D Miller et al. (1999) International C. elegans Meeting "Two-color GFP expression in C. elegans"

D Piston, D Hardin, A Fire, J Fleenor, G Patterson, D Miller, N Desai

[International C. elegans Meeting, 1999]

The Green Fluorescent Protein (GFP) is now widely used for imaging cells and organelles in living animals. GFP variants with distinct excitation and emission spectra have been developed and should prove highly useful for simultaneous imaging of separate proteins labeled with different colored GFPs. Here we describe expression vectors and fluorescence filter sets that can be used to obtain bright, distinct images of "Cyan" GFP (CFP) and "Yellow" GFP (YFP) in C. elegans (1). GFP vectors specifically designed for expression in C. elegans (2) were modified to include amino acid substitutions appropriate for CFP (Y66W, N146I, M153T, V163A) or YFP (S65G, V68A, S72A, T203Y) (see www.ciwemb.edu ). Previously described promoter regions and localization signals were employed to create trangenic animals expressing CFP and YFP either in different cells or in separate intracellular compartments. In these experiments, CFP appears "Blue" and the YFP signal is "Green." For example, an unc-4 promoter element drives expression in VA motor neurons whereas a del-1 upstream region is specific for the adjacent VB motor neurons. Transgenic animals expressing both unc-4::CFP and del-1::YFP display side-by-side VA (Blue) and VB (Green) motor neurons in the ventral nerve cord. In another experiment, cytoplasmically localized YFP and nuclear-localized CFP were expressed under the control of the muscle-specific unc-54 promoter to produce Green bodywall muscle cells with Blue nuclei. It should now be possible to utilize these vectors for a wide variety of two-color labeling experiments. The fluorescence filter sets used in the work were developed in collaboration with Chroma Technology Corp. For CFP: excitation = 436/10 nm; dichroic = 450 nm; emission = 485/50 nm For YFP: excitation = 500/20 nm; dichroic = 515 nm; emission = 520 nm long pass. (1) Miller, et al. (1999) BioTechniques, in press. (2) Fire, et al. (1998). p. 153-168. In M. Chalfie and S. Kain (Eds.), GFP Strategies and Applications. John Wiley and Sons, NY

Elizabeth B Rex et al. (2003) International Worm Meeting "Tyramine receptor (SER-2) isoforms in Caenorhabditis elegans:splicing within the third intracellular loop dictates octopamine affinity."

Elizabeth B Rex, Richard W Komuniecki

[International Worm Meeting, 2003]

Octopamine (OA) regulates essential processes in nematodes, such as pharyngeal pumping, locomotion and egg laying; however, little is known about the physiological role, if any, of its precursor, tyramine (TA). In the present study we have identified an alternatively-spliced tyramine receptor isoform, SER-2S, that differs from SER-2 and lacks 23 amino acids within the midregion of the third intracellular loop, 49 amino acids from TM5. RT-PCR using a C. elegans mixed stage cDNA pool suggests that both SER-2 isoforms are expressed at similar levels physiologically. Both SER-2 and SER-2S bind [3H] LSD in the low nM range and exhibit highest affinity for TA with Kis of 80 +/- 20 nM and 190 +/- 60 nM, respectively. Similarly, both isoforms exhibit nearly identical Kis for a number of octopaminergic/ adrenergic antagonists and TA reduces forskolin-stimulated cAMP levels in HEK293 cells expressing either SER-2 or SER-2S with nearly identical IC50s of 363 +/- 99 nM and 403 +/- 100 nM, respectively. In contrast, SER-2S exhibits a significantly lower affinity than SER-2 for other physiologically relevant biogenic amines, such as OA, dopamine and serotonin. Treatment of cells expressing SER-2 or SER-2S with pertussis toxin (PTX) has no effect on affinity for phentolamine, but dramatically reduces affinity for OA to similar levels for both SER-2 isoforms, suggesting that the mid-region of the third intracellular loop is involved in G-protein interactions and agonist affinity. Most importantly, these results suggest that C. elegans contains TA-specific receptors and that tyraminergic signaling may be important in the regulation of key processes in nematodes.

