Carbon Chapter 85 GFP-MCP

Green fluorescent protein (GFP) is a fluorescent protein that was originally isolated from the luminous organ of the jellyfish Aequorea victoria

Abstract

Green fluorescent protein (GFP) was discovered, purified, and characterized by Shimomura in a jellyfish beginning in 1962. Prasher cloned the gene for GFP and gave it to Tsien and Chalfie who made color mutants of GFP and expressed it in other organisms, respectively. Natural red and other color fluorescent proteins and those that could change color were discovered by Matz, the Lukyanov brothers, Miyawaki, and Salih. In vivo imaging using GFP was pioneered by Hoffman's group. GFP and other fluorescent proteins have revolutionized cell biology and led to the new field of in vivo cell biology.

9 Brain Metastasis

GFP-expressing B16 mouse malignant melanoma cells and the LOX human melanoma formed in the brain were visualized by GFP fluorescence of the cancer cells.17

Lewis lung carcinoma-GFP implanted orthotopically resulted in rapid tumor growth in nude mice and extensive metastasis formation visualized by GFP expression. Brain metastases were visualized in 30% of the animals.18

Nasopharyngeal carcinoma stably expressing GFP was orthotopically injected into the nasopharynx and a tumor formed brain metastasis.19

A craniotomy open window was used to image single Lewis lung cancer cells expressing GFP in the nucleus and RFP in the cytoplasm in the brain. The double labeling of cancer cells with GFP and RFP enabled mitosis and apoptosis of single cells to be imaged at the subcellular level through the craniotomy open window in live mice in real time.20–22 After chemotherapy, dual-color cancer cells with fragmented nuclei were visualized, indicating apoptosis. GFP-expressing apoptotic bodies and the destruction of RFP-expressing cytoplasm were also visualized in real time in nude mice through the craniotomy open window.

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New Models of the Cell Nucleus: Crowding, Entropic Forces, Phase Separation, and Fractals

Miho Yanagisawa, ... Kenichi Yoshikawa, in International Review of Cell and Molecular Biology, 2014

4.1 Cell-sized confinement accelerates gene expression

Green fluorescent protein (GFP) gene expression has been also tested within the liposomes (Ishikawa et al., 2004; Murtas et al., 2007; Noireaux and Libchaber, 2004; Nomura et al., 2003; Oberholzer et al., 1999). Interestingly, gene expression is reported to be enhanced in small W/O droplets coated by a lipid layer (Fiordemondo and Stano, 2007), or by adding liposomes outside (Bui et al., 2008). Using the size-controllable droplet system, we report a remarkable acceleration of gene expression in smaller droplets entrapping a cell-free translation system to synthesize GFP. Figure 7.13A shows confocal images of DOPG droplets entrapping a GFP expression system 3 h after encapsulation. The fluorescence image (Fig. 7.13A, left) shows the distribution of GFP fluorescence. The merged image (Fig. 7.13A, right) shows that GFP fluorescence was homogeneously distributed in each droplet. Interestingly, the intensity of GFP fluorescence in the droplets strongly depended on the droplet size. We calibrated the GFP intensity per volume and obtained the GFP concentration, CGFP, based on normalization by the value of the bulk solution after 3 h. As shown in Fig. 7.13B, CGFP decreased with an increase in the droplet size. In the case of a small droplets with radius R ~ 10 μm (Fig. 7.13B, 1), the value of CGFP was approximately four times larger than that for a large droplet with a radius ~ 45 μm (Fig. 7.13B, 5). On the other hand, CGFP values in droplets with almost the same R ~ 15 μm were similar (Fig. 7.13B, 2, 3). We have confirmed that there was no size dependence of CGFP among the droplets after encapsulation of GFP already expressed in the bulk solution. Furthermore, this result indicates that the membrane surface and/or the total volume significantly affect the GFP expression, since the surface-to-volume ratio is greater in a smaller droplet. If the lipid membrane contributed to the size dependence of GFP expression, the lipid species used should affect the relation between the droplet size and expression level. Thus, in the following experiments, we examined the effect of different lipids on the confinement effect.

Figure 7.13. Confinement to smaller space exhibits higher production of GFP. (A) Distribution of GFP fluorescent in droplets coated with a DOPG layer. (B) Profiles of fluorescent intensities along the diameter CGFP (Kato et al., 2012).

