Chalfie M et al. (1994) Science "Green fluorescent protein as a marker for gene expression."

Euskirchen G, Tu Y, Chalfie M, Prasher DC, Ward WW

[Science, 1994]

A complementary DNA for the Aequorea victoria green fluorescent protein (GFP) produces a fluorescent product when expressed in prokaryotic (Escherichia coli) or eukaryotic (Caenorhabditis elegans) cells. Because exogenous substrates and cofactors are not required for this fluorescence, GFP expression can be used to monitor gene expression and protein localization in living organisms.

Tirera, Fouley et al. (2019) International Worm Meeting "Roles of TCN-1 transmembrane protein in C. elegans osmosensation."

Hendricks, Michael, Penalva, Ylauna, Posner, Rachel, Tirera, Fouley, Tse, Victoria

[International Worm Meeting, 2019]

Maintaining a stable intracellular osmolarity is fundamental for the survival of all organisms. Extracellular osmotic changes perturb cellular functions, thus most organisms adopt physiologic and behavioural responses to adapt to and avoid osmotic stress. In mammals, members of the transient receptor potential vanilloid, also known as TRPV channels, are expressed in the osmosensory brain regions, however the underlying molecular mechanisms are not fully understood. In Caenorhabditis elegans, the OSM-9 TRPV channel is expressed in ASH sensory neurons and is involved in high osmolarity avoidance. On contact with a hyperosmotic stimulus, the nematode generates reversals to escape the high osmolarity region. Additionally, a previous study revealed that through RIM interneuron inhibition, food-deprived worms are willing to cross a high osmolarity barrier to reach an attractant, a food odour. Here we investigated the role of the uncharacterized transmembrane protein TCN-1 in osmosensation. We found that tcn-1 mutants display increased sensitivity to hyperosmotic barriers. Unlike wild type animals, food-deprived tcn-1 mutants will not cross a hyperosmotic barrier to reach an attractive odour, despite exhibiting normal locomotion and chemotaxis in the absence of a barrier. Therefore, our study demonstrates the TCN-1 transmembrane protein plays a role in osmosensation and might contribute to the regulation of intracellular osmolarity.

Miller DM et al. (1999) Biotechniques "Two-color GFP expression system for C. elegans."

Desai NS, Piston DW, Hardin DC, Xu SQ, Miller DM, Fleenor J, Fire A, Patterson GH

[Biotechniques, 1999]

We describe the use of modified versions of the Aequora victoria green fluorescent protein (GFP) to simultaneously follow the expression and distribution of two different proteins in the nematode, Caenorhabditis elegans. A cyan-colored GFP derivative, designated CFP, contains amino acid (aa) substitutions Y66W, N146I, M153T and V163A relative to the original GFP sequence and is similar to the previously reported "W7" form. A yellow-shifted GFP derivative, designated YFP, contains aa substitutions S65G, V68A, S72A and T203Y and is similar to the previously described "I0C" variant. Coding regions for CFP and YFP were constructed in the context of a high-activity C. elegans expression system. Previously characterized promoters and localization signals have been used to express CFP and YFP in C. elegans. Filter sets designed to distinguish YFP and CFP fluorescence spectra allowed visualization of the two distinct forms of GFP in neurons and in muscle cells. A series of expression vectors carrying CFP and YFP have been constructed and are being made available to the scientific community.

Galindo-Malagn XA et al. (2022) Zootaxa "New species, synonymies and records in the genus Rhagovelia Mayr, 1865 (Hemiptera: Heteroptera: Veliidae) from Colombia."

Moreira FFF, Morales I, Mondragn-F SP, Galindo-Malagn XA

[Zootaxa, 2022]

Rhagovelia medinae sp. nov., of the hambletoni group (angustipes complex), and R. utria sp. nov., of the hirtipes group (robusta complex), are described, illustrated, and compared with similar congeners. Based on the examination of type specimens, six new synonymies are proposed: R. elegans Uhler, 1894 = R. pediformis Padilla-Gil, 2010, syn. nov.; R. cauca Polhemus, 1997 = R. azulita Padilla-Gil, 2009, syn. nov., R. huila Padilla-Gil, 2009, syn. nov., R. oporapa Padilla-Gil, 2009, syn. nov, R. quilichaensis Padilla-Gil, 2011, syn. nov.; and R. gaigei, Drake Hussey, 1947 = R. victoria Padilla-Gil, 2012 syn. nov. The first record from Colombia is presented for R. trailii (White, 1879), and the distributions of the following species are extended in the country: R. cali Polhemus, 1997, R. castanea Gould, 1931, R. cauca Polhemus, 1997, R. gaigei Drake Hussey, 1957, R. elegans Uhler, 1894, R. femoralis Champion, 1898, R. malkini Polhemus, 1997, R. perija Polhemus, 1997, R. sinuata Gould, 1931, R. venezuelana Polhemus, 1997, R. williamsi Gould, 1931, and R. zeteki Drake, 1953.

