Bainbridge, Chance et al. (2017) International Worm Meeting "Investigation of the behavioral and cellular basis for magnetotaxis in C. elegans."

Benefield, Zachary, Bainbridge, Chance, McDonald, Jocelyn, Jackson, Janelle, Nguyen, Ken, Padia, Samantha, Hall, David, Vidal-Gadea, Andres, Barickman, Lucas

[International Worm Meeting, 2017]

The magnetic field of the Earth provides directional information to organisms across many taxa (from bacteria to mammals). However, the cellular and molecular mechanisms necessary for magnetic field detection in animals remain poorly understood, in part, because of the genetic, behavioral, and neurological intractability of the species studied. We previously showed that C. elegans readily orients to magnetic fields of earth-strength through a pair of identified sensory neurons (AFDs). This opens the door for the use of this behaviorally and genetically tractable species in the study of magnetotransduction. Much remains to be understood regarding how C. elegans transduces magnetic information, how this information is encoded, and how it ultimately affects the animal's behavior. Our lab studies the behavioral, cellular, and molecular basis of magnetotransduction in C. elegans. By recording animals orienting to magnetic stimuli, we determined that C. elegans orients to magnetic fields by modulating the frequency of deep bends during locomotion. We found that the integrity and number of AFD sensory villi are critical for magnetic orientation in C. elegans. Magnetotransduction and magnetic orientation in C. elegans function light-independently. We used streptavidin coated magnetic beads and atomic force microscopy to track the potential presence of endogenous magnetic particles in C. elegans. The behavioral, cellular, and genetic tractability of C. elegans offer a unique valuable opportunity to elucidate the mechanism through which animals harness the magnetic field of the earth to enhance their survival.

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.

Yochem JJ (2000) Midwest Worm Meeting "Toward a genetic understanding of molting"

Yochem JJ

[Midwest Worm Meeting, 2000]

The question of molting is being analyzed by three genetic approaches. One is a study of mutations in lrp-1, the product of which resembles gp330/megalin, a large member of the LDL receptor family. Worms mutant of LRP-1 are often encased in old cuticle or have a train of old cuticle that remains attached to the tail. Growth of wild-type worms in cholesterol-deficient media phenocopies lrp-1 mutations, suggesting that LRP-1 endocytoses dietary sterols that are required for the synthesis of a proper cuticle or for the shedding of old cuticle during ecdysis. These propositions, however, remain unproved. A second approach is therefore the isolation of mutations in genes other than in lrp-1 that confer a similar defect in the completion of molting. Besides the potential to increase our understanding of molting, these mutations may define genes that interact with lrp-1. A third approach is the isolation of mutations that disrupt molting in ways different from lrp-1 mutations. Thus far, three such mutations have been analyzed. Located near unc-29 on LGI, sv15 confers dumpiness, molting defects, and blistering of the cuticle. Present at low penetrance are deformed worms that have large protuberances. Cosmid rescue of the mutation will be described. sv3 X and sv9 ? both confer an unusual phenotype: homozygotes arrest growth in the L3 stage and are constricted by unshed cuticle for most of the middle part of the body. Cuticle is shed from the extremities, producing worms that resemble dumbbells. Attempts at cosmid rescue of sv3 will be described. The support of Bob Herman, Simon Tuck, Min Han, and Jocelyn Shaw is gratefully acknowledged.

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.

Tedesco PM et al. (1992) Worm Breeder's Gazette "Integrated Transgenic Lines Rescue Low Fertility in Age Strains"

Johnson TE, Tedesco PM

[Worm Breeder's Gazette, 1992]

Our main focus is on the positional cloning of age-1 .We based our initial cloning strategy on the cosegregation of Age and a reduced fertility (Fer) phenotype in backcrosses of age-1 to N2 ,making the assumption that the same mutational event gave rise to both traits. This assumption simplified the cloning because it is much easier to identify cosmids complementing the Fer trait than the Age trait. We showed that the fer-15 region was responsible for the reduced fertility by deficiency mapping and presented the corresponding physical map (WBG 11.4:14,15). Cosmid T13F9 detected end points of deficiencies immediately to the left and right of fer-15 ;thus T13F9 contains fer-15 ,while age-1 maps closer to unc-4 (WBG 11.4:74). As a final proof that the Age phenotype is separable from Fer we made a series of transgenic animals using the rol-6 coinjection strategy; we also included an actin-4 plasmid (pA4 Gal,Jocelyn Shaw) that was reported to increase the frequency of line formation (Mello, WBG 11.3:12). The injections were successful when we coinjected T13F9 and we saw complementation for fer-15 (b26)at 25 C, although the level of complementation was low. At 20 C little effect on fertility was observed (Table 1). We isolated two independent integrated lines starting with one of the cosmid/Rol-6 arrays and have found that one of these lines complements fertility quite well at 20 C and at 25 C. The other integrated line fails to complement at 20 C and has lower levels of complementation at 25 C. We were not able to get complementation using C26D10 which overlaps extensively with T13F9 and which was reported by Brister and L'Hernault (WBG 11.4:21) to contain fer-15 . These results show that integrated arrays work better for complementation than do extrachromosomal arrays. The quantitative approach we've employed is useful because, unlike qualitative analyses, quantitative analysis can provide better insight into the the level of function of integrated arrays positioned in different chromosomal milieus. We are planning life span assays of these integrated transgenics and hope that they will confirm the fact that the Age and Fer traits are genetically separable. [See Figure 1]