Frank Schecherer et al. (2005) International Worm Meeting "Proteome WormPD and the BioKnowledge Library: Integrating biological knowledge with genomics and proteomics"

Edgar Wingender, Angela Winnier, Sarah Ratsch, Frank Schecherer

[International Worm Meeting, 2005]

Genomics and proteomics, when combined with traditional research approaches, offer new opportunities to identify and characterize important biological pathways and molecular interactions. WormPD, as well as the other volumes of the Proteome BioKnowledge Library (BKL), are information resources that integrate biological knowledge with genomics and proteomics. The BKL contains information for species ranging from unicellular organisms (e.g. budding yeast and pathogenic fungi) to multicellular model organisms (e.g. nematode worms) to mammals (e.g. human, mouse, and rat). Individual report pages for each species-specific protein is interlinked to facilitate searching across multiple proteins and species, providing researchers the opportunity to develop a new understanding of related protein families with orthologous functions. In addition, we have developed a proprietary process, termed BioKnowledge Transfer (BKT), for the systematic analysis of protein sequences to generate descriptive title lines and predict properties, such as molecular function and biological role, based on similarity to characterized proteins in the BKL.WormPD currently contains information, either characterized from the literature or predicted via our BKT process, for over 20,000 protein sequences. We have curated more than 250,000 annotation lines, describing protein function or attributes, and over 95,000 Gene Ontology (GO) vocabulary terms (defining molecular function, biological processes, and cellular localizations) for WormPD. In addition, almost 16,000 mutant phenotype descriptions have been annotated to the protein reports. Proteins within WormPD are linked by sequence similarity, regulatory relationships, and genetic and physical interactions, as well as shared pathway memberships. A novel tool called the BioKnowledge Retriever now allows users to easily perform sophisticated, detailed queries in WormPD and the BKL. Furthermore, the vast body of information provided in WormPD is interlinked to all of the resources of the BKL, which include data for over 216,000 references characterizing 93,000 proteins of 24 different species. Examples of the utilities of WormPD and the BKL will be presented.

Brian Head et al. (2005) International Worm Meeting "Identification of Drp-1 Interacting Proteins"

Alexander M van der Bliek, Brian Head

[International Worm Meeting, 2005]

Mitochondrial division is essential for the development and survival of multicellular organisms. It is necessary for cell growth, homeostasis of energy production, and proper distribution of mitochondria during mitosis. Furthermore, experiments in mammalian tissue culture and C. elegans have shown that mitochondrial division plays a central role in programmed cell death, or apoptosis (Frank et al., 2001; Jagasia et al., 2005). Identification of the fundamental players Drp-1, Mdv-1, and Fis-1 has been useful in extending our basic knowledge concerning this essential process. However, it is certain that these components do not comprise an exhaustive list of mitochondrial division proteins. In the lab, we are using a combined biochemical/genetic approach in C. elegans to identify new components of the mitochondrial division apparatus. Biochemical purifications of TAP-tagged Drp-1, a GTPase that acts at mitochondrial division sites, will allow for the identification of Drp-1 binding partners. These proteins may be regulators of division or they may be novel components of the division complex. We are also conducting open-ended screens for enhancers of Drp-1 loss of function and using feeding RNAi to reveal genetic interaction with selected proteins.

Roumen Voutev et al. (2005) International Worm Meeting "Pro mutants, germline tumors, sheath cells, and ribosome biogenesis"

Roumen Voutev, E Jane Albert Hubbard, Darrell J Killian, James Hyungsoo Ahn

[International Worm Meeting, 2005]

