Anton Karabinos et al. (2002) European Worm Meeting "Essential roles for four cytoplasmic intermediate filament proteins in Caenorhabditis elegans development"

Ralf Schnabel, Anton Karabinos, Klaus Weber, Henning Schmidt, Jens Harborth

[European Worm Meeting, 2002]

The structural proteins of the cytoplasmic intermediate filaments (IF) arise in the nematode C. elegans from eight previously reported genes and an additional three genes now identified in the complete genome. Using double stranded RNA interference (RNAi) for all 11 C. elegans genes encoding cytoplasmic IF proteins we observe phenotypes for the five genes A1, A2, A3, B1 and C2. These range from embryonic lethality (B1) and embryonic/larval lethality (A3) to larval lethality (A1 and A2) and to a mild dumpy phenotype of adults (C2). Phenotypes A2 and A3 involve displaced body muscles and paralysis. They probably arise by reduction of hypodermal IF which participate in the transmission of force from the muscle cells to the cuticle. The B1 phenotype has multiple morphogenetic defects while the A1 phenotype arrested at the L1 stage. Thus, at least four IF genes are essential for C. elegans development. Their RNAi phenotypes describe the first lethal defects due to silencing of single IF genes. In contrast to C. elegans no IF genes have been identified in the complete Drosophila genome, posing the question of how Drosophila can compensate for the lack of these proteins which are essential in mammals and C. elegans. We speculate that the lack of IF proteins in Drosophila can be viewed as cytoskeletal alteration in which, for instance, stable microtubules, often arranged as bundles, substitute for cytoplasmic IF.

Ingo Buessing et al. (2003) International Worm Meeting "Functional analysis of the single calmodulin and the four calmodulin like genes of C. elegans by RNA interference and 4D microscopy"

Ralf Schnabel, Ekkehard Schulze, Ingo Buessing, Jian Wang, Anton Karabinos, Klaus Weber

[International Worm Meeting, 2003]

Calmodulin (CaM), a small calcium binding protein, is a key mediator of numerous calcium induced changes in cellular activity. Its ligands include enzymes, cytoskeletal proteins and ion channels, identified in large part by biochemical and cell biological approaches. Little is known about the functions of CaM-like genes. Thus far it has been difficult to assess the function of CaM genetically, because of the maternal supply in Drosophila and the presence of at least three nonallelic genes in vertebrates. Here we have used RNAi to analyse the functional importance of the single calmodulin gene (cmd-1) and the four calmodulin-like genes (cal-1 to cal-4) present in C. elegans. We find that RNAi leads to embryonic lethality for the calmodulin gene cmd-1 and an uncoordinated phenotype for the cal-4 gene. By detailed analysis of the cmd-1 RNAi embryos with the 4D-Microscope system we observed disturbed morphogenesis, aberrant cell migration patterns and a striking hyperproliferation of cells in the treated embryos. Normal apoptosis is suppressed in some instances, but in addition ectopic cell deaths occur and engulfment of cell corpses is inhibited. RNAi also allows several genes to be inactivated simultaneously. The triple RNAi approach for the cal-1, cal-2 and cal-3 genes gives rise to a complex phenotype while individual inactivations do not lead to a scorable phenotype.

(1982) Worm Breeder's Gazette "Goettingen worm group activities 1981 and prospects 1982"

[Worm Breeder's Gazette, 1982]

