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[
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.
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[
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.
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[
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.
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[
West Coast Worm Meeting,
2004]
In C. elegans epidermal intermediate filaments (IFs) and their associated structures, the trans-epidermal attachments, are essential for embryonic epidermal elongation (Woo et al 2004). The formation of muscle contractile units and trans-epidermal attachments are mutually dependent during epidermal elongation. To understand how the connection between epidermis and muscle is established and how the two tissues communicate during organogenesis, we performed a screen for epidermal elongation-defective mutants. One locus identified in this screen was defined by three lethal alleles and mapped to the cluster of LG II. Subsequent analysis showed that these mutations were allelic to
vab-13 and
ven-3 . By genetic mapping and allele sequencing we showed that all these mutations affect F10E7.4, which encodes the C. elegans member of the F-spondin family of secreted proteins. F-spondin has been shown to play roles in axon guidance, cell migration, and angiogenesis. Our genetic analysis shows that in C. elegans F-spondin is required for epidermal elongation and muscle attachment, as well as for proper positioning of neuronal processes. Using GFP reporters, we found that F-spondin is expressed in body muscle cells and is a secreted protein. Thus, F-spondin may function in embryogenesis in communication between muscle and epidermis. Immunostaining of F-spondin mutants suggest that F-spondin may indirectly affect the organization of epidermal actin microfilaments and trans-epidermal attachments . We are examining the expression patterns of muscle and basement membrane components in F-spondin mutants. To study the signaling pathways regulated by F-spondin, we are testing mutations in candidate receptor genes for genetic interactions with F-spondin mutations.
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[
C.elegans Neuronal Development Meeting,
2008]
Release of neurotransmitters from neurons is highly regulated. Several proteins play roles in this process, including UNC-13, and decreased release of neurotransmitters in
unc-13 mutants results in paralysis. We identified an F-box protein that interacts with UNC-13. F-box proteins participate in ubiquitin ligase complexes and in Drosophila, DUNC-13 is degraded via the ubiquitin proteasome pathway. This UNC-13/F-box interaction may therefore indicate that UNC-13 is tagged for proteasomal degradation with ubiquitin by the ligase complex in C. elegans. The C. elegans knockout consortium isolated a strain with a large deletion in the coding region of the gene that codes for the F-box protein. If the F-box protein is indeed involved in the degradation of UNC-13, this strain would be expected to have higher levels of UNC-13, which could result in changes in phenotypes. We characterized the F-box deletion mutant by assaying brood size, developmental rate, and body bends per minute. Aldicarb assays were used to determine whether a deletion in the gene coding for the F-box protein alters the response to inhibitors of acetylcholinesterase. We found that the deletion resulted in some changes in developmental rate and in aldicarb sensitivity. We are continuing to study strains with mutations in both the gene coding for the F-box protein and in
unc-13.
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[
International Worm Meeting,
2005]
Actin has been shown to play key roles during early development in the C. elegans embryo (Schneider and Bowerman, 2003). However, it has been difficult to faithfully reproduce F-actin dynamics in vivo during early embryogenesis. To visualize F-actin we have generated a transgenic line that uses the F-actin binding domain of Drosophila moesin to decorate endogenous actin filaments with GFP (GFP::Moe). The GFP::Moe fusion line appears to be specific for filamentous actin and allows the visualization of F-actin dynamics in C. elegans in embryos. Preliminary evidence shows that F-actin is very dynamic in many cellular processes from prior to fertilization through the first mitotic cellular division. During fertilization a posterior actin cap forms and then dissipates. Prior to the first cell division F-actin becomes enriched in the anterior, similar to the anterior PAR proteins. As seen in
par-3 mutants (Kirby et. al., 1990), depleting the embryo of PAR-6 with RNAi prevents this enrichment, suggesting that PAR-6 and the other anterior PARs may be required for F-actin to accumulate in the anterior. We have also observed the presence of highly dynamic actin comets in the early embryo. Preliminary evidence suggests that these comets may be involved with endocytic processes. Using results from large-scale RNAi surveys, we are employing the GFP::Moe line to perform secondary screening of genes associated with pseudocleavage defects to further characterize this process. We will present our progress in using this GFP::Moe line to study genetic interactions involving actin dynamics in early development.
-
[
International C. elegans Meeting,
2001]
The ubiuquitin-proteasome pathway is a key mechanism for substrate-specific degradation to control the abundance of a number of proteins. SCF complex, one of ubiquitin-protein ligases (E3s), regulates cell cycle progression, signal transduction, and many other biological systems. The SCF complex consists of invariable components, such as Skp1, Cul-1 and Rbx1, and variable components called F-box proteins that bind to Skp1 through the F-box motif. F-box proteins are substrate-specific adaptor subunits that recruit substrates to the SCF complex. Surprisingly, we found that the genome of Caenorhabditis elegans ( C. elegans ) contains at least 20 Skp1-like sequenses, whereas one or a few Skp1 is present in humans. Therefore, we studied C. elegans Skp1-like proteins (CeSkp1) that are likely to be variable components of SCF complex in addition to F-box proteins. At least, seven CeSkp1s were associated with C. elegans Cul-1 (CeCul-1) in yeast two-hybrid system as well as co-immunoprecipitation assay in mammalian cells, and these expression patterns were different in C. elegans . By RNA interference (RNAi), two of these CeSkp1s showed embyonic lethality and four showed the phenotype of slow growth. There were differences among CeSkp1s in ability to interact with F-box proteins. These results suggest that CeSkp1s, like F-box proteins, act as variable components of SCF complex in C. elegans .