Melendez A et al. (1997) International C. elegans Meeting "PROGRESS REPORT ON LIN-13. A ZINC-FINGER PROTEIN THAT ACTS IN VULVAL DEVELOPMENT"

Greenwald IS, Melendez A

[International C. elegans Meeting, 1997]

Two heat sensitive alleles of lin-13 were identified in screens for vulval mutants and genetically characterized (Ferguson and Horvitz, 1985; Ferguson et al. 1987). lin-13(n387) hermaphrodites raised at 25oC are sterile and display a Multivulva phenotype. lin-13 homozygotes raised at 15oC are fertile and non-Muv but display a zygotically rescuable grandchildless phenotype. Deficiency studies suggest that the existing lin-13 alleles are hypomorphs, and that the lin-13 null phenotype is likely to be zygotic lethal. Therefore, lin-13 appears to have several roles in development. Along with Gautam Kao, we rescued the Muv and sterility phenotypes of lin-13 with cosmids CO3B8 and KO4F4. We have narrowed the rescuing activity to a 12-kb (MluI-SalI) genomic fragment that encodes a predicted zinc finger protein, and a frameshift mutation introduced into this coding region abolishes rescuing activity. We have isolated cDNAs corresponding to this genomic region and have started characterizing them. The lin-13 transcript appears to be SL1-spliced and of approximately 7-kb in length, encoding a large protein of about 2,100 amino acids with 14 zinc-fingers of the C-X(2-4)-C-X(3)-F-X(5)-L-X(2)-H-X(3)-H class. lin-13 has been grouped with the synMuv genes, which define two functionally redundant pathways that negatively regulate vulval development (Ferguson et al. 1987; Ferguson and Horvitz, 1989). Studies of synMuv genes have suggested that some act in hyp7 and at least one acts in the VPCs (Herman and Hedgecock, 1990; Hedgecock and Herman, 1995; J. Thomas and R. Horvitz, personal communication). We have begun a genetic mosaic analysis to determine the cellular focus of lin-13 in vulval development (lin-13 reporter constructs suggest that it is expressed in both hyp7 and the VPCs). We are also screening for lin-13 null alleles to understand further the role of lin-13 in development.

Hujber, Edward J. et al. (2015) International Worm Meeting "Three-dimensional super-CLEM."

Hobson, Robert J., Merrill, Sean A., Jorgensen, Erik M., Hujber, Edward J.

[International Worm Meeting, 2015]

Correlative light and electron microscopy (CLEM) can reveal a protein's localization relative to cellular features on the nano-scale. We have made several improvements to our protocol to improve imaging resolution in all three dimensions. Among the most important changes has been using organic dyes as fluorophores, instead of fluorescent proteins. This allows us to localize tagged proteins to high resolution in three dimensions using a biplane imaging approach. Optimizing embedding resins, fixatives, and staining protocols has enabled use of transmission electron tomography instead of scanning electron microscopy, providing us with sub-nanometer cellular context in three dimensions. Using a fiducial-based alignment, we have correlated proteins with cellular structures at ~30 nm lateral and ~50 nm axial resolution.

Choi, Shin Sik et al. (2009) International Worm Meeting "Size- and tissue-dependent endocytic transport of oral-administered nanoparticles."

Choi, Shin Sik, Rothman, Joel

[International Worm Meeting, 2009]

Recent attention has been focused on nanomaterials owing to their broad potential for biological and medical applications. Nanoparticles fabricated from metals or polymers have been used to enhance the quality of biomedical images by increasing contrast of specific targets. Moreover, nanomaterials have been combined with medicines in an effort to prolong their in vivo half-lives. Most research into medical applications of nanoparticles has involved injecting them into an animal. We have found that polystyrene nanoparticles can undergo in vivo dispersion in C. elegans through oral administration. We found that orally administered nano-size polystyrene beads pass through the pharynx and are taken up into the intestine. When we fed animals with 50, 100, 200, and 500 nm polystyrene beads, only 50 and 100 nm particles were mobilized beyond the gut and were found to accumulate in the gonad and epidermis. Interestingly, nanoparticles were not detected in muscles or neurons. At a certain concentration, we found that nanoparticles induced toxic effects accompanied by pathological events, including defects in egg laying and disruption of the intestine. To explore the requirements for nanoparticle transport, we tested uptake of 50 and 100 nm particles in worms defective for genes involved in endocytosis and found that movement from the intestine to other sites was blocked in such mutants. Thus, inter-organ transport of nanoparticles from the intestine to the gonad and other tissues appears to involve endocytosis. These findings suggest that the upper limit in this size of nanoparticles that can undergo endocytic transport from the gut to other organs may be less than 200 nm.

Robby Weimer et al. (2005) International Worm Meeting "Identification of functional subsynaptic domains by high-pressure freezing and immuno-electron microscopy in C. elegans."

