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At least 307 records · Page 17Linked to original sources
PubMed2003

A GFP-based system to uncouple mRNA transport from translation in a single living neuron.

An inducible fluorescent system based on GFP is presented that allows for the uncoupling of dendritic mRNA transport from subsequent protein synthesis at the single cell level. The iron-responsive element (IRE) derived from ferritin mRNA in the 5'-UTR of the GFP reporter mRNA renders translation of its mRNA dependent on iron. The addition of the full-length 3'-UTR of the Ca(2+)/calmodulin-dependent protein kinase II alpha (CaMKIIalpha) after the stop codon of the GFP reading frame targets the reporter mRNA to dendrites of transfected fully polarized hippocampal neurons. As we show by time-lapse videomicroscopy, iron specifically turns on GFP reporter protein synthesis in a single transfected hippocampal neuron. We investigate whether GFP expression is affected--in addition to iron--by synaptic activity. Interestingly, synaptic activity has a clear stimulatory effect. Most importantly, however, this activity-dependent protein synthesis is critically dependent on the presence of the full-length 3'-UTR of CaMKIIalpha confirming that this sequence contains translational activation signals. The IRE-based system represents a new convenient tool to study local protein synthesis in mammalian cells where mRNA localization to a specific intracellular compartment occurs.

5' Untranslated Regions↗
PubMed2004

GFP-like proteins as ubiquitous metazoan superfamily: evolution of functional features and structural complexity.

Homologs of the green fluorescent protein (GFP), including the recently described GFP-like domains of certain extracellular matrix proteins in Bilaterian organisms, are remarkably similar at the protein structure level, yet they often perform totally unrelated functions, thereby warranting recognition as a superfamily. Here we describe diverse GFP-like proteins from previously undersampled and completely new sources, including hydromedusae and planktonic Copepoda. In hydromedusae, yellow and nonfluorescent purple proteins were found in addition to greens. Notably, the new yellow protein seems to follow exactly the same structural solution to achieving the yellow color of fluorescence as YFP, an engineered yellow-emitting mutant variant of GFP. The addition of these new sequences made it possible to resolve deep-level phylogenetic relationships within the superfamily. Fluorescence (most likely green) must have already existed in the common ancestor of Cnidaria and Bilateria, and therefore GFP-like proteins may be responsible for fluorescence and/or coloration in virtually any animal. At least 15 color diversification events can be inferred following the maximum parsimony principle in Cnidaria. Origination of red fluorescence and nonfluorescent purple-blue colors on several independent occasions provides a remarkable example of convergent evolution of complex features at the molecular level.

Animals↗
PubMed2001

A GFP-equipped bidirectional expression module well suited for monitoring tetracycline-regulated gene expression in mouse.

Doxycycline (Dox)-sensitive co-regulation of two transcriptionally coupled transgenes was investigated in the mouse. For this, we generated four independent mouse lines carrying coding regions for green fluorescent protein (GFP) and beta-galactosidase in a bicistronic, bidirectional module. In all four lines the expression module was silent but was activated when transcription factor tTA was provided by the alpha-CaMKII-tTA transgene. In vivo analysis of GFP fluorescence, beta-galactosidase and immunochemical stainings revealed differences in GFP and beta-galactosidase levels between the lines, but comparable patterns of expression. Strong signals were found in neurons of the olfactory system, neocortical, limbic lobe and basal ganglia structures. Weaker expression was limited to thalamic, pontine and medullary structures, the spinal cord, the eye and to some Purkinje cells in the cerebellum. Strong GFP signals were always accompanied by intense beta-galactosidase activity, both of which could be co-regulated by Dox. We conclude that the tTA-sensitive bidirectional expression module is well suited to express genes of interest in a regulated manner and that GFP can be used to track transcriptional activity of the module in the living mouse.

Animals↗
PubMed2001

Dynamics of intracellular granules with CD63-GFP in rat basophilic leukemia cells.