After Gunter von Ehrenstein's death of a heart attack December 26, 1980, the 6 directors of the Max-Planck-Institute for Experimental Medicine decided to close the Department of Molecular Biology as of the end of 1982. We are continuing work to finish up and publish work in progress with gradual attrition as people find new positions elsewhere and with correspondingly diminishing budget. A list of our recent papers and manuscripts is given below. Eddi Isnenghi completed his paper on the phenotypes of the Gottingen emb mutants (including TSP's and maternal tests) and is trying to microinject cloned DNA to rescue worm mutants. He is also trying to transform yeast mutants with worm DNA. Randy Cassada is still trying to map embs on LG III with deficiencies and duplications and is looking for new strains of C. elegans in the wild. Eddi has received a 6-months' extension of his DFG (= NSF) fellowship until July. Ken Denich is writing up his work on emb mutant lineages in Gottingen for a paper and as PhD Thesis for the Department of Zoology, University of Manitoba and will be returning there this summer to finish up PhD requirements. Einhard Schierenberg has continued with laser-induced cell fusion experiments. If the membrane between 2 cells is disrupted at the right time, the division of both nuclei results in 4 more or less normal cells. Development continues to a twitching lima bean monster. Fusing cells in later stages results in uncoordinated animals. All treated embryos show timing defects in the E-cell line similar to emb mutants, perhaps due to remixing of cytoplasm. A copy of Einhard's film on embryogenesis is available (with commentary) from Scott Emmons for those who want to show it to students. Einhard is going to Bill Wood's lab this fall. Khosro Radnia has left for a job in industry. Ulli Certa and Franz Scharfenberg are characterizing histone H2A variants in emb mutants. They have also identified several histone H4 DNA clones in a C. elegans genomic library using a sea urchin probe obtained from Mike Grunstein. Ulli has finished his PhD and plans to go to Grunstein's lab at UCLA this spring for a post- doc on yeast histones. Ulli and the other gene cloners have been working successfully with similar minded members of the Institute's Department of Chemistry. Franz hopes to do his PhD in that department. Shahid Siddiqui has just joined Joe Culotti at Northwestern. Tom Cole is very happy with his new Zeiss EM 109 electron microscope. Our computer-aided reconstruction program with color display now works for LM and EM input. We are trying to transfer the programs and data to the MRC, so they will still be available after our department has closed. Chris Carlson has gone to the EMBL in Heidelberg as of Jan 1 to work on computer graphics of molecules. Ursula Reuter also has a position at EMBL as of Jan 1 as an EM technician. Ed Hedgecock visited for 3 weeks this fall to show us microinjection, antibody staining (with anti-actin from Mary Osborne and Klaus Weber), post- embryonic lineage techniques and the like. It was a very useful and pleasant visit. We hope to have some of you as visitors in 1982!

Jun Liu et al. (2001) International C. elegans Meeting "Essential roles for C. elegans lamin gene in nuclear organization, cell cycle progression and spatial organization of nuclear pore complexes"

Perah Spann, Jun Liu, Klaus Weber, Tom Rolef Ben-Shahar, Millet Treinin, Yosef Gruenbaum, Dieter Riemer, Andrew Fire

[International C. elegans Meeting, 2001]

We are interested in using C. elegans as a model system to study the function of nuclear lamin and lamin-associated proteins during development. C. elegans has a single lamin gene, designated lmn-1 (previously termed CeLam-1). Antibodies raised against the lmn-1 product (Ce-lamin) detected a 64 kD nuclear envelope protein. Ce-lamin was detected in the nuclear periphery of all cells except sperm and was found in the nuclear interior in embryonic cells and in a fraction of adult cells. Reductions in the amount of Ce-lamin protein produce embryonic lethality. Although the majority of affected embryos survive to produce several hundred nuclei, defects can be detected as early as the first nuclear divisions. Abnormalities include rapid changes in nuclear morphology during interphase, loss of chromosomes, unequal separation of chromosomes into daughter nuclei, abnormal condensation of chromatin, an increase in DNA content, and abnormal distribution of nuclear pore complexes (NPCs). Under conditions of incomplete RNA interference a fraction of embryos escaped embryonic arrest and continue to develop through larval life. These animals exhibit additional phenotypes including sterility and defective segregation of chromosomes in germ cells. Our observations show that lmn-1 is an essential gene in C. elegans , and that the nuclear lamins are involved in chromatin organization, cell cycle progression, chromosome segregation, and correct spacing of NPCs.

Redemann S et al. (2014) Methods Mol Biol "The segmentation of microtubules in electron tomograms using Amira."

Verbavatz JM, Muller-Reichert T, Hyman AA, Weber B, Prohaska S, Moller M, Redemann S, Baum D

[Methods Mol Biol, 2014]

The development of automatic tools for the three-dimensional reconstruction of the microtubule cytoskeleton is crucial for large-scale analysis of mitotic spindles. Recently, we have published a method for the semiautomatic tracing of microtubules based on 3D template matching (Weber et al., J Struct Biol 178:129-138, 2012). Here, we give step-by-step instructions for the automatic tracing of microtubules emanating from centrosomes in the early mitotic Caenorhabditis elegans embryo. This approach, integrated in the visualization and data analysis software Amira, is applicable to tomographic data sets from other model systems.

Chalfie M (2010) J Vis Exp "The 2009 Lindau Nobel Laureate meeting: Martin Chalfie, Chemistry 2008."