Increasing evidence suggests that general cellular processes influence development in specific ways, likely reflecting tissue-specific differences in cellular demands. Gonadogenesis is proving an excellent model system to explore the intersection of ribosome biogenesis and development. Our data suggest that certain somatic gonad functions that influence germline proliferation are very sensitive to perturbations in ribosome biogenesis.Our analysis of pro (PROximal PROliferation) mutants led us to consider the intersection of development and ribosome biogenesis. The Pro phenotype is characterized by a proximal germline tumor. Our results support a model whereby the pro-1(na48) Pro phenotype stems from a cascade of events starting with an early reduction in germline proliferation due to functional defects in the distal sheath. This germline proliferation defect retards gonad arm extension, which in turn, causes a delay in meiotic entry. The delay in meiotic entry inappropriately juxtaposes mitotic germ cells with cells of the proximal sheath lineage and thereby allows these germ cells to respond to a latent proliferation-promoting activity of the proximal sheath lineage. Thus, due to developmental asynchrony between the somatic gonad and germ line, a proximal germline tumor eventually develops in the initially under-proliferated germ line (Killian and Hubbard, 2005).Several lines of evidence suggest that ribosome biogenesis is a limiting process for the germline proliferation-promoting activity of the distal sheath. PRO-1 is a conserved WD-repeat protein that acts in the sheath lineage of the somatic gonad (Killian and Hubbard, 2004). The budding yeast ortholog of PRO-1, IPI3, has been implicated in several processes including pre-rRNA processing (Peng et al., 2003; Krogan et al., 2004). Several C. elegans orthologs of direct IPI3 interactors that are also involved in ribosome biogenesis cause a Pro phenotype when depleted by RNAi. Reduction of ncl-1 increases rDNA transcription (Frank and Roth, 1998; Frank et al., 2002) and suppresses the pro-1 Pro phenotype. Reduction of lin-35/Rb in the soma also suppresses the Pro phenotype. This result is particularly intriguing since Rb negatively regulates rDNA transcription in mammals. In addition, the relative levels of rRNA processing intermediates are altered in pro-1(na48) mutants. Finally, pro-2, as defined by pro-2(na27), likely encodes an ortholog of yeast NOC2 which is implicated in nucleolar/nucleoplasmic transport of the large ribosomal subunit.

Brian P Head et al. (2004) West Coast Worm Meeting "Identification of Novel Mitochondrial Division Proteins"

Alexander M van der Bliek, Brian P Head

[West Coast Worm Meeting, 2004]

Mitochondrial division is essential for the survival of eukaryotic organisms. It is necessary for cell growth, homeostasis of energy production, and proper distribution of mitochondria during mitosis. Furthermore, experiments in mammalian tissue culture have shown that mitochondrial division has a role in programmed cell death, or apoptosis (Frank, 2001). Identification of the fundamental players - Drp-1, Mdv-1, and Fis-1 - has been useful in extending our basic knowledge concerning this essential process. However, it is certain that these components do not comprise an exhaustive list of mitochondrial division proteins. In the lab, we are using a combined genetic/biochemical approach in C. elegans to identify new components of the mitochondrial division apparatus. Biochemical purifications of TAP-tagged Drp-1, a GTPase that acts at mitochondrial division sites, will allow for the identification of binding partners that also act in division. Erp-1, a protein identified by homology to components of the endocytic pit, is unlike Drp-1 because its' role in division is still hotly contested. In order to shed light on the true function of this highly conserved protein, we will purify Erp-1 using the TAP-tag system. Genetically, we will identify Drp-1 alleles and Drp-1 interacting proteins using various enhancer and lethal screens. Finally, in collaboration with other lab members, we are attempting to determine the role that phospholipids play in regulating mitochondrial division.

Koehler, Fabian et al. (2015) International Worm Meeting "The loss of mma-1 LRPPRC function induces the mitochondrial unfolded protein response."