CD63 is located on the basophilic granule membranes in resting basophils, mast cells, and platelets, and is also located on the plasma membranes of the cells. We constructed a CD63-GFP (green fluorescent protein) plasmid and introduced it into rat basophilic leukemia (RBL-2H3) cells to observe the movements of CD63 on degranulation. The movements of CD63-GFP were studied in living RBL cells by confocal laser scanning microscopy (CLSM). CD63-GFP, in which GFP was conjugated to the C-terminus of CD63, was located on both the granule membranes and the plasma membranes of RBL cells. The diameter of the fluorescent granules in the cytoplasm varied from 0.5 to 1.5 microm. Before antigen stimulation most granules with CD63-GFP hardly moved in RBL cells. However, after antigen stimulation the plasma membranes ruffled violently and the granules moved dramatically. They reached the plasma membranes in a few minutes and fused with them instantaneously. Analysis of the movement of each granule provided a new insight into the elementary process of degranulation. The velocity of the granule movement toward the plasma membranes on antigen stimulation was calculated to be 0.1+/-0.02 microm/s. This shows that the granules are able to reach the plasma membranes in 2 or 3 min if the diameter of the cells is 20 microm.

Animals↗
PubMed2000

Visualization of neurogenesis in the central nervous system using nestin promoter-GFP transgenic mice.

Neurons are generated from neural progenitor cells not only during development but also in the mature brain. To develop an in vivo system for analyzing neurogenesis, we generated transgenic mice expressing green fluorescent protein (GFP) under the control of regulatory regions of the nestin gene. GFP fluorescence was observed in areas and during periods connected with neurogenesis, including embryonic neuroepithelium, neonatal cerebellum, and hippocampal dentate gyrus and rostral migratory pathway from the subventricular zone to the olfactory bulb in the adult. GFP-positive cells in the adult brain included immature neuronal cells expressing polysialylated NCAM. BrdU labeling experiments revealed that newly generated interneurons which migrated rostrally from the subventricular zone expressed GFP until they reached the olfactory bulb. These results indicate that nestin promoter-GFP transgenic mice can be utilized to visualize the regions of neurogenesis throughout the life of the animals and to follow the migration and differentiation of newly generated neurons.

Aging↗
PubMed2000

Heterogeneity of iron bioavailability on plants assessed with a whole-cell GFP-based bacterial biosensor.

Ferric iron is an essential element for microbial growth but its water solubility in aerobic environments is considered to be low. Thus it is a limiting resource for which microbes must compete in natural habitats. Since competition for iron occurs at the level of individual cells, knowledge of the variability in iron bioavailability to such individuals is required to assess the nature of the competition in these habitats. Ferric iron availability to cells of Pseudomonas syringae was assessed by quantifying the fluorescence intensity of single cells harbouring a plasmid-borne transcriptional fusion of an iron-regulated promoter from a locus encoding a membrane receptor for a pyoverdine siderophore with a reporter gene encoding green fluorescent protein (GFP) following fluorescence microscopy. Cells of this iron biosensor exhibited iron-dependent GFP fluorescence that was inversely proportional to the amount of iron added to the media, and which differed by over 20-fold in iron-replete compared to iron-deplete culture media. Cells cultured in a medium of a given iron content exhibited a very narrow range of fluorescence intensities. In contrast, the fluorescence intensity of cells of the biosensor strain recovered from the rhizosphere or phylloplane of inoculated bean plants varied greatly. The distribution of fluorescence intensities was strongly right-hand skewed, with about 10% of the cells exhibiting substantially higher GFP fluorescence than that of the median cell. Cells of a positive control strain, harbouring a fusion of the constitutive nptII promoter with the gfp reporter gene, exhibited uniform GFP fluorescence both in culture media and on plants. These results indicate that there is substantial heterogeneity of iron biovailability to cells of P. syringae on plants, with only a small subset of cells experiencing low iron availability. Such heterogeneity places constraints on models of interactions of bacteria in natural habitats that are based on competition for limited iron.

Biological Availability↗
PubMed2006

The Lem2 gene promoter of barley directs cell- and development-specific expression of gfp in transgenic plants.

