Demarco R S et al. (2010) Neuronal Development, Synaptic Function and Behavior, Madison, WI "An Axon’s Journey to Find Its Path – How TIAM-1, a GEF for Rac GTPases, Controls Axon Pathfinding Downstream of CDC-42"

Demarco R S, Lundquist E A

[Neuronal Development, Synaptic Function and Behavior, Madison, WI, 2010]

In the developing nervous system, neurons extend axonal processes in order to communicate with other cells. When extending its axons, a neuron forms a dynamic, actin-rich growth cone that senses the surrounding environment for the proper migration cues through lamellipodial and filopodial structures.Racs are small GTPases from the Rho sub-family that are involved in the regulation of actin-binding proteins that can modulate the actin cytoskeleton. Racs are active when bound to GTP, and inactive when bound to GDP. GEFs - guanosine exchange factors - are molecules that aid Racs in the exchange of GDP for GTP, hence activating them. UNC-73/Trio is a well characterized GEF for the two redundant Racs CED-10/Rac1 and MIG-2/RhoG. However, a loss-of-function (lof) double mutant of mig-2 and ced-10 had more severe pathfinding defects than a lof of unc-73. When screening through the other 18 Double-Homology (DH) containing GEFs in the genome, a lof of tiam-1 displayed axon pathfinding defects when in combination with mig-2 or ced-10. TIAM-1 is a GEF for Rac1 in mammals, and has been implicated in vitro in working downstream of Cdc-42. In order to test if TIAM-1 is indeed working upstream of the Racs, a constitutively activated (CA) form of this molecule was created (with its DH-PH domains amplified and membrane tagged) and crossed into the lof Racs. Both lof mig-2 and ced-10 were able to suppress the ectopic lamellipodia caused by tiam-1(CA). To verify in vivo if TIAM-1 is downstream of CDC-42, we crossed tiam-1(lof) into a CA form of CDC-42 that causes neuronal developmental defects. tiam-1(lof) was able to significantly suppress such defects. In addition, tiam-1(lof) does not add in the number of defects caused by cdc-42(lof).We also sought to determine biochemically if TIAM-1 mechanistically works as a GEF for the Racs. We have generated purified TIAM-1(DH-PH) protein and performed a mant-GTP based GEF assay to measure the specificity of TIAM-1. Our results show TIAM-1 is specific for Rac but not Cdc-42 nor Rho. Our data suggest that in axon pathfinding, the previously uncharacterized GEF TIAM-1 works upstream of the Rac GTPases. Moreover, we have good evidence TIAM-1 could be the link between the CDC-42 and Rac GTPase pathways. Our next step in understanding the role of TIAM-1 in axon pathfinding is to determine of which receptor TIAM-1 is working downstream. Preliminary data suggest TIAM-1 works downstream of UNC-40 in ventral axon guidance. Future studies with guidance receptors will further dissect the role of TIAM-1 in axon pathfinding.

Galindo-Malagn XA et al. (2022) Zootaxa "New species, synonymies and records in the genus Rhagovelia Mayr, 1865 (Hemiptera: Heteroptera: Veliidae) from Colombia."

Moreira FFF, Morales I, Mondragn-F SP, Galindo-Malagn XA

[Zootaxa, 2022]

Rhagovelia medinae sp. nov., of the hambletoni group (angustipes complex), and R. utria sp. nov., of the hirtipes group (robusta complex), are described, illustrated, and compared with similar congeners. Based on the examination of type specimens, six new synonymies are proposed: R. elegans Uhler, 1894 = R. pediformis Padilla-Gil, 2010, syn. nov.; R. cauca Polhemus, 1997 = R. azulita Padilla-Gil, 2009, syn. nov., R. huila Padilla-Gil, 2009, syn. nov., R. oporapa Padilla-Gil, 2009, syn. nov, R. quilichaensis Padilla-Gil, 2011, syn. nov.; and R. gaigei, Drake Hussey, 1947 = R. victoria Padilla-Gil, 2012 syn. nov. The first record from Colombia is presented for R. trailii (White, 1879), and the distributions of the following species are extended in the country: R. cali Polhemus, 1997, R. castanea Gould, 1931, R. cauca Polhemus, 1997, R. gaigei Drake Hussey, 1957, R. elegans Uhler, 1894, R. femoralis Champion, 1898, R. malkini Polhemus, 1997, R. perija Polhemus, 1997, R. sinuata Gould, 1931, R. venezuelana Polhemus, 1997, R. williamsi Gould, 1931, and R. zeteki Drake, 1953.