Chalfie M

[J Vis Exp, 2010]

American Biologist Martin Chalfie shared the 2008 Nobel Prize in Chemistry with Roger Tsien and Osamu Shimomura for their discovery and development of the Green Fluorescent Protein (GFP). Martin Chalfie was born in Chicago in 1947 and grew up in Skokie Illinois. Although he had an interest in science from a young age--learning the names of the planets and reading books about dinosaurs--his journey to a career in biological science was circuitous. In high school, Chalfie enjoyed his AP Chemistry course, but his other science courses did not make much of an impression on him, and he began his undergraduate studies at Harvard uncertain of what he wanted to study. Eventually he did choose to major in Biochemistry, and during the summer between his sophomore and junior years, he joined Klaus Weber's lab and began his first real research project, studying the active site of the enzyme aspartate transcarbamylase. Unfortunately, none of the experiments he performed in Weber's lab worked, and Chalfie came to the conclusion that research was not for him. Following graduation in 1969, he was hired as a teacher Hamden Hall Country Day School in Connecticut where he taught high school chemistry, algebra, and social sciences for 2 years. After his first year of teaching, he decided to give research another try. He took a summer job in Jose Zadunaisky's lab at Yale, studying chloride transport in the frog retina. Chalfie enjoyed this experience a great deal, and having gained confidence in his own scientific abilities, he applied to graduate school at Harvard, where he joined the Physiology department in 1972 and studied norepinephrine synthesis and secretion under Bob Pearlman. His interest in working on C. elegans led him to post doc with Sydney Brenner, at the Medical Research Council Laboratory of Molecular Biology in Cambridge, England. In 1982 he was offered position at Columbia University. When Chalfie first heard about GFP at a research seminar given by Paul Brehm in 1989, his lab was studying genes involved in the development and function of touch-sensitive cells in C. elegans. He immediately became very excited about the idea of expressing the fluorescent protein in the nematode, hoping to figure out where the genes were expressed in the live organism. At the time, all methods of examining localization, such as antibody staining or in situ hybridization, required fixation of the tissue or cells, revealing the location of proteins only at fixed points in time. In September 1992, after obtaining GFP DNA from Douglas Prasher, Chalfie asked his rotation student, Ghia Euskirchen to express GFP in E. coli, unaware that several other labs were also trying to express the protein, without success. Chalfie and Euskirchen used PCR to amplify only the coding sequence of GFP, which they placed in an expression vector and expressed in E.coli. Because of her engineering background, Euskirchen knew that the microscope in the Chalfie lab was not good enough to use for this type of experiment, so she captured images of green bacteria using the microscope from her former engineering lab. This work demonstrated that GFP fluorescence requires no component other than GFP itself. In fact, the difficulty that other labs had encountered stemmed from their use of restriction enzyme digestions for subcloning, which brought along an extra sequence that prevented GFP's fluorescent expression. Following Euskirchen's successful expression in E. coli, Chalfie's technician Yuan Tu went on to express GFP in C. elegans, and Chalfie published the findings in Science in 1994. Through the study of C. elegans and GFP, Chalfie feels there is an important lesson to be learned about the importance basic research. Though there has been a recent push for clinically-relevant or patent-producing (translational) research, Chalfie warns that taking this approach alone is a mistake, given how "woefully little" we know about biology. He points out the vast expanse of the unknowns in biology, noting that important discoveries such as GFP are very frequently made through basic research using a diverse set of model organisms. Indeed, the study of GFP bioluminescence did not originally have a direct application to human health. Our understanding of it, however, has led to a wide array of clinically-relevant discoveries and developments. Chalfie believes we should not limit ourselves: "We should be a little freer and investigate things in different directions, and be a little bit awed by what we're going to find."

Jegatheeswaran S et al. (2023) ACS Appl Mater Interfaces "Encapsulation of Caenorhabditis elegans in Water-in-Water Microdroplets to Study the Worm Viability: Alternative Avenue to Manipulate Microdroplet Environment."

