The Experts below are selected from a list of 51993 Experts worldwide ranked by ideXlab platform
Joseph G. Culotti - One of the best experts on this subject based on the ideXlab platform.
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The Wnt Frizzled Receptor MOM-5 Regulates the Unc-5 Netrin Receptor through Small GTPase-Dependent Signaling to Determine the Polarity of Migrating Cells
PLOS Genetics, 2015Co-Authors: Naomi Levy-strumpf, Meghan Krizus, Hong Zheng, Louise Brown, Joseph G. CulottiAbstract:Wnt and Netrin signaling regulate diverse essential functions. Using a genetic approach combined with temporal gene expression analysis, we found a regulatory link between the Wnt receptor MOM-5/Frizzled and the UNC-6/Netrin receptor Unc-5. These two receptors play key roles in guiding cell and axon migrations, including the migration of the C. elegans Distal Tip Cells (DTCs). DTCs migrate post-embryonically in three sequential phases: in the first phase along the Antero-Posterior (A/P) axis, in the second, along the Dorso-Ventral (D/V) axis, and in the third, along the A/P axis. Loss of MOM-5/Frizzled function causes third phase A/P polarity reversals of the migrating DTCs. We show that an over-expression of Unc-5 causes similar DTC A/P polarity reversals and that Unc-5 deficits markedly suppress the A/P polarity reversals caused by mutations in mom-5/frizzled. This implicates MOM-5/Frizzled as a negative regulator of Unc-5. We provide further evidence that small GTPases mediate MOM-5’s regulation of Unc-5 such that one outcome of impaired function of small GTPases like CED-10/Rac and MIG-2/RhoG is an increase in Unc-5 function. The work presented here demonstrates the existence of cross talk between components of the Netrin and Wnt signaling pathways and provides further insights into the way guidance signaling mechanisms are integrated to orchestrate directed cell migration.
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UNC-129 regulates the balance between UNC-40 dependent and independent Unc-5 signaling pathways
Nature Neuroscience, 2009Co-Authors: Lesley T Macneil, W Rod Hardy, Tony Pawson, Jeffrey L Wrana, Joseph G. CulottiAbstract:The Unc-5 receptor mediates axon repulsion from UNC-6/netrin through UNC-40 dependent (Unc-5 + UNC-40) and independent (Unc-5 alone) signaling pathways. It has been shown that UNC-40–dependent signaling is required for long-range repulsion of UNC-6/netrin; however, the mechanisms used to regulate distinct Unc-5 signaling pathways are poorly understood. We found that the C. elegans transforming growth factor β (TGF-β) family ligand UNC-129, graded opposite to UNC-6/netrin, functions independent of the canonical TGF-β receptors to regulate Unc-5 cellular responses. Our observations indicates that UNC-129 facilitates long-range repulsive guidance of UNC-6 by enhancing Unc-5 + UNC-40 signaling at the expense of Unc-5 alone signaling through interaction with the Unc-5 receptor. This increases the set point sensitivity of growth cones to UNC-6/netrin as they simultaneously migrated up the UNC-129 gradient and down the UNC-6 gradient. Similar regulatory interactions between oppositely graded extracellular cues may be a common theme in guided cell and axon migrations. In the worm, netrin and the TGFβ-related molecule UNC-129 form opposing dorsoventral gradients. Motor axons are repelled by netrin and attracted to UNC-129, but no TGFβ receptors appear to be involved in the attraction. This study shows that UNC-129 enhances repulsive signaling from the netrin receptor complex Unc-5/UNC-40, via a direct interaction with Unc-5.
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C. elegans seu-1 encodes novel nuclear proteins that regulate responses to UNC-6/netrin guidance cues.