Jean-Louis BESSEREAU, Olivier Meyrignac, Robby Weimer

[International Worm Meeting, 2005]

Chemical synapses are specialized sites of cell contact capable of relaying information from one cell to another quickly and with high fidelity. These biophysical properties are due, in part, to the spatial relationships between the synaptic vesicle exocytosis machinery and the postsynaptic neurotransmitter-sensing apparatus. To characterize the precise spatial organization of the synapse in the living state, we used high-pressure freezing combined with morphological and immuno-electron microscopy in the nematode Caenorhabditis elegans.High-pressure freezing is capable of converting liquid water, to a depth of approximately 0.2mm, into amorphic ice in less than 10 ms. Because the adult C. elegans hermaphrodite is ~0.1mm at its widest girth, an entire living adult animal can be physically immobilized nearly instantaneously by high-pressure freezing thus, providing a unique opportunity to examine cellular structures, specifically synapses, caught in suspended animation. After cryosubstitution and resin embedding, immunogold staining of ultrathin sections was used to localize proteins involved in different steps of the synaptic vesicle cycle. Specifically, we were able to visualize the localization of RAB-3, SYD-2, UNC-10, DYN-1 and UNC-49. Distribution of pre- and postsynaptic antigens was quantitatively analyzed using the electron-dense presynaptic density as landmarks.Our analysis suggests that neurotransmitter release sites are restricted to an area within 40 nm of presynaptic densities, which appear as modular structures of roughly 30 nm in diameter. Postsynaptic receptor fields are aligned with these release sites offset by a 40 nm cleft. Synaptic vesicle exocytosis likely proceeds through a primed hemi-fused intermediate that were observed within 40 nm of presynaptic densities, while vesicle recycling occurs at distance to release sites.

Franks CJ et al. (1998) European Worm Meeting "The Effect of 5-HT and Octopamine on Electrical Potentials Recorded from the Pharynx of Wild-Type C. elegans and a goa-1 mutant."

Chad JE, Franks CJ, Holden-Dye L, Walker RJ

[European Worm Meeting, 1998]

The C. elegans G protein subunit G-alpha-0 is encoded by the gene G-alpha-0. It has been implicated in mediating the effect of 5-HT on locomotion, defecation and egg laying (S!galat et al., 1995). 5-HT also stimulates pharyngeal muscle but goa-1 mutations do not alter this response suggesting that 5-HT signaling in this muscle does not involve G-alpha-0. However, G-alpha-0 is expressed in the pharyngeal muscle and goa-1 mutations, such as n1134, cause defective pharyngeal pumping. This indicates that G-alpha-0 is involved in the regulation of pumping, but by a neurotransmitter other than 5-HT. Here we present electrophysiological data indicating that octopaminergic modulation of pharyngeal pumping differs between wild-type and goa-1 mutants. The anterior region of C. elegans was sectioned from the body and placed in a perfusion chamber on an inverted microscope stage in modified Dent!s saline (composition in mM: NaCl 144, MgCl2 10, CaCl2 1, KCl 6, HEPES 5, pH 7.4). Intracellular recordings were made from pharyngeal muscle (60-100 MOhm microelectrodes; 3M KAcetate), connected to an Axoclamp 2A amplifier. Drugs were applied by perfusion . Firstly we made a quantitative comparison of the increase in action potential frequency elicited by 5-HT in the pharynxes of wild-type and n1134 C. elegans, to confirm the results of the behavioural studies that suggested that the 5-HT response was not mediated by Go. The 5-HT concentration-response curves were not different: EC50 in wild type was 124 nM (95% confidence limits 28-558 nM; n=6) and for n1134 it was 90 nM (95% confidence limits, 21-383 nM; n=6). We then went on to look at the wild-type response to octopamine. The preparation was perfused with 500 nM 5-HT to maintain pumping. Octopamine (threshold 500 nM) decreased action potential frequency (n=10). In some preparations this inhibition was followed by a transient excitation. For n1134, octopamine was significantly less effective at reducing action potential frequency compared to wild type (n=6). We are currently investigating responses elicited in a second goa-1 mutant, n363 and in a goa-1(xs) mutant. Reference: S!galat, L., D. A. Elkes and J. M. Kaplan. 1995. Modulation of serotonin-controlled behaviors by Go in Caenorhabditis elegans. Science 267: 1648-1651. Acknowledgments: Many thanks to Laurent S!galat for providing the n1134 C. elegans. Chris Franks is funded by the BBSRC.

Kato Y et al. (2000) Japanese Worm Meeting "Immune defense of worms: In vitro antimicrobial properties of recombinant ASABF-type antimicrobial peptides isolated from Caenorhabditis elegans and Ascaris suum"

Suzuki M, Kawano K, Miyazawa M, Zhang H, Matsuura A, Aizawa T, Kato Y, Nitta K, Yoshida S, Murakami R, Koganezawa N

[Japanese Worm Meeting, 2000]

