Snyder PM et al. (1994) J Biol Chem "Membrane topology of the amiloride-sensitive epithelial sodium channel."

Welsh MJ, Stokes JB, Snyder PM, McDonald FJ

[J Biol Chem, 1994]

The amiloride-sensitive epithelial sodium channel (ENaC) is involved in fluid and electrolyte absorption across a number of epithelia, and cloning of several ENaC subunits has begun to facilitate investigation of the structure, function, and regulation of this channel. Analysis of the amino acid sequence has revealed two potential membrane-spanning domains, but little else is known about the structure of ENaC. To investigate the membrane topology of one subunit, alpha rENaC, we used in vitro transcription, translation, and translocation into microsomal membranes. This generated a glycosylated protein of 93 kDa. Sequence analysis also revealed eight potential sites for N-glycosylation, six of which were found to be glycosylated (Asn190, Asn259, Asn320, Asn339, Asn424, and Asn538), indicating that they are extracellular. The C terminus was localized as intracellular based on antibody recognition and protease sensitivity of a tagged epitope at the C terminus. The N terminus was also found to be intracellular, based on its protease sensitivity. Similar results were obtained by expression in Xenopus oocytes. Together, these results support a model of alpha rENaC consisting of an intracellular N terminus and C terminus, a large N-glycosylated extracellular domain, and two membrane-spanning domains that each pass once through the plasma membrane. Because of their sequence similarity, it is likely that this structure is shared by other ENaC subunits and possibly the degenerins of Caenorhabditis elegans as well.

Fabig G et al. (2019) Methods Cell Biol "In situ analysis of male meiosis in C. elegans."

Muller-Reichert T, Laue M, Schwarz A, Fabig G, Striese C

[Methods Cell Biol, 2019]

We describe a routine method to locate cells of appropriate meiotic stages in the gonad of Caenorhabditis elegans males prior to 3D reconstruction of meiotic spindles by electron tomography. For this, serial semi-thick (300nm) sections of whole worms are pre-screened and recorded at low magnification by transmission electron microscopy. Cells of interest are identified in aligned image stacks showing the entire proximal region of male gonads at low magnification. Tilt series of selected cells are then recorded at higher magnification to reconstruct meiotic spindles of selected cells in 3D. Our approach allows a routine staging of spermatocytes without the use of anesthetics or the application of physical immobilization of worms. We also describe a modification of a previously published protocol (Muller-Reichert, Srayko, Hyman, O'Toole, & McDonald, 2007) by using polyvinylpyrrolidone (PVP) instead of bovine serum albumin (BSA) as a "filler" for specimen loading in high-pressure freezing.

Paupard M-C et al. (2000) East Coast Worm Meeting "Improved Tissue Preservation Using Metal Mirror Fixation or High Pressure Freezing for TEM"

Macaluso F, Stephney G, Paupard M-C, Hall DH

[East Coast Worm Meeting, 2000]

Recent reports (see below) showed that high pressure freezing (HPF) followed by freeze substitution is superior to chemical immersion fixation for C. elegans. HPF captures a more "life-like" view of the worm's ultrastructure. We compared HPF and a related technique, rapid freezing onto a metal mirror (MMF). For MMF, live animals on a small piece of filter paper are plunged against a metal mirror in liquid nitrogen. Freezing damage is often a problem, but some animals seem to be well frozen throughout. For HPF, we have tried two methods to concentrate live animals into a small metal planchette (see Lavin and McDonald ref's below). Further processing is the same for both methods. While holding at very low temperatures, the samples are freeze substituted into 1% osmium tetroxide in acetone, then embedded into plastic resin and cured for thin sectioning. By TEM fast-frozen worms reveal excellent views of membrane events and organelles. For instance, we see active endocytosis events that are not captured by chemical fixation. The microtubule network is better preserved and the basal laminae look strikingly different. Sample images are shown at www.aecom.yu.edu/wormem/new.html. HPF and MMF also hold promise for high resolution immunoEM. By reducing the osmium content and adding a dilute aldehyde fixative to the freeze substitution medium, we can better preserve structure than by our microwave technique (Paupard et al., submitted). We have successfully localized epitopes in thin sections from HPF samples. We are conducting HPF trials with Stan Erlandson and Ya Chen at the U. of Minnesota. MMF equipment is available here at Einstein and elsewhere. HPF machines are available to outside users in Madison, Berkeley, Minneapolis, and Albany. As our skills improve, we will offer such services to the C. elegans community. For further information on HPF, we recommend the following sources: Colleen Lavin's website at www.geology.wisc.edu/~uwmr/coating.html Martin Muller's website at www.em.biol.ethz.ch/ Kent McDonald, Methods in Molecular Biology, vol 117, pp. 77-97 (Humana Press) 1999.

