KA McDonald et al. (1999) International C. elegans Meeting "Constitutively activated integrin during muscle development"

JZ Schwarz, KA McDonald, BD Williams

[International C. elegans Meeting, 1999]

Integrins are heterodimeric transmembrane proteins that bind components of the extracellular matrix (ECM). Recent advances have shown that integrin-mediated signaling can control fundamental cell properties such as motility, cytoskeletal remodeling, differentiation, and gene expression. We are investigating integrin function during muscle cell development in C. elegans. Body wall muscle cells express the integrin a subunit PAT-2 and b subunit PAT-3, both of which are located in regularly patterned cytoskeletal anchorage structures analogous to vertebrate muscle Z and M lines. pat-2 and pat-3 mutations disrupt cytoskeletal assembly, resulting in the Pat developmental arrest phenotype. The integrin heterodimer can modulate its affinity for ECM ligands and the ability of its cytoplasmic domain to interact with the cytoskeleton and associated signaling molecules. The "non-activated" heterodimer has a low affinity for extracellular ligands and does not show cytoskeletal association and related signaling. Conversely, the "activated" heterodimer has high affinity for ligand and does associate with the cytoskeleton. In our working model of body wall muscle development, integrin is activated by binding to an ECM ligand, and in response, initiates cytoskeletal assembly. To test this model, we have introduced a mutation into a functional pat-3::gfp construct that is predicted to constitutively "activate" the integrin heterodimer (Hughes et al., 1996, JBC 271: 6571). This mutation disrupts a highly conserved potential salt bridge between the a and b subunits. When expressed transgenically in a wild-type background the "activated" PAT-3::GFP protein localizes normally to muscle cell attachment structures. We have observed mispositioned sex muscle insertions in these animals, indicating dominant effects on cell migration and/or insertion. Surprisingly, the "activated" pat-3::gfp transgene rescues pat-3 loss-of-function homozygotes, but only at very low frequency. Most animals expressing the transgene arrest as Pats. We are currently characterizing the defects in muscle cell development that occur in these animals. The relatively few rescued adults often show examples of mispositioned body wall and sex muscles, again consistent with migration/insertion defects.

McDonald KA et al. (1998) Midwest Worm Meeting "Effects of constitutively activated and ligand binding deficient integrin beta subunits on muscle development in C. elegans."

McDonald KA, Williams BD

[Midwest Worm Meeting, 1998]

Muscle is a model paradigm for the assembly of cytoskeletal elements and cytoskeletal filament anchorages that are found in all cells. A crucial molecule involved in these processes is the transmembrane heterodimeric molecule, integrin. This family of adhesion molecules is virtually ubiquitous, and has proven to be necessary and regulatory in the differentiation of muscle tissue. Analysis of integrin's mode of action during muscle development has relied mainly upon the analysis of cultured cells. C. elegans muscle development provides an opportunity to study integrin function in vivo within intact embryos. An integrin beta subunit (PAT-3) and two alpha subunits (PAT-2, INA-1) have been identified in the nematode C. elegans. In body wall muscle cells the PAT-2 and PAT-3 subunits are located in regularly patterned cytoskeletal anchorage structures called dense bodies (analogs to the vertebrate striated muscle cell Z-lines) and M-lines. In mutants lacking either PAT-2 or PAT-3 the cytoskeleton of the body wall muscle cells does not assemble properly. These animals arrest development with the PAT phenotype (paralyzed, arrested elongation at two-fold). We are interested in dissecting the role that integrin plays during muscle development and cytoskeletal assembly. Analysis of vertebrate integrins has identified several highly conserved amino acids within the beta subunit that can be mutated to cause very specific defects in integrin function. One specific amino acid substitution in the extracellular domain blocks the binding of extracellular ligands. A second substitution within the short beta subunit cytoplasmic domain locks the integrin heterodimer into an activated state that is normally induced by ligand binding. Activation allows the cytoplasmic domain of the integrin heterodimer to interact with cytoskeletal proteins and assemble a solid-state signaling complex that has been associated with integrin-mediated cell signaling events. To investigate integrin function during muscle assembly in vivo, we first constructed a PAT-3::GFP translational fusion, and have found that is able to rescue pat-3 mutants. We then introduced the ligand-binding and the constitutive activation mutations into separate constructs. We have also made a construct that contains both mutations. To date, we have tested the ability of each of the mutant subunits to localize properly in the presence of the endogenous wild-type PAT-3 subunit in transgenic animals. We plan shortly to test whether the mutant constructs can rescue any or all aspects of muscle development in a pat-3 loss-of-function background. Our results so far indicate that if expressed at sufficient levels, the mutant subunit with defective ligand binding function causes the detachment of the dense bodies and M-lines from the basal surface of body wall muscle cells, and subsequent concentration of the contractile apparatus into the ends of affected cells. This result supports the conclusion, contrary to evidence in cultured vertebrate cells, that integrin is capable of associating with the cytoskeleton in absence of ligand binding (activation). The constitutive activation of the integrin beta subunit has no apparent affect on muscle structure in the presence of wild type protein. The affect of a constitutively active integrin on development and muscle structure is presently being assessed in developing pat-3 embryos.

