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.

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.

Rand JB et al. (2000) East Coast Worm Meeting "UNC-13 in the C. elegans Nervous System"

Kohn RE, Rand JB, McManus JR, Moulder GL, Duke A, Barstead RJ, Duerr JS

[East Coast Worm Meeting, 2000]

C. elegans unc-13 and its homologues in vertebrates and Drosophila are involved in neurotransmitter release. UNC-13 has several regions homologous to PKC regulatory domains; these domains confer it with calcium, phorbol ester and phospholipid binding properties (Maruyama and Brenner, PNAS 88, 1991). A 5.9kb transcript coding for a 200kDa protein was initially identified (now designated L-R for left and right regions). We have identified two additional types of transcripts. One transcript includes a 1kb novel exon (L-M-R, M for middle region) and another transcript lacks the 5' region included in the other two transcripts (M-R). All three transcripts are identical at the 3' end (R). C. elegans with mutations in the 5' end of the gene (L) alter two types of transcripts (L-R and L-M-R) resulting in an uncoordinated coily phenotype and resistance to the anti-cholinesterease, aldicarb. A 2.7kb deletion near the 3' end (R) (identified by Bob Barstead using PCR analysis) affects all unc-13 transcripts and results in a lethal phenotype. Antibodies recognizing the N-terminal region of UNC-13 (L) label synapses, but not synaptic vesicles, of most or all neurons; many mutations in L and R remove staining with this antibody. Supported by grants from the NIH and OCAST.

Abergel, Z. et al. (2017) International Worm Meeting "Adaptation of the nematode C. elegans to hypoxia and reoxygenation stress reveals an unexpected function of the neuroglobin GLB-5 in innate immunity."

Zuckerman, B., Zelmanovich, V., Abergel, Z., Abergel, R., Gross, E., Smith, Y., Romero, L., Livshits, L.

[International Worm Meeting, 2017]

Deprivation of oxygen (hypoxia) followed by reoxygenation (H/R stress) is a major component in several pathological conditions such as vascular inflammation, myocardial ischemia, and stroke. However how animals adapt and recover from H/R stress remains an open question. Previous studies showed that the neuroglobin GLB-5(Haw) is essential for the fast recovery of the nematode Caenorhabditis elegans (C. elegans) from H/R stress. Here, we characterize the changes in neuronal gene expression during the adaptation of worms to hypoxia and recovery from H/R stress. Our analysis shows that innate immunity genes are differentially expressed during both adaptation to hypoxia and recovery from reoxygenation stress. Moreover, we reveal that the prolyl hydroxylase EGL-9, a known regulator of both adaptation to hypoxia and the innate immune response, inhibits the fast recovery from H/R stress through its activity in the O2-sensing neurons AQR, PQR, and URX. Finally, we show that GLB-5(Haw) acts in AQR, PQR, and URX to increase the tolerance of worms to bacterial pathogenesis. Together, our studies suggest that innate immunity and recovery from H/R stress are regulated by overlapping signaling pathways.

A V Bicknell et al. (2002) European Worm Meeting "The C. elegans F47F2.1 gene encodes a cyclic AMP-dependent protein kinase (PK-A) catalytic subunit"

A V Bicknell, M J Fisher, M Tabish, R A Clegg, H H Rees

[European Worm Meeting, 2002]

PK-A mediates all known effects of cyclic AMP on cellular activity in eukaryotes. The holoenzyme is an inactive tetramer of two regulatory (R) subunits and two catalytic (C) subunits. Following binding of cyclic AMP to the R subunits, dissociation of active C-subunits occurs. In mammals, , and isoforms of C-subunit, encoded by different genes, have been identified.

Angelo RG et al. (1997) International C. elegans Meeting "DISCOVERY OF A POTENTIAL ANCHOR/SCAFFOLD PROTEIN FOR C. ELEGANS PROTEIN KINASE A (PKA)"

Angelo RG, Rubin CS

[International C. elegans Meeting, 1997]

C. elegans PKA is composed exclusively of catalytic and regulatory (R) subunits encoded by the kin-1 and kin-2 genes. Since C. elegans lacks other PKA isoforms, this enzyme must disseminate signals carried by cAMP to all cell compartments. Approximately 60% of total R (PKA) is in the particulate fraction of disrupted C. elegans, indicating that protein/protein interactions may diversify PKA signaling by anchoring the kinase at specific intracellular locations. Co-localization of PKA with upstream activators and/or downstream effectors can create target sites for cAMP action. To understand the mechanism of PKA localization in C. elegans, a cDNA expression library was screened with recombinant radiolabeled-R (0.5 nM) to obtain high-affinity binding proteins. A cDNA encoding a novel, 143 kDa A kinase anchor protein (AKAP1) was retrieved and sequenced. The corresponding gene (rap-1) is located in LGII and contains 17 exons. AKAP1 is an acidic protein (pI=4.4) that is unrelated to previously characterized proteins. C. elegans R is avidly bound by both soluble and immobilized fragments of AKAP1. Competition binding studies indicate that C. elegans R is a preferred ligand, whereas mammalian RII and RI isoforms are only weakly sequestered. Scatchard analysis yielded a Kd value of ~10 nM for the R/AKAP1 complex. Deletion mutagenesis, coupled with protein expression in E. coli and in vitro assays, demonstrated that residues 235 to 255 govern high-affinity binding of R by AKAP1. A hydrophobic surface generated by amino acids with branched aliphatic side chains may be a key determinant of R binding activity. Site directed mutagenesis will pinpoint essential residues involved in PKA binding. Studies aimed at identifying the AKAP1 binding site on R are also in progress. High-affinity anti-AKAP1 IgGs are being used to determine (a) whether AKAP1 expression is developmentally-regulated and cell- specific and (b) the relationship between R (PKA) and AKAP1 in vivo.