Transgenic approaches to combating fungal pathogens, such as Fusarium graminearum, require the targeting of antifungal gene expression in tissues of developing seed spikes of cereal grains, especially lemmas and epicarps. The Lem2 gene of barley encodes a lectin-like protein that is strongly up-regulated by salicylic acid and is preferentially expressed in lemmas, paleas (lemma/palea) and coleoptiles. Transient expression studies have indicated that the proximal -75/+70 region (relative to the transcription start site) determines organ specificity. In the present study, Golden Promise barley stably transformed with Morex Lem2 promoter/gfp reporter constructs displayed cell- and development-specific expression of gfp (green fluorescent protein gene). This expression corresponded to the expression seen in Northern blots of Morex organs. Under the full-length promoter, strong GFP fluorescence was observed in the lemma/palea, glumes, coleoptile, auricle and ligule. Weak GFP fluorescence was also observed in the rachis, tips of primary leaves and the leaf sheath. Unexpectedly, strong expression occurred in the epicarp, even though Lem2 is not expressed in this organ in Morex. Studies showed that the Lem2 promoter is more highly methylated in the epicarp than in the lemma of Morex. In the lemma/palea, gfp underwent a temporal shift in expression from the mesophyll to specialized epidermal cork cells. Similar to the lemma/palea, expression in the leaf sheath was localized in the cork cells. Progressive 5' deletions of the promoter to nucleotide -75 gradually reduced the level of gfp expression, but tissue- and cell-specific expression was retained.

5' Flanking Region↗
PubMedDate not supplied

Expression of GFP-actin leads to failure of nuclear elongation and cytokinesis in Tetrahymena thermophila.

Green fluorescent protein (GFP)-tagged actin was used to investigate the distribution and function of actin in Tetrahymena. A strain that expresses both GFP-actin and endogenous actin was developed by transformation of Tetrahymena thermophila with a ribosomal DNA-based replicative vector. Confocal microscopy of living cells and immunogold electron microscopy confirmed localization of GFP-actin to basal bodies and the contractile ring. Incorporation of the fusion protein into these and other actin-related structures correlated with severe impairment of macronuclear elongation and cytokinesis. At 30 degrees C macronuclear elongation failed to occur in 25% of the transformants despite completion of micronuclear division. At 20 degrees C macronuclear elongation failed to occur in 2% of the population. Arrest of cytokinesis coincided with failure of macronuclear elongation. Arrested cells developed into homopolar doublets with two sets of oral structures. This study indicates a requirement for actin in nuclear elongation and cytokinesis. Although GFP-actin can interfere with the functioning of actin-containing structures, the GFP-actin transformant strain can be used to monitor actin distribution and dynamics and is therefore an important new tool for further studies of Tetrahymena actin.

Actins↗
PubMed1999

GFP-aided confocal laser scanning microscopy can monitor Agrobacterium tumefaciens cell morphology and gene expression associated with infection.

We tagged Agrobacterium tumefaciens cells with a mini-Tn5 transposon containing a promoterless gene encoding a green fluorescent protein (GFP). Some of the GFP-tagged individual bacterial cells exhibited strong green fluorescence, which reflected the expression levels of the GFP-tagged genes. Those cells could be readily detected with a confocal laser scanning microscope (CLSM). We observed that the fluorescence and morphology of A. tumefaciens cells grown in plant tissues resembled those grown in a minimal medium of low pH, which is required for expression of the virulence genes responsible for tumorigenesis. This suggests that GFP-aided CLSM can be used to determine which growth medium is more representative of the nutritional conditions that a pathogen encounters in plant tissues. We also observed that the fluorescence and morphology of A. tumefaciens cells changed dramatically during the course of infection. Our data suggested that A. tumefaciens cells were probably better fed upon successful colonization. We believe that GFP-aided CLSM can help study the fate of A. tumefaciens cells inside plant tissues by monitoring cell morphology and gene expression associated with the infection process in situ.

Agrobacterium tumefaciens↗
PubMed2005

Analysis of developmental potentials of dental pulp in vitro using GFP transgenes.

BACKGROUND: In recent years there has been increasing progress in identifying stem cells from adult tissues and their potential application for tooth replacement/regeneration. Our previous in vivo studies show that pOBCol3.6GFP and pOBCol2.3GFP transgenic animals provide a unique model to gain insight into progenitor/stem cells in the dental pulp capable of giving rise to odontoblasts. OBJECTIVES: To characterize the behavior of dental pulp cells derived from pOBCol3.6GFP animals in vitro. EXPERIMENTAL DESIGN: Primary cultures were established from the coronal portions of the pulps isolated first molars from 5-day-old pOBCol3.6GFP heterozygous mice and grown for 21 days. In these cultures proliferation, clonogenic capacity, activation of 3.6-GFP and mineralization were examined. RESULTS: Our observations show that dental pulp cells derived from 3.6-GFP contain a population of proliferative, clonogenic cells with the ability to mineralize. We also show the stage specific activation/upregulation of 3.6-GFP in primary cultures derived from dental pulp. In these cultures, expression of Col1a1-3.6-GFP occurs prior to the appearance of mineralized nodules and is unregulated in mineralized nodules. CONCLUSIONS: Col1a1-GFP transgenes appear to fulfill many of the requirements of a marker gene for cell lineage studies in intact tooth and primary cultures derived from dental pulp.