Solorzano E et al. (2011) BMC Evol Biol "Shifting patterns of natural variation in the nuclear genome of caenorhabditis elegans."

Bergeron RD, Sung W, Okamoto K, Thomas WK, Solorzano E, Datla P

[BMC Evol Biol, 2011]

BACKGROUND: Genome wide analysis of variation within a species can reveal the evolution of fundamental biological processes such as mutation, recombination, and natural selection. We compare genome wide sequence differences between two independent isolates of the nematode Caenorhabditis elegans (CB4856 and CB4858) and the reference genome (N2). RESULTS: The base substitution pattern when comparing N2 against CB4858 reveals a transition over transversion bias (1.32:1) that is not present in CB4856. In CB4856, there is a significant bias in the direction of base substitution. The frequency of A or T bases in N2 that are G or C bases in CB4856 outnumber the opposite frequencies for transitions as well as transversions. These differences were not observed in the N2/CB4858 comparison. Similarly, we observed a strong bias for deletions over insertions in CB4856 (1.44: 1) that is not present in CB4858. In both CB4856 and CB4858, there is a significant correlation between SNP rate and recombination rate on the autosomes but not on the X chromosome. Furthermore, we identified numerous significant hotspots of variation in the CB4856-N2 comparison.In both CB4856 and CB4858, based on a measure of the strength of selection (ka/ks), all the chromosomes are under negative selection and in CB4856, there is no difference in the strength of natural selection in either the autosomes versus X or between any of the chromosomes. By contrast, in CB4858, ka/ks values are smaller in the autosomes than in the X chromosome. In addition, in CB4858, ka/ks values differ between chromosomes. CONCLUSIONS: The clear bias of deletions over insertions in CB4856 suggests that either the CB4856 genome is becoming smaller or the N2 genome is getting larger. We hypothesize the hotspots found represent alleles that are shared between CB4856 and CB4858 but not N2. Because the ka/ks ratio in the X chromosome is higher than the autosomes on average in CB4858, purifying selection is reduced on the X chromosome.

Ju J et al. (2023) Nucleic Acids Res "Structure of the Caenorhabditis elegans m6A methyltransferase METT10 that regulates SAM homeostasis."

Tomita K, Ju J, Kuroyanagi H, Aoyama T, Yashiro Y, Yamashita S

[Nucleic Acids Res, 2023]

In Caenorhabditis elegans, the N6-methyladenosine (m6A) modification by METT10, at the 3'-splice sites in S-adenosyl-l-methionine (SAM) synthetase (sams) precursor mRNA (pre-mRNA), inhibits sams pre-mRNA splicing, promotes alternative splicing coupled with nonsense-mediated decay of the pre-mRNAs, and thereby maintains the cellular SAM level. Here, we present structural and functional analyses of C. elegans METT10. The structure of the N-terminal methyltransferase domain of METT10 is homologous to that of human METTL16, which installs the m6A modification in the 3'-UTR hairpins of methionine adenosyltransferase (MAT2A) pre-mRNA and regulates the MAT2A pre-mRNA splicing/stability and SAM homeostasis. Our biochemical analysis suggested that C. elegans METT10 recognizes the specific structural features of RNA surrounding the 3'-splice sites of sams pre-mRNAs, and shares a similar substrate RNA recognition mechanism with human METTL16. C. elegans METT10 also possesses a previously unrecognized functional C-terminal RNA-binding domain, kinase associated 1 (KA-1), which corresponds to the vertebrate-conserved region (VCR) of human METTL16. As in human METTL16, the KA-1 domain of C. elegans METT10 facilitates the m6A modification of the 3'-splice sites of sams pre-mRNAs. These results suggest the well-conserved mechanisms for the m6A modification of substrate RNAs between Homo sapiens and C. elegans, despite their different regulation mechanisms for SAM homeostasis.

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.

Rose JK et al. (2022) Front Behav Neurosci "Neuroligin Plays a Role in Ethanol-Induced Disruption of Memory and Corresponding Modulation of Glutamate Receptor Expression."

Liang J, Lin CHS, Rose JK, Parvand M, Butterfield M, Rankin CH

[Front Behav Neurosci, 2022]

Exposure to alcohol causes deficits in long-term memory formation across species. Using a long-term habituation memory assay in Caenorhabditis elegans, the effects of ethanol on long-term memory (> 24 h) for habituation were investigated. An impairment in long-term memory was observed when animals were trained in the presence of ethanol. Cues of internal state or training context during testing did not restore memory. Ethanol exposure during training also interfered with the downregulation of AMPA/KA-type glutamate receptor subunit (GLR-1) punctal expression previously associated with long-term memory for habituation in C. elegans. Interestingly, ethanol exposure alone had the opposite effect, increasing GLR-1::GFP punctal expression. Worms with a mutation in the C. elegans ortholog of vertebrate neuroligins (nlg-1) were resistant to the effects of ethanol on memory, as they displayed both GLR-1::GFP downregulation and long-term memory for habituation after training in the presence of ethanol. These findings provide insights into the molecular mechanisms through which alcohol consumption impacts memory.

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.