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Synaptic Specificity is Generated by the Synaptic Guidepost Protein SYG-2 and it’s Receptor, SYG-1

Synaptic Specificity is Generated by the Synaptic Guidepost Protein SYG-2 and it’s Receptor, SYG-1. Kang Shen, Richard D. Fetter and Cornelia I. Bargmann Cell, Vol. 116, 869 – 881, March 19, 2004. C. elegans II, pg 613. wormatlas.org. Caenorhabditis elegans.

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Synaptic Specificity is Generated by the Synaptic Guidepost Protein SYG-2 and it’s Receptor, SYG-1

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  1. Synaptic Specificity is Generated by the Synaptic Guidepost Protein SYG-2 and it’s Receptor, SYG-1 Kang Shen, Richard D. Fetter and Cornelia I. Bargmann Cell, Vol. 116, 869 – 881, March 19, 2004

  2. C. elegans II, pg 613 wormatlas.org Caenorhabditis elegans Understand the cellular and molecular mechanisms that underlie synapse formation in vivo C. elegans Well Characterized Nervous System  En Passant Synapses  Presynaptic Specializations  Postsynaptic Specializations Studied en passant synapses formed by HSNL near the C. elegans vulva

  3. devgen.com/.../productoffering/c_elegans.html Figure adapted fr www.eb.tuebingen.mpg.de/.../wurm_entw.html biol.unt.edu/~ppadilla/WormLarvae.jpg Postembryonic Development

  4. 2)Early L3  cell – cell interactions specify the fates of VPCs 1)Late L1 , the VPCs are born • signaling events specify • P5.p, P6.p & P7.p to adopt vulval fates • P3.p, P4.p & P8.p to adopt non-vulval fates P3 P4 P5 P6 P7 P8 3)VPCs execute the specified fates (generate # & types of vulval cells) 4)Vulval cells undergo cell movements, cell fusion and eversion to form mature vulva NB NB NB NB NB NB L1 S S S S S S L L T N T T T T L L T N L2 L3 4 Stages of Vulval Development

  5. HSNR VC6 VC3 VC5 VC4 HSNL Vulval Cell Neurons (VC) L1  born L3  send out processes (late L3) L4  branch in the region of the vulva HSN’s are born in the embryo and migrate near gonadal primordium (mid-body) prior to hatching L2 & L3  axon outgrowth L3 & L4  synapse formation Egg Laying Neurons wormatlas.org .veryfunnypics.com/ pics/food/images/eggs.jpg

  6. HSNL forms en passant synapses onto the VC4, 5 motor neurons and the vulval muscles near the vulva region synaptic vesicle clustering in HSNL is not dependent on postsynaptic neurons and muscles something is contacting HSNL and acting as synaptic guidepost to directing HSNL presynaptic vesicles to adjacent regions wormatlas.org HSN In Wild Type Animals

  7. Isolated the Mutant syg-1(ky652) Encodes an Ig superfamily protein that acts in the presynaptic HSNL axon HSNL synaptic vesicles fail to accumulate at normal synaptic locations and form ectopic anterior clusters Defects in synapse formation similar to animals lacking vulval epithelial cells SYG-1 protein is localized to the site of future synapses. Elimination of vulval epithelial cells results in diffuse localization of SYG-1 on HSNL axon The Ig Superfamily Protein SYG-1 Determines the Location of Specific Synapses in C. elegans(Cell, Vol. 112, 619-630, March 7, 2003) Anterior displacement of the vulvaSYG-1 localization is displaced anteriorly matching the position of the ectopic vulva MUV’s SYG-1 localizes to each of the vulva

  8. Goal of this Paper wormatlas.org Suggest a SYG-1/SYG-2 interaction stimulates the formation of appropriate synapses while suppressing inappropriate synapses syg-2 encodes the guidepost signal in the vulval epithelial cells Hypothesis Synapses made by the C. elegans HSNL neuron are initiated by the guidepost role of the primary vulval epithelial cells, acting through the TM Ig superfamily protein SYG-1 on HSNL

  9. WT animals syg-1(ky652) lin-11(n566) syg-2(ky673) syg-2(ky671) ↓ accumulation of SNB-1::YFP near the vulva SNB-1::YFP clusters shifted to ectopic anterior positions lin-11 mutants have defects in synaptic localization similar to those of syg-1 and syg-2 mutants Mutations in syg-2 disrupt synaptic vesicle clustering in HSNL SNB-1 (synaptobrevin)  synaptic vesicle protein that labels synaptic vesicle clusters in C. elegans and other animals lin-11(n566) is associated with cell lineage defects in the 1° & 2° vulval cell lineage 1° vulval cells are generated but fail to function normally

