Wilm TP et al. (1997) International C. elegans Meeting "sud-1, a candidate for an embryonic cell cycle suppressor gene in the nematode C. elegans"

Schnabel R, Schnabel H, Wilm TP

[International C. elegans Meeting, 1997]

Our lab undertook several large scale screens for maternal effect embryonic lethal mutations in the nematode C. elegans using balancer chromosomes for LG's II-V. Complementation analysis revealed that the 875 mutations isolated define 350 genes. Beside genes involved in cell fate determination a large group of genes was found that has cell biological functions in the embryo. Many of these may represent functions in initiating or regulating cell division producing either embryos that arrest at the one cell stage or at the 24 cell stage at the onset of gastrulation. Four independent recessive strict maternal-effect lethal mutations in a single gene, sud-1 (LG II), result in an opposite phenotype. Supernumerary cell divisions are observed. Embryos from homozygous sud-l hermaphrodites produce more than 1000 cells by undergoing additional cell division rounds after the end of the proliferation phase of development when morphogenesis normally starts. Differentiation processes are strongly reduced and only rarely detectable at varying cellular positions and the formation of tissues and organs fails to occur. This excessive proliferation of cells could be due to either a general failure in early cell fate determination or the failure of cell cycle arrest at the end of proliferation. In double mutants of sud-1 with a gene that generally suppresses cell cycles the terminal number of cells is reduced back to roughly 500 and terminal cell differentiation reappears. We therefore suggest that the primary function of wildtype SUD-1 is the suppression of cell cycling at the end of the proliferative phase. We have identified and cloned the gene sud-1 by phenotypic rescueing and sequenced the full- length cDNA. The single ORF predicts a protein of 130 kDa with no similarity to any database entry so far. The molecular analysis of sud-1 is in progress.

Douglas A Fantz et al. (2001) International C. elegans Meeting "Docking sites on substrate proteins direct extracellular signal-regulated kinase (ERK) to phosphorylate specific residues."

Kerry Kornfeld, Dave Jacobs, Douglas A Fantz, Danielle Glossip

[International C. elegans Meeting, 2001]

During vulval development the primary cell fate is induced by a signaling pathway that includes mpk-1 , the C. elegans homolog of extracellular signal-regulated kinase (ERK), a member of the mitogen activated protein (MAP) kinase family. ERK phosphorylates S/TP sites in many different protein substrates including C. elegans LIN-1. We identified gain-of-function mutations in lin-1 that affect an FQFP motif that functions as a docking site for ERK. We characterized the sequence of amino acids that can constitute the FXFP motif using peptide and protein substrates. Substitutions of the phenylalanines at positions 1 and 3 had significant effects, indicating that these phenylalanines provide substantial binding affinity, whereas substitutions of the residues at positions 2 and 4 had less effect. LIN-1 also contains a different ERK docking site called the D-domain. The FXFP and D-domain docking sites were analyzed in a variety of positions and arrangements in the proteins Elk-1 (a vertebrate homolog of C. elegans LIN-1) and C. elegans KSR-1. Our results indicate that the FXFP and D-domain docking sites form a flexible, modular system that has two functions. First, the affinity of a substrate for ERK can be regulated by the number, type, position, and arrangement of docking sites. Second, in substrates with multiple potential phosphorylation sites, docking sites can direct phosphorylation of specific S/TP residues. In particular, the FQFP motif of Elk-1 is necessary and sufficient to direct phosphorylation of serine 383, a residue that is important for Elk-1 transcriptional activation, whereas the D-domain directs phosphorylation of other S/TP sites in Elk-1.

Wender, Nora et al. (2011) International Worm Meeting "Analysis of cellular toxicity mechanisms of alpha-synuclein in C. elegans."