Koehler, Fabian, Rolland, Stephane, Mueller-Rischart, Kathrin, Conradt, Barbara

[International Worm Meeting, 2015]

In a genetic screen for Caenorhabditis elegans mutants with abnormal mitochondrial morphology, we identified mma-1 (mitochondrial morphology abnormal-1), a C. elegans homolog of the French Canadian Leigh Syndrome gene LRPPRC (leucine-rich pentatricopeptide repeat containing). Inactivation of mma-1 in C. elegans or LRPPRC in mammalian cells results in a decrease in the production of mitochondria-encoded subunits of cytochrome c oxidase (COX) and consequently a reduction in COX activity. We have previously shown that in this context, mitochondria form a hyperfused network that transiently maintains cellular ATP levels (Rolland et al, 2013). We now demonstrate that the inactivation of mma-1 LRPPRC also leads to an imbalance between mitochondria- and nuclear-encoded COX subunits, which triggers a conserved mitochondrial unfolded protein response (UPRmt). We also show that the activation of UPRmt leads to the restoration of mitochondrial proteostasis. Finally, we present evidence that UPRmt and the mitochondrial hyperfusion response are coordinated but mediated by genetically distinct pathways. We propose that in the context of mma-1 LRPPRC knock-down, mitochondrial hyperfusion helps to transiently maintain cellular ATP levels enabling UPRmt to restore mitochondrial proteostasis.Rolland, S.G., Motori, E., Memar, N., Hench, J., Frank, S., Winklhofer, K.F. , and Conradt, B. (2013). Impaired complex IV activity in response to loss of LRPPRC function can be compensated by mitochondrial hyperfusion. PNAS 110, E2967-2976.Barbara Conradt and Stephane Rolland are co-corresponding authors.

Jacques Pecreaux et al. (2006) European Worm Meeting "Mechanical Oscillations during Asymmetric Spindle Positioning Require a Threshold Number of Active Cortical Force Generators"

Jonathon Howard, Frank Julicher, Stephan W. Grill, Anthony A. Hyman, Karsten Kruse, Jens-Christian Roper, Jacques Pecreaux

[European Worm Meeting, 2006]

Jacques Pecreaux1, Jens-Christian Rper2, Karsten Kruse3, Frank Julicher3, Anthony A. Hyman1, Stephan W. Grill, Jonathon Howard1 Background. Asymmetric division of the C. elegans zygote is due to the posterior-directed movement of the mitotic spindle during metaphase and anaphase. During this movement along the anterior-posterior axis, the spindle oscillates transversely. A theoretical analysis indicates that oscillations might occur as a result of the concerted action of many cortical force generators that pull on astral microtubules in a tug-of-war situation. This model predicts a threshold of motor activity below which no oscillations occur. Results: We have tested the existence of a threshold by using RNA interference to gradually reduce the levels of GPR-1 and GPR-2 that are involved in the G-protein-mediated regulation of the force generators. We found an abrupt cessation of oscillations as expected if the activity drops below a threshold. Furthermore, we could account for the complex choreography of the mitotic spindle - the precise temporal coordination of the build-up and die-down of the transverse oscillations with the posterior displacement - by a gradual increase in the processivity of the force generators during metaphase and anaphase. Conclusions: The agreement between our results and modeling suggests that the same motor machinery underlies two different spindle motions in the embryo: the equal and opposite motors on each side of the AP axis drive oscillations whereas the imbalanced motors in the two halves of the embryo drive posterior displacement.

Karla Gutierrez et al. (2004) West Coast Worm Meeting "A forward genetic screen for new genes that regulate asymmetric neuroblast divisions in C. elegans"

Gian Garriga, Shaun Cordes, Karla Gutierrez

[West Coast Worm Meeting, 2004]