Tan JH, Jegatheeswaran S, Hwang DK, Fraser AG

[ACS Appl Mater Interfaces, 2023]

Due to the great biocompatibility of the aqueous two phase system (ATPS), biological cells have been widely encapsulated in ATPS microdroplets (diameter &lt; 50 <i>&#x3bc;m)</i>. However, the immobilization of relatively large multicellular organisms such as <i>Caenorhabditis elegans</i> in ATPS droplets remains challenging as the spontaneous generation of droplets greater than 200 &#x3bc;m is difficult without external perturbations. In this study, we utilize a microneedle-assisted coflow microfludic channel to passively form ATPS microdroplets larger than 200 &#x3bc;m and successfully entrap <i>C. elegans</i> in the microdroplets. We monitor the worm viability and its temporal stroke frequency up to 6 h. We study the effects of dextran (DEX)-to-polyethylene glycol (PEG) flow ratios and worm concentration on the droplet diameter, worm encapsulation efficiency, and the number of droplets containing individual worms. Larger ATPS microdroplets (&gt;200 &#x3bc;m) form in the ranges of capillary number (<i>Ca</i>) between 0.020 to 0.20 and Weber number (<i>We</i>) between 10^-5 and 10^-3. An ATPS with the encapsulation ability and biocompatibility can offer an alternative immobilization tool for multicellular organisms to existing platforms such as water/oil droplets.

Memar, Nadin et al. (2015) International Worm Meeting "Never change a running system? A surprising similarity in nematode embryogenesis."

Schiemann, Sabrina, Conradt, Barbara, Chiesielski, Hanna, Memar, Nadin, Luthe, Katharina, Hennig, Christian, Schnabel, Ralf

[International Worm Meeting, 2015]

An important justification of genome projects in general is the notion that there should be a strong correlation between genotype and phenotype. C. elegans and C. briggsae show more differences at the genomic level than humans compared to mice [1]. However, the behavior and anatomy of these nematodes are overall very similar. C. elegans and C. briggsae shared the last common ancestor 80-100 million years ago [2]. Here we present a detailed analysis of the embryogenesis of C. elegans, C. briggsae, C. remanei and C. brenneri. Microscopic 4D analyses, including the terminal differentiation patterns and bioinformatical quantifications of cell behavior show that the embryogenesis of these nematodes cannot be distinguished. Thus, significantly differing genomes lead to morphological processes that are almost completely identical. To determine whether this process is also almost identical in more distantly related nematodes, we analyzed Pristionchus pacificus, which separated from C. elegans more than 280-430 million years ago [3]. We found that up to the pre-morphogenetic stage, when embryos contain 400 cells and are about to undergo morphogenesis, the Caenorhabditis species and P. pacificus share (beside some minor differences such as, for example, an additional cell death after the 9th cleavage round) identical cell positions and fates. However, starting with the pre-morphogenetic stage, development in P. pacificus and the Caenorhabditis species analyzed takes two different paths. We propose that in nematodes, the pre-morphogenetic stage may be equivalent to the so called 'phylotypic stage' that Klaus Sander proposed to be a conserved embryonic stage in other animals [4]. 1. Stein, L.D., et al., The genome sequence of Caenorhabditis briggsae: a platform for comparative genomics. PLoS Biol, 2003. 1(2): p. E45.2. Hillier, L.W., et al., Comparison of C. elegans and C. briggsae genome sequences reveals extensive conservation of chromosome organization and synteny. PLoS Biol, 2007. 5(7): p. e167.3. Dieterich, C., et al., The Pristionchus pacificus genome provides a unique perspective on nematode lifestyle and parasitism. Nat Genet, 2008. 40(10): p. 1193-8.4. Sander, K., Specification of the Basic Body Pattern in Insect Embryogenesis. Advances in Insect Physiology, 1976. 12: p. 125-238.

Oswald , Mackenzi et al. (2020) MicroPubl Biol

Dahlberg, Caroline (Lina), Oswald , Mackenzi, Hulsey-Vincent, Heino

[MicroPubl Biol, 2020]