Animals↗
PubMed2005

Binding of PLCdelta1PH-GFP to PtdIns(4,5)P2 prevents inhibition of phospholipase C-mediated hydrolysis of PtdIns(4,5)P2 by neomycin.

AIM: To investigate the effects of the pleckstrin homology (PH) domain of phospholipase C(delta1) (PLC(delta1)PH) on inhibition of phospholipase C (PLC)-mediated hydrolysis of phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] by neomycin. METHODS: A fusion construct of green fluorescent protein (GFP) and PLC (delta1)PH (PLC(delta1)PH-GFP), which is known to bind PtdIns(4,5)P2 specifically, together with laser-scanning confocal microscopy, was used to trace PtdIns(4,5)P2 translocation. RESULTS: Stimulation of the type 1 muscarinic receptor and the bradykinin 2 receptor induced a reversible PLC(delta1)PH-GFP translocation from the membrane to the cytosol in COS-7 cells. PLC inhibitor U73122 blocked the translocation. Wortmannin, a known PtdIns kinase inhibitor, did not affect the translocation induced by ACh, but blocked recovery after translocation, indicating that PtdIns(4,5)P2 hydrolysis occurs through receptor-mediated PLC activation. Neomycin, a commonly used phospholipase C blocker, failed to block the receptor-induced PLCd1PH-GFP translocation, indicating that neomycin is unable to block PLC-mediated PtdIns(4,5)P2 hydrolysis. However, in the absence of PLCd1PH-GFP expression, neomycin abolished the receptor-induced hydrolysis of PtdIns(4,5)P2 by PLC. CONCLUSION: Although PLCd1PH and neomycin bind to PtdIns(4,5)P2 in a similar way, they have distinct effects on receptor-mediated activation of PLC and PtdIns(4,5)P2 hydrolysis.

Acetylcholine↗
PubMed2001

Ren1d and Ren2 cooperate to preserve homeostasis: evidence from mice expressing GFP in place of Ren1d.

To distinguish the contributions of Ren1(d) and Ren2 to kidney development and blood pressure homeostasis, we placed green fluorescent protein (GFP) under control of the Ren1(d) renin locus by homologous recombination in mice. Homozygous Ren1(d)-GFP animals make GFP mRNA in place of Ren1(d) mRNA in the kidney and maintain Ren2 synthesis in the juxtaglomerular (JG) cells. GFP expression provides an accurate marker of Ren1(d) expression during development. Kidneys from homozygous animals are histologically normal, although with fewer secretory granules in the JG cells. Blood pressure and circulating renin are reduced in Ren1(d)-GFP homozygotes. Acute administration of losartan decreases blood pressure further, suggesting a role for Ren2 protein in blood pressure homeostasis. These studies demonstrate that, in the absence of Ren1(d), Ren2 preserves normal kidney development and prevents severe hypotension. Chronic losartan treatment results in compensation via recruitment of both Ren1(d)- and Ren2-expressing cells along the preglomerular vessels. This response is achieved by metaplastic transformation of arteriolar smooth muscle cells, a major mechanism to control renin bioavailability and blood pressure homeostasis.

Angiotensin Receptor Antagonists↗
PubMed2002

Embryonic stem cells and mice expressing different GFP variants for multiple non-invasive reporter usage within a single animal.