We are interested in studying asymmetric cell division (ACD), the process by which a mother cell divides to produce daughter cells that adopt different fates. Our lab has shown that mutations in two genes, ham-1 and pig-1 , disrupt ACD in many neuroblast lineages including the HSN/PHB neuroblast lineage. In wild-type embryos, the HSN/PHB neuroblast division produces a smaller anterior cell that dies and a larger posterior cell that divides to produce the HSN and PHB neurons. In ham-1 and pig-1 mutants, the anterior daughter of the HSN/PHB neuroblast is frequently transformed into its sister, dividing to produce extra HSNs and PHBs. ham-1 and pig-1 mutations interact synergistically with mutations in ced-3 and ced-4 to control the penetrance of extra HSN and PHB neurons (Guenther and Garriga, 1996; Cordes, Frank and Garriga, in preparation). This observation suggests that both ham-1 and pig-1 act in parallel to the programmed cell death pathway and regulate both cell death and cell fate in the HSN/PHB lineage. The mechanism by which ham-1 and pig-1 control ACD in the HSN/PHB and other neuroblast lineages is not understood. To identify additional genes that work with ham-1 and pig-1 to regulate ACD in the nervous system, we are screening for mutants with extra neurons. Specifically, we are mutagenizing mec-4::gfp; ced-3(n2436) hermaphrodites with EMS and screening for mutants with extra AVMs, PVMs, and PLMs. ( ham-1 mutants have extra PLMs and pig-1 mutants have extra AVMs, PVMs, and PLMs). Since mutations in ham-1 and pig-1 interact synergistically with mutations in ced-3 to control the penetrance of extra neurons, we are using the ced-3 mutation in our screen to sensitize the genetic background. So far, we have identified eight mutant strains that produce extra PLMs, and four of these strains also produce extra AVMs and PVMs.

Joshua W Ziel et al. (2007) International Worm Meeting "UNC-6/Netrin orients the Anchor Cell for Invasion."

David R Sherwood, Joshua W Ziel, Anjon Auydha

[International Worm Meeting, 2007]

Understanding the cellular circuitry governing cell-invasive behavior and basement membrane degradation is critical for designing strategies to delay or prevent tumor cell metastasis in vivo. Analysis of Anchor Cell (AC) invasion into the vulval epithelium allows us to address this question using the high-resolution genetic and cell-biological strengths of C. elegans. Our previous work has shown that AC invasion is correlated with the production of a migratory cue by the primary Vulval Precursor Cells (VPC). To extend these findings, we have characterized the intra-cellular distribution of F-actin and the membrane lipid PI(4,5)P2, known markers of a migratory leading edge, over the time-course of AC invasion. In the AC, both markers are strikingly concentrated along the basal membrane and define an invasive cellular surface prior to frank degradation of the basement membrane. In addition, we find that known regulators of the actin cytoskeleton such as the Rac orthologs MIG-2 and CED-10, as well as UNC-34/Enabled also localize to the invasive membrane, clearly demonstrating that migratory activity is under tight spatial control in wild-type ACs. To better understand the signals that polarize the migratory response within the AC, we have examined mutations in known guidance pathways. Null mutations in unc-6/Netrin or unc-40/DCC result in penetrant AC invasion defects. Genetically these mutations enhance the phenotypes of mutants unable to produce the vulval cue, and thus comprise a second pathway that collaborates with the vulval signal to promote invasion. Intriguingly, the UNC-6 signal and the vulval cue behave differently with respect to the localization leading edge markers, including MIG-2/Rac. We find that UNC-6 is required for early establishment the invasive basal surface, whereas the vulval cue is dispensable, at least during early stages, for leading edge formation. These data suggest that UNC-6/Netrin and the vulval stimulus are qualitatively distinct while informing AC migration. We describe models that may explain directed migration during AC invasion and experiments we hope will distinguish them.