Woog I et al. (2012) Methods Cell Biol "Correlative light and electron microscopy of intermediate stages of meiotic spindle assembly in the early Caenorhabditis elegans embryo."

Buchner M, Muller-Reichert T, Srayko M, White S, Woog I

[Methods Cell Biol, 2012]

This chapter is an update of the previously published book chapter "Correlative Light and Electron Microscopy of Early C. elegans Embryos in Mitosis" (Muller-Reichert, Srayko, Hyman, O'Toole, & McDonald, 2007). Here, we have adapted and improved the protocol for the isolated meiotic embryos, which was necessary to meet the specific challenges a researcher faces while investigating the development of very early Caenorhabditis elegans embryos ex-utero. Due to the incompleteness of the eggshell assembly, the meiotic embryo is very fragile and much more susceptible to changes in the environmental conditions than the mitotic ones. To avoid phototoxicity associated with wide-field UV illumination, we stage the meiotic embryos primarily using transmitted visible light. Throughout the staging and high-pressure freezing, we incubate samples in an isotonic embryo buffer. The ex-utero approach allows precise tracking of the developmental events in isolated meiotic embryos, thus facilitating the comparison of structural features between wild-type and mutant or RNAi-treated samples.

Stephney G et al. (2000) Midwest Worm Meeting "Improved Tissue Preservation Using Metal Mirror Fixation or High Pressure Freezing for TEM"

Hall DH, Macaluso F, Paupard M-C, Stephney G

[Midwest Worm Meeting, 2000]

Recent reports (see below) showed that high pressure freezing (HPF) followed by freeze substitution is superior to chemical immersion fixation for C. elegans. HPF captures a more "life-like" view of the worm's ultrastructure. We compared HPF and a related technique, rapid freezing onto a metal mirror (MMF). For MMF, live animals on a small piece of filter paper are plunged against a metal mirror in liquid nitrogen. Freezing damage is often a problem, but some animals seem to be well frozen throughout. For HPF, we have tried two methods to concentrate live animals into a small metal planchette (see Lavin and McDonald ref's below). Further processing is the same for both methods. While holding at very low temperatures, the samples are freeze substituted into 1% osmium tetroxide in acetone, then embedded into plastic resin and cured for thin sectioning. By TEM fast-frozen worms reveal excellent views of membrane events and organelles. For instance, we see active endocytosis events that are not captured by chemical fixation. The microtubule network is better preserved and the basal laminae look strikingly different. Sample images are shown at www.aecom.yu.edu/wormem/new.html. HPF and MMF also hold promise for high resolution immunoEM. By reducing the osmium content and adding a dilute aldehyde fixative to the freeze substitution medium, we can better preserve structure than by our microwave technique (Paupard et al., submitted). We have successfully localized epitopes in thin sections from HPF samples. We are conducting HPF trials with Stan Erlandson and Ya Chen at the U. of Minnesota. MMF equipment is available here at Einstein and elsewhere. HPF machines are available to outside users in Madison, Berkeley, Minneapolis, and Albany. As our skills improve, we will offer such services to the C. elegans community. For further information on HPF, we recommend the following sources: Colleen Lavin's website at www.geology.wisc.edu/~uwmr/coating.html Martin Muller's website at www.em.biol.ethz.ch/ Kent McDonald, Methods in Molecular Biology, vol 117, pp. 77-97 (Humana Press) 1999.

Paupard M-C et al. (2000) West Coast Worm Meeting "Improved Tissue Preservation Using Metal Mirror Freezing or High Pressure Freezing for TEM"

Macaluso F, Hall DH, Paupard M-C, Stepheney G

[West Coast Worm Meeting, 2000]