BACKGROUND: Non-invasive autofluorescent reporters have revolutionized lineage labeling in an array of different organisms. In recent years green fluorescent protein (GFP) from the bioluminescent jellyfish Aequoria Victoria has gained popularity in mouse transgenic and gene targeting regimes 1. It offers several advantages over conventional gene-based reporters, such as lacZ and alkaline phosphatase, in that its visualization does not require a chromogenic substrate and can be realized in vivo. We have previously demonstrated the utility and developmental neutrality of enhanced green fluorescent protein (EGFP) in embryonic stem (ES) cells and mice 2. RESULTS: In this study we have used embryonic stem (ES) cell-mediated transgenesis to test the enhanced cyan fluorescent protein (ECFP) and enhanced yellow fluorescent protein (EYFP), two mutant and spectrally distinct color variants of wild type (wt) GFP. We have also tested DsRed1, the novel red fluorescent protein reporter recently cloned from the Discostoma coral by virtue of its homology to GFP. To this end, we have established lines of ES cells together with viable and fertile mice having widespread expression of either the ECFP or EYFP GFP-variant reporters. However, we were unable to generate equivalent DsRed1 lines, suggesting that DsRed1 is not developmentally neutral or that transgene expression cannot be sustained constitutively. Balanced (diploid <-> diploid) and polarized (tetraploid <-> diploid) chimeras comprising combinations of the ECFP and EYFP ES cells and/or embryos, demonstrate that populations of cells expressing each individual reporter can be distinguished within a single animal. CONCLUSIONS: GFP variant reporters are unique in allowing non-invasive multi-spectral visualization in live samples. The ECFP and EYFP-expressing transgenic ES cells and mice that we have generated provide sources of cells and tissues for combinatorial, double-tagged recombination experiments, chimeras or transplantations.

Animals↗
PubMed2000

Lumenal targeted GFP, used as a marker of soluble cargo, visualises rapid ERGIC to Golgi traffic by a tubulo-vesicular network.

The mechanism by which soluble proteins without sorting motifs are transported to the cell surface is not clear. Here we show that soluble green fluorescent protein (GFP) targeted to the lumen of the endoplasmic reticulum but lacking any known retrieval, retention or targeting motifs, was accumulated in the lumen of the ERGIC if cells were kept at reduced temperature. Upon activation of anterograde transport by rewarming of cells, lumenal GFP stained a microtubule-dependent, pre-Golgi tubulo-vesicular network that served as transport structure between peripheral ERGIC-elements and the perinuclear Golgi complex. Individual examples of these tubular elements up to 20 microm in length were observed. Time lapse imaging indicated rapid anterograde flow of soluble lumenal GFP through this network. Transport tubules, stained by lumenal GFP, segregated rapidly from COPI-positive membranes after transport activation. A transmembrane cargo marker, the temperature sensitive glycoprotein of the vesicular stomatitis virus, ts-045 G, is also not present in tubules which contained the soluble cargo marker lum-GFP. These results suggest a role for pre-Golgi vesicular tubular membranes in long distance anterograde transport of soluble cargo. http://www. biologists.com/JCS/movies/jcs1334.html

Animals↗
PubMed2001

Green fluorescent protein (GFP) as a marker of aryl hydrocarbon receptor (AhR) function in developing zebrafish (Danio rerio).

We developed an inducible in vivo reporter system to examine expression of the aryl hydrocarbon receptor (AhR) during development in zebrafish (Danio rerio). AhR is a ligand-activated transcription factor that mediates the toxic actions of environmental contaminants such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Induction of cytochrome P4501A1 (CYP1A1) is an early biomarker of AhR activation. A 1905 base pair region of the human CYP1A1 promoter/enhancer region was regulated by AhR in zebrafish liver cells after exposure to TCDD (10 nM) in a transient transfection assay. This regulatory region was fused to the cDNA sequence encoding green fluorescent protein (GFP) of jellyfish (Aequorea victoria). Transgenic zebrafish were generated to express this AhR-regulated GFP construct. Injected fish exposed to TCDD exhibited induction of GFP in the eye, nose, and vertebrae of zebrafish embryos (48 and 72 hr after fertilization) compared to vehicle controls (DMSO), which did not express GFP. To investigate whether AhR-regulated GFP expression correlated with sites of TCDD toxicity, we exposed wild-type zebrafish to DMSO or TCDD and examined them for morphologic abnormalities. By 5 days after fertilization, TCDD-exposed fish exhibited gross dysmorphogenesis in cranio-facial and vertebral development.

Animals↗
PubMed2004

Comparison of the action of transient and continuous PTH on primary osteoblast cultures expressing differentiation stage-specific GFP.

