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Caenorhabditis elegans •Free living nematode •1mm long and transparent •Lives in soil •Feed on microorganisms •E.coli in laboratory •Hermaphrodite sex •Rare males (0.05%) •Crossing •Eggs •Life span 2-3 weeks •Generation time 4 days •C.elegans field started in 1965 with Sydney Brenner •2002 Nobel prize •2003 C.elegans survived the Space Shuttle Columbia’s disintegration Embryogenesis first mitosis gastrulation Cell lineage The developmental fate of every single somatic cell (959-1031) has mapped out first mitosis gastrulation Life Cycle dauer C.elegans anatomy cuticle spermatheca WHY C.elegans? •Cheap •Life cycle is 4 days •Genome completely sequenced (100X6 bps) •A lot of information is available in web •19.000 genes: very little genes redundancy •Low complexity but with organs and tissue specifications •Transparent (anatomy) •Hermaphroditic lifestyle, males available for crossing •Biochemistry difficult •No cell lines available •dissection of specific tissues is unrealistic Methods C.elegans and the Web Information about C.elegans is stored in the database ACeDB. Several windows: 1) Sequence window (genome as a string of nucleotide bases) 2) Physical map window (genome as a set of DNA clones) 3) Genetic window (genes as detected by mutation) In addition in AceDB there are information about: 1) Cell lineage and development 2) ESTs, gene structure and homologies 3) Genetic rearrangement and mutants available 4) C.elegans meetings’ abstracts and publications. deletion: 848 bp deletion CTCGATTT/ACCCCTGAAC Mutant phenotype: homozygous viable CGC center WT and mutant stocks of C.elegans are available from the Caenorhabditis Genetic Center (University of Missouri, Columbia) Long term storage of C.elegans in liquid nitrogen (or -80C) is possible through the use of glycerol-containing media Transformation Transformation was introduced in the early 1980s. DNA is injected into the cytoplasm of the gonads. The DNA can pass through the germline in the form of extrachromosomal array Purposes: 1) identification of genes by rescuing a mutant phenotype using a WT copy of the gene 2) Expression pattern using the gene of interest with reporter 3) Interference of a biological process by overexpression of WT or mutated gene gonad 40X DIC Gene expression 3 approaches to study gene expression in C.elegans: 1) Reporter-gene fusion with transformation ( GFP, LacZ) 2) In situ hybridization using mRNA 3) Immunofluorescence with specific antibody Genetics in C.elegans Forward genetic 1) R. Mutagenesis 2) Transposons Phenotypes genes Reverse Genetics 1) RNAi 2) PCR identification of rearrangements Gene phenotypes Forward Genetic Gene targeting techniques based on Homologous Recombination are not available in C.elegans Random mutagenesis •Random mutagen (EMS/TMP-UV) to generate point mutations or small deletions •Analysis of F2 for the selected phenotypes •Mapping using visible markers and polymorphic DNA sequences Forward Genetic Transposons •Transposons: discrete segment of DNA moving in the genome, encoding a transposase •Normally present in C.elegans in different copies (strain-dependent) •Activated by forced expression of transposases •Most common:Tc1 (“cut and past mechanism”) •Insertional mutagenesis with Tc1 will generate mutant alleles tagged by the transposon that can be used for identify the mutated gene Problems: 1) Other Tc elements are mobilized in the mutator strain 2) Several copies of the Tc1 (identification the mutagenic insertion) 3) Transposition cannot be controlled Solution: mobilization of Mos-1 element (a Tc1 absent in C.elegans) achieved by conditional expression of the Mos-1 transposase Reverse genetics KO/RNAi gene Phenotype(s) • • RNA interference Specific KO Specific KO: EMS/TMP mutagenesis (deletions) PCR to identified the mutated gene http://celeganskoconsortium.omrf.org/ http://shigen.lab.nig.ac.jp/c.elegans/index.jsp RNA interference Double stranded RNA is used Genome-scale 3 ways to interfere: RNAi analysis 1) Injection of dsRNA in gonads 2) Soaking animals in dsRNA 3) Feeding animal with bacteria producing dsRNA