Several recent reports (see below) have demonstrated that C. elegans tissues can be very well preserved for electron microscopy by high pressure freezing (HPF) followed by freeze substitution, perhaps substantially better than by standard chemical immersion fixation. HPF shows the potential to capture a more "life-like" view of the worm's ultrastructure. We have been testing both HPF and a related technique, rapid freezing on a metal mirror (MMF) followed by freeze substitution. Both methods obtain similar high quality fixation, although there are some freezing artifacts using the metal mirror device that are eliminated in HPF. For MMF, live animals are concentrated on a small piece of filter paper and plunged against a metal mirror at liquid nitrogen temperature. While freezing damage often occurs about 5-15 microns into the worms, some animals are very well frozen throughout. The frozen samples are held at low temperature and freeze substituted into 1% osmium tetroxide in acetone, then embedded into plastic resin and cured for thin sectioning. For HPF, we have tried two methods to concentrate live animals into small metal planchette, either holding the animals within fine strands of dialysis tubing (C. Lavin, pers. comm.), or mixing them into a slurry of yeast paste to form a space-filling solid support (McDonald, 1999). Examination of fast-frozen specimens by TEM reveals excellent views of membrane events and organelles. For instance, we see many omega figures on coelomocytes which are indicative of active endocytosis, events which are not commonly captured by chemical fixation. Synaptic active zones and vesicles are well preserved, as are their relationships to microtubules. A network of microtubules can also been seen extending to the periphery of hypodermis. Basal laminae look strikingly different, much looser and more mesh-like when compared to chemical fixation. Sample images are shown on our website [www.aecom.yu.edu/wormem/new.html]. These two preparation methods, HPF and MMF, also hold great promise for high resolution immuno-EM. By reducing the osmium content and adding a dilute aldheyde fixation to the freeze substitution medium, we can obtain better resolution than is currently possible by our microwave technique. We have successfully localized epitopes in thin sections from HPF samples. MMF equipment is available here at Einstein campus. We are conducting HPF trials with the help of Stan Erlandson and Ya Chen at the University of Minnesota. As our skills improve, we will be happy to offer such services to the C. elegans community. For further information on HPF, we recommend the following sources: Colleen Lavin's website at www.geology.wisc.edu/~uwmr/caoting.html Martin Muller's website at www.em.bio.ethz.ch/ Kent McDonald, Methods in Molecular Biology, vol 117, pp. 77-97 (Human Press) 1999. In the U.S., there are HPF machines open to the outside users in Madison, Berkeley, Minneapolis and Albany.

Hardaway JA et al. (2014) J Neurosci Methods "An open-source analytical platform for analysis of C. elegans swimming-induced paralysis."

Fleming KA, Zhang B, Snarrenberg CL, Nackenoff A, Blakely RD, Wang J, Fleming PA, Whitaker SM, Hardie SL, Hardaway JA

[J Neurosci Methods, 2014]

BACKGROUND: The nematode Caenhorhabditis elegans offers great power for the identification and characterization of genes that regulate behavior. In support of this effort, analytical methods are required that provide dimensional analyses of subcomponents of behavior. Previously, we demonstrated that loss of the presynaptic dopamine (DA) transporter, dat-1, evokes DA-dependent Swimming-Induced Paralysis (Swip) (Mcdonald et al., 2007), a behavior compatible with forward genetic screens (Hardaway et al., 2012). NEW METHOD: Here, we detail the development and implementation of SwimR, a set of tools that provide for an automated, kinetic analysis of C. elegans Swip. SwimR relies on open source programs that can be freely implemented and modified. RESULTS: We show that SwimR can display time-dependent alterations of swimming behavior induced by drug-treatment, illustrating this capacity with the dat-1 blocker and tricyclic antidepressant imipramine (IMI). We demonstrate the capacity of SwimR to extract multiple kinetic parameters that are impractical to obtain in manual assays. COMPARISON WITH EXISTING METHODS: Standard measurements of C. elegans swimming utilizes manual assessments of the number of animals exhibiting swimming versus paralysis. Our approach deconstructs the time course and rates of movement in an automated fashion, offering a significant increase in the information that can be obtained from swimming behavior. CONCLUSIONS: The SwimR platform is a powerful tool for the deconstruction of worm thrashing behavior in the context of both genetic and pharmacological manipulations that can be used to segregate pathways that underlie nematode swimming mechanics.

David H Hall et al. (2001) International C. elegans Meeting "New! Improved! Fixation protocols for TEM"

David H Hall, Tylon Stephney, Gloria Stephney, Ya Chen, Frank Macaluso

[International C. elegans Meeting, 2001]

We continue to test alternate methods for preparing worms for transmission electron microscopy. We will describe new protocols, and will demonstrate what makes them better [or different] in comparison to previous methods (Hall, 1995). We still like simple immersion fixation and chopping open the animals by knife blade, and have made minor changes in the starting solutions to get optimum results. For early larval stages, which have never fixed well by immersion, and which are too little to chop open easily, we have adapted a new microwave protocol which gives very good results on intact worms. The resulting fixation looks equivalent to our immersion preparations of adults. Microwave fixation is proving very useful in the analysis of arrested animals from RNAi preparations, and should be excellent for looking at late embryos or dauers. Fast freezing methods offer a quite different approach, and the quality of tissue preservation can be superb. Both metal mirror freezing and high pressure freezing can produce excellent results, and they are achieving wider use over the past few years (Mohler et al., 1998; Rappleye et al., 1999). The inherent contrast after freeze substitution is often much greater, in part because the primary fixation contains only osmium, or a combination of osmium and aldehyde together. These methods allow much more rapid fixation. We can capture more "life-like" views of biological events in action, particularly for events such as vesicle fusions at the plasma membrane. Delicate cytoskeletal elements such as microtubules are also well preserved. We continue to try new combinations of fixatives and solvents to improve the appearance of nerve processes and synapses by fast freezing. Kent McDonald has been very helpful in suggesting improvements to these protocols. Laserhole fixations of embryos are technically rather difficult to accomplish, but can facilitate the passage of fixatives and embedding resins through the eggshell. We are continuing to use the protocol worked out by Carolyn Norris. See our website for details.