Melissa Harrison et al. (2001) International C. elegans Meeting "FUNCTIONAL STUDIES OF THE CLASS B SYNMUVS REQUIRED FOR THE NEGATIVE REGULATION OF VULVAL INDUCTION AND CHARACTERIZATION OF A NOVEL CLASS B SYNMUV GENE"

Melissa Harrison, Bob Horvitz

[International C. elegans Meeting, 2001]

A receptor tyrosine kinase/Ras pathway necessary for vulval induction in C. elegans has been shown to be negatively regulated by two redundant pathways, defined by the synthetic Multivulva (synMuv) class A and B genes. Mutations in members of either class alone do not result in a Multivulva phenotype. However, animals containing loss-of-function mutations in both a class A and a class B gene display a synMuv phenotype. While most of the identified class A genes encode novel proteins, many of the class B synMuv genes, including lin-35 Rb, dpl-1 DP, efl-1 E2F, lin-53 RbAp48, hda-1 HDAC, and let-418 Mi-2, have homologs in other organisms known to be involved in chromatin modification complexes. Three mutations that define a new class B synMuv were identified in a recent screen for additional class B genes performed with Craig Ceol and Frank Stegmeier in our laboratory. We mapped these mutations to the far left of the X chromosome and are currently attempting to clone the gene by cosmid rescue. Several mammalian homologs of the class B synMuv genes have been shown to be components of the NuRD chromatin remodelling complex. Additionally, some of the proteins in the synMuv B pathway have been shown to physically interact either in vitro (1,2) or in yeast two-hybrid assays (3). We are attempting to use co-immunoprecipitation experiments to expand the current knowledge of the presumptive class B protein complex in C. elegans . Analysis of the effects of various mutations on co-immunoprecipitation may allow us to assign functionality to some novel class B genes. Currently, there is little understanding of how the class A and B synMuvs redundantly regulate vulval induction. Class A and B synMuvs might play redundant roles in the formation of a chromatin modifying complex, and such physical interactions could also be demonstrated through co-immunoprecipitaion experiments. (1) Ceol, C. and H.R. Horvitz (in press). (2) Lu and Horvitz. (1998) Cell 95 : 981. (3) Walhout et al. (2000) Science 287 :116.

Craig Ceol et al. (2002) East Coast Worm Meeting "CHARACTERIZATION OF lin-54 AND OTHER GENES THAT NEGATIVELY REGULATE let-60 Ras SIGNALING DURING VULVAL DEVELOPMENT"

Craig Ceol, Bob Horvitz

[East Coast Worm Meeting, 2002]

The synthetic multivulva (synMuv) class A and class B genes act redundantly to negatively regulate Ras signaling during vulval development. The class B synMuv genes encode proteins that likely function to target the histone deacetylase HDA-1 to specific DNA sequences. Some of the proteins involved in this proposed targeting, including LIN-35 RB, DPL-1 DP and EFL-1 E2F, are conserved in mammals, and a similar targeting function has been described for their mammalian counterparts. To better understand this conserved pathway, we cloned the class B synMuv gene lin-54 and identified new synMuv genes in a genetic screen. lin-54 was originally identified and mapped to a small region of LGIV by Jeff Thomas, a former graduate student in our laboratory. We cloned lin-54 and found that it encodes a protein with two copies of a cysteine-rich domain. A similar domain is found in proteins in other species; however, the function of this domain is not understood. We observed partial maternal rescue of the Muv phenotype of lin-54 mutants, indicating that maternally-provided lin-54 can regulate the vulval cell fate specification decisions that occur during postembryonic development. However, the stage-specific ectopic expression of lin-54 under the control of heat shock promoters indicated that lin-54 need not function prior to vulval induction to regulate this process. In addition, we conducted in vitro protein interaction experiments using LIN-54 and other class B synMuv proteins. We will present preliminary data concerning these studies. With graduate students Frank Stegmeier and Melissa Harrison, we screened approximately 6500 haploid genomes and recovered 95 new synMuv mutations. The mutations we assigned to complementation groups fall into three general classes: 1) mutations that affect new and previously characterized class B synMuv genes, 2) mutations that affect ark-1, sli-1 and gap-1, genes that are thought to directly regulate Ras pathway components, and 3) mutations that synergize with class A and class B synMuv genes and define previously uncharacterized genes (see abstract by Ceol and Horvitz). We will present our characterization of these new mutations.