Green Fluorescent protein (GFP), used as a cellular tag, provides researchers with a valuable method of measuring gene expression and cell tracking. However, there is evidence to suggest that the ...immunogenicity and cytotoxicity of GFP potentially confounds the interpretation of
in vivo
experimental data. Studies have shown that GFP expression can deteriorate over time as GFP tagged cells are prone to death. Therefore, the cells that were originally marked with GFP do not survive and cannot be accurately traced over time. This review will present current evidence for the immunogenicity and cytotoxicity of GFP in
in vivo
studies by characterizing these responses.
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EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
Summary
The localization of proteins in specific domains or compartments in the 3D cellular space is essential for many fundamental processes in eukaryotic cells. Deciphering spatial organization ...principles within cells is a challenging task, in particular because of the large morphological variations between individual cells. We present here an approach for normalizing variations in cell morphology and for statistically analyzing spatial distributions of intracellular compartments from collections of 3D images. The method relies on the processing and analysis of 3D geometrical models that are generated from image stacks and that are used to build representations at progressively increasing levels of integration, ultimately revealing statistical significant traits of spatial distributions. To make this methodology widely available to end‐users, we implemented our algorithmic pipeline into a user‐friendly, multi‐platform, and freely available software. To validate our approach, we generated 3D statistical maps of endomembrane compartments at subcellular resolution within an average epidermal root cell from collections of image stacks. This revealed unsuspected polar distribution patterns of organelles that were not detectable in individual images. By reversing the classical ‘measure‐then‐average’ paradigm, one major benefit of the proposed strategy is the production and display of statistical 3D representations of spatial organizations, thus fully preserving the spatial dimension of image data and at the same time allowing their integration over individual observations. The approach and software are generic and should be of general interest for experimental and modeling studies of spatial organizations at multiple scales (subcellular, cellular, tissular) in biological systems.
Significance Statement
Deciphering spatial organization within cells is challenging, because of the large morphological variations between cells. Here we present an algorithmic pipeline for normalizing variations in cell morphology and for statistically analyzing the spatial distributions of intracellular compartments from collections of 3D images, using endomembrane compartments in epidermal root cell cells as an example. Our analyses uncovered unsuspected polar distribution patterns not detectable in individual images. The approach and software should be generally applicable for modeling spatial organizations at multiple scales.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
GFP and its derivatives revolutionized the study of proteins. Spinach is a recently reported in vitro-evolved RNA mimic of GFP, which as genetically encoded fusions makes possible live-cell, ...real-time imaging of biological RNAs without resorting to large RNA-binding protein-GFP fusions. To elucidate the molecular basis of Spinach fluorescence, we solved the cocrystal structure of Spinach bound to its cognate exogenous chromophore, showing that Spinach activates the small molecule by immobilizing it between a base triple, a G-quadruplex and an unpaired G. Mutational and NMR analyses indicate that the G-quadruplex is essential for Spinach fluorescence, is also present in other fluorogenic RNAs and may represent a general strategy for RNAs to induce fluorescence of chromophores. The structure guided the design of a miniaturized 'Baby Spinach', and it provides a foundation for structure-driven design and tuning of fluorescent RNAs.
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DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, UILJ, UKNU, UL, UM, UPUK
Single crystals of optoelectronic materials that respond to external stimuli, such as mechanical, light, or heat, are immensely attractive for next generation smart materials. Here we report single ...crystals of a green fluorescent protein (GFP) chromophore analogue with irreversible mechanical bending and associated unusual enhancement of the fluorescence, which is attributed to the strained molecular packing in the perturbed region. Soft crystalline materials with such fluorescence intensity modulations occurring in response to mechanical stimuli under ambient pressure conditions will have potential implications for the design of technologically relevant tunable fluorescent materials.