Three different species of lipids, anionic DOPG, zwitterionic DOPC, and zwitterionic DOPE, were used to encapsulate the GFP gene expression system within droplets. The time-courses of GFP concentration per unit volume, CGFP, were monitored for 45 h beginning soon after encapsulation. Identical droplets with a radius R = 10–100 μm were monitored. As shown in Fig. 7.14, the value of CGFP increased linearly with time up to 20 h, and reached a saturation point at ~ 40 h. Among all the three types of lipids, CGFP in a small droplet with R ~ 20 μm was higher than that in a large droplet with R ~ 50 μm. Additionally, CGFP at a given time and the production rate (VGFP = dCGFP/dt) were both dependent on the type of lipid. VGFP in each droplet remained almost constant for at least 1 day, which implies 0th-order reaction kinetics. Interestingly, VGFP was inversely proportional to the radius of the droplets (R) when R was under 50 μm, with VGFP in droplets with R = 10 μm being more than 10 times higher than that in the bulk. As shown in Fig. 7.14, the acceleration rates of GFP production in cell-sized droplets strongly depend on the lipid types. These results demonstrate that the membrane surface has the significant effect to facilitate protein production, especially when the scale of confinement is in the order of the cell size. Additionally, such time development of GFP expression can be analyzed within liposomes by using the droplet-transfer method (Section 5.1) (Saito et al., 2009).

Figure 7.14. Size dependence of the GFP concentration per unit volume, CGFP, at 25 h and the production rate (VGFP = dCGFP/dt) from 0 to 20 h (Kato et al., 2012).

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Cell Markers: Green Fluorescent Protein (GFP)

M. Chalfie, in Encyclopedia of Genetics, 2001

Uses of GFP

GFP and its variants have been used in organisms from bacteria and yeast to mice and human cells. One of the most common uses of GFP is in promoter and protein fusion constructs. Promoter fusions with GFP can document patterns of gene expression. Given the dynamics of GFP production (the fluorophore takes some time to form) and stability (the protein appears to be long-lived), detailed studies of the onset and cessation of gene expression (with a resolution of minutes) are not possible. Protein fusions are useful in determining the subcellular localization of a protein of interest and whether that localization changes during development, with different growth conditions, or in different genetic backgrounds. The most useful fusions are those that also rescue the mutant phenotype, because the rescue indicates that the fusion protein functions appropriately.

Sometimes these fusion constructs are used to analyze a protein or promoter of interest. At other times these fusions mark cells or cellular compartments so that biological phenomena can be examined or manipulated. Nuclei, endoplasmic reticulum, Golgi, mitochondria, peroxisomes, and synaptic endings have all been labeled using GFP. Once organisms have been labeled, they can be subjected to various conditions or they can be mutated to obtain mutants with altered or absent expression. For example, we have used GFP-labeled neurons in the nematode Caenorhabditis elegans as the basis of a screen for mutations that alter cell fate, cell migration, or neuronal outgrowth.

GFP can also indicate the presence of viruses and microorganisms. In molecular biology research, the labeling of viral proteins makes GFP a useful transfection marker. Since GFP labels living cells, the labeling of microorganisms may be particularly important in studying interactions between and within populations, e.g., symbiosis and host–parasite interactions. GFP can also be used to monitor infectious processes in plants and animals.

Recently several groups have produced GFP fusion proteins that couple the fluorescence of GFP to particular biological conditions. Such hybrid molecules respond with altered fluorescence to differences in membrane potential, calcium concentration, and pH. These molecules and others like them promise to greatly expand the usefulness of GFP into the realm of biological sensors.

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Methods of Analysis

E. Liu, ... P.V. Moghe, in Comprehensive Biomaterials II, 2017

3.19.3.1.3 Green fluorescent protein and variants

Green fluorescent protein (GFP) was originally derived from the jellyfish Aequorea victoria (Prendergast and Mann, 1978). It has 238 amino acid residues and a green fluorophore, which is comprised of only three amino acids: Ser65-Tyr66-Gly67. The stable protein structure is formed by beta sheets, which have a conformation that makes up an 11-stranded drum-like structure (Chalfie, 1995). The stability of GFP allows it to withstand pH levels ranging from mildly acidic (pH=5.5) to extremely basic (pH=12), and can also resist temperatures of up to 65°C. GFP has major and minor excitation peaks at wavelengths of 395 nm and 475 nm, respectively. Several modifications have been made from the original GFP, most notably the reduction of the dual excitation peaks of 395 nm and 475 nm down to one excitation peak of 488 nm, which is in the visible blue-light range. The emission peak of original and modified GFP is detected at 509 nm, which is in the visible green region of the electromagnetic spectrum (Chalfie, 1995; Prendergast and Mann, 1978). GFP variants with spectra that range from blue to red (shown in Table 2) can be used for live cell–biomaterial interaction imaging.