Maintaining proteostasis, or protein homeostasis, is an important cellular function because misfolded proteins can aggregate and contribute to neurodegenerative diseases. One way that cells preserve proteostasis is through the Endoplasmic Reticulum Associated Degradation pathway (ERAD). ERAD relies on interactions between E2 ubiquitin-conjugating enzymes and E3 ubiquitin ligases to ubiquitylate misfolded proteins in order to signal for their destruction via the proteasome (Vembar and Brodsky 2008). We investigated if missense mutations in genes that encode two E2 ubiquitin-conjugating enzymes in C. elegans, UBC-6 and UBC-7, affect spontaneous reversal frequency (Brockie et al. 2001; Jones et al. 2002; Stewart et al. 2016; Zheng et al. 1999). UBC-6 and UBC-7 are conserved across Eukaryotes (Figure 1C). We obtained strains of animals from the Million Mutation Project with the missense mutations that confer the amino acid changes T158I in UBC-6 and P79L in UBC-7. The analogous residues in human Ube2j1 or Ube2j2 and Ube2g2 are Asp160 or 153 and Proline 78, respectively, which were mapped onto the available structures of the human enzymes (Figure 1C). In Ube2j2 (C. elegans UBC-6) Asp153 makes contact with Phe156, and mutating this residue decreases the efficiency of the enzyme (Tobi Ritterhoff, personal communication). Structural alignments suggest that Pro78 (mutated in UBC-7) is part of the well-conserved HPN (Histadine-Proline-Asparagine) motif, so mutation of the analogous residue was hypothesized to abrogate enzymatic activity (Cook and Shaw 2012; Wu et al. 2003).Our results show that the E2 ubiquitin-conjugating enzyme mutations did not significantly affect C. elegans spontaneous reversal frequency (Figure 1A). glr-1 animals were used as a control and reversed significantly less frequently than wild-type animals, as previously reported (Kowalski et al. 2011). Both ubc-6 and ubc-7 animals had less frequent spontaneous reversals than WT animals and more frequent spontaneous reversals than glr-1 animals, but neither of these results were significant.One clear reason for the insignificant change in reversal frequency in the ubc-6 and ubc-7 mutant animals could be that the missense mutations in these strains we observed do not affect gene function. It is also possible that UBC-6 and UBC-7 are able to compensate for each other in C. elegans. In S. cerevisiae Ubc6p and Ubc7p work cooperatively and sequentially to target substrates in coordination with the E3 ligases Doa10p (orthologous to C. elegans MARC-6), and Hrd1p (orthologous to C. elegans HRD-1 and paralogous to HRDL-1) (Sasagawa et al. 2007; Weber et al. 2016). It could be that this mechanism of tandem ubiquitylation is not conserved in C. elegans despite its conservation for human UBE2J2 (Weber et al. 2016). The human orthologs HRD1 and UBE2J1 (an additional ortholog of Ubc6p) also work together to target substrate proteins for degradation (Bays et al. 2001; Burr et al. 2011). Therefore it is possible that C. elegans E3s could collaborate with either UBC-6 or UBC-7 to target substrate proteins for degradation. In the future, we plan to address these possibilities using double ubc-6; ubc-7 point mutants and CRISPR-Cas9-targeted deletion mutants for UBC-6 and UBC-7, in combination with E3 ligase mutants.

De Henau S et al. (2009) European Worm Neurobiology Meeting "Wide diversity in structure and expression profiles among members of the Caenorhabditis elegans globin protein family"

Hoogewijs D, De Henau S, Vanfleteren JR, Braeckman BP

[European Worm Neurobiology Meeting, 2009]

Globins are small globular proteins, with a characteristic 3-over-3 alpha-helical sandwich structure that encloses an iron-containing heme group. Organisms can express multiple globin molecules that have variant properties and functions (Weber and Vinogradov 2001; Vinogradov and Moens, 2008). In silico analysis of the genome of Caenorhabditis elegans revealed an unexpectedly high number of globin genes featuring a remarkable diversity in gene structure, amino acid sequence and expression profiles (Hoogewijs et al., 2004). To reveal tissue-specific expression profiles of this protein family, we constructed gene fusions of the promoter and enhancer regions for all 33 globin genes to the coding region of GFP. The majority of these fusion products is expressed in neuronal cells in the head and tail portions of the body, as well as in the nerve cord. The other globin genes seem to be expressed in non-neuronal tissues, including body wall muscle, vulval muscle and the pharynx. Based on these results, most globin genes are likely to have a cell-specific function (Hoogewijs et al., 2008). At this moment, we are extending this expression analysis by including the complete coding region of each globin gene. By doing this we will be able to determine whether the globin gene introns and 3.UTR contain additional cis-regulatory information. Secondly, we used real-time PCR (qPCR) to analyze expression patterns of this globin family under hypoxia stress condition. C. elegans, as a soil nematode, frequently encounters severe hypoxic conditions in its natural environment. We found that oxygen shortage (0,1% O2) modulates the expression of several globin-like genes. This seems to indicate that these globins are involved in the protection of tissues from hypoxia. Strikingly, there is little overlap in genes responsive to hypoxia and genes responsive to anoxia (12h <0,001% O2) (Hoogewijs et al., 2007), which might illustrate functional diversity in this protein family. In conclusion, the strong variation in expression localization and stress responses of this protein family might indicate that the majority of these globins are not simple oxygen carriers. Rather, they could be involved in a variation of signaling and stress responses.