Wender, Nora, Hegermann, Jan, Eimer, Stefan

[International Worm Meeting, 2011]

Aggregation of a-synuclein (aS) in Lewy bodies is one of the pathological hallmarks of Parkinson's disease (PD). Mutations in aS as well as increased aS expression levels have been associated with PD. As the PD mutants of aS are more prone to forming fibrillar aggregates, these fibrils were initially considered to be causing toxicity. However, in different model systems no clear correlation between toxicity and fibril formation was found. This indicates that small oligomeric precursors rather than fibrils might be the toxic species. In order to investigate which form of aS is causing toxicity, we were making use of a designed variant of aS that stops at the stage of soluble oligomers. This variant is called TP aS (triple proline aS) as it was derived from wt aS by introduction of three proline residues. In vitro NMR studies as well as in vivo analysis of the aggregation of aS variants tagged with YFP in C. elegans muscle cells confirmed that the occurrence of aS fibrils was strongly reduced in TP aS. Next we were assessing toxicity of TP aS in comparison to wt aS as well as PD mutants of aS by investigation of neurite degeneration and impairment of dopamine-associated behavior upon expression of different aS variants in C. elegans dopaminergic neurons. We found that TP is the most toxic aS variant, exhibiting the most severe effects on dopaminergic neuron morphology and function. This suggests that indeed the small oligomers rather than aS fibrils are causing toxicity. So far, the cellular function of aS and the mechanisms causing toxicity remain unclear. It is known that aS binds to intracellular membranes including the mitochondrial membrane. Furthermore, mitochondrial impairment upon expression of wt and mutant aS was reported. Recently it has been reported that several PD-associated genes play a role in mitochondrial dynamics and that loss of function or overexpression leads to morphological changes in the mitochondrial network. Therefore we aimed in understanding how expression of different variants of aS affects mitochondrial dynamics. Thus we were analyzing mitochondrial morphology and distribution by Electron Microscopy as well as Spinning Disk Microscopy. We found that expression of aS in C. elegans muscles and neurons leads to mitochondrial fragmentation and severe changes in mitochondrial morphology. Remarkably, similar changes could be observed in aged worms not expressing aS. It is therefore tempting to suggest that increased expression levels of aS accelerate mitochondrial aging.

Matthias Muller et al. (2003) International Worm Meeting "What is the function of sud-1?"

Matthias Muller, Ralf Schnabel

[International Worm Meeting, 2003]

We described earlier four maternal effect mutations of the gene sud-1 (see Wilm et al. IWM 1997). These mutations cause supernumerary cell divisions in the embryo. Expression of terminal differentiation markers is generally reduced to approximately 20% of the normal cell number. A lineage analysis using a 4D-microscope system also shows no recognisable lineage transformations, however cells do not sort to their normal position but stay put at their place of birth. Cloning of the gene did not reveal any information about its function. We now search for interacting partners using a Y2H approach to place the gene in a molecular network whose function may be known. To learn more about the possible cellular function of the protein we proceed now with several approaches. Polyclonal antibodies raised against a small peptide of the putative ORF as well as a GFP fusion protein indicates a membrane association in the embryo. In vitro cell culture experiments hint to cell adhesion defects in blastomeres derived from mutant embryos. It is now an interesting question how an interference with cell adhesion in the early embryo could cause a lack of cell fate specification in all somatic founder cells and even the germline of the embryo.

Kornfeld K et al. (2000) Midwest Worm Meeting "ERK MAP Kinase Recognition of Substrates is Mediated by a Modular System of Docking Sites that Directs Phosphorylation at Specific Residues"

Fantz DA, Kornfeld K, Jacobs D

[Midwest Worm Meeting, 2000]

