Zimmer M (2009) Chem Soc Rev "GFP: from jellyfish to the Nobel prize and beyond."

Zimmer M

[Chem Soc Rev, 2009]

On December 10, 2008 Osamu Shimomura, Martin Chalfie and Roger Tsien were awarded the Nobel Prize in Chemistry for "the discovery and development of the green fluorescent protein, GFP". The path taken by this jellyfish protein to become one of the most useful tools in modern science and medicine is described. Osamu Shimomura painstakingly isolated GFP from hundreds of thousands of jellyfish, characterized the chromophore and elucidated the mechanism of Aequorean bioluminescence. Martin Chalfie expressed the protein in E. coli and C. elegans, and Roger Tsien developed a palette of fluorescent proteins that could be used in a myriad of applications.

Cohen SM et al. (2015) Biochem Biophys Res Commun "Evolutionary and functional perspectives on signaling from neuronal surface to nucleus."

Li B, Cohen SM, Ma H, Tsien RW

[Biochem Biophys Res Commun, 2015]

Reliance on Ca(2+) signaling has been well-preserved through the course of evolution. While the complexity of Ca(2+) signaling pathways has increased, activation of transcription factors including CREB by Ca(2+)/CaM-dependent kinases (CaMKs) has remained critical for long-term plasticity. In C. elegans, the CaMK family is made up of only three members, and CREB phosphorylation is mediated by CMK-1, the homologue of CaMKI. CMK-1 nuclear translocation directly regulates adaptation of thermotaxis behavior in response to changes in the environment. In mammals, the CaMK family has been expanded from three to ten members, enabling specialization of individual elements of a signal transduction pathway and increased reliance on the CaMKII subfamily. This increased complexity enables private line communication between Ca(2+) sources at the cell surface and specific cellular targets. Using both new and previously published data, we review the mechanism of a CaMKII-CaM nuclear translocation. This intricatepathway depends on a specific role for multiple Ca(2+)/CaM-dependent kinases and phosphatases: /CaMKII phosphorylates CaMKII to trap CaM; CaN dephosphorylates CaMKII to dispatch it to the nucleus; and PP2A induces CaM release from CaMKII so that CaMKK and CaMKIV can trigger CREB phosphorylation. Thus, while certain basic elements have been conserved from C. elegans, evolutionary modifications offer opportunities for targeted communication, regulation of key nodes and checkpoints, and greater specificity and flexibility in signaling.

Shownkeen R et al. (1995) International C. elegans Meeting "THE CAENORHABDITIS ELEGANS PHYSICAL MAP"

Coulson AR, Chissoe SL, Waterston RH, Sulston JE, Fraser A, Shownkeen R

[International C. elegans Meeting, 1995]

Twelve contigs of cosmids and yeast artificial chromosomes (YACs) span more than 95Mb of the 100Mb C.elegans genome. 650 markers link the physical and genetic maps.Hybridisation of tag-sequenced cDNA clones to a map-representative set of YACs indicates that the map incorporates in excess of 99.8% of genes. The map is accessible in ACeDB. We (S.C.) are investigating the representation by bacterial artificial chromosomes (BACs) of regions of the genome not represented by cosmids. Two grids of YACs, of 958 clones ('Poly2') and 223 clones ('Suppoly') are available on request. The latter represents regions of the genome that have been characterised or better defined since the selection of clones for the former. Cosmid clones and YAC grids are available from the Sanger Centre (requests to alan@sanger.ac.uk; FAX 01223 494919). YAC clones and 'cm' series cDNA clones are available from the Sanger Centre or Washington University (rw@nematode. wustl.edu; FAX 314 362 2985).

Jin YS et al. (1995) Worm Breeder's Gazette "News on GFP variants"

Jin YS, Horvitz HR

[Worm Breeder's Gazette, 1995]