Crystals of a green fluorescent protein chromophore analogue show enhanced fluorescence emission in a plastically deformed mechanically bent state owing to strained molecular packing.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
Aquaporin-2 (AQP2) fine tunes urine concentration in response to the antidiuretic hormone vasopressin. In addition, AQP2 has been suggested to promote cell migration and epithelial morphogenesis. A ...cell system allowing temporal and quantitative control of expression levels of AQP2 and phospho-mimicking mutants has been missing, as has a system allowing expression of fluorescently tagged AQP2 for time-lapse imaging. In the present study, we generated and validated a Flp-In T-REx Madin-Darby canine kidney cell system for temporal and quantitative control of AQP2 and phospho-mimicking mutants. We verified that expression levels can be temporally and quantitatively controlled and that AQP2 translocated to the plasma membrane in response to elevated cAMP, which also induced S256 phosphorylation. The phospho-mimicking mutants AQP2-S256A and AQP2-S256D localized as previously described, primarily intracellular and to the plasma membrane, respectively. Induction of AQP2 expression in combination with transient, low expression of enhanced green fluorescent protein-tagged AQP2 enabled expression without aggregation and correct translocation in response to elevated cAMP. Interestingly, time-lapse imaging revealed AQP2-containing tubulating endosomes and that tubulation significantly decreased 30 min after cAMP elevation. This was mirrored by the phospho-mimicking mutants AQP2-S256A and AQP2-S256D, where AQP2-S256A-containing endosomes tubulated, whereas AQP2-S256D-containing endosomes did not. Thus, this cell system enables a multitude of cell-based assays warranted to provide deeper insights into the mechanisms of AQP2 regulation and effects on cell migration and epithelial morphogenesis.
Efficient selection and production of antibody fragments in microbial systems remain to be a challenging process. To optimize microbial production of single-chain variable fragments (scFvs), we have ...chosen five model targets, 1) a hapten, Zearalenone (ZEN) mycotoxin, along with infectious agents 2) rabies virus, 3)
Propionibacterium acnes
, 4)
Pseudomonas aeruginosa
, and a cancer cell 5) acute myeloid leukemia cell line (HL-60). The scFv binders were affinity selected from a non-immunized human phage display scFv antibody library and genetically fused to the N-terminus of emerald green fluorescent protein (EmGFP). The scFv-EmGFP fusion constructs were subcloned into an expression vector, under the control of T7 promoter, C-terminally tagged with hexa-histidine and expressed in different
Escherichia coli
(
E. coli
) hosts. This enabled the detection of cells that expressed the correct scFv-EmGFP fusion, termed fluorobody, via bright fluorescent signal in the cytoplasm. Among the three
E. coli
hosts tested, an engineered
E. coli
B strain called SHuffle B that promotes disulfide bond formation in the cytoplasm appeared to be the most appropriate host. The recombinant fluorobodies were well expressed (2–8 mg/L), possessed the fluorescence property of EmGFP, and retained the ability to bind to their cognate targets. Their specific bindings were demonstrated by ELISA, fluorescence-linked immunosorbent assay (FLISA), flow cytometry, and fluorescent microscope imaging. The fluorobody expression platform in this study could be further adopted as a one-step immunostaining technique based on scFv, isolated from phage display library to numerous desired targets.
Key points
•
E. coli SHuffle express T7 is a suitable expression host for scFv-EmGFP (fluorobody)
•
Only the clones harboring scFv-EmGFP plasmid will show bright fluorescent signal
•
This platform can be used to produce fluorobodies for numerous purposes
Graphical abstract
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CEKLJ, DOBA, EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, IZUM, KILJ, KISLJ, MFDPS, NLZOH, NUK, OILJ, PILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UILJ, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
Due to a significant proportion of glycans binding to the peptide (constituting approximately 50-90% of the molecular weight), analyzing the interaction between the entire mucin molecule and its ...recognition protein (lectin) can be challenging. To address this, we propose a semiquantitative approach for measuring the interaction between mucin and lectin, which involves immobilizing mucin in a 96-well plate and subsequently adding lectin tagged with green fluorescent protein.