Developmental biology, 2007Co-Authors: Hong Zheng, Joseph G. Culotti, Antonio Colavita, Ludivine Coudiere, Cheryl Camia, David C MerzAbstract:In C. elegans, ectopic expression of the Unc-5 netrin receptor is sufficient to cause repulsion of growth cones and cells away from ventral sources of the UNC-6/netrin guidance cue. A genetic suppressor screen identified the seu-1 gene as required for repulsion of touch neuron growth cones ectopically expressing Unc-5. We report here that seu-1 mutations also enhance the frequency of distal tip cell migrations of Unc-5 or unc-40 mutants. The seu-1 gene encodes two novel proteins (SEU-1A and SEU-1B) containing a charged central domain and several regions of low amino acid complexity. Transgenic rescue experiments indicate that seu-1 can act cell autonomously in the touch neurons and distal tip cells and that SEU-1 function requires both the SEU-1A and SEU-1B isoforms. A GFP fusion construct was expressed in a dynamic pattern throughout development and localized in the nuclei of neuronal and non-neuronal cells, including gonadal leader cells. These results implicate nuclear SEU-1 in the interpretation of UNC-6/netrin directional information by migrating growth cones and cells.
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Unc-5 Function Requires Phosphorylation of Cytoplasmic Tyrosine 482, but Its UNC-40-Independent Functions also Require a Region between the ZU-5 and Death Domains
Developmental biology, 2002Co-Authors: Marie T. Killeen, Tony Pawson, Aldis Krizus, Jeifei Tong, Robert Steven, Ian Richard Scott, Joseph G. CulottiAbstract:Members of the Unc-5 protein family are transmembrane receptors for UNC-6/netrin guidance cues. To analyze the functional roles of different Unc-5 domains, we sequenced mutations in seven severe and three weak alleles of Unc-5 in Caenorhabditis elegans. Four severe alleles contain nonsense mutations. Two weak alleles are truncations of the cytodomain, but one is a missense mutation in an extracellular immunoglobulin domain. To survey the function of different regions of Unc-5, wild-type and mutant Unc-5::HA transgenes were tested for their ability to rescue the Unc-5(e53) null mutant. Our data reveal partial functional requirements for the extracellular domains and identify a portion of the cytoplasmic juxtamembrane (JM) region as essential for rescue of migrations. When nine cytodomain tyrosines, including seven in the JM region, are mutated to phenylalanine, Unc-5 function and tyrosine phosphorylation are largely compromised. When F482 in the JM region of the mutant protein is reverted to tyrosine, Unc-5 tyrosine phosphorylation and in vivo function are largely recovered, suggesting that Y482 phosphorylation is critical to Unc-5 function in vivo. Our data also show that part of the ZU-5 motif is required for UNC-40-independent signaling of Unc-5.
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Netrin Stimulates Tyrosine Phosphorylation of the Unc-5 Family of Netrin Receptors and Induces Shp2 Binding to the RCM Cytodomain
Journal of Biological Chemistry, 2001Co-Authors: Jiefei Tong, Joseph G. Culotti, Marie T. Killeen, Robert Steven, Kathleen L. Binns, Tony PawsonAbstract:Abstract Caenorhabditis elegans Unc-5 and its mammalian homologues such as RCM are receptors for the secreted axon guidance cue UNC-6/netrin and are required to mediate the repulsive effects of UNC-6/netrin on growth cones. We find that C. elegans Unc-5 and mouse RCM are phosphorylated on tyrosinein vivo. C. elegans Unc-5 tyrosine phosphorylation is reduced in unc-6 null mutants, and RCM tyrosine phosphorylation is induced by netrin-1 in transfected HEK-293 cells, demonstrating that phosphorylation of Unc-5 proteins is enhanced by UNC-6/netrin stimulation in both worms and mammalian cells. An activated Src tyrosine kinase induces phosphorylation of RCM at multiple cytoplasmic tyrosine residues creating potential binding sites for cytoplasmic signaling proteins. Indeed, the NH2-terminal SH2 domain of the Shp2 tyrosine phosphatase bound specifically to a Tyr568 RCM phosphopeptide. Furthermore, Shp2 associated with RCM in a netrin-dependent manner in transfected cells, and co-immunoprecipitated with RCM from an embryonic mouse brain lysate. A Y568F mutant RCM receptor failed to bind Shp2 and was more highly phosphorylated on tyrosine than the wild type receptor. These results suggest that netrin-stimulated phosphorylation of RCM Tyr568 recruits Shp2 to the cell membrane where it can potentially modify RCM phosphorylation and function.
Yishi Jin - One of the best experts on this subject based on the ideXlab platform.
