Calvi, Ida et al. (2021) International Worm Meeting "A GSP-2/PKC-3 balance is required for polarity establishment in C. Elegans"

Gotta, Monica, Calvi, Ida

[International Worm Meeting, 2021]

Cell polarity is a fundamental property of many cells and proper establishment of polarity is required for the development of organisms and for the function and integrity of cells and tissues. The one-cell C. elegans embryo is polarized along the anterior-posterior axis and this is essential for asymmetric cell division, a specialized division that results in two cells with a different size and fate. PAR proteins, a network of highly dynamic proteins with scaffold and/or enzyme function, are the main actors involved in cell polarity in the one-cell C. elegans embryos. Just after the fertilization, the anterior PAR proteins (aPARs), PAR-3/PAR-6/PKC-3, are uniformly distributed at the cortex, whereas the posterior PAR proteins (pPARs), PAR-1/PAR-2, are in the cytoplasm. Uniform cortical PKC-3 phosphorylates PAR-2 inhibiting its posterior localization and polarity establishment. A cortical flow, triggered by the sperm, initiates the segregation of the aPARs to the anterior side of the embryo and liberates the posterior pole, allowing the loading of PAR-2 (and PAR-1) at the posterior. How PAR-2 is relieved from PKC-3 phosphorylation to ensure PAR-2 cortical localization and hence polarity establishment is not known. We find that the PP1 phosphatase GSP-2 antagonizes PKC-3 phosphorylation, allowing PAR-2 posterior cortical localization and polarity establishment. Depletion of GSP-2 via RNAi rescues the PAR-2 localization in the pkc-3 (ts) strain, suggesting that PKC-3 and GSP-2 are involved in the same process, but with opposing function. Moreover, mutation of the PP1 binding site in PAR-2 abolishes the interaction between PAR-2 and GSP-2 in the Y2H system. We are now introducing the mutation in the endogenous PAR-2 to investigate its localization in the C. elegans embryo. Our data unravel how the phosphorylation state of PAR-2 is properly balanced in the one-cell C. elegans embryo to ensure robust establishment and maintenance of polarity.

Dorlean, Shad-Lee et al. (2021) International Worm Meeting "Investigating the connection between the DAF-7/TGF-beta signaling pathway and the dense core vesicle protein IDA-1"

McLaughlin, Shannon, Rojas Rodriguez, Paola A, McGehee, Annette, Dorlean, Shad-Lee

[International Worm Meeting, 2021]

The DAF-7/TGF-beta signaling pathway in C. elegans is affected by the environment. This includes factors such as inadequate food supply, high temperature, or overpopulation. We previously found that the DAF-7/TGF-beta signaling pathway regulates the abundance of the glutamate receptor GLR-1. Mutants in the DAF-7/TGF-beta signaling pathway have increased levels of GLR-1 and an increased rate of spontaneous reversals, a behavior which is regulated by GLR-1. However, the exact mechanism of how DAF-7/TGF-beta signaling regulates GLR-1 is unknown. We tested to see whether ida-1, which is expressed in cells that express glr-1, might be involved in this regulation. IDA-1 is normally found on the surface of dense core vesicles (DCV's) and is involved in normal neuropeptide and DCV signaling. To test the involvement of ida-1 in the DAF-7/TGF-beta-dependent regulation of GLR-1 we assessed spontaneous reversal frequency and GLR-1::GFP levels in single and double mutants. The increased rate of spontaneous reversals and the increased GLR-1::GFP levels that are detected in daf-7 mutants are blocked in daf-7 ida-1 double mutants. These results support the idea that IDA-1 does indeed play a role in the DAF-7/TGF-beta-dependent regulation of GLR-1. We then focused on testing whether IDA-1 expression and localization are affected in daf-7 mutants. We investigated this question using strains expressing IDA-1::GFP. We imaged neurons in the head of C. elegans worms of the appropriate genotypes at both the cell body and neurite in order to measure the intensity of the IDA-1::GFP. We found increased levels of IDA-1::GFP in both cell bodies and neuronal puncta in the daf-7 mutant as compared to wildtype. These results suggest that the DAF-7 pathway regulates levels of IDA-1. The exact mechanism however, is yet to be discovered.

