Wu, Monica Z. et al. (2013) International Worm Meeting "Characterization of the AGO protein VSR-1 in small RNA-mediated gene silencing pathways in the worm."

Wu, Monica Z., Claycomb, Julie M.

[International Worm Meeting, 2013]

Argonautes (AGOs) are the key effector components of RNA interference (RNAi) and related endogenous small RNA pathways. C. elegans possesses 26 AGO family proteins and although deletion mutant strains for each of the C. elegans AGOs have been generated, the functions of only a handful of these proteins are understood. The AGOs that have been characterized thus far have been shown to perform distinct functions. Therefore, further investigation of the remaining AGOs can lead to novel insights into small RNA mediated gene-silencing functions. We have been investigating the role that a relatively uncharacterized, but well-conserved AGO protein, which we have named VSR-1 (Versatile Small RNAs), plays in small RNA-mediated gene regulation in C. elegans. mRNA and protein expression analysis demonstrates that VSR-1 is expressed throughout development, but is enriched in later stages when the germline develops, implicating VSR-1 in germline small RNA functions. Immunolocalization studies show that VSR-1 localizes to embryonic P granules, consistent with localization patterns of other Argonautes, including WAGO-1 and CSR-1. VSR-1 also localizes to oocyte and embryonic chromatin, which has been confirmed through biochemical fractionation experiments, suggesting a role for this AGO in transcriptional regulation or other nuclear functions. To identify with which small RNAs VSR-1 interacts, we have cloned and Illumina sequenced small RNAs in both the vsr-1 mutant background and from VSR-1 complexes. Consistent with VSR-1 playing a role in the biogenesis of particular small RNAs, the small RNAs enriched in VSR-1 complexes are depleted in the mutant. Our initial analyses indicate that VSR-1 associates with particular subsets of small RNAs spanning multiple classes. These small RNAs have been implicated in playing key roles in germline and embryonic development. These findings are exciting and point to a novel activity for VSR-1 in multiple small RNA pathways, as all other AGOs studied thus far have been shown to associate with one particular class of small RNAs in the worm. Our ongoing studies will reveal additional insights into the roles of VSR-1 in worm development.

Khan, Munzareen et al. (2021) International Worm Meeting "

Khan, Munzareen, Sengupta, Piali, Dwyer, Noelle, Hartmann, Anna, Bargmann, Cori, Piccione, Madeline

[International Worm Meeting, 2021]

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Malinow, R.A. et al. (2019) International Worm Meeting "Functional dissection of C. elegans bZip-protein CEBP-1."

Kim, K.W., Koorman, T., Boxem, M., Ying, P., Malinow, R.A., Jin, Y.

[International Worm Meeting, 2019]

C. elegans CEBP-1 is a C/EBP bZIP transcription factor and essential for axon regeneration. Previous studies have focused on the role of bZIP domain at the C-terminus of CEBP-1 in axon regeneration (Yan et al., 2009 Cell). However, CEBP-1 also has a long N-terminal fragment that contains no known protein motifs and how it is involved in CEBP-1 function remains unknown. Here, we report novel structural motifs in the N-terminus of CEBP-1 for axon regeneration and nuclear import. Using two types of forward genetic screens, we have identified a unique domain in the N-terminus that is critical for CEBP-1's in vivo functions. This N' domain also has a predicted propensity to form alpha helices. Furthermore, using yeast two-hybrid screening with N-terminal CEBP-1 fragments as baits, we have identified IMA-3/Importin-? as a CEBP-1 binding partner. In transgenic expression studies, we characterized the subcellular localization of CEBP-1 fragments and identified three nuclear localization signals (NLS) in CEBP-1, one which interacts with IMA-3. Lastly, we showed that ima-3 is required for axon regeneration. Taken together, our findings uncover new protein structural elements for CEBP-1, and further reinforce the importance of Importin-mediated nuclear import system for promoting axon regeneration. Yan, D., Wu, Z., Chisholm, A.D., and Jin, Y. (2009). The DLK-1 kinase promotes mRNA stability and local translation in C. elegans synapses and axon regeneration. Cell 138, 1005-1018.

Ji Wu et al. (2002) East Coast Worm Meeting "Regulation of Touch Cell Functional Genes"

Ji Wu, Martin Chalfie

[East Coast Worm Meeting, 2002]

We have previously shown that egl-44 and egl-46 are two transcription regulators involved in the development of several types of neurons in C. elegans, with significant homology in other organisms, and that they repress the expression of touch cell-specific features in FLP neurons (Wu et al., 2001,Genes. Dev., 15: 789).

Wu S-L et al. (1996) East Coast Worm Meeting "CHARACTERIZATION OF AN ATYPICAL PROTEIN KINASE C IN C. Elegans."