Magalska A et al. (2000) European Worm Meeting "The worm is not enough - the construction of cDNA and gDNA libraries from Ancylostoma ceylanicum"

Kapulkin V, Ciesielski A, Wedrychowicz HZ, Magalska A

[European Worm Meeting, 2000]

We are interested in usage of C. elegans as system for expression of hookworm genes. Hookworms (Ancylostomatideae ) are economically and medically important blood feeding nematodes which cause intestinal infections in humans and domestic animals. It has been reported recently that above 1 billion of human beings are currently infected with these worms. The application of C. elegans as a heterologous host for hookworm genes is limited mainly by the lack of hookworm genomic data. For this purpose we have constructed cDNA and gDNA libraries from the Ancylostoma ceylanicum -important human/animal parasite related to C. elegans. The cDNA library is based on PCR mediated amplification of spliced leader of parasite cDNA reported previously ( Martin SA, Thompson FJ, Devaney E. Mol. Biochem. Parasitol. 1995 Mar;70(1-2):241-5.). PCR amplified cDNA has been gel fractionated and cloned. The most abundant library is available now from adult mixed sex A. ceylanicum (above 6000 clones), smaller library was constructed from infective L3 larvae. The large inserts gDNA libraries were constructed by ligation of PFGE fractionated Ancylostoma genomic DNA into the pBACe3.6 (provided by Pieter deJong), both digested with EcoRI. Ligation mixture was precipitated with ethanol in the presence of yeast tRNA prior electroporation into DH10B host. Both cDNA and gDNA libraries were arrayed in 96 well format, and stored at -70C. Randomly picked clones were sequenced from both ends, blastn and blastx searches were performed. For cDNA clones our sequencing results show clear similarities to C. elegans genes : gene F38E11.2 hsp-12.6; similar to Human Alpha crystallin B chain, gene C15C7.6; similar to syntaxin-6, gene T20F5.2; similar to the S25B family of peptidases. For BAC clones, end sequencing was also performed ( in collaboration with M. obocka, Institute of Biochemistry and Biophysics ) , however no significant similarities were found (yet!).

Carolina Ibanez-Ventoso et al. (2007) International Worm Meeting "MicroRNAs in Aging and Healthspan."

Carolina Ibanez-Ventoso, Monica Driscoll, Mehul Vora

[International Worm Meeting, 2007]

Over 200 mutations that affect C. elegans longevity have been identified. To date, much of the research on C. elegans aging has focused on factors that extend lifespan with less emphasis on their effects on healthspan (the period of mid life vigor that precedes decline). Our lab has been interested in identifying genes that extend healthspan, conferring longer periods of youthful behavior. We have identified genetic and pharmacological manipulations that can extend healthspan with the use of new aging biomarkers and assays we''ve developed: decline of muscle integrity (physical markers and automated computer vision swimming analysis), age-associated accumulation of autofluorescent cellular material (lipofuscin and age pigments), and micoRNA (miRNA) abundance. miRNAs are small molecules prevalent in metazoans that regulate cellular expression by binding to partially homologus sites on gene transcripts to down-regulate translation. Our interest in these broad conserved regulators has been influenced by the suggestion that they might function in biology to limit stochastic biological processes—lost of robustness and increase in stochastic events is associated with aging. To address whether miRNAs could influence the aging process, we conducted the first genome wide-analysis of how miRNAs change with age in any organism. This work showed that about half of C. elegans miRNAs change significantly during adult lifespan and could thus contribute to healthspan [1]. Indeed the miRNA lin-4 influences lifespan and the expression of healthspan indicators like lipofuscin [2], work we have confirmed. Our preliminary work supports that several other miRNAs also impact lifespan and the quality of aging. We are exploiting the powerful experimental advantages of the nematode C. elegans to decipher roles of miRNAs in nematode healthspan and to test whether engineered expression of these small molecules can confer anti-aging therapies in C. elegans. Since many miRNAs are conserved in expression pattern and function, we anticipate that our exploratory work in this area may suggest specific working hypotheses for mammalian aging, perhaps influencing design of novel means of clinical intervention. 1.Ibanez-Ventoso et al. (2006) Modulated microRNA expression during adult lifespan in C. elegans. Aging Cell 5: 235-246. 2.Boehm M, Slack FJ (2005) A developmental timing microRNA and its target regulate life span in C. elegans. Science 310: 1954-1957.