The use of GFP has enabled the visualization of biological processes in living animals (Chalfie et al., 1994; see reviews in TIG 11 320-329). Recently, several mutations in GFP were found to alter the wavelength spectrum of excitation and emission of the protein and to increase the intensity of fluorescence (Heim et al. 1994; 1995). In particular, Ser65Cys or Ser65Thr, and Ile167Thr mutations resulted in an increased intensity of fluorescence and in red-shifting of the excitation and emission wavelengths. We have made two GFP variants containing mutations at both amino acid 65 and 167: GFP-TT with Ser65Thr and Ile167Thr, and GFP-CT with Ser65Cys and Ile167Thr. These GFP variants are still fluorescent. The intensity of fluorescence of these new GFP variants appeared stronger than that of either the original wild-type GFP or the GFP containing single mutations such as Ser65Cys and Ser65Thr when the expression of each GFP was driven by the promoters of the unc-25 and vab-3 genes. These new GFP variants seemed to have a broader spectrum of excitation and emission than the wild-type GFP and the GFPs with single mutations, since we could detect fluorescence using the rhodoamine filter from these new GFP variants but not from the wild-type or other GFPs when driven by the unc-25 promoter; the maximal intensity was still obtained using the fluorescein filter. These double mutant GFPs also gave stronger signals than the wild-type GFP in premorphogenesis embryos (Andrew Chisholm, personal comunication). We have not performed any biochemical or biophysical analyses of these new GFP proteins. Although GFP-CT and GFP-TT result in similarly strong fluorescence, when fused to the VAMP protein for targeting to synaptic vesicles (Nonet, 1995), GFP-CT produced a much weaker signal than GFP-TT or the wild-type GFP, which suggests that local factors, such as pH, may not be optimal for fluorescence from a Ser65Cys mutant GFP in synaptic vesicles. These GFP variants are available. We would appreciate feedback from anyone who uses these constructs. Chalfie, M., Tu, Y., Euskirchen, G., Ward, W. W. and Prasher, D. C. (1994). Green fluorescent protein as a marker for gene expression. Science 263, 802-805. Heim, R., Cubitt, A. B. and Tsien, R. Y. (1995). Improved green fluorescence. Nature 373, 663-664. Heim, R., Prasher, D. C. and Tsien, R. Y. (1994). Wavelength mutations and posttranslational autoxidation of green fluorescent protein. PNAS 91, 12501-12504. Nonet, M. L. (1995). Visualization of presynaptic terminals using GFP. WBG 13 #5, 40.

Millan MI et al. (2009) Medicina (B Aires) "[The green fluorescent protein that glows in bioscience]."

Millan MI, Becu-Villalobos D

[Medicina (B Aires), 2009]

Green fluorescent protein (GFP) is a protein produced by the jellyfish Aequorea victoria, that emits bioluminescence in the green zone of the visible spectrum. The GFP gene has been cloned and is used in molecular biology as a marker. The three researchers that participated independently in elucidating the structure and function of this and its related proteins, Drs. Shimomura, Chalfie and Tsien were awarded the Nobel Prize in Chemistry 2008. Dr. Shimomura discovered and studied the properties of GFP. Using molecular biological techniques, Chalfie succeeded in introducing the GFP gene into the DNA of the small, almost transparent worm C. elegans, and initiated an era in which GFP would be used as a glowing marker for cellular biology. Finally, Dr.Tsien found precisely how GFP's structure produces the observed green fluorescence, and succeeded in modifying the structure to generate molecules that emit light at slightly different wavelengths, which gave tags of different colors. Fluorescent proteins are very versatile and are being used in many areas, such as microbiology, biotechnology, physiology, environmental engineering, development, etc. They can, for example, illuminate growing cancer tumours; show the development of Alzheimer's disease, or detect arsenic traces in water. Finding the key to how a marine organism produces light unexpectedly ended up providing researchers with a powerful array of tools with which to visualize cell biology in action.

Peng H et al. (2014) Nat Commun "Virtual finger boosts three-dimensional imaging and microsurgery as well as terabyte volume image visualization and analysis."

Long F, Iannello G, Gonzalez-Bellido PT, Zhou Z, Xiao H, Myers E, Bria A, Peng H, Chen J, Mitra A, Oh SW, Zeng H, Ascoli GA, Butler V, Hawrylycz M, Tsien RW, Zhou J, Tang J

[Nat Commun, 2014]

Three-dimensional (3D) bioimaging, visualization and data analysis are in strong need of powerful 3D exploration techniques. We develop virtual finger (VF) to generate 3D curves, points and regions-of-interest in the 3D space of a volumetric image with a single finger operation, such as a computer mouse stroke, or click or zoom from the 2D-projection plane of an image as visualized with a computer. VF provides efficient methods for acquisition, visualization and analysis of 3D images for roundworm, fruitfly, dragonfly, mouse, rat and human. Specifically, VF enables instant 3D optical zoom-in imaging, 3D free-form optical microsurgery, and 3D visualization and annotation of terabytes of whole-brain image volumes. VF also leads to orders of magnitude better efficiency of automated 3D reconstruction of neurons and similar biostructures over our previous systems. We use VF to generate from images of 1,107 Drosophila GAL4 lines a projectome of a Drosophila brain.