Table 2. List of common fluorescence proteins

Species/sourceFluorescence proteinsExcitation (nm)Emission (nm)GreenEGFP489508CyanECFP434/453477/501YellowEYFP514527RedDsRed558583

In order for biomaterial scientists to utilize GFP fusion proteins to capture cellular and subcellular responses to biomaterials, the construction and expression of GFP can be easily accomplished via standard molecular biology techniques. While a complete discussion is beyond the scope of this article, the readers are referred to molecular biology protocols, and in particular to the book by Goldman and Spector (2005). By introducing GFP into host cells, one can visualize GFP-tagged whole-cells, subcellular organisms, and cytoskeletal structure/organization. This form of targeting allows microscopist and biomaterial scientists alike to study cellular behavior by observing GFP-tagged proteins and capture information at a level that was previously inaccessible, both spatially and temporally (March et al., 2003). Apart from aforementioned GFP-based markers, phytochromes, light-sensitive photoreceptor, along with other proteins in the phytochrome signaling network, have been utilized to reversibly control the translocation of proteins to the cell membrane (Levskaya et al., 2009).

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Optical Molecular Imaging

R.M. Hoffman, in Comprehensive Biomedical Physics, 2014

4.03.1 The Green Fluorescent Protein Nude Mouse

The green fluorescent protein (GFP) nude mouse was obtained by crossing nontransgenic nude mice with the transgenic C57/B6 mouse in which the β-actin promoter drives GFP expression in essentially all tissues (Okabe et al., 1997). In crosses between nu/nu GFP male mice and nu/+ GFP female mice, the embryos fluoresced green. Approximately 50% of the offspring of these mice were GFP nude mice. Newborn mice and adult mice fluoresced very bright green and could be detected with a simple blue light-emitting diode flashlight with a central peak of 470 nm and a bypass emission filter. In the adult mice, the organs brightly expressed GFP, including the heart, lungs, spleen, pancreas, esophagus, stomach, and duodenum. The following systems were dissected out and shown to have brilliant GFP fluorescence: the entire digestive system from tongue to anus; the male and female reproductive systems; brain and spinal cord; and the circulatory system, including the heart and major arteries and veins. The skinned skeleton highly expressed GFP. Pancreatic islets showed GFP fluorescence. The spleen cells were also GFP positive. The liver is relatively dim. Red fluorescent protein (RFP)-expressing human cancer cell lines, including PC-3-RFP prostate cancer, HCT-116-RFP colon cancer, MDA-MB-435-RFP breast cancer, and HT1080-RFP fibrosarcoma, were transplanted to the transgenic GFP nude mice. All of these human tumors grew extensively in the transgenic GFP nude mouse. Dual-color fluorescence imaging enabled visualization of human tumor–host interaction by whole-body imaging and at the cellular level in fresh and frozen tissues (Yang et al., 2004).

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Caenorhabditis elegans: Cell Biology and Physiology

Hanna Fares, Alexander M van der Bliek, in Methods in Cell Biology, 2012

1 Steady-State Analysis of GFP Uptake

GFP secreted into the body cavity provides a convenient marker for fluid-phase uptake. Secreted GFP is made by body-wall muscles under the control of the highly active myo-3 promoter. Transgenic pmyo-3::ssGFP worms are made with a signal sequence attached to GFP for translocation into the ER (Fig. 3A). This GFP is secreted from body-wall muscles into the body cavity and is then taken up by coelomocytes (Fig. 3A) (Fares and Greenwald, 2001). GFP then accumulates in the lysosomes of coelomocytes (Treusch et al., 2004).

Effects on endocytic transport in coelomocytes will become apparent when endocytosis mutants are crossed with a strain containing the pmyo-3::ssGFP transgene (Fig. 3B). Mutations that disrupt early stages of endocytosis in coelomocytes cause GFP to accumulate in the body cavity and reduce the sizes of endosomal and lysosomal compartments in coelomocytes (as an example see the effects of the cup-4 mutation on endocytosis in Fig. 3B). Mutations that disrupt later stages of endocytosis or lysosomal transport cause GFP to accumulate in enlarged endosomal or lysosomal compartments (as an example see the effects of the cup-5 mutation on endocytosis in Fig. 3B). This method shows steady-state distributions, which sometimes makes it difficult to identify the primary defect cause by a particular mutation.

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Tools of the Cell Biologist

In Medical Cell Biology (Third Edition), 2008

Genetic Tagging

Green Fluorescent Protein

GFP was first identified and purified from the jellyfish Aequorea victoria, where it acts in conjunction with the luminescent protein aequorin to produce a green fluorescence color when the organism is excited. In brief, excitation of Aequorea results in the opening of membrane Ca2+ channels; cytosolic Ca2+ activates the aequorin protein and aequorin, in turn, uses the energy of ATP hydrolysis to produce blue light. By quantum mechanical resonance, blue light energy from aequorin excites adjacent molecules of GFP; these excited GFP molecules then produce a bright green fluorescence. Thus, the organism can "glow green in the dark" when excited. The resonant energy transfer between excited aequorin and GFP is an example of a naturally occurring fluorescence resonance energy transfer (FRET) process (see later).