Hicks, Tara et al. (2021) International Worm Meeting "R-loop-induced irreparable DNA damage in C. elegans meiosis"

Hicks, Tara, Koury, Emily, Williams, Cameron, Smolikove, Sarit

[International Worm Meeting, 2021]

Most DNA-RNA hybrids are formed naturally during transcription and are composed of a nascent RNA strand hybridized to DNA as part of R-loops. The accumulation of these structures in S-phase can result in replication-transcription conflict, an outcome which can lead to the formation of double strand breaks (DSBs). While R-loops' role in mitotically dividing cells has been characterized, there are only a handful of studies describing the effect of R-loops in meiosis and these studies present a complex picture of the outcome of R-loop formation on germ cells. Here we show that DSBs formed by R-loops trigger an altered cellular response to DNA damage. RNase H is an enzyme responsible for degradation of the RNA strand in DNA-RNA hybrids and plays an essential role in preventing this outcome and its deleterious consequences. Using null mutants for the two Caenorhabditis elegans genes encoding for RNase H1 and RNase H2 (hereby rnh mutants), our studies explore the effects of replication stress-induced DNA-RNA hybrid accumulation on meiosis. As expected, rnh mutants exhibit an increase in R-loop formation. Consequently, an elevation of DSBs in germline nuclei is evidenced by the accumulation of RAD-51 foci. Despite no repair mechanism abrogation, rnh mutants fail to repair all DSBs generated, leading to a fragmentation of chromosomes in diakinesis oocytes. By combining our double mutant with a spo-11 null mutation, we show that although replicative defects are the main contributor to the phenotype, R-loops formed in meiosis are likely contributors as well. We present evidence that while rnh mutants accumulate DNA-RNA hybrids and subsequent DSBs may signal a degree of checkpoint activation in mitosis, some damaged nuclei prevail past the checkpoint, enter into meiosis, and remain unrepaired throughout. Moreover, we find no evidence of an increase in apoptosis, which indicates that DNA damage generated by R-loops remain undetected by an apoptotic checkpoint. This data altogether points to DSBs initiated by R-loops representing an irreparable type of DNA damage that evades cellular machineries designed for damage recognition.

Birsoy B et al. (2010) Biology of the C. elegans Male, Madison, WI "Novel Left-Right Asymmetries of Male C.elegans"

Rothman J H, Choi S, Birsoy B, Downes J C, Bloss T A

[Biology of the C. elegans Male, Madison, WI, 2010]

While the mechanism that breaks left-right (L-R) anatomical asymmetry has been described in C. elegans, we have found that at least one additional mechanism must exist to generate L-R differences in the deployment of alternative cell death pathways in the male tail, male mating behavior and patterning of the male gonad. Upon recognizing a hermaphrodite, males initiate backward locomotion and continuously scan along the hermaphrodite's body. When their tails reach either the head or tail of the hermaphrodite, males turn to maintain contact and then continue backward locomotion on the opposite side of the hermaphrodite. We found that this turning behavior shows a distinct right-handed bias: when individual males were allowed to mate with lin-2(e1309) vulvaless hermaphrodites, >70% of the turns when made over the hermaphrodites' body (away from the agar surface) and >55% of turns when made under the hermaphrodites' body (toward the agar surface) are right-handed. To determine whether this bias in turning direction correlated with L-R asymmetry in the male mating structure, which results from stochastic EGL-1-dependent loss of sensory rays, we examined the turning bias in egl-1(n1084n3082) mutants. Wild-type males lack rays more frequently on the right and egl-1 mutant males have no ray loss but still display the right-hand turning bias. To assess whether this L-R behavioral bias is dependent on anatomical asymmetry, we analyzed gpa-16(it143) mutants in which the L-R asymmetry of the internal organs is reversed as a result of the reversal in the early embryonic symmetry break. Males with reversed anatomical asymmetry showed a virtually identical right hand bias in turning, demonstrating that an asymmetry-determining system that is independent of the previously described L-R symmetry breaking event must control this behavior. During the characterization of the gonad reversals of males, we also observed a previously unreported temperature sensitive reversal of L-R asymmetry of the male gonads in N2 (Bristol) and a Hawaiian wild isolate. We will describe our recent findings on these novel L-R asymmetries of the C.elegans male anatomy and behavior.

L C Bowen et al. (2002) European Worm Meeting "The C. elegans cyclic AMP-dependent protein kinase (PK-A) catalytic subunit gene (kin-1)"

L C Bowen, M Tabish, M J Fisher, H H Rees, R A Clegg

[European Worm Meeting, 2002]

PK-A is one of the major multifunctional ser/thr protein kinases found in eukaryotic organisms. The holoenzyme is comprised of two catalytic (C) and two regulatory (R) subunits. Both R and C subunits occur in multiple isoforms encoded by distinct genes. In many organisms, the genes that encode the C subunits also show alternative splicing behaviour. This generates an even broader array of C subunit heterogeneity. The diversity of C subunit polypeptides may enable this protein kinase to selectively target substrate polypeptides for phosphorylation.