Hung, Wesley L. et al. (2013) International Worm Meeting "Attenuation of insulin signaling contributes to FSN-1-mediated regulation of synapse development."

Gao, Shangbang, Li, Hang, Hung, Wesley L., Hwang, Christine, Bessereau, Jean-Louis, Wang, Ying, Zhen, Mei, Liao, Edward H, Chitturi, Jyothsna, Stigloher, Christian

[International Worm Meeting, 2013]

The conserved neuronal F-box protein FSN-1 regulates neuromuscular junction development by negatively regulating DLK-mediated MAPK signaling in the presynaptic terminal (Liao et al., 2004; Nakata et al., 20005; Wu et al., 2007). We show here that attenuation of insulin/IGF signaling also contributes to FSN-1-dependent synaptic development and function. Loss of fsn-1 leads to aberrant synapse growth and a significant decrease in spontaneous vesicle release frequency from the neuromuscular junctions. These synaptic defects are partially and specifically rescued by decreasing insulin/IGF signaling activity in postsynaptic muscles, as well as by reducing the activity of EGL-3, a proprotein convertase that processes neuronal agonistic insulin/IGF ligands INS-4 and INS-6. FSN-1 interacts with, and potentiates the ubiquitination of EGL-3 in vitro, and reduces the level of EGL-3 in vivo. A constitutively activated MAPK, MKK-4(DD), can revert the suppression effect of fsn-1 by daf-2, indicating that MAPK pathway may act genetically downstream of the insulin pathway. We propose that FSN-1 negatively regulates insulin/IGF signaling and MAPK pathways to coordinate synapse growth signals from both pre- and postsynaptic terminals. Liao, EH, Hung, W, Abrams, B, Zhen, M. (2004) Nature 430:345 Nakata, K, Abrams, B, Grill, B, Goncharov, A, Huang, X, Chisolm, A, Jin, Y (2005) Cell 120:407 Wu, C, Daniels, RW, DiAntonio, A (2007) Neural Dev. 2:16.

Coulson AR et al. (1995) Methods Cell Biol "The physical map of the Caenorhabditis elegans genome."

Kozono Y, Coulson AR, Huynh C, Shownkeen R

[Methods Cell Biol, 1995]

The clone-based physical map of the 100-Mb Caenorhabditis elegans genome has evolved over a number of years. Although the detection of clone overlaps and construction of the map have of necessity been carried out centrally, it has been essentially a community project. Without the provision of cloned markers and relevant map information by the C. elegans community as a whole, the map would lack the genetic anchor points and coherent structure that make it a viable entity. Currently, the map consists of 13 mapped contigs totaling in excess of 95 Mb and 2 significant unmapped contigs totaling 1.3 Mb. Telomeric clones are not yet in place. The map carries 600 physically mapped loci, of which 262 have genetic map data. With one exception, the physical extents of the remaining gaps are not known. The exception is the remaining gap on linkage group (LG) II. This has been shown to be bridged by a 225-kb Sse83871 fragment. Because the clones constituting the map are a central resource, there is essentially no necessity for individuals to construct cosmid and yeast artificial chromosome (YAC) libraries. Consequently, such protocols are not included here. Similarly, protocols for clone fingerprinting, which forms the basis of the determination of cosmid overlaps and the mapping of clones received from outside sources and has to be a centralized operation, and YAC linkage are not give here. What follows is essentially a "user's guide" to the physical map. Details of map construction are given where required for interpretation of the map as distributed. The physical mapping has been a collaboration between the MRC Laboratory of Molecular Biology, Cambridge, United Kingdom (now at The Sanger Centre, Cambridge, UK) and Washington University School of Medicine, St. Louis, Missouri. Inquiries regarding map interpretation, information, and materials should be addressed to alan@sanger.ac.uk or rw@nematode.wustl.edu.