Tobias R Zahn et al. (2003) International Worm Meeting "Deletion mutants of the neuroendocrine protein tyrosine phosphatase-like ida-1 gene."

Cindi Schwartz, Erin Domke, Margaret A MacMorris, Tobias R Zahn, John C Hutton

[International Worm Meeting, 2003]

In mammals, the closely related protein tyrosine phosphatase-like type 1 transmembrane proteins ICA512 and phogrin are restricted to neuroendocrine tissues including brain, pituitary and pancreatic beta-cells, where they are localized to dense core vesicles (DCVs) of the regulated secretory pathway. Both are of clinical relevance as they represent major autoantigens in type 1 diabetes. Their physiological role remains enigmatic, though strong evolutionary conservation and functional studies suggest a conserved regulatory role in hormone and neuropeptide secretion. IDA-1 is the single C. elegans ortholog of ICA512 and phogrin and is also expressed in a subset of neurons and the neuroendocrine-like uv1 cells in worms, suggesting functional conservation (Zahn et al., J Comp Neurol 429:127). Anti-IDA-1 immunostaining and immuno-electron microscopy show that IDA-1 is localized intracellularly to small punctate organelles identified as DCVs. The C. elegans Gene Knockout Consortium recently made available two deletion alleles within the ida-1 locus, tm334 and ok409. tm334 is a small deletion within the N-terminus of the protein, while ok409 represents a larger deletion. ok409 but not tm334 animals are moderately egg-laying defective, consistent with a function for ida-1 in the neuropeptidergic control of egg-laying. We are currently characterizing the phenotypes of these deletion mutants. The C. elegans model will aid in elucidating the physiological role of ida-1 and its mammalian orthologs.

Tobias R Zahn et al. (2001) International C. elegans Meeting "Movement of IDA-1::GFP-tagged vesicles in C. elegans nerve processes"

Margaret A MacMorris, Tobias R Zahn, Joseph K Angleson, John C Hutton

[International C. elegans Meeting, 2001]

In mammals, the closely related protein tyrosine phosphatase-like type 1 transmembrane proteins ICA512 (IA-2) and phogrin (IA-2beta) are restricted to neuroendocrine tissues including brain, pituitary and pancreatic beta-cells, where they are localized to dense core vesicles (DCVs) of the regulated secretory pathway. Phogrin has been shown to undergo Ca 2+ -dependent phosporylation in response to secretagogues and ICA512 to interact with betaIV spectrin and beta2-syntrophin. These findings together with the restricted expression pattern, intracellular localization and the strong conservation between species suggest a role of these proteins in regulated secretion. They are of additional interest as major targets of autoimmunity in type 1 diabetes. We recently reported the existence of a single ortholog of phogrin and ICA512 in C. elegans , IDA-1 ( i slet cell d iabetic a utoantigen). Using GFP reporter constructs, we localized ida-1 gene expression to a subset of about 30 mostly sensory neurons and the vulval neurosecretory-like uv1 cells. Expression was sex-specific and included the hermaphrodite-specific vulval motoneurons and many male-specific neurons. In contrast to synaptic vesicles (SVs) in C. elegans , very little is known about the larger ‘dark-cored’ vesicles which have been seen in the electron-microscopic analysis of the worm’s nervous system. We hypothesize that IDA-1 in C. elegans like ICA512 and phogrin in mammals is specific to DCVs which are distinct from synaptic vesicles and secrete neuropeptides or hormones in a regulated fashion. SNB-1::GFP-tagged SVs have proven very useful tools, and more recently Scholey and colleagues showed that in live C. elegans animals movement of GFP-tagged molecular rafts in amphid dendrites can be observed with a fluorescent light microscope. We generated transgenic C. elegans strains expressing an IDA-1::GFP fusion protein, showing punctate staining within neuronal cell bodies and their processes. The distribution of IDA-1::GFP in nerve processes is reminiscent of the bouton-like staining of SV markers. In contrast to the reported subcellular distribution of SNB-1::GFP, IDA-1::GFP strongly stains cell bodies though is excluded from the nucleus consistent with DCV biogenesis and recycling at the trans-Golgi network and endosomal compartments. Small IDA-1::GFP punctae shuttle between larger immobile GFP accumulations distributed periodically along axons and dendrites. Our initial analysis of vesicle movement in PHC dendrites reveals rates of transport of ~ 1.2 microm/s in retrograde and more variable ~ 2.6 microm/s in anterograde direction indicative of fast microtubule-based transport. We generated IDA-1::GFP transgenic mutants defective in motor proteins including unc-104 , osm-3 , and che-3 to directly test a requirement of IDA-1::GFP-tagged vesicle transport by those molecular motors. In an attempt to define similarities and differences between C. elegans SVs and the IDA-1::GFP-tagged structures, we introduced the IDA-1::GFP transgene into a variety of mutant backgrounds that are known to severely disrupt the localization of synaptic vesicles and proper formation of synapses, such as unc-11 , syd-2 , and rpm-1 . The observed subcellular localization and movements of IDA-1::GFP are consistent with a vesicular localization of IDA-1 and indicate that the nematode will be a useful model to study the molecular mechanisms involved in DCV transport and recycling.

