned far-red biosensors at the forefront of non-invasive visualization techniques for observing intracellular activities and intercellular behaviors. In this review, far-red FPs and their applications in living systems are mainly discussed. Firstly, various far-red FPs, characterized by emission peaks spanning from 600 nm to 650 nm, are introduced. This is followed by a detailed presentation of the fundamental principles enabling far-red biosensors to detect biomolecules and environmental changes. Furthermore, the review accentuates the superiority of far-red FPs in multi-color imaging. In addition, significant emphasis is placed on the value of far-red FPs in improving imaging resolution, highlighting their great contribution to the advancement of in vivo imaging.

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The detection of mercury ions is an important task in both environmental monitoring and cell biology research. However, existing genetically encoded sensors for mercury ions have certain limitations, such as negative fluorescence response, narrow dynamic range, or the need for cofactor supplementation. To address these limitations, we have developed novel sensors by fusing a circularly permutated version of the mNeonGreen green fluorescent protein with the merP mercury-binding protein from Gram-negative bacteria Shigella flexneri. The developed NeMeHg and iNeMeHg sensors responded to mercury ions with positive and negative fluorescence changes, respectively. We characterized their properties in vitro. Using the developed biosensors, we were able to successfully visualize changes in mercury ion concentration in mammalian cultured cells.

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Peri-nuclear clustering (PNC) of chloroplasts has largely been described in senescent and pathogen- or ROS- stressed cells. Stromules, tubular plastid extensions are also observed under similar conditions. Coincident observations of PNC and stromules associate the two phenomena in facilitating retrograde signaling between chloroplasts and the nucleus. However, PNC incidence in non-stressed cells under normal growth and developmental conditions, when stromules are usually not observed, remains unclear. Using transgenic Arabidopsis expressing different organelle-targeted fluorescent proteins we show that PNC is a dynamic subcellular phenomenon that continues in the absence of light and is not dependent on stromule formation. PNC is facilitated by tandem plastid-ER dynamics created through membrane contact sites between the two organelles. While PNC increases upon ER-membrane expansion, some plastids may remain in the peri-nuclear region due to their localization in ER-lined nuclear indentions. Moreover, some PNC plastids may sporadically extend stromules into ER-lined nuclear grooves. Our findings strongly suggest that PNC is not an exclusive response to stress caused by pathogens, high light or exogenous-H2O2 treatment and does not require stromule formation. However, morphological and behavioural alterations in ER and concomitant changes in tandem, plastid-ER dynamics play a major role in facilitating the phenomenon.

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We present mCLIFY: a monomeric, bright, yellow, and long-lived fluorescent protein (FP) created by circular permutation of YPet, the brightest yellow FP from Aequorea Victoria for use in cellular and in vitro single molecule studies. mCLIFY retains the enhanced photophysical properties of YPET as a monomer at concentrations ≤ 40 µM. In contrast, we determined that YPet has a dimerization dissociation constant (K D 1-2) of 3.4 µM. Dimerization of YPet can cause homo-FRET, which underlies quantitative errors due to dimerization and homo-FRET. We determined the atomic structure of mCLIFY at 1.57 Å resolution and used its similarity with Venus for guided chromophore-targeted substitution studies to provide insights into its enhanced photophysical properties. The mutation V58L within the chromophore pocket improved quantum yield and extinction coefficient, making mCLIFY ~30% brighter than Venus. The extensive characterization of the photophysical and structural properties of YPet and mCLIFY presented here allowed us to reveal the basis of their long lifetimes and enhanced brightness and the basis of YPet's dimerization.

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Proteins often show alterations in their subcellular localization with changing environmental conditions; transcription factors enter the nucleus or are actively removed from the nucleus; some even bind to endo-membranes by conditional membrane anchors; and other proteins and mRNA arrange in RNA granules. These are some examples of the complex regulation of subcellular localization, which often depends on posttranslational modifications and is triggered by environmental stressors. The challenge is the precise identification of the compartments, the quantitative analysis of proteins, which reside in multiple compartments, and their transport dynamics. Therefore, appropriate compartment markers and routines for a reproducible quantitative workflow are required.