http://www.geneservice.co.uk/products/rnai/Celegans.jsp C.elegans RNAi library of about 16.000 genes It is a “transient” KO Works fine but not always Can give interesting phenotypes when the KO is lethal Is C.elegans a good model system to study endocytosis? oocytes Nerve system Also: coelomocytes for Fluid fase endocytosis Forward Genetic screenings to identify endocytic proteins 1) Yolk-GFP uptake in oocytes Identification of rme genes QuickTime™ and a TIFF (L ZW) d eco mpre ssor are nee ded to s ee this picture . QuickTime™ and a TIFF (LZW) decompressor are needed to see this picture. QuickTime™ and a TIFF (LZW) decompressor are needed to see this picture. Identification of several rme genes (receptor-mediated endocytosis) Several genes were not identified in human (rme-1, rme-6, rme-8) Still several to be identified Forward Genetic screenings to identify endocytic proteins 2) Compensatory endocytosis at presynaptic level: Identification of rics genes (resistant to inhibitor of cholinesterase) aldicarb cholinesterase Screening in presence of aldicarb allowed the identification of endocytic proteins such AP180, Synaptojanin, Endophilin, Synaptotagmin... More complex screening because it targets proteins involved also in exocytosis (synaptotagmin, unc 13-18, syntaxin...) and production/transport of acetylcholine (kinesins) Reverse genetic screening to identify genes required for synapse structure and function (Nature 2005) • Pre-selected 2027 genes on the basis of sequence and domain and involvement in signal transduction, membrane trafficking synaptic localization • Screening for aldicarb resistance by feeding RNAi, using eri-1 or eri-1;dgk-1 strains (aldicarb hypersensitive) • 185 genes identified to be RIC (resistant to inhibitors of cholinesterase), 132 not known to be involved in synaptic transmission • Expression pattern of 100 genes using transgenic animals (26 axonal proteins) • presynaptic localization: Co-localization with synaptobrevin (24/26) • Synaptic structure: distribution of GFP::SNB in the mutants • Molecular mechanisms???? EHS-1 is the C.e homologue of Eps15 Eps15 EHS-1 EH1 EH2 EH1 EH3 EH2 EH3 COIL COIL EHs ehs-1 DPFs DPFs COIL DPFs SL1 ATG TGA EHS-1 is a neuronal protein and localizes in synaptic vesicle-rich regions Generating EPSF... Generating EPSF... Characterization of ehs-1 mutant ehs-1(ok146) 12077 13807 SL1 ATG 1 2 3 4 5 7 8 TGA WT ehs-1 Aldicarb: acetylcholinesterase inhibitor larval stage ehs-1 No Aldicarb geno type L2 L4 0.2mM 0.5 mM 1.0 mM Aldicarb Aldicarb Aldicarb +/+ .98 (176) .52 (204) .16 (215) 0 (218) -/- .97 (165) .70 (172) * .66 (165) * .06 (142) * +/+ .98 (505) .58 (564) .21 (600) .03 (169) -/- .98 (512) .80 (494) * .43 (534) * .15 (177) * ehs-1 is involved in synaptic transmission TS Uncoordinate phenotype of ehs-1 mutant ehs-1 animals show: •aldicarb resistance •Unc phenotype at high temperature (at 30°C they become paralyzed) •Depletion of synaptic vesicles at not permissive temperature Electron-microscopy of WT, ehs-1 mutants and DN transgenic worms at 15°C (left) and 30°C (right). Arrows indicate presynaptic zones where vesicles are recycled. Genetic and Physical interaction with dynamin The TS uncoordinate phenotype of ehs-1 KO worms is similar to the dynamin mutant phenotype •ehs-1;dyn-1 double mutant is almost lethal •EHS-1 interacts with DYN-1 •hEps15 interacts with hDynamin Dyn input PC12 lys GST-Grb COS lys WB a-dyn ehs-1 is a positive regulator of dynamin function ehs-1 + Lesson from C.elegans: 1) EHS-1 (and EPS 15) is a neuronal protein 2) Involved in synaptic transmission 3) Partner of dynamin Conclusions Great model system for genetic analysis (rapid life cycle,small size,easy to grow in lab, self fertilization, crossing with males) Small genome(no redundancy) and simple anatomy (1000 cells, transparent) Constant cell number in the same position make the animal suitable for studying development For RME and fluid fase endocytosis several mutants were identified by genetic screening, at least 20 are still without name and identity....... BRIC Biotech Research & Innovation Centre University of Copenhagen Simon Rose Claudia Krag Anna Schultz