Lawson, Hannah N. et al. (2021) International Worm Meeting "Molecular mechanisms regulating organization of sensory neuron cilia"

Lawson, Hannah N., Sengupta, Piali

[International Worm Meeting, 2021]

Primary cilia are present on all sensory neurons and are critical for their functions. The organization of neurons and their processes within neuropils has been shown to be important for their functions in multiple organisms. However, whether the organization of cilia within a sense organ is similarly important for sensory neuron function is unclear. In C. elegans, the cilia of eight chemosensory neurons of the head amphid sense organs are present within a channel formed by surrounding glia. Previous work from the Sengupta lab showed that these cilia are stereotypically arranged within this channel and form specific contacts with one another. We found that sax-7 mutants, which have altered amphid neuron dendrite order, retain stereotypical cilia organization within this channel, suggesting cilia arrangement is not a consequence of dendrite order. Cilia-cilia contact appears to be dependent on the presence of ciliary distal segments. Because flagella adhesion during Chlamydomonas mating is mediated by glycoproteins, we asked whether glycoproteins might control ciliary organization and cilia-cilia contact in C. elegans. Our preliminary results indicate that mutations in an enzyme involved in synthesizing N-linked glycoproteins alters ciliary organization and cilia-cilia contact. In a screen of candidate regulators of cilia organization, we identified the conserved phogrin homolog IDA-1. Mutations in ida-1 alter cilia position within the amphid channel. IDA-1 localizes to the ciliary base; however, mutations in the BBSome protein BBS-7 mislocalize IDA-1 to the cilia. To determine whether IDA-1 localization is critical for regulating ciliary position, we examined the cilia of bbs-7 mutants. Our preliminary results indicate that cilia in these animals are also disorganized. Additionally, the cilia-cilia contacts typically seen in wild-type animals may be altered in bbs-7 mutants. IDA-1 is a transmembrane protein localized to the membrane of dense-core vesicles (DCVs) and is implicated in regulating DCV cargo release. Mutations in unc-31 disrupt DCV exocytosis, however, the cilia of unc-31 mutants appear largely wild-type, suggesting that ida-1 control of ciliary organization is independent of its role in DCV signaling. Our current experiments are aimed at further exploring the molecular mechanisms underlying ciliary organization and establishing the contribution of cilia-cilia contact for regulating sensory neuron functions.

Tao Cai et al. (2003) International Worm Meeting "IA-2, a Vesicle Membrane-Associated Protein, Regulates Synaptic Transmission in C. elegans"

Michael Krause, Abner L Notkins, Tao Cai, Tetsunari Fukushige

[International Worm Meeting, 2003]

IA-2, a major autoantigen in type 1 diabetes, is a receptor-tyrosine phosphatase-like protein associated with the membrane of secretory granules of neural and endocrine-specific cells. IA-2 lacks phosphatase activity due to an amino acid substitution within the catalytic domain and the function of IA-2 remains unclear. In order to gain insight into the cellular functions of IA-2, we have studied the C. elegans homolog, CeIA-2 encoded by the ida-1 gene. Using two independent, putative null, deletion alleles of ida-1 we show that animals lacking CeIA-2 are viable with only subtle visible phenotypes. Pharmacological studies show loss of CeIA-2 results in presynaptic neuronal defects involving neurotransmitter release. Furthermore, ida-1 interacts genetically with unc-31 and unc-64, two genes encoding factors required for neuronal vesicle function. These results suggest that CeIA-2 is a novel regulator of synaptic transmission that regulates vesicle release.