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the jip3 scaffold protein unc 16 regulates rab 5 dependent membrane trafficking at c elegans synapses
Developmental Neurobiology, 2009Co-Authors: Heather M Brown, Alexandr Goncharov, Barth D Grant, Yishi Jin, Heather Van EppsAbstract:How endosomes contribute to the maintenance of vesicular structures at presynaptic terminals remains controversial and poorly understood. Here, we have investigated synaptic endosomal compartments in the presynaptic terminals of C. elegans GABAergic motor neurons. Using RAB reporters, we find that several subsynaptic compartments reside in, or near, presynaptic regions. Loss of function in the C. elegans JIP3 protein, UNC-16, causes a RAB-5-containing compartment to accumulate abnormally at presynaptic terminals. Ultrastructural analysis shows that synapses in unc-16 mutants contain reduced number of synaptic vesicles, accompanied by an increase in the size and number of cisternae. FRAP analysis revealed a slow recovery of RAB-5 in unc-16 mutants, suggestive of an impairment of RAB-5 activity state and local vesicular trafficking. Overexpression of RAB-5:GDP partially suppresses, whereas overexpression of RAB-5:GTP enhances, the synaptic defects of unc-16 mutants. Our data demonstrate a novel function of UNC-16 in the regulation of synaptic membrane trafficking and suggest that the synaptic RAB-5 compartment contributes to synaptic vesicle biogenesis or maintenance.
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MAX-1, a novel PH/MyTH4/FERM domain cytoplasmic protein implicated in netrin-mediated axon repulsion.
Neuron, 2002Co-Authors: Xun Huang, Marc Tessier-lavigne, Hwai-jong Cheng, Yishi JinAbstract:The netrin UNC-6 repels motor axons by activating the Unc-5 receptor alone or in combination with the UNC-40/DCC receptor. In a genetic screen for C. elegans mutants exhibiting partial defects in motor axon projections, we isolated the max-1 gene (required for motor neuron axon guidance). max-1 loss-of-function mutations cause fully penetrant but variable axon guidance defects. Mutations in Unc-5 and unc-6, but not in unc-40, dominantly enhance the mutant phenotypes of max-1, whereas overexpression of Unc-5 or unc-6, but not of unc-40, bypasses the requirement for max-1. MAX-1 proteins contain PH, MyTH4, and FERM domains and appear to be localized to neuronal processes. Human MAX-1 and UNC5H2 colocalize in discrete subcellular regions of transfected cells. Our results suggest a possible role for MAX-1 in netrin-induced axon repulsion by modulating the Unc-5 receptor signaling pathway.
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max 1 a novel ph myth4 ferm domain cytoplasmic protein implicated in netrin mediated axon repulsion
Neuron, 2002Co-Authors: Xun Huang, Hwai-jong Cheng, Marc Tessierlavigne, Yishi JinAbstract:The netrin UNC-6 repels motor axons by activating the Unc-5 receptor alone or in combination with the UNC-40/DCC receptor. In a genetic screen for C. elegans mutants exhibiting partial defects in motor axon projections, we isolated the max-1 gene (required for motor neuron axon guidance). max-1 loss-of-function mutations cause fully penetrant but variable axon guidance defects. Mutations in Unc-5 and unc-6, but not in unc-40, dominantly enhance the mutant phenotypes of max-1, whereas overexpression of Unc-5 or unc-6, but not of unc-40, bypasses the requirement for max-1. MAX-1 proteins contain PH, MyTH4, and FERM domains and appear to be localized to neuronal processes. Human MAX-1 and UNC5H2 colocalize in discrete subcellular regions of transfected cells. Our results suggest a possible role for MAX-1 in netrin-induced axon repulsion by modulating the Unc-5 receptor signaling pathway.
Marie T. Killeen - One of the best experts on this subject based on the ideXlab platform.
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The ENU-3 protein family members function in the Wnt pathway parallel to UNC-6/Netrin to promote motor neuron axon outgrowth in C. elegans.