MAP kinases are signaling molecules that phosphorylate S/TP motifs in a wide variety of substrate proteins. The mechanisms that enable MAP kinases to identify substrate proteins and phosphorylate functionally important residues are not well understood. We are investigating how MAP kinases interact with substrate proteins by focusing on the C. elegans proteins, LIN-1 and KSR-1, and the human ETS transcription factor, Elk-1. LIN-1 and KSR-1 are part of the Ras signaling pathway required for vulval formation in C. elegans. Genetic studies of lin-1 indicate that it is negatively regulated by the Ras pathway and functions downstream of mpk-1 ERK MAP kinase. KSR-1 is a protein kinase that positively mediates Ras signaling. We have shown that LIN-1, KSR-1, and Elk-1 can function as high-affinity substrates for ERK MAP kinase in vitro. All three of these proteins possess an ERK MAP kinase docking site defined by an FXFP motif. The ETS transcription factors LIN-1 and Elk-1 also possess a second type of docking site called the D-box which appears to mediate binding to ERK and JNK MAP kinase. FXFP and the D-box can function independently or in combination as docking sites to form a modular recognition system for MAP kinases. In vitro assays have been used to define the primary sequence that is necessary for FXFP function and to determine the protein context required for FXFP and D-box function. Peptide inhibition assays have demonstrated that the FXFP and D-box sites bind different pockets on the ERK MAP kinase enzyme and possess distinct spatial requirements between the docking site and potential phosphorylation sites. Further in vitro experiments have been performed to characterize a third docking site found in ribosomal S6 kinase that has recently been described to mediate interactions between ERK and p38 MAP kinases and ribosomal S6 kinase. We have also found evidence suggesting that these distinct docking sites mediate preferential phosphorylation of specific S/TP motifs in a substrate. It is known that phosphorylation of Elk-1 at Serine-383 is required for its transcriptional activation. We have shown using phospho-specific antibodies that an FXFP docking site in its wild-type context is necessary for highly efficient phosphorylation at Serine-383 of Elk-1. This suggests that MAP kinases may utilize the modular nature of FXFP and the D box to differentially regulate substrates by directing phosphorylation to functionally distinct sites. We have also demonstrated the relevance of FXFP and D box docking sites as in vivo regulators of ERK MAP kinase/substrate interactions in vertebrate cells by analyzing the transactivation potential of wild-type Elk-1 and variants containing mutations in FXFP and/or the D box. The combination of in vitro enzyme assays and in vivo functional assays have provided a set of guidelines for the prediction of potential in vivo substrates of ERK MAP kinases by defining the sequence and context necessary for the function of MAP kinase docking sites.

Andrew Papp et al. (2001) International C. elegans Meeting "The Low-Cost Worm Lab"

William Rhoades, Dan Houchin, Andrew Papp

[International C. elegans Meeting, 2001]

The equipment for Molecular Biology and Genetics research traditionally has been expensive, due to a variety of factors, including: low production volumes, high precision requirements, and complex sales/distribution systems. We have tried to improve this situation through the use of innovative or alternative technologies, and the adaptation of consumer and industrial mass-produced equipment for use in the biology laboratory. While a variety of approaches have been pursued toward the efficient DNA-mediated transformation of C. elegans (Papp et al., 1997; Wilm et al. 1999), so far, no method has rivaled the effectiveness of direct microinjection (Mello et al., 1995). A major "difficulty" with microinjection is the cost of an inverted microscope with Differential Interference Contrast (DIC) optics and a Microinjection Controller. In response to this, we developed a low-cost electronic foot pedal controlled Microinjection Controller (Papp et al., 1991), and more recently, coupled it to a low-cost inverted microscope that utilizes custom-made Hoffman Modulation Contrast (HMC) optics, along with an inexpensive glide stage and micromanipulator. These high numerical aperture HMC optics yield a DIC-like image of oocytes and syncytial nuclei. While not ideal for demanding cell lineage studies, this system is more than adequate for microinjection. We can retail our complete system for $10,000, about one-third the cost of traditional systems, making it a nice dedicated microinjection station. Proper maintenance of C. elegans cultures, particularly temperature-sensitive mutants, requires a precisely temperature-controlled incubator. Incubators with the ability to both heat and cool are traditionally expensive devices employing both Freon compressors and heating coils that fight against each other to control the temperature. In response to this, we developed a low-cost microprocessor-controlled incubator, based on the Peltier thermoelectric effect, which is now commonly employed in consumer-grade coolers. This unit quickly learns just the right amount of power to apply to the heat pump, in order to maintain the set temperature with about 0.1 degree Celsius precision, and it has sophisticated alarms to inform the user if anything has gone wrong. These incubators are available for less than $800, about one-third the cost of traditional heating and cooling incubators. C. elegans genetics experiments require many media-filled Petri dishes. Traditional Sterile Media Dispensers utilize unreliable peristaltic pumps, or unreliable syringe based pumps, both of which are expensive and often cumbersome. In response to this, we developed a low-cost, all-digital microprocessor-controlled Sterile Media Dispenser based on a miniature autoclavable electromagnetic pump. This unit has a variety of advantages included memory storage for common sizes of Petri dishes, and retails for $650, about one-third the cost of other Sterile Media Dispensers. Samples of this equipment, as well as a discussion of ideas in the pipeline will be at the Poster Session. Mello C, Fire A. 1995. DNA transformation. "Methods in Cell Biology. Caenorhabditis elegans : Modern Biological Analysis of an Organism." H.F. Epstein and D.C. Shakes (eds), Academic Press, Inc., San Diego. 48: 451-482. Papp A, Lee J, Farris K. 1997. WORM ZAPPING II: C. ELEGANS ELECTROPORATION? 1997 International Worm Meeting abstract 466. Papp A, Mancillas J. 1991. Improved Injection Setup! Worm Breeder's Gazette 12(1): 52. Wilm T, Demel P, Koop HU, Schnabel H, Schnabel R. 1999. Ballistic transformation of Caenorhabditis elegans . Gene 229: 31-35.