Although invertebrate model animals are thought to be useful for studying innate immunity, the immune defense of C. elegans still remains unclear. Previously, we suggested that ASABF-type antimicrobial peptides, CS alpha beta-type antimicrobial peptides containing four intramolecular disulfide bridges, should contribute to the immune defense in worms. To date, six members of the ASABF-type antimicrobial peptides have been identified, i.e., ABF-1 and 2 in C. elegans, and ASABF-alpha, beta, gamma, and delta in A. suum. In this study, a recombinant ABF-2 and ASABF-alpha were produced using a yeast expression system, and their antimicrobial activity was characterized in detail. The recombinant ASABF-alpha was effective against all tested Gram+ bacteria (7/7, 50% bactericidal or microbicidal concentration (BC50) = 0.01-0.7 nM except for Leuconostoc mesentreroides), part of the Gram- bacteria (8/14, BC50 > 70 nM), and part of the yeasts (3/9, BC50 > 10 nM). Hemolytic activity against human erythrocytes was almost negligible. Less than a 1-min contact was enough to kill Staphylococcus aureus ATCC6538P. The bactericidal activity against S. aureus was inhibited by salts. The antimicrobial property of the recombinant ABF-2 was similar to that of ASABF-alpha. These results suggest that ASABF-type antimicrobial peptides should contribute to worm immunity against various prokaryotic and eucaryotic microbes.

Melendez A et al. (1996) East Coast Worm Meeting "lin-13 encodes a zinc finger protein"

Melendez A, Greenwald IS

[East Coast Worm Meeting, 1996]

Two heat sensitive alleles of lin-13 were identified in screens for vulval mutants and genetically characterized (Ferguson and Horvitz, 1985; Ferguson et al. 1987). lin-13(n387) hermaphrodites raised at 25oC are Multivulva (Muv) and sterile. lin-13 homozygotes raised at 15oC are fertile and non-Muv but display a zygotically rescuable grandchildless phenotype. Deficiency studies suggest that the existing lin-13 alleles are hypomorphs, and that the lin-13 null phenotype is likely to be zygotic lethal. Therefore, lin-13 appears to have several roles in development. lin-13 maps very close and to the left of mab-5 (M. Costa and C. Kenyon, personal communication). Pools of cosmids for the implicated genomic region were tested in germline transformation experiments, and cosmids C03B8 and K04F4 can rescue both the sterility and Muv phenotype of lin-13 (G. Kao, A. Mele'ndez and I. Greenwald, unpublished observations). A 16-kb genomic (Mlu I) fragment rescued the Muv and sterility phenotypes of lin-13 mutants. The genomic DNA corresponding to the lin-13 rescuing Mlu I fragment contains at least two predicted open reading frames: a protein containing many zinc finger motifs and a protein with ankyrin repeats (A. Coulson, J. E. Sulston and R. H. Waterson, personal communication). We found that a 12-kb (BstE II-Mlu I) fragment, excluding the 5' end of the predicted zinc finger containing protein, no longer rescued. By contrast a 12-kb (Mlu I-Sal I) fragment that included the predicted zinc finger protein and eliminated most of the genomic region predicted to encode the ankyrin repeat protein still rescued. We have also introduced a frameshift mutation into the predicted zinc finger protein coding region of the 16-kb Mlu I genomic fragment and it no longer rescued. All of these results suggest that lin-13 encodes a zinc finger containing protein. lin-13 has been grouped with the SynMuv genes (Ferguson et al. 1987). Studies of SynMuv genes have suggested that some act in hyp7 and at least one acts in the VPCs (Herman and Hedgecock, 1990; Hedgecock and Herman, 1995; J. Thomas and R. Horvitz, personal communication). We therefore plan to determine the cellular focus of lin-13 activity by using reporter genes and genetic mosaic analysis.

Jessica Rivera et al. (2002) West Coast Worm Meeting "Visualizing synapses in the motor neuron circuit."

Sam Wells, Christina Gleason, David M Miller III, Jessica Rivera

[West Coast Worm Meeting, 2002]

A major emphasis of this laboratory is to unravel the mechanism whereby the UNC-4 homeoprotein regulates synaptic specificity in the motor neuron circuit. In unc-4(e120) mutants, the usual inputs to VA motor neurons from AVD, AVD, and AVE command interneurons are replaced with gap junctions from AVB, which are normally reserved for VB motor neurons. The unc-4 wiring defect was initially detected by John White and colleagues by EM reconstruction of serial sections. We are now developing an alternative approach to detect motor neuron-specific synapses in living animals in the confocal microscope. The idea is to label presynaptic and postsynaptic proteins with different colored GFPs and then to use specific promoters to drive expression of these GFP-tagged markers in the command interneurons and in their postsynaptic motor neuron partners. The Zeiss LSM 510 confocal microscope provides argon laser lines that are ideal for discrete excitation of CFP (458 nm) and YFP (514 nm). An authentic synapse in the C. elegans motor neuron circuit labeled in this manner should appear as superimposed CFP and YFP "spots" in the ventral nerve cord.