David Alan Wharton (2010) Evolutionary Biology of Caenorhabditis and Other Nematodes "Panagrolaimus davidi, an Antarctic nematode model for the survival of extreme environmental stress"

David Alan Wharton

[Evolutionary Biology of Caenorhabditis and Other Nematodes, 2010]

Nematodes are found in almost all environments, including those where they are often exposed to extreme environmental stress. Panagrolaimus davidi is an Antarctic nematode living associated with moss and algae in terrestrial habitats on the Victoria Land coast that are free of snow and ice for part of the year. It has to survive very variable thermal and hydric environments where liquid water and temperatures suitable for growth are only periodically available. P. davidi can survive complete water loss (anhydrobiosis) and is the only organism that has been shown to survive intracellular ice formation throughout its tissues. It has several cold tolerance strategies, including; freeze avoidance, cryoprotective dehydration, freezing tolerance and anhydrobiosis. The mechanisms involved may include the production of trehalose, ice active proteins and the control of ice nucleation. Do the different survival strategies of P. davidi represent the expression of different gene sets or does the production of stress-related compounds provide protection against a variety of environmental challenges? Other nematodes, including Caenorhabditis elegans, are not so resistant to desiccation and freezing. Comparing the genomes of P. davidi and C. elegans may thus highlight the adaptations that are necessary for the survival of extreme environmental stress.

Thomas BJ et al. (2019) PLoS One "CemOrange2 fusions facilitate multifluorophore subcellular imaging in C. elegans."

Cole FS, Silverman GA, Thomas BJ, Chou WYY, Wambach JA, Kim H, Buland JR, Jia H, Homayouni A, Moreno M, Luke CJ, Pak SC, Huang H, Wight IE, Dawson Z

[PLoS One, 2019]

Due to its ease of genetic manipulation and transparency, Caenorhabditis elegans (C. elegans) has become a preferred model system to study gene function by microscopy. The use of Aequorea victoria green fluorescent protein (GFP) fused to proteins or targeting sequences of interest, further expanded upon the utility of C. elegans by labeling subcellular structures, which enables following their disposition during development or in the presence of genetic mutations. Fluorescent proteins with excitation and emission spectra different from that of GFP accelerated the use of multifluorophore imaging in real time. We have expanded the repertoire of fluorescent proteins for use in C. elegans by developing a codon-optimized version of Orange2 (CemOrange2). Proteins or targeting motifs fused to CemOrange2 were distinguishable from the more common fluorophores used in the nematode; such as GFP, YFP, and mKate2. We generated a panel of CemOrange2 fusion constructs, and confirmed they were targeted to their correct subcellular addresses by colocalization with independent markers. To demonstrate the potential usefulness of this new panel of fluorescent protein markers, we showed that CemOrange2 fusion proteins could be used to: 1) monitor biological pathways, 2) multiplex with other fluorescent proteins to determine colocalization and 3) gain phenotypic knowledge of a human ABCA3 orthologue, ABT-4, trafficking variant in the C. elegans model organism.

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.

Zhang, Donglei et al. (2013) International Worm Meeting "The lipid binding and GAP domains of CYK-4 are essential for cytokinesis."

Glotzer, Michael, Loria, Andy, Zhang, Donglei

[International Worm Meeting, 2013]

Cytokinesis is a fundamental biological processes in which the parent cell physically separates and sequesters the duplicated genetic materials into two daughter cells. RhoA is a central regulator of cytokinesis, whose activity is redundantly controlled by centralspindlin and NOP-1 [1, 2]. Centralspindlin is a heterotetramer of a kinesin protein ZEN-4 and a Rho family GAP CYK-4. In addition to organizing the central spindle, this complex also plays key roles during furrow initiation, ingression and completion. CYK-4 contains numerous functional domains, including a coiled-coil domain, a C1 domain, and a GAP domain [3]. In order to better investigate functions of these domains of CYK-4 on cytokinesis, we generated strains carrying RNAi-resistant alleles of CYK-4::GFP by MosSCI. The RNAi-resistant alleles of CYK-4 allow us to examine transgenic CYK-4 variants by time-lapse fluorescence microscopy while specifically depleting endogenous CYK-4 by RNAi. We found that wild type CYK-4::GFP was properly localized in cyk-4(RNAi) embryos and fully compensated for depletion of endogenous CYK-4 in wild type and nop-1 mutants. Although CYK-4(DC1)::GFP also localized to the central spindle, furrow regression occurred in cyk-4(RNAi) strains, and it failed to induce a furrow in nop-1; cyk-4(RNAi) strains. These results suggest that the C1 domain plays a role at an early stage of cytokinesis (i.e. RhoA activation). Consistent with this, we found that CYK-4::GFP was recruited to the equatorial membrane, where RhoA is activated, while CYK-4(DC1)::GFP was not. Finally, we found that a catalytically inactivated allele, CYK-4(R459A)::GFP, failed to support full furrow ingression in cyk-4(RNAi) embryos. The defect was observed as a late regression phenotype as the furrow ingressed to ~90% of embryo width. These results suggest that CYK-4 GAP activity is required at a late stage of cytokinesis. 1. Glotzer, M., Science (2005); 2. Tse, Y.C. et al., Mol Biol Cell (2012); 3. White, E.C. et al., Cytoskeleton (2012).