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Large Stokes shift red fluorescent proteins (LSS-RFPs) are genetically encoded and exhibit a significant difference of a few hundreds of nanometers between their excitation and emission peak maxima (i.e., the Stokes shift). These LSS-RFPs (absorbing blue light and emitting red light) feature a unique photocycle responsible for their significant Stokes shift. The photocycle associated with this LSS characteristic in certain RFPs is quite perplexing, hinting at the complex nature of excited-state photophysics. This article provides a brief review on the fundamental mechanisms governing the photocycle of various LSS-RFPs, followed by a discussion on experimental results on mKeima emphasizing its relaxation pathways which garnered attention due to its >200 nm Stokes shift. Corroborating steady-state spectroscopy with computational studies, four different forms of chromophore of mKeima contributing to the cis-trans conformers of the neutral and anionic forms were identified in a recent study. Furthering these findings, in this account a detailed discussion on the photocycle of mKeima, which encompasses sequential excited-state isomerization, proton transfer, and subsequent structural reorganization involving three isomers, leading to an intriguing temperature and pH-dependent dual fluorescence, is explored using broadband femtosecond transient absorption spectroscopy.

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BACKGROUND: Fluorescent proteins (FPs) are pivotal reagents for flow cytometry analysis or fluorescent microscopy. A new generation of immunoreagents (fluobodies/chromobodies) has been developed by fusing recombinant nanobodies to FPs. METHODS: We analyzed the quality of such biomolecules by a combination of gel filtration and SDS-PAGE to identify artefacts due to aggregation or material degradation. RESULTS: In the SDS-PAGE run, unexpected bands corresponding to separate fluobodies were evidenced and characterized as either degradation products or artefacts that systematically resulted in the presence of specific FPs and some experimental conditions. The elimination of N-terminal methionine from FPs did not impair the appearance of FP fragments, whereas the stability and migration characteristics of some FP constructs were strongly affected by heating in loading buffer, which is a step samples undergo before electrophoretic separation. CONCLUSIONS: In this work, we provide explanations for some odd results observed during the quality control of fluobodies and summarize practical suggestions for the choice of the most convenient FPs to fuse to antibody fragments.

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Standardized and thoroughly characterized genetic tools are a prerequisite for studying cellular processes to ensure the reusability and consistency of experimental results. The discovery of fluorescent proteins (FPs) represents a milestone in the development of genetic reporters for monitoring transcription or protein localization in vivo. FPs have revolutionized our understanding of cellular dynamics by enabling the real-time visualization and tracking of biological processes. Despite these advancements, challenges remain in the appropriate use of FPs, specifically regarding their proper application, protein turnover dynamics, and the undesired disruption of cellular functions. Here, we systematically compared a comprehensive set of 15 FPs and assessed their performance in vivo by focusing on key parameters, such as signal over background ratios and protein stability rates, using the Gram-negative model organism Salmonella enterica as a representative host. We evaluated four protein degradation tags in both plasmid- and genome-based systems and our findings highlight the necessity of introducing degradation tags to analyze time-sensitive cellular processes. We demonstrate that the gain of dynamics mediated by the addition of degradation tags impacts the cell-to-cell heterogeneity of plasmid-based but not genome-based reporters. Finally, we probe the applicability of FPs for protein localization studies in living cells using standard and super-resolution fluorescence microscopy. In summary, our study underscores the importance of careful FP selection and paves the way for the development of improved genetic reporters to enhance the reproducibility and reliability of fluorescence-based research in Gram-negative bacteria and beyond.