Tobias R Zahn et al. (2002) West Coast Worm Meeting "Dense core vesicle trafficking in neurons visualized in vivo"

John C Hutton, Tobias R Zahn, Joseph K Angleson, Margaret A MacMorris

[West Coast Worm Meeting, 2002]

In mammals, the closely related protein tyrosine phosphatase-like type 1 transmembrane proteins ICA512 and phogrin are restricted to neuroendocrine tissues including brain, pituitary and pancreatic beta-cells, where they are localized to dense core vesicles (DCVs) of the regulated secretory pathway. IDA-1 is the single C. elegans ortholog of ICA512 and phogrin and is also expressed in a subset of neurons and the neuroendocrine-like uv1 cells. Immunocytochemistry showed that IDA-1 is localized intracellularly to small punctate organelles identified as DCVs. An IDA-1::GFP fusion protein proved a useful tool to investigate the in vivo trafficking of DCVs in C. elegans neurons. Real-time imaging demonstrated that vesicle movements occurred bidirectionally with frequent saltatory movements and occasional reversals. Transport was blocked by nocodazole and azide, suggesting the involvement of distinct microtubule-based molecular motors of the kinesin and/or dynein superfamilies. Movements were complex and occurred at multiple distinct velocities of up to 4 um/s, implying the presence of multiple motor proteins with different properties. Furthermore, transport velocities were different for axons and dendrites, demonstrating a polarized distribution of the molecular transport machinery.

Tiewen Liu et al. (2004) East Coast Worm Meeting "Neuropeptide modulation of C. elegans male mating behavior"

Tiewen Liu, Maureen M Barr

[East Coast Worm Meeting, 2004]

The C. elegans male mating behavior comprises the steps of response, backing, turning, location of vulva, spicule insertion and sperm transfer. Cell ablation experiments have identified sensory neurons responsible for each step (1), making this behavior an attractive model for studying synaptic transmission in a defined neural circuit. Neuropeptides represent a large and diverse set of non-classical neural transmitters. They can modulate synaptic transmission by regulating presynaptic neurotransmitter release and/or postsynaptic neurotransmitter receptor responsiveness. The C. elegans genome is predicted to encode 23 FMRFamide-like neuropeptide ( flp ) genes (2). To determine the function of these flp genes in male mating behavior, we characterized their expression patterns in adult C. elegans males with GFP reporters. Several flp genes are expressed in male-specific neurons, suggesting a role in the male nervous system. We also characterized the male mating behavior of a number of flp mutants. Four of them, flp-8 , flp-10 , flp-12 and flp-20 , exhibit an abnormal 'stutter' turning phenotype. Currently, we are trying to determine the cellular basis of this turning defect. We are also interested in the biosynthesis and secretion of neuropeptides in C. elegans . To this end, we have found that C. elegans proprotein convertase egl-3 (3) has overlapping expression pattern with some of the flp genes and egl-3 mutant males also have a stutter turning phenotype. We also found that ida-1 , the C. elegans homolog of mammalian dense core vesicle IA-2 (4), is expressed in a subset of FMRFamide-like neuropeptide expressing neurons, and ida-1 mutant males exhibit stutter turning phenotype. We are currently investigating the mechanisms by which EGL-3 and IDA-1 regulate male turning behavior. We are grateful to Dr. Chris Li for the flp ::GFP strains and flp mutant strains. 1) Liu and Sternberg 1995, Neuron 14, 79-89. 2) Li et al 1999, Ann. NY Acad. Sci. 897, 239-252. 3) Thacker and Rose 2000, Bioessays 22, 545-553. 4) Zahn et al 2001, J. Comp. Neurol. 429, 127-143.

Bird, David (2015) International Worm Meeting "A model for feeding-site formation by root-knot nematode."