Zhu X et al. (1995) International C. elegans Meeting "INTEGRIN EXPRESSION PATTERN IN C. elegans"

Zhu X, Hedgecock EM

[International C. elegans Meeting, 1995]

Integrins are a family of extracellular matrix receptors involved in cell adhesion, signal transduction and cytoskeletal organization. Three genes for integrin subunits have been identified in C. elegans to date. Two a-subunit genes were discovered by the genome sequencing project. One b-subunit gene was cloned by degenerate PCR based on the known insect and vertebrate sequences, later identified as the product of pat-3 (Gettner et al., 1992). Genetic and immunohistochemical studies indicate that pat-3 is essential for muscle development, canal outgrowth and some cell migrations. These and other studies in higher organisms have led us to speculate that pat-3 might play diverse roles in development, both embryonically and post- embryonically. To further study bPAT-3 function, we sought to establish the spatial and temporal expression pattern in more detail using A. victoria GFP as a tag. We have inserted GFP into the bPAT-3 reading frame immediately upstream of the normal terminator. When this construct was injected into pat-3 null mutants, the animals were fully rescued by a heritable extrachromosomal array. Rescued animals had bright green fluorescence in specific patterns including most sites previously detected by immunofluorescence with anti-integrin antibodies. Hence, the fusion protein bPAT-3::GFP is functional and behaves much like the wildtype protein. bPAT-3::GFP is expressed continuously in muscle cells in larvae and adults. In body wall muscles, it is localized at Z-disc (dense body), M-line and dense plaque attachments. Vulval, uterine , anal depressor and sphincter muscles, but not pharynx, also express strongly. Muscle arms are also seen projecting to the nerve cords and ring. In addition, sheet-like structures lining the inner surface of the nerve ring are visible. Tentatively, these are the processes of mesoglial cells GLR. bPAT-3::GFP is also expressed transiently in many tissues. Larval neuroblasts such as ALM and Q descendants express during migration. Weaker signals are seen in the gonad, including distal tip cells and sheath cells. Finally, various cells express transiently in the late embryo. In conclusion, our data shows that both transient and continuous expression of pat-3 play roles in many different cell types.

Papp, Andy et al. (2009) International Worm Meeting "Investigation of Low-cost GFP Microscopy."

Papp, Andy, Aldrich, Chris, Bangalore, Srikanth, Perry, David

[International Worm Meeting, 2009]

The Green Fluorescent Protein (GFP) gene from the fluorescent jellyfish Aequorea victoria, and its variants (such as EGFP), are used extensively to study the location and timing of gene expression in transgenic animals and plants (Chalfie et al, 1994). Resultant GFP fusion proteins are especially well-suited to studying in vivo gene expression patterns in the transparent nematode, C. elegans and the transparent larva of the fly D. melanogaster. Perhaps one of the most significant limitations to its use in large-scale genetic screens is the high cost of equipment needed to observe GFP microscopically - namely the Fluorescence Dissecting Stereomicroscope for screening and picking mutants and upright and inverted epi-fluorescent compound microscopes for more detailed studies. Commercially available Fluorescence Dissecting Stereomicroscopes typically sell in the midrange between US$10,000 and US$20,000. They are offered by only the "high-end" microscope companies, based upon their most expensive dissecting scopes, and incorporate their premium-priced mercury arc-lamp illuminators, power supplies and epi-fluorescence modules. This leads us to wonder whether it is possible to cut corners without sacrificing utility, and which corners can be cut. Since the inception of GFP-based expression screens, a variety of researchers have produced custom-made and home-made GFP dissecting scopes. These include, for example, Welcome Bender (Harvard Medical School, pers. comm.) and Ian Chin-Sang (Chin-Sang, 2004). There are several possibilities to consider toward lowering the cost of a Fluorescence Dissecting Stereomicroscope. It may be possible to get sufficient light from relatively inexpensive, long-lived, lower-power-consuming Light Emitting Diodes (LEDs) vs. mercury arc lamps. Depending upon the spectral specificity of the LEDs, filters or dichroic mirrors might be omitted. Getting enough light intensity of the precise wavelengths that excite GFP fluorescence focused onto the sample is important. The exciting beam can be provided directly or focused backward through the microscope using "epi-illumination". We will explore these possibilities and report (and possibly demonstrate) the results. Chin-Sang, Ian. (2004) GFP Stereoscope Using LED light source. Queen''s University, Kingston, ON, Canada. http://130.15.90.245/gfp_stereoscope.htm.