Developmental biology, 2017Co-Authors: Roxana Oriana Florica, Victoria E. B. Hipolito, Stephen Bautista, Homa Anvari, Chloe Rapp, Suzan El-rass, Alimohammad Asgharian, Costin N. Antonescu, Marie T. KilleenAbstract:The axons of the DA and DB classes of motor neurons fail to reach the dorsal cord in the absence of the guidance cue UNC-6/Netrin or its receptor Unc-5 in C. elegans. However, the axonal processes usually exit their cell bodies in the ventral cord in the absence of both molecules. Strains lacking functional versions of UNC-6 or Unc-5 have a low level of DA and DB motor neuron axon outgrowth defects. We found that mutations in the genes for all six of the ENU-3 proteins function to enhance the outgrowth defects of the DA and DB axons in strains lacking either UNC-6 or Unc-5. A mutation in the gene for the MIG-14/Wntless protein also enhances defects in a strain lacking either Unc-5 or UNC-6, suggesting that the ENU-3 and Wnt pathways function parallel to the Netrin pathway in directing motor neuron axon outgrowth. Our evidence suggests that the ENU-3 proteins are novel members of the Wnt pathway in nematodes. Five of the six members of the ENU-3 family are predicted to be single-pass trans-membrane proteins. The expression pattern of ENU-3.1 was consistent with plasma membrane localization. One family member, ENU-3.6, lacks the predicted signal peptide and the membrane-spanning domain. In HeLa cells ENU-3.6 had a cytoplasmic localization and caused actin dependent processes to appear. We conclude that the ENU-3 family proteins function in a pathway parallel to the UNC-6/Netrin pathway for motor neuron axon outgrowth, most likely in the Wnt pathway.
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ENU-3 is a new player in UNC-6/Netrin directed axon outgrowth and guidance in C. elegans
Receptors and clinical investigation, 2014Co-Authors: Marie T. KilleenAbstract:UNC-6/Netrin is an axon guidance cue that works through UNC-40/DCC/Frazzled to attract cells and neurons and through Unc-5 and UNC-40 to repulse cells and neurons during nervous system development. The gene encoding the novel protein ENU-3 was identified in a mutant that enhanced the axon outgrowth defects of the DA and DB classes of motor neurons in a strain lacking functional Unc-5. Mutant ENU-3 also enhanced the axon outgrowth and guidance defects of the ventrally directed processes of the AVM and PVM touch receptor neurons in a strain lacking functional UNC-40. ENU-3 also plays a role in adhesion of axons to the substratum as does UNC-40. The protein is a putative single pass transmembrane protein expressed throughout the nervous system whose possible biochemical roles are currently unknown.
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enu 3 functions in an unc 6 netrin dependent pathway parallel to unc 40 dcc frazzled for outgrowth and guidance of the touch receptor neurons in c elegans
Developmental Dynamics, 2014Co-Authors: Callista Yee, Roxana Oriana Florica, Jeffrey Fillingham, Marie T. KilleenAbstract:Background: UNC-6 and SLT-1 guide the migrations of the ventrally directed processes of the AVM and PVM touch receptor neurons and UNC-6 guides the axons of the DA and DB classes of motor neurons in C. elegans. The UNC-6 receptors are Unc-5 and UNC-40. The axon outgrowth defects of a subset of the DB motor neurons in the absence of Unc-5 are enhanced by mutations in enu-3. Results: An enu-3 mutation enhances defects in ventral guidance of the processes of the AVM and PVM touch receptor neurons, the dorsal guidance of the distal tip cell and causes additional architectural defects in axons in unc-40 mutant strains in an UNC-6 dependent manner. These observations suggest that ENU-3 and UNC-40 function in parallel pathways dependent on UNC-6. ENU-3 depends on the presence of UNC-40 for its full effect on motor neuron axon outgrowth. Conclusions: ENU-3 works in an UNC-6 dependent pathway parallel to UNC-40 in ventral guidance of AVM and PVM and in dorsal guidance of the distal tip cells. Motor neuron axon outgrowth defects are caused by the presence of UNC-40 and the absence of functional Unc-5 or UNC-6 and defects are enhanced by the absence of functional ENU-3. Developmental Dynamics 243:459–467, 2014. © 2013 Wiley Periodicals, Inc.
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ENU-3 functions in an UNC-6/netrin dependent pathway parallel to UNC-40/DCC/frazzled for outgrowth and guidance of the touch receptor neurons in C. elegans.