Bird, David

[International Worm Meeting, 2015]

Root-knot nematode (RKN: Meloidogyne spp.) is an obligate parasite of land plants and causes severe yield reduction in crops world-wide. Characteristic of the parasitic interaction is the induction of a permanent feeding site, composed of several Giant Cells (GC) surrounded by a gall. Interrogation of the M. hapla genome revealed that the nematode encodes replicas of plant peptide hormone families, including 12 CEP, 8 CLE, IDA, RALF, and RGF. In planta, peptide hormones work as powerful developmental and physiological regulators at the cell-to-cell level and as plant-wide signals. These hormones are not found in other animals. Thus, by encoding a full gamut of these peptides, which I term "xenomones," RKN is unique.Based on the assumption that the role played by the M. hapla xenomones will recapitulate that of the cognate, indigenous plant-peptide hormones, I propose a model of infection by RKN. Central is the sequential release of specific xenomones by the nematode to facilitate/execute the necessary plant response:1) Mechanical penetration of root by L2 dauer;2) intercellular migration: secretion of IDA xenomone3) cytokinin release by L2 to initiate development of root stem cells4) induction of cell expansion: secretion of RALF xenomone5) suppression of cytokinesis: secretion of CEP xenomone6) suppression of differentiation: secretion of CLE xenomoneTo establish the relationship of M. hapla xenomones with their receptor(s), we have solved the structure of 8 xenomones to atomic resolution, and modeled their docking onto the 380 predicted Arabidopsis trans-membrane receptors. These docking experiments are double blinded and reveal distinct classes of receptor::ligand relationships.

Edwards, Stacey et al. (2011) International Worm Meeting "Forward Genetic Analysis of Dense Core Vesicle Maturation and Function."

Hoover, Christopher, Miller, Kenneth, Edwards, Stacey

[International Worm Meeting, 2011]

Recent studies suggest close interactions between the synaptic Ga signaling pathways and the functions of neuronal dense core vesicles (DCVs). We have found that the function of the neuronal Gas pathway broadly overlaps with the function of UNC-31 (CAPS), which facilitates DCV docking and exocytosis.1 A large forward genetic screen for suppressors of goa-1 (Gao) null mutants recovered the expected mutants with impaired Gaq signaling but also produced mutations in proteins involved in DCV function (UNC-31) and maturation (UNC-108/ RAB2).2 Interestingly, Rab2 is the most highly conserved Rab in the animal kingdom, and yet very little is known about its precise function. Recent studies have shown that unc-108 null mutants send the same number of DCVs to axons as wild type; however, their vesicles are missing up to two-thirds of their soluble and transmembrane cargoes.2,3 As a result of the loss of the DCV transmembrane cargo IDA-1-GFP in unc-108 mutant somas, IDA-GFP accumulates in somas at levels about 2-fold higher than wild type. We have used this as the basis of a genetic screen to identify other players that act with UNC-108 in dense core vesicle maturation. We mutagenized a strain that expresses IDA-1-GFP in motor neurons as an integrated transgene. We then screened its grandprogeny for rare mutants with brighter cell somas. We also included another transgene in the strain that expresses soluble RFP from the same promoter to rule out gene expression changes. To screen animals we plated L4-stage F2s on 24-well culture plates (40 animals per well) and screened them using a fluorescence stereomicroscope with a high resolution objective. In a pilot screen (~20,000 F2s; ~4-fold coverage) we isolated 17 mutants of interest and have sorted them into complementation groups and mapped many of the new mutations. Among the interesting genes identified so far are three proteins that appear to be conserved in all organisms with a nervous system, but whose function is largely unknown. One of these proteins is HID-1, which was recently implicated in dense core vesicle maturation.4,5 Since some of the genes are represented by only 1 or 2 alleles, we are doing more cycles. 1Charlie et al., 2006. Genetics. 172(2), 943-961. 2Edwards et al., 2009. J. Cell Biology.186(6):881-895. 3Sumakovic et al., 2009. J. Cell Biology.186(6):897-914. 4Mesa et al., 2011. Genetics. 187(2): 467-483. 5Yu et al., 2011. Biochem J. 434(3):383-90.