UNLABELLED: Primary calvarial osteoblast cultures derived from type I collagen promoter-GFP reporter transgenic mice were used to examine progression of the osteoblast lineage. This system was validated by assessing the effect of PTH on osteoblast growth in real time. The anabolic effect of PTH seemed to be the result of enhanced osteoblast differentiation rather than expansion of a progenitor population. INTRODUCTION: Activation of green fluorescent protein (GFP) marker genes driven by Col1a1 promoter fragments has been associated with the level of osteoblast differentiation. GFP-marked cultures provide an approach to continuously monitor the level of osteoblast differentiation in real time without the termination of cultures. MATERIALS AND METHODS: Neonatal calvarial cells transgenic for pOBCol2.3GFP and pOBCol3.6GFP were used to establish calvarial osteoblast cultures. Parathyroid hormone (PTH) was added either continuous (days 1-21) or transient (days 1-7) to examine its diverse effect on osteoblast differentiation in cultures for 21 days. Three fluorescent markers were used: (1) pOBCol3.6GFP, which is activated in preosteoblastic cells; (2) pOBCol2.3GFP, which is restricted to differentiated osteoblasts; and (3) xylenol orange (XO), which stains the mineralized nodules. Progression of osteoblast differentiation indicated by fluorescent markers was documented throughout the entire period of culture. Recorded fluorescent images were analyzed in the patterns of expression and quantitated in the area of expression. RESULTS: Continuous PTH blocked osteoblast differentiation, which was evident by the attenuation of pOBCol3.6GFP and an absence of pOBCol2.3GFP. In contrast, transient PTH inhibited the initial osteoblast differentiation but ultimately resulted in a culture with more mineralized nodules and enhanced osteoblast differentiation expressing strong levels of pOBCol3.6GFP and pOBCol2.3GFP. Quantitative analysis showed that transient PTH first decreased then later increased areas of GFP expression and XO staining, which correlated with results of Northern blot and alkaline phosphatase activity. Transient PTH caused a decrease in DNA content during the treatment and after the removal of PTH. CONCLUSION: GFP-marked cultures combined with fluorescent image analysis have the advantage to assess the effect of PTH on osteoblast differentiation in real time. Results suggest that the anabolic effect of transient PTH is caused by an enhancement in osteoblast differentiation rather than an increase in the population of progenitor cells.

Animals↗
PubMed2005

Behavior of transplanted bone marrow-derived GFP mesenchymal cells in osteochondral defect as a simulation of autologous transplantation.

To elucidate the behavior of autologously transplanted mesenchymal cells in osteochondral defects, we followed transplanted cells using green fluorescent protein (GFP) transgenic rats, in which all cells express GFP signals in their cytoplasm and nuclei as transplantation donors. Bone marrow-derived mesenchymal cells, which contain mesenchymal stem cells (MSCs), were obtained from transgenic rats. Then, dense mesenchymal cell masses created by hanging-drop culture were transplanted and fixed with fibrin glue into osteochondral defects of wild-type rats. At 24 weeks after surgery, the defects were repaired with hyaline-like cartilage and subchondral bone. GFP positive cells, indicating transplanted mesenchymal-derived cells, were observed in the regenerated tissues for 24 weeks although GFP positive cells decreased in number with time. Because GFP causes no immunological rejection and requires no chemicals for visualization, transplantation between transgenic and wild-type rats can be regarded as a simulation of autologous transplantation, and the survivability of transplanted cells are able to be followed easily and reliably. Thus, the behavior of transplanted mesenchymal cells was able to be elucidated in vivo by this strategy, and the results could be essential in future tissue engineering for the regeneration of osteochondral defects with original hyaline cartilage and subchondral bone.

Animals↗
PubMed2006

Understanding the folding of GFP using biophysical techniques.

Green fluorescent protein (GFP) and its many variants are probably the most widely used proteins in medical and biological research, having been extensively engineered to act as markers of gene expression and protein localization, indicators of protein-protein interactions and biosensors. GFP first folds, before it can undergo an autocatalytic cyclization and oxidation reaction to form the chromophore, and in many applications the folding efficiency of GFP is known to limit its use. Here, we review the recent literature on protein engineering studies that have improved the folding properties of GFP. In addition, we discuss in detail the biophysical work on the folding of GFP that is beginning to reveal how this large and complex structure forms.

Green Fluorescent Proteins↗