-
[
C. elegans: Development and Gene Expression, EMBL, Heidelberg, Germany,
2010]
Morphogenesis requires dynamic interaction between the adherens junctions and the actin cytoskeleton. This interaction is mediated by ?-catenin, which was proposed to be a stable bridge between the junctions and F-actin at the apical regions of polarized epithelial cells. However, ?-catenin cannot bind to F-actin and its adherens junction partner, ?-catenin, simultaneously, suggesting the bridge is dynamic and complex. ?-catenin was thought to inhibit Arp2/3-based branched actin at the junctions for normal maturation of adherens junctions. Our research suggests a new positive role for Arp2/3-dependent branched actin in junctional maturation and during embryonic cell movements of C. elegans . In this study we investigate how progressive WAVE/Scar and Arp2/3-depen dent accumulation of ?-catenin at the adherens junctions contributes to the accumulation of F-actin at apical regions of epithelial cells. We find that removing Arp2/3-dependent actin nucleation disrupts junctional maturation and results in altered levels of adherens junctional proteins in epithelial tissues. In particular, there is a drop in both ?-catenin and F-actin levels at the apical intestine, and altered organization of the apical intestine. Subcellular fractionation reveals that loss of Arp2/3-dependent actin nucleation reduces the amount of ?-catenin in the membrane-associated pool. These data demonstrate an essential role for Arp2/3 to dynamically remodel F-actin to support adherens junctions and polarized F-actin during cell migration and tissue morphogenesis.
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[
Mid-west Worm Meeting,
2004]
P-granules are complexes of proteins and RNA found surrounding the nuclei of C. elegans germ cells and germ cell precursors. GLH (germline RNA helicase) proteins are components of the germline specific P-granules, which are necessary for fertility in C. elegans . PAN-1, a P-granule associated novel protein, was identified as a GLH-binding protein in yeast two hybrid assays. PAN-1 contains some conserved amino acids of N-terminal F-box motifs, as well as sixteen leucine-rich repeats and a weak FOG-2 homology (FTH) motif, each found in F-box proteins. F-box proteins, in the SCF (SKP-1, Cullin, F-box) complex, utilize ubiquitin-mediated substrate degradation. When
pan-1 is eliminated by RNA interference (RNAi), the larvae arrest between the L1 and L2 stages and can survive eight days at 20 0 C. A
pan-1(
gk142) deletion strain exhibits the same 'forever-young' phenotype. mRNA analysis and protein expression show that PAN-1 is not germline specific but is germline enhanced. Experiments are ongoing to separate potential germline and somatic functions of PAN-1. If PAN-1 belongs to the family of F-box proteins, it may be implicated in regulating GLH protein levels, as are two other GLH binding proteins, CSN-5 and KGB-1.
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Van Damme, Sara, Schoofs, Liliane, Watteyne, Jan, De Fruyt, Nathan, Beets, Isabel, Fadda, Melissa
[
International Worm Meeting,
2021]
Neuropeptides are an evolutionarily conserved group of neuromodulators that regulate a wide range of adaptive behaviors, such as learning. Yet, unravelling the molecular and circuit mechanisms underlying this neuropeptidergic modulation is challenging due to the diversity of neuropeptide signaling pathways, and their 'wireless' extrasynaptic mode of action. Using reverse pharmacology, we have constructed a molecular map of the C. elegans neuropeptide-receptor network. Phylogenetic reconstruction of the evolutionary history of nematode neuropeptide systems across bilaterian animals revealed several nematode-specific diversifications of neuropeptide signaling in addition to evolutionarily ancient neuropeptide pathways. One of these ancient, conserved pathways is a neuropeptide Y/F (NPY/F)-like signaling system that is an important regulator of learning behavior both in Proto- and Deuterostomia. We found that NPY/F-like FLP-34 neuropeptides are required in serotonergic neurons for aversive olfactory associative learning, which is functionally similar to the role of NPY in vertebrate learning as well as to the role of NPF in invertebrate learning. NPY/F-like neuropeptides are released from serotonergic neurons and signal through the G protein-coupled receptor NPR-11 in the excitatory AIA interneurons to facilitate olfactory aversive learning. In addition, signaling through NPY/F-like receptor NPR-11 also affects learning in salt gustatory plasticity, a gustatory associative learning paradigm. NPY/F-like signaling is not the only neuropeptidergic signaling system affecting learning behavior; we discovered additional neuropeptides that appear to be important to the learning process as well, including peptides that are expressed in non-neuronal cells. Our current research focuses on unravelling the functions of such non-neuronal neuropeptide messengers in learning and other types of behavioral plasticity.