Developmental dynamics : an official publication of the American Association of Anatomists, 2013Co-Authors: Callista Yee, Roxana Oriana Florica, Jeffrey Fillingham, Marie T. KilleenAbstract:Background: UNC-6 and SLT-1 guide the migrations of the ventrally directed processes of the AVM and PVM touch receptor neurons and UNC-6 guides the axons of the DA and DB classes of motor neurons in C. elegans. The UNC-6 receptors are Unc-5 and UNC-40. The axon outgrowth defects of a subset of the DB motor neurons in the absence of Unc-5 are enhanced by mutations in enu-3. Results: An enu-3 mutation enhances defects in ventral guidance of the processes of the AVM and PVM touch receptor neurons, the dorsal guidance of the distal tip cell and causes additional architectural defects in axons in unc-40 mutant strains in an UNC-6 dependent manner. These observations suggest that ENU-3 and UNC-40 function in parallel pathways dependent on UNC-6. ENU-3 depends on the presence of UNC-40 for its full effect on motor neuron axon outgrowth. Conclusions: ENU-3 works in an UNC-6 dependent pathway parallel to UNC-40 in ventral guidance of AVM and PVM and in dorsal guidance of the distal tip cells. Motor neuron axon outgrowth defects are caused by the presence of UNC-40 and the absence of functional Unc-5 or UNC-6 and defects are enhanced by the absence of functional ENU-3. Developmental Dynamics 243:459–467, 2014. © 2013 Wiley Periodicals, Inc.
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The dual role of the ligand UNC-6/Netrin in both axon guidance and synaptogenesis in C. elegans
Cell Adhesion & Migration, 2009Co-Authors: Marie T. KilleenAbstract:The extracellular cue UNC-6/Netrin is a well-known axon guidance molecule and recently it has also been shown to be involved with localization of pre-synaptic complexes. Via the UNC-40/DCC/Fra, UNC-6/Netrin promotes the formation of pre-synaptic terminals between the pre-synaptic AIY interneuron and its post-synaptic partner, the RIA interneuron. In the DA9 motor neuron, UNC-6/Netrin has an alternate role promoting the exclusion of pre-synaptic components from the dendrite via its Unc-5-receptor. Surprisingly, the requirement for Unc-5 persists even after DA9 axon migration is complete, because synapses become mis-localized after it is depleted. This observation provides at least a partial explanation for the persistence of UNC-6/Netrin and Unc-5 in the adult nervous system. These activities parallel the previously known bi-functional axon guidance effects of UNC-6/Netrin, since it can attract cells and axons expressing UNC-40/DCC/Fra and repel those expressing Unc-5 alone or in combination with UNC-40. U...
Alexandr Goncharov - One of the best experts on this subject based on the ideXlab platform.
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the jip3 scaffold protein unc 16 regulates rab 5 dependent membrane trafficking at c elegans synapses
Developmental Neurobiology, 2009Co-Authors: Heather M Brown, Heather Van Epps, Alexandr Goncharov, Barth D GrantAbstract:How endosomes contribute to the maintenance of vesicular structures at presynaptic terminals remains controversial and poorly understood. Here, we have investigated synaptic endosomal compartments in the presynaptic terminals of C. elegans GABAergic motor neurons. Using RAB reporters, we find that several subsynaptic compartments reside in, or near, presynaptic regions. Loss of function in the C. elegans JIP3 protein, UNC-16, causes a RAB-5-containing compartment to accumulate abnormally at presynaptic terminals. Ultrastructural analysis shows that synapses in unc-16 mutants contain reduced number of synaptic vesicles, accompanied by an increase in the size and number of cisternae. FRAP analysis revealed a slow recovery of RAB-5 in unc-16 mutants, suggestive of an impairment of RAB-5 activity state and local vesicular trafficking. Overexpression of RAB-5:GDP partially suppresses, whereas overexpression of RAB-5:GTP enhances, the synaptic defects of unc-16 mutants. Our data demonstrate a novel function of UNC-16 in the regulation of synaptic membrane trafficking and suggest that the synaptic RAB-5 compartment contributes to synaptic vesicle biogenesis or maintenance. © 2008 Wiley Periodicals, Inc. Develop Neurobiol, 2009.