GM Stanfield et al. (1999) International C. elegans Meeting "Analysis of downstream events in the pathway for programmed cell death"

GM Stanfield, HR Horvitz, E Hartwieg

[International C. elegans Meeting, 1999]

We are genetically and molecularly analyzing genes that may be involved in the downstream events of programmed cell death, such as the adoption of cell-corpse morphology and the engulfment and degradation of cell corpses. To facilitate phenotypic analyses of cell-death mutants, we are interested in developing markers that identify different stages in the cell-death process. Toward this end, we have developed methods for performing TUNEL ( T dT-mediated d U TP N ick E nd L abeling) assays on C. elegans . Our analyses of wild-type, DNA degradation-defective and engulfment-defective embryos indicate that there are at least three steps of DNA degradation in cell corpses: an initiation step in which TUNEL-reactive ends are generated, a nuc-1 -mediated step in which TUNEL-reactive ends are degraded (or masked) and an engulfing cell-mediated step (Hedgecock et al., Science 220, 1277-9, 1983) in which DNA degradation is completed. ced-8 and ced-11 are candidate downstream cell-death genes. In ced-8 mutant embryos, refractile cell corpses appear later than in wild-type embryos. However, TUNEL detects more dying cells at early embryonic stages in ced-8 embryos than are visible using Nomarski microscopy, suggesting that DNA degradation is less delayed than is the onset of refractility. Thus, ced-8 may couple cell killing to morphological changes in the dying cell. In ced-11 embryos, dying cells do not assume the refractile, electron-dense characteristic of normal cell corpses. TUNEL assays indicate that ced-11 embryos have slightly increased numbers of TUNEL-reactive nuclei relative to the wild type. Thus, mutations in ced-11 might result in slowed DNA degradation in addition to altered cell-corpse morphology.

Lee, Myon-Hee et al. (2009) International Worm Meeting "FOG-3 phosphorylation by MAPK controls the C. elegans sperm fate."

Lee, Myon-Hee, Nykamp, Keith, Kimble, Judith

[International Worm Meeting, 2009]

FOG-3 TOB/BTG family protein is essential for sperm fate specification in C. elegans (Chen et al. 2000) and the primary C. elegans ERK/MAPK, known as MPK-1, has also been implicated in sperm fate specification (Lee et al. 2007). We have obtained several lines of genetic evidence that suggest a key role for MPK-1 in controlling the sperm fate. To learn how MPK-1 might function in this important cell fate decision, we examined the FOG-3 amino acid sequence and found a potential MAPK-docking site and four predicted MAPK phosphorylation sites (i.e S/TP). We then test the idea that MPK-1 might control the sperm fate by FOG-3 phosphorylation. In vitro, we have found that FOG-3 can be directly phosphorylated by murine ERK/MAP kinase; in vivo, we have generated a battery of FOG-3 transgenes and found that FOG-3 phosphorylation is critical for sperm fate specification. Importantly mammalian Tob is also a substrate of MAPK (Maekawa et al. 2002; Suzuki et al. 2002). We suggest therefore that the C. elegans germline may use an ancient regulatory cassette for control of the sperm/oocyte decision. References: Lee et al. (2007) Multiple functions and dynamic activation of MPK-1 extracellular signal-regulated kinase signaling in Caenorhabditis elegans germline development. Genetics 177, 2039-62. Maekawa et al. (2002) Identification of the anti-proliferative protein Tob as a MAPK substrate. J Biol Chem 277, 37783-7. Suzuki et al. (2002) Phosphorylation of three regulatory serines of Tob by Erk1 and Erk2 is required for Ras-mediated cell proliferation and transformation. Genes Dev 16, 1356-70. Chen et al. (2000) A novel member of the Tob family of proteins controls sexual fate in Caenorhabditis elegans germ cells. Dev Biol 217, 77-90.