The gene for GFP has been cloned and engineered in various was to permit the optimal expression and fluorescence efficiency of GFP in a wide variety of organisms and cell types. Cloning has furthermore permitted the GFP coding sequence to be used in protein expression vectors such that a chimeric construct is expressed, consisting of GFP fused onto the amino- or carboxylterminal end of the protein of interest. Variant GFP proteins and related proteins from different organisms are now available that extend the range of fluorescence colors that are produced: blue (cyan) fluorescent protein (CFP), yellow fluorescent protein, and red fluorescent protein.

GFP is a β-barrel protein (its structure is shown in Fig. 1-2). Within an hour or so after synthesis and folding, a self-catalyzed maturation process occurs in the protein, whereby adjacent serine, glycine, and tyrosine side chains in the interior of the barrel react with each other and with oxygen to form a fluorophore covalently attached to a through-barrel α-helical segment, near the center of the β-barrel cavity. The GFP fluorophore thus produced is excited by the absorption of blue light from the fluorescence microscope, and then decays with the release of green fluorescence.

Figure 1-2. The structure of green fluorescent protein (GFP). GFP is an 11-strand β-barrel, with an α-helical segment threaded up through the interior of the barrel. The amino- and carboxyl-terminal ends of the protein are free and do not participate in forming the stable β-barrel structure. Within an hour or so after synthesis and folding, a self-catalyzed maturation process occurs in the protein, whereby side chains in the interior of the barrel react with each other and with oxygen to form a fluorophore covalently attached to the through-barrel α-helical segment, near the center of the β-barrel cavity.

Because the amino- and carboxyl-terminal ends of GFP are free and do not contribute to the β-barrel structure, the coding sequence for GFP can be incorporated into expression vector constructs, such that chimeric fusion proteins can be expressed with a GFP domain located at either the amino- and carboxylterminal ends of the protein of interest. As mentioned earlier, the great advantage of genetic tagging of proteins with fluorescent molecules such as GFP is that this technique permits one to visualize the subcellular location of the protein of interest in a living cell. Consequently, one can observe not only the location of a protein but also the path it takes to arrive at that location. For example, using a GFP-tagged human immunodeficiency virus (HIV) protein, it was discovered that after entry into cells, the HIV reverse transcription complex travels via microtubules from the periphery of the cell to the nucleus.

The FRET technique can be used to monitor the interaction of one protein with another inside a living cell. As discussed earlier in this chapter, in Aequorea, blue light energy from aequorin is used to excite GFP by the quantum mechanical process of resonance energy transfer. Energy transfer like this can occur only when donor and acceptor molecules are close to each other (within 10nm). Investigators are able to take advantage of this process to detect when or if two proteins in the cell bind each other under some circumstance. Both proteins of interest need merely be tagged with a pair of complementary (donor-acceptor) fluorescent proteins, such as CFP and GFP, and then coexpressed in the cell. CFP is excited by violet light, and then emits blue fluorescence. If the two proteins do not bind each other in the cell, only blue fluorescence will be emitted on violet light excitation; if, however, the two proteins do bind each other, resonant energy transfer from the donor CFP will be captured by the GFP-tagged partner, and green fluorescence will be detected (Fig. 1-3).

Figure 1-3. Fluorescence resonance energy transfer (FRET). A: The two proteins of interest are expressed in cells as fusion proteins with either blue fluorescent protein (BFP) (protein X) or GFP (protein Y). Excitation of BFP with violet light results in the emission of blue fluorescent light by BFP; excitation of GFP with blue light yields green fluorescence. B: If the two proteins do not bind each other inside the cell, excitation of the BFP molecule with violet light results simply in blue fluorescence. If, however, (C) the two proteins do bind each other, they will be close enough to permit resonant energy transfer between the excited BFP molecule and the GFP protein, resulting in green fluorescence after violet excitation.

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Regulators and Effectors of Small GTPases

Yan Feng, ... Angela Wandinger-Ness, in Methods in Enzymology, 2001

Background.

GFP chimeras have proven extremely useful for monitoring molecular dynamics in live cells.8 GFP–Rab7 chimeras were generated as probes for purposes of analyzing late endosome-cytoskeleton connections. The GFP–Rab7 chimeras are concurrently being used to study late endocytic transport in real time by tracing the flux of fluorescently tagged endocytic tracers through Rab7-positive endosomes. It is well established that intact actin and microtubule networks are critical for endocytosis.9 Although the Rab proteins have classically described roles in membrane docking and fusion (reviewed in Ref. 10), recent studies using GFP–Rab5 chimeras have uncovered a direct role for rab proteins in the regulation of membrane transport along the cytoskeleton.11 The known dependence of early to late endocytic transport on functional Rab72 and an intact microtubule network12 warranted an evaluation of the translocation of Rab7-positive endosomes along the cytoskeleton as detailed below.

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Chapter 85 - GFP-MCP

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