The flexibility and versatility of self-complementing split fluorescent proteins (FPs) have enabled a wide range of applications. In particular, the FP1-10/11 split system contains a small fragment ...that facilitates efficient generation of endogenous-tagged cell lines and animals as well as signal amplification using tandem FP11 tags. To improve the FP1-10/11 toolbox we previously developed, here we used a combination of directed evolution and rational design approaches, resulting in two mNeonGreen (mNG)-based split FPs (mNG3A1-10/11 and mNG3K1-10/11) and one mClover-based split FP (CloGFP1-10/11). mNG3A1-10/11 and mNG3K1-10/11 not only enhanced the complementation efficiency at low expression levels, but also allowed us to demonstrate signal amplification using tandem mNG211 fragments in mammalian cells.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Because of their low levels of expression and the inadequacy of current research tools, CB
cannabinoid receptors (CB
R) have been difficult to study, particularly in the brain. This receptor is ...especially relevant in the context of neuroinflammation, so novel tools are needed to unveil its pathophysiological role(s).
We have generated a transgenic mouse model in which the expression of enhanced green fluorescent protein (EGFP) is under the control of the cnr2 gene promoter through the insertion of an Internal Ribosomal Entry Site followed by the EGFP coding region immediately 3' of the cnr2 gene and crossed these mice with mice expressing five familial Alzheimer's disease (AD) mutations (5xFAD).
Expression of EGFP in control mice was below the level of detection in all regions of the central nervous system (CNS) that we examined. CB
R-dependent-EGFP expression was detected in the CNS of 3-month-old AD mice in areas of intense inflammation and amyloid deposition; expression was coincident with the appearance of plaques in the cortex, hippocampus, brain stem, and thalamus. The expression of EGFP increased as a function of plaque formation and subsequent microgliosis and was restricted to microglial cells located in close proximity to neuritic plaques. AD mice with CB
R deletion exhibited decreased neuritic plaques with no changes in IL1β expression.
Using a novel reporter mouse line, we found no evidence for CB
R expression in the healthy CNS but clear up-regulation in the context of amyloid-triggered neuroinflammation. Data from CB
R null mice indicate that they play a complex role in the response to plaque formation.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
The spectrophotometric properties of the green fluorescent protein (GFP) result from the post‐translationally cyclized chromophore composed of three amino acids including a tyrosine at the center of ...the β‐barrel protein. Altering the amino acids in the chromophore or the nearby region has resulted in numerous GFP variants with differing photophysical properties. To further examine the effect of small atomic changes in the chromophore on the structure and photophysical properties of GFP, the hydroxyl group of the chromophore tyrosine was replaced with a nitro or a cyano group. The structures and spectrophotometric properties of these superfolder GFP (sfGFP) variants with the unnatural amino acids (UAAs) 4‐nitro‐l‐phenylalanine or 4‐cyano‐l‐phenylalanine were explored. Notably, the characteristic 487 nm absorbance band of wild‐type (wt) sfGFP is absent in both unnatural amino‐acid‐containing protein constructs (Tyr66pNO2Phe‐sfGFP and Tyr66pCNPhe‐sfGFP). Consequently, neither Tyr66pNO2Phe‐sfGFP nor Tyr66pCNPhe‐sfGFP exhibited the characteristic emission of wt sfGFP centered at 511 nm when excited at 487 nm. Tyr66pNO2Phe‐sfGFP appeared orange due to an absorbance band centered at 406 nm that was not present in wt sfGFP, while Tyr66pCNPhe‐sfGFP appeared colorless with an absorbance band centered at 365 nm. Mass spectrometry and X‐ray crystallography confirmed the presence of a fully formed chromophore and no significant structural changes in either of these UAA‐containing protein constructs, signaling that the change in the observed photophysical properties of the proteins is the result of the presence of the UAA in the chromophore.
This work explores the structural and spectrophotometric implications of replacing a tyrosine residue in the chromophore of a fluorescent protein with two different unnatural amino acids.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
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