Rubin CS, Wu S-L

[East Coast Worm Meeting, 1996]

Protein kinase Cz (PKCz) is an atypical member of the PKC superfamily. PKCz contains Cys-rich zinc binding and catalytic domains that are homologous with corresponding regions in other PKCs. Unlike other PKCs, the zeta isoform in not activated by diacyl glycerol, TPA or calcium. However, PKCz appears to be a target for activation by alternative lipid second messengers (3,4,5-phosphoinositol, ceramide) generated by PI-3-kinase and/or sphingomyelinase. Activated PKCz has been implicated in the transmission of regulatory signals from the cytoplasm to the nucleus, but its precise physiological roles remain to be rigorously determined. We have initiated studies on the structure, function and regulation of an atypical C. elegans PKC (designated PKC-Z) that is related to mammalian PKCz. A cDNA that encodes full-length PKC-Z was cloned and sequenced. The predicted PKC-Z protein (598 amino acid residues, molecular weight = 68,000) is only ~55% identical with mammalian PKCz. N-terminal and central portions of the two proteins are highly divergent. However, the catalytic, psuedosubstrate and Cys-rich domains are highly conserved. Comparison of the cDNA sequence with data from the C. elegans genome project indicates that the PKC-Z gene (LGII) encompasses 3.6 kbp of DNA and contains 9 exons. A C terminal fragment (120 amino acids) of PKC-Z was expressed as a His-tagged fusion protein in E. coli, purified and injected into rabbits to produce antiserum. Western immunoblot analysis revealed that PKC-Z is most abundantly expressed in the soluble fraction of embryos. The kinase is also evident in L1-L4 larvae and adult nematodes, but the bulk of the enzyme is tightly associated with the particulate fractions of post-embryonic animals. Consistent with the latter observation, immunostaining and confocal microscopy of permeabilized C. elegans revealed that PKC-Z is localized and concentrated in discrete intracellular clusters. The nature of the intracellular structure at which PKC-Z accumulates is not yet known. PKC-Z is being expressed in baculovirus/Sf9 cells and in mammalian cells for enzymic characterization. In addition, injected anti-sense oligonucleotides are being used to probe the function(s) of PKC-Z in embryogenesis.

Piggott, C.A. et al. (2017) International Worm Meeting "Investigating the roles of membrane contact site proteins in axon regeneration."

Jin, Y., Piggott, C.A., Wu, Z.

[International Worm Meeting, 2017]

Axons are vital for the function of the nervous system as they transmit neuronal signals to their targets. During development of the nervous system, axons readily grow and make precise connections, but that ability is lost as animals mature into adults. It is unclear which signaling pathways promote and restrain axon growth throughout development into adulthood. Through a systematic genetic screen using laser axotomy of PLM neurons, we identified several genes that are predicted to function at interorganellar membrane contacts. Loss of function in these genes display differential effects on PLM axon regeneration. Among them, we have focused on esyt-2, a member of the extended synaptotagmin family, which have been shown to maintain membrane contact sites between the endoplasmic reticulum (ER) and plasma membrane. We found that ESYT-2 localizes to putative ER-plasma membrane contact sites in neurons. Inducing injury by laser axotomy causes an immediate relocalization of ESYT-2, which may represent a change in membrane contact sites in response to injury. We are currently investigating the mechanism of how membrane contact site protein may play an important role in axon regeneration.

Ghosh Roy, A. et al. (2009) International Worm Meeting "Dissecting Calcium/cAMP mediated axon regeneration pathways."

Wu, Z., Chisholm, A.D., Ghosh Roy, A., Goncharov, A., Jin, Y.

[International Worm Meeting, 2009]

We are interested in the molecular pathways that promote the regenerative response of axons after injury. Injury of axons of cultured neurons causes a transient elevation of intracellular calcium. Pharmacological elevation of calcium and cAMP are long known to enhance the ability of axons to regenerate in vitro and in vivo. However the molecular mechanisms that govern the Ca/cAMP mediated axonal regeneration are not clearly understood. We previously reported that the lateral touch neurons ALM and PLM show robust regenerative responses after injury (Wu et al., PNAS, 2007). We report here that calcium and cAMP play critical roles in the regenerative responses of these neurons. Using genetically encoded Ca sensors we have found that laser axotomy triggers a rapid local elevation of intracellular calcium that propagates bidirectionally away from the axotomy site. The amplitude of this calcium transient correlates with the extent of total axonal regrowth. Further, gain of function in the voltage-gated calcium channel, egl-19 increases the amplitude of the calcium transient and accelerates regenerative growth, suggesting calcium levels are a critical determinant of regrowth kinetics. We find that genetic elevation of calcium enhances regeneration in several ways. The injured axon enters the regeneration phase earlier compared to controls, extends faster, and more often fuses with the severed distal fragment. Our ultrastructural analysis shows that in these cases the regrowing axon has physically fused membranes with the distal fragment. This fusion process appears not to require known fusogens such as EFF-1 or AFF-1. Mutations that chronically elevate neuronal cAMP promote regeneration in a similar manner with the exception that the regenerating process also has an increased tendency to extend ventral synaptic branches. Reduction of neuronal cAMP by overexpression of PDE-4 blocked this ventral branch regrowth, indicating that cAMP functions cell autonomously to promote growth. Activation of protein kinase A has similar effects on regeneration, suggesting PKA is the major effector of cAMP. We further find that different bZip transcription factors are required for distinct aspects of the regenerative response, suggesting calcium/cAMP acts via multiple transcription factors. Our studies indicate that calcium and cAMP have conserved signaling roles and are rate limiting for regeneration. We are currenty exploring the relationship between calcium/cAMP signaling and other regeneration pathways.