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The topology of the transcription factor network (TFN) of Escherichia coli is far from uniform, with 22 global regulator (GR) proteins controlling one-third of all genes. So far, their production rates cannot be tracked by comparable fluorescent proteins. We developed a library of fluorescent reporters for 16 GRs for this purpose. Each consists of a single-copy plasmid coding for green fluorescent protein (GFP) fused to the full-length copy of the native promoter. We tracked their activity in exponential and stationary growth, as well as under weak and strong stresses. We show that the reporters have high sensitivity and specificity to all stresses tested and detect single-cell variability in transcription rates. Given the influence of GRs on the TFN, we expect that the new library will contribute to dissecting global transcriptional stress-response programs of E. coli. Moreover, the library can be invaluable in bioindustrial applications that tune those programs to, instead of cell growth, favor productivity while reducing energy consumption.IMPORTANCECells contain thousands of genes. Many genes are involved in the control of cellular activities. Some activities require a few hundred genes to run largely synchronous transcriptional programs. To achieve this, cells have evolved global regulator (GR) proteins that can influence hundreds of genes simultaneously. We have engineered a library of Escherichia coli strains to track the levels over time of these, phenotypically critical, GRs. Each strain has a single-copy plasmid coding for a fast-maturing green fluorescent protein whose transcription is controlled by a copy of the natural GR promoter. By allowing the tracking of GR levels, with sensitivity and specificity, this library should become of wide use in scientific research on bacterial gene expression (from molecular to synthetic biology) and, later, be used in applications in therapeutics and bioindustries.

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Genetically encoded fluorescent metal ion sensors are powerful tools for elucidating metal dynamics in living systems. Over the last 25 years since the first examples of genetically encoded fluorescent protein-based calcium indicators, this toolbox of probes has expanded to include other essential and non-essential metal ions. Collectively, these tools have illuminated fundamental aspects of metal homeostasis and trafficking that are crucial to fields ranging from neurobiology to human nutrition. Despite these advances, much of the application of metal ion sensors remains limited to mammalian cells and tissues and a limited number of essential metals. Applications beyond mammalian systems and in vivo applications in living organisms have primarily used genetically encoded calcium ion sensors. The aim of this Perspective is to provide, with the support of historical and recent literature, an updated and critical view of the design and use of fluorescent protein-based sensors for detecting essential metal ions in various organisms. We highlight the historical progress and achievements with calcium sensors and discuss more recent advances and opportunities for the detection of other essential metal ions. We also discuss outstanding challenges in the field and directions for future studies, including detecting a wider variety of metal ions, developing and implementing a broader spectral range of sensors for multiplexing experiments, and applying sensors to a wider range of single- and multi-species biological systems.

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Vasorin (VASN), a transmembrane protein heavily expressed in endothelial cells, has garnered recent interest due to its key role in vascular development and pathology. The oligomeric state of VASN is a crucial piece of knowledge given that receptor clustering is a frequent regulatory mechanism in downstream signaling activation and amplification. However, documentation of VASN oligomerization is currently absent. In this brief report, we describe the measurement of VASN oligomerization in its native membranous environment, leveraging a class of fluorescence fluctuation spectroscopy. Our investigation revealed that the majority of VASN resides in a monomeric state, while a minority of VASN forms homodimers in the cellular membrane. This result raises the intriguing possibility that ligand-independent clustering of VASN may play a role in transforming growth factor signaling.

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Wild-type SAASoti and its monomeric variant mSAASoti can undergo phototransformations, including reversible photoswitching of the green form to a nonfluorescent state and irreversible green-to-red photoconversion. In this study, we extend the photochemistry of mSAASoti variants to enable reversible photoswitching of the red form. This result is achieved by rational and site-saturated mutagenesis of the M163 and F177 residues. In the case of mSAASoti it is M163T substitution that leads to the fastest switching and the most photostable variant, and reversible photoswitching can be observed for both green and red forms when expressed in eukaryotic cells. We obtained a 13-fold increase in the switching efficiency with the maximum switching contrast of the green form and the appearance of comparable switching of the red form for the C21N/M163T mSAASoti variant. The crystal structure of the C21N mSAASoti in its green on-state was obtained for the first time at 3.0 Å resolution, and it is in good agreement with previously calculated 3D-model. Dynamic network analysis reveals that efficient photoswitching occurs if motions of the 66H residue and phenyl fragment of chromophore are correlated and these moieties belong to the same community.