  10. (Pre/Post)synapse dense core vesicle vesicle electron dense presynaptic specializations C. elegans II, pg 614 in some cases see thickening of post synaptic membrane (difficult to visualize)

  11. syg-2(ky673) syg-2(ky671) syg-1(ky652) Wild Type triangles represent active zone; diamonds represent # of synaptic vesicles Synapse Characterization in syg-1 & syg2 Mutants Scanning EM; 150μm region centered on the vulva 1 WT L4; 1 L4 syg-1(ky652); 2 L4 syg-2(ky673); 1 L4 syg-2(ky671)

  12. 7 Ig Domains TypeII PDZ Fibronectin typeIII syg-2(ky671) G to A mutation at SL1 splice acceptor site at the first exon of syg-2 gene  LOF mutation in the syg-2 gene syg-2(ky673) 32kb deletion (affects 4 predicted ORF) SYG-2 Encodes an Immunoglobulin Superfamily Protein

  13. SYG-1 SYG-2 4 Drosophila play a role in Drosophila muscle function NEPH1 and nephrin are the mammalian orthologs which play a role in kidney development

  14. SYG-1::GFP SYG-2::GFP Mid L3 Mid L3 SYG-2::GFP expressed in 1° epithelial cells SYG-1::GFP clusters at the same time that SYG-2::GFP expression begins Mid L4 Mid L4 ↑ SYG-2::GFP expression in 1° vulval epithelial cells YAD YAD SYG-2::GFP expression disappeared within the first 6hrs of adulthood Disappearance of SYG-2::GFP correlates to more diffuse SYG-1::GFP distribution SYG-2Expression Across Development SYG-2 required for early synaptic specification, not for maintenance of synaptic function

  15. Does the SYG-2 protein correspond to this vulval signal? Examined synaptic localization of SYG-1 in a syg-2 Mutant Wild Type WT animals SYG-1 was localized to the segment of the axon contacting the primary vulval epithelial cells syg-2(ky671) & syg-2(ky673) SYG-1 was diffusely distributed on the HSNL axon, syg-2(ky673) syg-2(ky671) Suggests that SYG-2 is essential for SYG-1 localization at synaptic sites. note unc-86 promoter expressed only in HSN neurons SYG-2 Expression is necessary to localize SYG-1 Synapses made by the C. elegans HSNL neuron are initiated by the guidepost role of the primary vulval epithelial cells, acting through the TM Ig superfamily protein SYG-1 on HSNL

  16. Does SYG-2 directly specify the location of SYG-1 & synaptic vesicles? Expressed SYG-2 in 2° vulval epithelial cells using an egl-17 promoter in syg-2(ky673) SYG-2 expression in the 2° vulval epithelial cells is sufficient to attract SYG-1 protein from HSNL axon Wild Type SYG-1::GFP The interaction of SYG-1 & SYG-2 is sufficient to trigger the clustering of SNB-1 vesicles Wild Type SNB-1::GFP egl-17 is expressed in a subset of vulval epithelial cells SYG-2 in secondary vulval epithelial cells relocalizes SYG-1 & SNB-1 labelled vesicles

  17. transfected cells tested for aggregation (homo or hetero) S2 cells were cotransfected  SYG-1 cDNA & GFP  SYG-2 cDNA & dsRED homophilic interactions of SYG-1 & SYG-2 are weak SYG-1 transfected cells specifically aggregated with SYG-2 transfected cells complete aggregation within 60min of incubation Do SYG-1 & SYG-2 Interact Directly? Drosophila S2 cell aggregation assay with SYG-1 & SYG-2

  18. direct interaction HSNL epithelial cell SYG-1 SYG-2 specifies the location of synapses synaptic specificity drives the selection of appropriate postsynaptic targets excludes inappropriate HSNL targets Summary Synapses made by the C. elegans HSNL neuron are initiated by the guidepost role of the primary vulval epithelial cells, acting through the TM Ig superfamily protein SYG-1 on HSNL Ig superfamily protein SYG-2 is the molecular guidpost cue on the epithelial cells

  19. SYG-1 SYG-1 VC axons & vulval muscles VC axons & vulval muscles contacts contacts SYG-2 SYG-2 HSNL HSNL contacts contacts 1° & 2° vulval epithelial cells PVQL, PVPR, AVKL axons body wall muscles hypodermal cells 1° & 2° vulval epithelial cells PVQL, PVPR, AVKL axons body wall muscles hypodermal cells ? ? ? ? Summary Wild-Type Animals syg-1 & syg-2 Mutants

  20. Thank You

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