McGurk P D et al. (2010) Neuronal Development, Synaptic Function and Behavior, Madison, WI "Protein Kinase C Signaling Acts With Rac GTPase Signaling in Axon Pathfinding"

Demarco R S, Lundquist E A, McGurk P D

[Neuronal Development, Synaptic Function and Behavior, Madison, WI, 2010]

The Rac GTPases CED-10/Rac and MIG-2/RhoG define parallel, redundant pathways that control axon pathfinding. During neuron development, actin-modulating proteins in the axonal growth cone rearrange the cytoskeleton to create projections that guide the axon to its target. The Arp2/3 complex and UNC-115/abLIM are actin-modulating proteins that act downstream of CED-10/Rac in this process. A two-hybrid screen with UNC-115 identified the Receptor for Activated C Kinase protein (RACK-1). In other systems, RACK proteins interact with protein kinase C (PKC) in diverse processes. To determine if PKC signaling acts with UNC-115/abLIM and Rac GTPases in axon pathfinding, we assayed axon pathfinding in pkc mutant backgrounds.C. elegans has four orthologs of protein kinase C – PKC-1, PKC-2, PKC-3, and TPA-1. We observed that mutations in each of the pkc genes caused variable defects in PDE axon pathfinding that were synergistically enhanced by mig-2. In contrast, ced-10; pkc double mutants did not display synergistic enhancement. These data suggest that PKC signaling might act in the CED-10/Rac and UNC-115/abLIM pathway in parallel to MIG-2/RhoG. Future studies will test the idea that CED-10/Rac regulates UNC-115/abLIM through RACK-1 and PKC signaling.

Wightman BC et al. (1997) International C. elegans Meeting "FAX-1, A NUCLEAR HORMONE RECEPTOR REQUIRED FOR AXON PATHFINDING"

Wightman BC, Garriga G, Kalola C, Slade DJ, DeMarco R

[International C. elegans Meeting, 1997]

We are identifying molecules that function in the guidance of neuron growth cones along particular axonal pathways. Mutations in the fax-1 (defective fasciculation of axons) gene cause disruptions in the pathfinding of growth cones along the ventral nerve cord (VNC). The AVKR, HSNL and PVQL growth cones normally extend along the left VNC bundle. In fax-1 mutants these three growth cones are unable to recognize the left VNC bundle pathway, and extend inappropriately along the right VNC bundle. This defect is not a secondary consequence of faulty pioneering of the left VNC bundle, because the pioneer axon PVPR is present and appears normal. In order to understand the role of fax-1 in directing growth cones, we cloned the gene. Genetic mapping localized fax-1 to a region of the X chromosome between unc-20 and unc-78. Transformation rescue experiments demonstrated that fax-1 resides on cosmid F56E3 and encodes a protein that bears a strong similarity to the retinoic acid receptor super-family of Zn-finger transcription factors. Therefore, fax-1 may regulate the transcription of genes that mediate axon pathfinding. We are building GFP reporter fusions to determine in which neurons fax-1 may function

Lu WX et al. (1990) J Biol Chem "Cloning, structure, and expression of the gene for a novel regulatory subunit of cAMP-dependent protein kinase in Caenorhabditis elegans."

Bagchi S, Lu WX, Rubin CS, Gross RE

[J Biol Chem, 1990]

The nematode Caenorhabditis elegans (C. elegans) expresses the regulatory subunit (R) of cAMP-dependent protein kinase at a level similar to the levels determined for R subunits in mammalian tissues. Approximately 60% of the C. elegans cAMP-binding protein is tightly associated with particulate structures by noncovalent interactions. Ionic detergents or 7 M urea solubilize particulate R. Solubilized and cytosolic R subunits have apparent Mr values of 52,000 and pI values of 5.5. cDNA and genomic DNA encoding a unique C. elegans R subunit were cloned and sequenced. The derived amino acid sequence contains 375 residues; carboxyl-terminal residues 145-375 are 69% identical with mammalian RI. However, residues 44-145 are markedly divergent from the corresponding regions of all other R sequences. This region might provide sufficient structural diversity to adapt a single R subunit for multiple functional roles in C. elegans. Antibodies directed against two epitopes in the deduced amino acid sequence of C. elegans R avidly bound nematode cytosolic and particulate R subunits on Western blots and precipitated dissociated R subunits and R2C2 complexes from solution. Immunofluorescence analysis revealed that the tip of the head, which contains chemosensory and mechanosensory neurons, and the pharyngeal nerve ring were enriched in R. The R subunit concentration is low during early embryogenesis in C. elegans. A sharp increase (approximately 6-fold) in R content begins several hours before the nematodes hatch and peaks during the first larval stage. Developmental regulation of R expression occurs at translational and/or post-translational levels. The 8-kilobase pair C. elegans R gene is divided into 8 exons by introns ranging from 46 to 4300 base pairs. The 5'-flanking region has no TATA box and contains preferred and minor transcription start sites.