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the jip3 scaffold protein unc 16 regulates rab 5 dependent membrane trafficking at c elegans synapses
Developmental Neurobiology, 2009Co-Authors: Heather M Brown, Alexandr Goncharov, Barth D Grant, Yishi Jin, Heather Van EppsAbstract:How endosomes contribute to the maintenance of vesicular structures at presynaptic terminals remains controversial and poorly understood. Here, we have investigated synaptic endosomal compartments in the presynaptic terminals of C. elegans GABAergic motor neurons. Using RAB reporters, we find that several subsynaptic compartments reside in, or near, presynaptic regions. Loss of function in the C. elegans JIP3 protein, UNC-16, causes a RAB-5-containing compartment to accumulate abnormally at presynaptic terminals. Ultrastructural analysis shows that synapses in unc-16 mutants contain reduced number of synaptic vesicles, accompanied by an increase in the size and number of cisternae. FRAP analysis revealed a slow recovery of RAB-5 in unc-16 mutants, suggestive of an impairment of RAB-5 activity state and local vesicular trafficking. Overexpression of RAB-5:GDP partially suppresses, whereas overexpression of RAB-5:GTP enhances, the synaptic defects of unc-16 mutants. Our data demonstrate a novel function of UNC-16 in the regulation of synaptic membrane trafficking and suggest that the synaptic RAB-5 compartment contributes to synaptic vesicle biogenesis or maintenance.
Erik A. Lundquist - One of the best experts on this subject based on the ideXlab platform.
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RHO-1 and the Rho GEF RHGF-1 interact with UNC-6/Netrin signaling to regulate growth cone protrusion and microtubule organization in Caenorhabditis elegans.
PLoS genetics, 2019Co-Authors: Mahekta R. Gujar, Aubrie M. Stricker, Erik A. LundquistAbstract:UNC-6/Netrin is a conserved axon guidance cue that directs growth cone migrations in the dorsal-ventral axis of C. elegans and in the vertebrate spinal cord. UNC-6/Netrin is expressed in ventral cells, and growth cones migrate ventrally toward or dorsally away from UNC-6/Netrin. Recent studies of growth cone behavior during outgrowth in vivo in C. elegans have led to a polarity/protrusion model in directed growth cone migration away from UNC-6/Netrin. In this model, UNC-6/Netrin first polarizes the growth cone via the Unc-5 receptor, leading to dorsally biased protrusion and F-actin accumulation. UNC-6/Netrin then regulates protrusion based on this polarity. The receptor UNC-40/DCC drives protrusion dorsally, away from the UNC-6/Netrin source, and the Unc-5 receptor inhibits protrusion ventrally, near the UNC-6/Netrin source, resulting in dorsal migration. Unc-5 inhibits protrusion in part by excluding microtubules from the growth cone, which are pro-protrusive. Here we report that the RHO-1/RhoA GTPase and its activator GEF RHGF-1 inhibit growth cone protrusion and MT accumulation in growth cones, similar to Unc-5. However, growth cone polarity of protrusion and F-actin were unaffected by RHO-1 and RHGF-1. Thus, RHO-1 signaling acts specifically as a negative regulator of protrusion and MT accumulation, and not polarity. Genetic interactions are consistent with RHO-1 and RHGF-1 acting with Unc-5, as well as with a parallel pathway, to regulate protrusion. The cytoskeletal interacting molecule UNC-33/CRMP was required for RHO-1 activity to inhibit MT accumulation, suggesting that UNC-33/CRMP might act downstream of RHO-1. In sum, these studies describe a new role of RHO-1 and RHGF-1 in regulation of growth cone protrusion by UNC-6/Netrin.