Swathi Arur et al. (2006) Development & Evolution Meeting "Functional Genomics Approach to Identify Targets of MPK-1 ERK Signaling in Germline Development"

Andy Golden, Tina Chang, Tim Schedl, Sudhir Nayak, Swathi Arur, Mitsue Ohmachi

[Development & Evolution Meeting, 2006]

RAS/ERK signaling is important for many aspects of normal development while inappropriate signaling can predispose to cancer. In C. elegans MPK-1 ERK is critical for spatial co-ordination of meiotic prophase progression and oogenesis and regulates nine different germline processes. The cellular response to signaling occurs as a consequence of ERK activating or inactivating target proteins by phosphorylation. However, targets of MPK-1 that mediate germline development are unknown. To identify downstream targets of MPK-1 signaling, we are using an integrated in silico, reverse genetic and biochemical approach. In silico searches identified >2000 proteins from the C. elegans proteome that contain putative ERK docking (e.g. D-domain) and phospho-acceptor (S/TP) sites. 156 were chosen for RNAi screening based on a) multiple docking sites, b) conservation of docking sites and c) germline expression. MPK-1 regulates a number of germline processes and for execution of an individual process it likely phosphorylates multiple targets. Thus RNAi based gene inactivation of each target is performed in sensitized genetic backgrounds to permit detection of small or partial changes in the pathway that controls a given cellular process. To date, we have identified 34 genes that behave genetically as mpk-1 targets for germline development. RNAi of 25 genes enhances specific phenotypes of an mpk-1 temperature sensitive (ts) loss-of-function mutant while RNAi of 9 genes enhance let-60 RAS ts gain-of-function. Biochemical analysis has thus far shown that 11 of 13 tested gene products identified by RNAi are direct in vitro substrates of activated mammalian ERK2. Together, this work has identified at least 11 gene products representing diverse functional categories that are likely direct targets that execute MPK-1 ERK regulated aspects of germline development. Given the conservation of the targets and their docking sites it is likely that the mouse/human orthologous proteins are previously unknown targets of ERK phosphorylation.

Dave Jacobs et al. (1999) International C. elegans Meeting "A modular system of docking sites mediates ERK map kinase recognition of substrate proteins such as LIN-1 and KSR-1"

Kerry Kornfeld, Dave Jacobs

[International C. elegans Meeting, 1999]

MAP kinases are signaling molecules that phosphorylate S/TP motifs in a wide variety of substrate proteins. The mechanisms that enable MAP kinases to identify substrate proteins and phosphorylate particular residues are not well characterized. We are investigating how MAP kinases interact with substrate proteins. We have focused on two C. elegans substrates, LIN-1 and KSR-1. LIN-1 contains 441 amino acids and is likely to be a DNA-binding transcription factor, since it contains an N-terminal ETS domain. lin-1(lf) mutations cause a multivulva phenotype, and lin-1(gf) mutations cause a vulvaless phenotype, suggesting lin-1 functions as a switch and lin-1 activity prevents Pn.p cells from adopting the 1deg vulval fate. Genes in the Ras signaling pathway promote the 1deg fate, and epistasis analyses indicate lin-1 functions downstream of these genes, including mpk-1 ERK MAP kinase, suggesting lin-1 is negatively regulated by the action of this signaling pathway. KSR-1 is a protein kinase that positively mediates Ras signaling.We showed that the C-terminal region of LIN-1 was sufficient to function as a high-affinity substrate for ERK MAP kinase in vitro . We analyzed six lin-1(gf) mutations; each alters or eliminates the C-terminal sequence FQFP, suggesting this motif is required for negative regulation of LIN-1. This motif is conserved in other ETS proteins that also mediate Ras signaling. Biochemical experiments demonstrated that FXFP is an evolutionarily conserved docking site that mediates ERK MAP kinase binding to these substrates. FXFP and the D box, a different docking site that is conserved in these ETS proteins, form a modular recognition system, since they can function independently or in combination. FXFP is specific for ERK, whereas the D box mediates binding to ERK and JNK MAP kinase, suggesting that the partially overlapping substrate specificities of ERK and JNK result from recognition of shared and unique docking sites. These findings enabled us to predict new ERK substrates, such as KSR-1, which contains a FXFP motif and is a high-affinity substrate for ERK. Furthermore, this information was used to design peptide inhibitors of ERK that functioned in vitro and in vivo .