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Here, we demonstrate the beneficial effect of surfactant-producing pseudomonads on Pantoea eucalypti 299R. We conducted a series of experiments in environments of increasing complexity. P. eucalypti 299R (Pe299R), and Pseudomonas sp. FF1 (Pff1) or Pe299R and surfactant-production deficient Pseudomonas sp. FF1::ΔviscB (Pff1ΔviscB) were co-inoculated in broth, on swarming agar plates, and on plants. In broth, there were no differences in the growth dynamics of Pe299R when growing in the presence of Pff1 or Pff1ΔviscB. By contrast, on swarming agar plates, Pe299R was able to co-swarm with Pff1 which led to a significant increase in Pe299R biomass compared to Pe299R growing with Pff1ΔviscB or in monoculture. Finally in planta, and using the single-cell bioreporter for reproductive success (CUSPER), we found a temporally distinct beneficial effect of Pff1 on co-inoculated Pe299R subpopulations that did not occur in the presence of Pff1ΔviscB. We tested three additional surfactant-producing pseudomonads and their respective surfactant knockout mutants on PE299R on swarming agar showing similar results. This led us to propose a model for the positive effect of surfactant production during leaf colonization. Our results indicate that co-motility might be common during leaf colonization and adds yet another facet to the already manyfold roles of surfactants.

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The objective of the current review is to summarize the current state of optical methods in redox biology. It consists of two parts, the first is dedicated to genetically encoded fluorescent indicators and the second to Raman spectroscopy. In the first part, we provide a detailed classification of the currently available redox biosensors based on their target analytes. We thoroughly discuss the main architecture types of these proteins, the underlying engineering strategies for their development, the biochemical properties of existing tools and their advantages and disadvantages from a practical point of view. Particular attention is paid to fluorescence lifetime imaging microscopy as a possible readout technique, since it is less prone to certain artifacts than traditional intensiometric measurements. In the second part, the characteristic Raman peaks of the most important redox intermediates are listed, and examples of how this knowledge can be implemented in biological studies are given. This part covers such fields as estimation of the redox states and concentrations of Fe-S clusters, cytochromes, other heme-containing proteins, oxidative derivatives of thiols, lipids, and nucleotides. Finally, we touch on the issue of multiparameter imaging, in which biosensors are combined with other visualization methods for simultaneous assessment of several cellular parameters.

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Fluorescence in marine animals has mainly been studied in Cnidaria but is found in many different phyla such as Annelida, Crustacea, Mollusca, and Chordata. While many fluorescent proteins and molecules have been identified, very little information is available about the biological functions of fluorescence. In this review, we focus on describing the occurrence of fluorescence in marine animals and the behavioural and physiological functions of fluorescent molecules based on experimental approaches. These biological functions of fluorescence range from prey and symbiont attraction, photoprotection, photoenhancement, stress mitigation, mimicry, and aposematism to inter- and intraspecific communication. We provide a comprehensive list of marine taxa that utilise fluorescence, including demonstrated effects on behavioural or physiological responses. We describe the numerous known functions of fluorescence in anthozoans and their underlying molecular mechanisms. We also highlight that other marine taxa should be studied regarding the functions of fluorescence. We suggest that an increase in research effort in this field could contribute to understanding the capacity of marine animals to respond to negative effects of climate change, such as rising sea temperatures and increasing intensities of solar irradiation.

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Flavin-based fluorescent proteins are oxygen-independent reporters that hold great promise for imaging anaerobic and hypoxic biological systems. In this study, we explored the feasibility of applying circular permutation, a valuable method for the creation of fluorescent sensors, to flavin-based fluorescent proteins. We used rational design and structural data to identify a suitable location for circular permutation in iLOV, a flavin-based reporter derived from A. thaliana. However, relocating the N- and C-termini to this position resulted in a significant reduction in fluorescence. This loss of fluorescence was reversible, however, by fusing dimerizing coiled coils at the new N- and C-termini to compensate for the increase in local chain entropy. Additionally, by inserting protease cleavage sites in circularly permuted iLOV, we developed two protease sensors and demonstrated their application in mammalian cells. In summary, our work establishes the first approach to engineer circularly permuted FbFPs optimized for high fluorescence and further showcases the utility of circularly permuted FbFPs to serve as a scaffold for sensor engineering.

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