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RHO-1 and the Rho GEF RHGF-1 interact with UNC-6/Netrin signaling to regulate growth cone protrusion and microtubule organization in C. elegans
2019Co-Authors: Mahekta R. Gujar, Aubrie M. Stricker, Erik A. LundquistAbstract:UNC-6/Netrin is a conserved axon guidance cue that directs growth cone migrations in the dorsal-ventral axis of C. elegans and in the vertebrate spinal cord. UNC-6/Netrin is expressed in ventral cells, and growth cones migrate ventrally toward or dorsally away from UNC-6/Netrin. Recent studies of growth cone behavior during outgrowth in vivo in C. elegans have led to a polarity/protrusion model in directed growth cone migration away from UNC-6/Netrin. In this model, UNC-6/Netrin first polarizes the growth cone via the Unc-5 receptor, leading to dorsally biased protrusion and F-actin accumulation. UNC-6/Netrin then regulates protrusion based on this polarity. The receptor UNC-40/DCC drives protrusion dorsally, away from the UNC-6/Netrin source, and the Unc-5 receptor inhibits protrusion ventrally, near the UNC-6/Netrin source, resulting in dorsal migration. Unc-5 inhibits protrusion in part by excluding microtubules from the growth cone, which are pro-protrusive. Here we report that the RHO-1/RhoA GTPase and its activator GEF RHGF-1 inhibit growth cone protrusion and MT accumulation in growth cones, similar to Unc-5. However, growth cone polarity of protrusion and F-actin were unaffected by RHO-1 and RHGF-1. Thus, RHO-1 signaling acts specifically as a negative regulator of protrusion and MT accumulation, and not polarity. Genetic interactions suggest that RHO-1 and RHGF-1 act with Unc-5, as well as with a parallel pathway, to regulate protrusion. The cytoskeletal interacting molecule UNC-33/CRMP was required for RHO-1 activity to inhibit MT accumulation, suggesting that UNC-33/CRMP might act downstream of RHO-1. In sum, these studies describe a new role of RHO-1 and RHGF-1 in regulation of growth cone protrusion by UNC-6/Netrin.
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Control of Growth Cone Polarity, Microtubule Accumulation, and Protrusion by UNC-6/Netrin and Its Receptors in Caenorhabditis elegans
Genetics, 2018Co-Authors: Mahekta R. Gujar, Lakshmi Sundararajan, Aubrie M. Stricker, Erik A. LundquistAbstract:UNC-6/Netrin has a conserved role in dorsal-ventral axon guidance, but the cellular events in the growth cone regulated by UNC-6/Netrin signaling during outgrowth are incompletely understood. Previous studies showed that, in growth cones migrating away from UNC-6/Netrin, the receptor Unc-5 regulates growth cone polarity, as observed by polarized F-actin, and limits the extent of growth cone protrusion. It is unclear how Unc-5 inhibits protrusion, and how UNC-40 acts in concert with Unc-5 to regulate polarity and protrusion. New results reported here indicate that Unc-5 normally restricts microtubule (MT) + end accumulation in the growth cone. Tubulin mutant analysis and colchicine treatment suggest that stable MTs are necessary for robust growth cone protrusion. Thus, Unc-5 might inhibit protrusion in part by restricting growth cone MT accumulation. Previous studies showed that the UNC-73/Trio Rac GEF and UNC-33/CRMP act downstream of Unc-5 in protrusion. Here, we show that UNC-33/CRMP regulates both growth cone dorsal asymmetric F-actin accumulation and MT accumulation, whereas UNC-73/Trio Rac GEF activity only affects F-actin accumulation. This suggests an MT-independent mechanism used by Unc-5 to inhibit protrusion, possibly by regulating lamellipodial and filopodial actin. Furthermore, we show that UNC-6/Netrin and the receptor UNC-40/DCC are required for excess protrusion in Unc-5 mutants, but not for loss of F-actin asymmetry or MT + end accumulation, indicating that UNC-6/Netrin and UNC-40/DCC are required for protrusion downstream of, or in parallel to, F-actin asymmetry and MT + end entry. F-actin accumulation might represent a polarity mark in the growth cone where protrusion will occur, and not protrusive lamellipodial and filopodial actin per se. Our data suggest a model in which UNC-6/Netrin first polarizes the growth cone via Unc-5, and then regulates protrusion based upon this polarity (the polarity/protrusion model). UNC-6/Netrin inhibits protrusion ventrally via Unc-5, and stimulates protrusion dorsally via UNC-40, resulting in dorsally-directed migration. The polarity/protrusion model represents a novel conceptual paradigm in which to understand axon guidance and growth cone migration away from UNC-6/Netrin.