Winge P et al. (1994) Worm Breeder's Gazette "R-ras 1 and R-ras 2 (TC21) Homologs."

Gobel V, Friend S, Winge P, Fleming JT

[Worm Breeder's Gazette, 1994]

R-ras I and R-ras 2 (TC21) homologs Per Winge*, Vercna Gobel*+, Stephen Friend*, and John Fleming*+. MGH Cancer Center and +DepL of Pediatrics, Boston, MA. Human r-ras 1 and r-ras 2 (TC21) belong to the closer relatives (>50% amino acid identity) of ras in the ras superfamily of GDP/GTP-binding proteins. They are the first members to exhibit transforming potential when mutated at some which render ras oncogenic and make it insensitive to GAP action (Graham & Der, 1994). These recent findings have led to current investigations of their role-in human cancer. Furthermore, r-ras 1 -- by immunoprecipitation and in the yeast-2-hybrid-system -- was shown to interact with bc1-2, the human homolog to ced-9 (Fernandez-Sarabia & Bischoff, 1993) and has thus been implicated as a possible effector of apoptosis. There is evidence that the r-ras proteins participate in some but not all aspects of the ras signal transduction pathway involving upstream tyrosinc kinases and downstream serine/threonine kinases. It has not yet been elucidated in the mammalian system (1) what alternative pathway the r-ras proteins may be utilizing and (2) what functional relevance is represented by the in vitro interaction of r-ras 1 and bc1-2. We are trying to address these questions in C elegans and have cloned the homologs of r-ras I and r-ras 2 using a degeneratc PCR approach. We have screened c-DNA and genomic libraries and obtamed and sequenced full length c-DNA and genomic clones of r-ras 1 and a full length c-DNA clone of r- ras 2. The genomic sequence of r-ras 2 was recently made available by the genome sequencing project. The amino acid comparison shows high homologyrldentity to thc human proteins for r-ras 1 and r-ras 2 (TC21). R-ras 1 was localizcd to chromosome II ncar lin-29, and r-ras 2 maps close to embS on chromosome m. To obtain r-ras germline deletions, we have screened a TCl insertion library which we constructed using the mutator strain MT 3126 (protocols kindly proYided by Jocl Rothman, Susan Mango and Ed Maryon), and have isolated transposon insertions in r-ras 1. We are currently in the proccss of sib sclection to purify the strains. To get some first appreciation of a functional role of r-ras towards apoptosis versus growth stimulating propertics, we have also started to inject a r-ras 1 hcat shock promotor expression construct to generatc strains in which r-ras can be overexpressed Ihis additional approach has been choscn since redundancy may be expected in thc ras related protcin familics and thus thc knockout of one of the proteins may not give clear results. We will screen the overexpressing strains for (1) apoptosis and (2) muv phcnotype. In collaboration with Bob Horvitz's laboratory r-ras GST fusion proteins will be generated to test the in vitro interacion with ccd-9. Finally, we are constructing r-ras 1 and r-ras 2 promotor expression vectors with GFP/betaGAL to define the expression patterns of both genes.

Wang B et al. (2021) Nat Commun "Pseudomonas aeruginosa PA14 produces R-bodies, extendable protein polymers with roles in host colonization and virulence."

Jo J, Wang B, Price-Whelan A, Brown LM, Sieben C, Vasquez-Rifo A, Lin YC, McDonald ST, Dietrich LEP

[Nat Commun, 2021]

R-bodies are long, extendable protein polymers formed in the cytoplasm of some bacteria; they are best known for their role in killing of paramecia by bacterial endosymbionts. Pseudomonas aeruginosa PA14, an opportunistic pathogen of diverse hosts, contains genes (referred to as the reb cluster) with potential to confer production of R-bodies and that have been implicated in virulence. Here, we show that products of the PA14 reb cluster associate with R-bodies and control stochastic expression of R-body structural genes.PA14 expresses reb genes during colonization of plant and nematode hosts, and R-body production is required for full virulence in nematodes. Analyses of nematode ribosome content and immune response indicate that P. aeruginosa R-bodies act via a mechanism involving ribosome cleavage and translational inhibition. Our observations provide insight into the biology of R-body production and its consequences during P. aeruginosa infection.