Westlund B et al. (1995) International C. elegans Meeting "let-42(oz36), A LATE-ONSET TUMOROUS GERMLINE MUTANT"

Westlund B, Schedl TB

[International C. elegans Meeting, 1995]

A polarity exists in hermaphrodite and male gonads such that germ cells at the very distal end are dividing mitotically while those more proximal cease proliferating and are in various stages of the meiotic pathway. We have identified a mutant (oz36 ) in which this germline polarity is established appropriately, but with time the distal mitotic zone expands and in extreme cases fills the entire gonad. Thus, oz36 hermaphrodites are initially self-fertile, but develop a late-onset tumorous germline phenotype. This phenotype is incompletely penetrant and exhibits both a temperature dependence (15oC>20oC>>25oC) and a maternal effect. oz36 males also can display a tumorous germline phenotype. Moreover, they are slightly feminized both in the germline (produce oocytes) and the soma (have truncated tail fans and produce vitellogenins). In addition to the tumorous germline phenotype, oz36 hermaphrodites display a maternal effect sterile (non-tumorous) phenotype that has a complementary temperature dependence (25oC>20oC>>15oC). Furthermore, at 25oC approximately 33% of the animals have a protruding vulva. oz36 fails to complement let-42(g38), an allele that displays a temperature sensitive (25.6oC) leaky maternal effect embryonic lethal phenotype (1). At 25oC some g38 hermaphrodite self-progeny are viable and develop to adulthood; they are sterile, frequently have a protruding vulva, and appear phenotypically indistinguishable from oz36 homozygotes at 25oC. g38 tumorous animals have not been observed at any temperature. let-42 is uncovered by nDf40. We are currently characterizing oz36/nDf40 and g38/nDf40 phenotypes, examining g38 males, and screening for new alleles of let-42. let-42 maps between stP127 (cosmid RW#127) and mua-3 (cosmid F55E6) (2) on LGIII. This interval spans 150-200 kb and its nucleotide sequence has been determined by the genome sequencing project. We are attempting to rescue the 25oC protruding vulva phenotype with cosmids from this region. 1. Cassada, R. et al. (1981) Devel. Biol. 84, 193-205. 2. Plenefisch, J. and E. Hedgecock (1994) WBG 13.3, 53.

V Centonze et al. (1999) International C. elegans Meeting "Excitation-contraction coupling in C. elegans muscle cells as monitored by multiphoton excitiation FRET imaging of calcium indicator proteins."

B Saari, D Wokosin, E Maryon, V Centonze, P Anderson

[International C. elegans Meeting, 1999]

Muscle contraction is initiated by membrane depolarization followed by a rapid elevation of cytoplasmic Ca 2+ . This process, called excitation-contraction coupling, is mediated by voltage gated Ca 2+ channels in the plasma membrane and intracellular Ca 2+ channels in the sarcoplasmic reticulum. Using a system similar to that described by Kerr, et al. (WCWM '98) we are characterizing Ca 2+ transients during pharyngeal pumping in wild-type animals, which we will compare to transients in animals having mutations in genes such as Ca 2+ channels. Multiphoton excitation is being used to elicit Ca 2+ -dependent fluorescence resonance energy transfer (FRET) emissions from a cameleon (ycam2) protein (provided by R. Tsien). ycam2 consists of two mutant GFP moieties having differing excitation and emission spectra, connected by a Ca 2+ -binding linker peptide. When ycam2 binds Ca 2+ it undergoes a conformational change that allows FRET between the 480nm-emitting moiety (cyan fluorescent protein-CFP) and the 535nm-emitting moiety (yellow fluorescent protein-YFP). Ca 2+ increases are thus detectable as an increase in the YFP/CFP emission ratio. We have expressed ycam2 in pharyngeal muscle cells using the myo-2 promoter. Imaging is performed on a prototype multidimensional multiphoton microscope. The instrument allows wide field direct detection of 3 channels: 1) bright field image; 2) YFP emission (535-550nm); 3) CFP emission (380-485nm). ycam2 fluorescence is imaged with a Ti-sapphire laser tuned to 800nm to primarily excite the CFP moiety. Time series are collected and data is subsequently analyzed using NIH-image. We have observed Ca 2+ transients during pharyngeal pumping by dissecting pharynxes in serotonin containing saline and imaging the terminal bulb. We observe 10% changes in YFP/CFP emission ratio from "baseline" to "peak" which correlate with opening of the terminal bulb lumen. Although our system does not yet allow "real time" resolution of Ca 2+ transients, we can correlate pharyngeal muscle contraction with calcium elevation in wild-type and mutant animals. This work was supported by RR00570.