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UNC-6/Netrin and its Receptors Unc-5 and UNC-40/DCC Control Growth Cone Polarity, Microtubule Accumulation, and Protrusion
2018Co-Authors: Mahekta R. Gujar, Lakshmi Sundararajan, Aubrie M. Stricker, Erik A. LundquistAbstract:Many axon guidance ligands and their receptors have been identified, but it is still unclear how these ligand-receptor interactions regulate events in the growth cone, such as protrusion and cytoskeletal arrangement, during directed outgrowth in vivo . In this work, we dissect the multiple and complex effects of UNC-6/Netrin on the growth cone. Previous studies showed that in C. elegans , the UNC-6/Netrin receptor Unc-5 regulates growth cone polarity, as evidenced by loss of asymmetric dorsal F-actin localization and protrusion in Unc-5 mutants. Unc-5 and another UNC-6/Netrin receptor UNC-40/DCC also regulate the extent of protrusion, with UNC-40/DCC driving protrusion and Unc-5 inhibiting protrusion. In this work we analyze the roles of UNC-6/Netrin, UNC-40/DCC, and Unc-5 in coordinating growth cone F-actin localization, microtubule organization, and protrusion that results in directed outgrowth away from UNC-6/Netrin. We find that a previously-described pathway involving the UNC-73/Trio Rac GEF and UNC-33/CRMP that acts downstream of Unc-5, regulates growth cone dorsal asymmetric F-actin accumulation and protrusion. Unc-5 and unc-33 mutants also display excess EBP-2::GFP puncta, suggesting that MT + end accumulation is important in growth cone polarity and/or protrusion. unc-73 Rac GEF mutants did not display excess EBP-2::GFP puncta despite larger and more protrusive growth cones, indicating a MT-independent mechanism to polarize the growth cone and to inhibit protrusion, possibly via actin. Finally, we show that UNC-6/Netrin and UNC-40/DCC are required for excess protrusion in Unc-5 mutants, but not for loss of F-actin asymmetry or MT + end accumulation, indicating that UNC-6/Netrin and UNC-40/DCC are required for protrusion downstream of F-actin asymmetry and MT + end entry. Our data suggest a model in which UNC-6/Netrin polarizes the growth cone via Unc-5, and then regulates a balance of pro- and anti-protrusive forces driven by UNC-40 and Unc-5, respectively, that result in directed protrusion and outgrowth.
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Flavin monooxygenases regulate Caenorhabditis elegans axon guidance and growth cone protrusion with UNC-6/Netrin signaling and Rac GTPases.
PLOS Genetics, 2017Co-Authors: Mahekta R. Gujar, Aubrie M. Stricker, Erik A. LundquistAbstract:The guidance cue UNC-6/Netrin regulates both attractive and repulsive axon guidance. Our previous work showed that in C. elegans, the attractive UNC-6/Netrin receptor UNC-40/DCC stimulates growth cone protrusion, and that the repulsive receptor, an Unc-5:UNC-40 heterodimer, inhibits growth cone protrusion. We have also shown that inhibition of growth cone protrusion downstream of the Unc-5:UNC-40 repulsive receptor involves Rac GTPases, the Rac GTP exchange factor UNC-73/Trio, and the cytoskeletal regulator UNC-33/CRMP, which mediates Semaphorin-induced growth cone collapse in other systems. The multidomain flavoprotein monooxygenase (FMO) MICAL (Molecule Interacting with CasL) also mediates growth cone collapse in response to Semaphorin by directly oxidizing F-actin, resulting in depolymerization. The C. elegans genome does not encode a multidomain MICAL-like molecule, but does encode five flavin monooxygenases (FMO-1, -2, -3, -4, and 5) and another molecule, EHBP-1, similar to the non-FMO portion of MICAL. Here we show that FMO-1, FMO-4, FMO-5, and EHBP-1 may play a role in UNC-6/Netrin directed repulsive guidance mediated through UNC-40 and Unc-5 receptors. Mutations in fmo-1, fmo-4, fmo-5, and ehbp-1 showed VD/DD axon guidance and branching defects, and variably enhanced unc-40 and Unc-5 VD/DD axon guidance defects. Developing growth cones in vivo of fmo-1, fmo-4, fmo-5, and ehbp-1 mutants displayed excessive filopodial protrusion, and transgenic expression of FMO-5 inhibited growth cone protrusion. Mutations suppressed growth cone inhibition caused by activated UNC-40 and Unc-5 signaling, and activated Rac GTPase CED-10 and MIG-2, suggesting that these molecules are required downstream of UNC-6/Netrin receptors and Rac GTPases. From these studies we conclude that FMO-1, FMO-4, FMO-5, and EHBP-1 represent new players downstream of UNC-6/Netrin receptors and Rac GTPases that inhibit growth cone filopodial protrusion in repulsive axon guidance.