Victoria Andriulis

Illinois State University; Normal IL, United States of America

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.

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.

Hughes, K. et al. (2019) International Worm Meeting "Physical exertion exacerbates deterioration in an animal model of Duchenne muscular dystrophy."

Rodriguez, Anjelica, Vemuri, Samantha, Barickman, Lucas, Andriulis, Victoria, Flatt, Kristen, Vidal-Gadea, Andres, Ray, Sneha, Niswonger, Dana, Gutta, Neha, Stein, Wolfgang, Hughes, K., Veerappan, Visalakshi, Schroeder, Nathan, Singhvi, Aakanksha, Kullman, Alex, Lim, Calis, Rodemoyer, Brian, Schuler, Andrew, Cuciarone, Kori

[International Worm Meeting, 2019]

Duchenne muscular dystrophy (DMD) is a genetic disorder caused by loss of dystrophin, responsible for connecting actin to the sarcolemma and transferring force into the extracellular matrix. In humans, DMD presents at a young age, resulting in developmental delays, muscle necrosis, increased sarcoplasmic calcium, loss of ambulation, and early death. Current animal models are unable to model the severity of DMD without the addition of sensitizing mutations. Thus, it remains elusive if increased sarcoplasmic calcium observed in dystrophic muscles follows or leads the mechanical insults caused by the muscle's disrupted contractile machinery. This knowledge has important implications for patients, as physiotherapeutic treatments may either help or exacerbate symptoms, depending on how dystrophic muscles differ from healthy ones. We observe that sarcoplasmic calcium dysregulation in dys-1 worms precedes overt structural phenotypes and can be mitigated by silencing calmodulin expression. Recently, we showed that burrowing dystrophic (dys-1) worms recapitulate many salient phenotypes of DMD. Here, we report dys-1 worms display early pathogenesis and increased lethality. To learn how dystrophic musculature responds to altered physical activity, we cultivated dys-1 animals in environments requiring either high intensity or high frequency muscle exertion during locomotion. We find that several muscular parameters (such as size) improve with increased activity. However, longevity in dystrophic animals was negatively associated with muscular exertion regardless of the duration of the effort. The high degree of phenotypic conservation between dystrophic worms and humans provides a unique opportunity to gain insights into DMD's underlying pathology and to assess potential treatment strategies.

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.