Gilleard JS et al. (1995) International C. elegans Meeting "REGULATION OF THE CUTICULAR COLLAGEN GENE dpy-7"

Barry JD, Gilleard JS, Johnstone IL

[International C. elegans Meeting, 1995]

We are interested in identifying trans acting factors which control cuticular collagen gene expression. Sequences sufficient for regulated expression of dpy-7 appear to reside relatively close to the initiator ATG since the wild type gene only requires 310bp of 5' flanking sequence for transformation repair of phenotype. We have analysed sequences necessary and sufficient for dpy-7 expression using lac-Z fusions and we have identified a 95bp fragment, encompassing the transcritional start sites, which is sufficient to produce lac-Z expression predominantly, or possibly exclusively, in hypodermal cells. This fragment includes a region in which 65 out of 72bp are conserved in the 5' flank of the C.briggsae dpy-7 homolog and interspecies transformation rescue and lac-Z fusion experiments have shown that the dpy-7 cis regulatory elements are functionally conserved. A 15bp deletion at the 5' end of this homology which removes a AGATAA motif, also present in C.briggsae, completely ablates lac-Z expression . We are attempting to identify transcription factors which bind to this motif by South Western expression library screening. We are also searching for new C.elegans GATA factor homologs with a particular interest in any which are expressed in hypodermal cells postembryonically.

Dolinski CM et al. (1997) International C. elegans Meeting "PHYLOGENETIC IMPLICATION OF BUCCAL CAPSULE DEVELOPMENT IN SOME BACTERIAL FEEDING NEMATODES"

Baldwin JG, Dolinski CM, Thomas WK

[International C. elegans Meeting, 1997]

Bacterial feeding nematodes including Zeldia punctata (Rhabditida: Nematoda), and Caenorhabditis elegans differ profoundly in feeding adaptations and specifically in the rhabdions (cuticular thickenings), and associated cells lining in the buccal capsule. We are carrying out a range of tests to determine what rhabdions are evolutionarly homologous between the two species. Reconstruction of the buccal capsule and procorpus (pharynx) with transmission electron microscopy indicates that the lining of the buccal capsule of Z. punctata includes four sets of muscular radial cells ma, mb, mc, md in contrast the same region in C. elegans which has two sets of epithelial radial cells (e1, e3) and two sets of radial muscle cells (m1, m2). In Z. punctata ma is a set of three radial muscle cells each with 2 nuclei. In C. elegans m1 has the identical arrangement. Similarly, in Z. punctata mb is a set of 3 muscle cells each with one nucleus, similar to m1 in C. elegans. These findings could contradict all previous hypotheses of homology, and suggest instead that ma and mb in Z. punctata are homologous respectively with m1 and m2 in C. elegans. Ongoing additional tests of homology include comparative cell lineages and screening mutants to recognize genes involved in buccal capsule expression.

Piggott, C.A. et al. (2019) International Worm Meeting "Roles of membrane contact site components in axon regeneration."

Piggott, C.A., Chisholm, A.D., Jin, Y., Wu, Z.

[International Worm Meeting, 2019]

The ability of axons to regenerate after injury is conserved across animals. However, the molecular processes regulating regrowth of mature axons are not well understood, in particular the mechanisms that generate new membranes. Studies using laser axotomy in C. elegans have revealed many conserved genes and pathways in axon regeneration. To gain new insights into how regulation of membrane generation and remodeling contributes to axon regeneration, we have focused on the roles of endoplasmic reticulum (ER) - plasma membrane (PM) contacts. Among orthologs of membrane contact site components, we have found that jph-1, the sole member of the junctophilin family in C. elegans, plays distinct roles in axon regeneration. Junctophilins are transmembrane proteins localized to ER-PM contacts in excitable cells, where they couple PM- and ER- localized calcium channels. Mice lacking muscle Junctophilins are embryonic lethal or die shortly after birth, whereas little is known about neuronal roles of Junctophilins. jph-1 null mutants grow slowly, and are small and thin compared to wild-type animals. These developmental defects are rescued by expressing a GFP-tagged wild-type transgene. GFP::JPH-1 is broadly expressed in muscles and neurons, and localizes to membrane contact site-like puncta. We previously reported that in PLM axon regeneration, loss of jph-1 causes reduced regrowth. Strikingly, jph-1 also inhibits fusion between the regrowing axon and distal fragment after axon injury (Kim et al. 2018). Our on-going studies aim to dissect how jph-1 interacts with other genes implicated in axon fusion. Additionally, by time-lapse imaging studies, we have observed that JPH-1 relocalizes rapidly in response to laser axotomy, similar to ESYT-2, a membrane of the extended synaptotagmin family. This finding suggests that membrane contact site dynamics are part of the regenerative response. We are currently investigating the mechanism of how ER-PM contact site components may play an important role in axon regeneration.