Buyannemekh K et al. (2024) Dev Biol "Left/right asymmetrically expressed ephrin and Flamingo proteins regulate lateralized axon growth in C. elegans."

Buyannemekh K, Bertrand V, Villoutreix P

[Dev Biol, 2024]

While the nervous system of bilaterian animals is mainly left-right (L-R) symmetric at the anatomical level, some molecular and functional L-R asymmetries exist. However, the extent of these molecular asymmetries and their functional consequences remain poorly characterized. C. elegans allows to study L-R asymmetries in the nervous system with single-neuron resolution. We have previously shown that a neural bHLH transcription factor, HLH-16/Olig, is L-R asymmetrically expressed in the AIY neuron lineage and regulates AIY axon projections in a L-R asymmetric manner. Here, by combining a candidate approach and single-cell RNA sequencing data analysis, we identify the ephrin protein EFN-2 and the Flamingo protein FMI-1 as downstream targets of HLH-16 that are L-R asymmetrically expressed in the AIY lineage. We show that EFN-2 and FMI-1 collaborate in the L-R asymmetric regulation of axonal growth. EFN-2 may act via a non-canonical receptor of the L1CAM family, SAX-7. Our study reveals novel molecular L-R asymmetries in the C. elegans nervous system and their functional consequences.

Pohl C (2011) Commun Integr Biol "Left-right patterning in the C. elegans embryo: Unique mechanisms and common principles."

Pohl C

[Commun Integr Biol, 2011]

The development of bilateral symmetry during the evolution of species probably 600 million years ago brought about several important innovations: It fostered efficient locomotion, streamlining and favored the development of a central nervous system through cephalization. However, to increase their functional capacities, many organisms exhibit chirality by breaking their superficial left-right (l-r) symmetry, which manifests in the lateralization of the nervous system or the l-r asymmetry of internal organs. In most bilateria, the mechanisms that maintain consistent l-r asymmetry throughout development are poorly understood. This review highlights insights into mechanisms that couple early embryonic l-r symmetry breaking to subsequent l-r patterning in the roundworm Caenorhabditis elegans. A recently identified strategy for l-r patterning in the early C. elegans embryo is discussed, the spatial separation of midline and anteroposterior axis, which relies on a rotational cellular rearrangement and non-canonical Wnt signaling. Evidence for a general relevance of rotational/torsional rearrangements during organismal l-r patterning and for non-canonical Wnt signaling/planar cell polarity as a common signaling mechanism to maintain l-r asymmetry is presented.

Vinci CR et al. (2007) J Biol Chem "Recognition of age-damaged (R,S)-adenosyl-L-methionine by two methyltransferases in the yeast Saccharomyces cerevisiae."

Vinci CR, Clarke SG

[J Biol Chem, 2007]

The biological methyl donor, S adenosylmethionine (AdoMet), can exist in two diastereoisomeric states with respect to its sulfonium ion. The "S" configuration, (S,S)AdoMet, is the only form that is produced enzymatically as well as the only form used in almost all biological methylation reactions. Under physiological conditions, however, the sulfonium ion can spontaneously racemize to the "R" form, producing (R,S)AdoMet. As of yet, (R,S)AdoMet has no known physiological function and may inhibit cellular reactions. In this study, two enzymes have been found in Saccharomyces cerevisiae that are capable of recognizing (R,S)AdoMet and using it to methylate homocysteine to form methionine. These enzymes are the products of the SAM4 and MHT1 genes, previously identified as homocysteine methyltransferases dependent upon AdoMet and S-methylmethionine respectively. We find here that Sam4 recognizes both (S,S) and (R,S)AdoMet, but its activity is much higher with the R,S form. Mht1 reacts with only the R,S form of AdoMet while no activity is seen with the S,S form. R,S-specific homocysteine methyltransferase activity is also shown here to occur in extracts of Arabidopsis thaliana, Drosophila melanogaster, and Caenorhabditis elegans, but has not been detected in several tissue extracts of Mus musculus. Such activity may function to prevent the accumulation of (R,S)AdoMet in these organisms.