scholarly journals 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

2019 ◽  
Author(s):  
Mahekta R. Gujar ◽  
Aubrie M. Stricker ◽  
Erik A. Lundquist
Keyword(s):  
Axon Guidance ◽  
Growth Cone ◽  
Neural Circuit ◽  
Growth Cones ◽  
Negative Regulator ◽  
Microtubule Organization ◽  
C Elegans ◽  
Guidance Cue ◽  
Rho Gef

AbstractUNC-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.Author SummaryNeural circuits are formed by precise connections between axons. During axon formation, the growth cone leads the axon to its proper target in a process called axon guidance. Growth cone outgrowth involves asymmetric protrusion driven by extracellular cues that stimulate and inhibit protrusion. How guidance cues regulate growth cone protrusion in neural circuit formation is incompletely understood. This work shows that the signaling molecule RHO-1 acts downstream of the UNC-6/Netrin guidance cue to inhibit growth cone protrusion in part by excluding microtubules from the growth cone, which are structural elements that drive protrusion.

scholarly journals UNC-6/Netrin and its Receptors UNC-5 and UNC-40/DCC Control Growth Cone Polarity, Microtubule Accumulation, and Protrusion

2018 ◽  
Author(s):  
Mahekta R. Gujar ◽  
Lakshmi Sundararajan ◽  
Aubrie Stricker ◽  
Erik A. Lundquist
Keyword(s):  
Axon Guidance ◽  
Growth Cone ◽  
Growth Cones ◽  
Microtubule Organization ◽  
C Elegans ◽  
Control Growth ◽  
Receptor Interactions ◽  
Independent Mechanism ◽  
Actin Localization

AbstractMany 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.

scholarly journals The PH/MyTH4/FERMmolecule MAX-1 inhibits UNC-5 activity in regulation of VD growth cone protrusion in Caenorhabditis elegans

2021 ◽  
Author(s):  
Snehal S. Mahadik ◽  
Erik A. Lundquist
Keyword(s):  
Spinal Cord ◽  
Plasma Membrane ◽  
Axon Guidance ◽  
Growth Cone ◽  
Growth Cones ◽  
Genetic Interactions ◽  
Wild Type ◽  
C Elegans ◽  
Guidance Cue ◽  
Mechanistic Insight

UNC-6/Netrin is a secreted conserved guidance cue that regulates dorsal-ventral axon guidance of C. elegans and in the vertebral spinal cord. In the polarity/protrusion model of VD growth cone guidance away from ventrally-expressed UNC-6 (repulsion), UNC-6 first polarizes the growth cone via the UNC-5 receptor such that filopodial protrusions are biased dorsally. UNC-6 then regulates a balance of protrusion in the growth cone based upon this polarity. UNC-5 inhibits protrusion ventrally, and the UNC-6 receptor UNC-40/DCC stimulates protrusion dorsally, resulting in net dorsal growth cone outgrowth. UNC-5 inhibits protrusion through the flavin monooxygenases FMO-1, 4, and 5 and possible actin destabilization, and inhibits pro-protrusive microtubule entry into the growth cone utilizing UNC-33/CRMP. The PH/MyTH4/FERM myosin-like protein was previously shown to act with UNC-5 in VD axon guidance utilizing axon guidance endpoint analysis. Here, we analyzed the effects of MAX-1 on VD growth cone morphology during outgrowth. We found that max-1 mutant growth cones were smaller and less protrusive than wild-type, the opposite of the unc-5 mutant phenotype. Furthermore, genetic interactions suggest that MAX-1 might normally inhibit UNC-5 activity, such that in a max-1 mutant growth cone, UNC-5 is overactive. Our results, combined with previous studies suggesting that MAX-1 might regulate UNC-5 levels in the cell or plasma membrane localization, suggest that MAX-1 attenuates UNC-5 signaling by regulating UNC-5 stability or trafficking. In summary, in the context of growth cone protrusion, MAX-1 inhibits UNC-5, demonstrating the mechanistic insight that can be gained by analyzing growth cones during outgrowth in addition to axon guidance endpoint analysis.

scholarly journals A requirement for filopodia extension toward Slit during Robo-mediated axon repulsion

2016 ◽  
Vol 213 (2) ◽  
pp. 261-274 ◽  
Author(s):  
Russell E. McConnell ◽  
J. Edward van Veen ◽  
Marina Vidaki ◽  
Adam V. Kwiatkowski ◽  
Aaron S. Meyer ◽  
...  
Keyword(s):  
Axon Guidance ◽  
Actin Polymerization ◽  
Growth Cones ◽  
Guidance Cues ◽  
Guidance Cue ◽  
3D Environments ◽  
Direct Binding ◽  
Mouse Dorsal Root Ganglion ◽  
Axon Repulsion

Axons navigate long distances through complex 3D environments to interconnect the nervous system during development. Although the precise spatiotemporal effects of most axon guidance cues remain poorly characterized, a prevailing model posits that attractive guidance cues stimulate actin polymerization in neuronal growth cones whereas repulsive cues induce actin disassembly. Contrary to this model, we find that the repulsive guidance cue Slit stimulates the formation and elongation of actin-based filopodia from mouse dorsal root ganglion growth cones. Surprisingly, filopodia form and elongate toward sources of Slit, a response that we find is required for subsequent axonal repulsion away from Slit. Mechanistically, Slit evokes changes in filopodium dynamics by increasing direct binding of its receptor, Robo, to members of the actin-regulatory Ena/VASP family. Perturbing filopodium dynamics pharmacologically or genetically disrupts Slit-mediated repulsion and produces severe axon guidance defects in vivo. Thus, Slit locally stimulates directional filopodial extension, a process that is required for subsequent axonal repulsion downstream of the Robo receptor.

scholarly journals Flavin Monooxygenases Regulate C. elegans Axon Guidance and Growth Cone Protrusion with UNC-6/Netrin signaling and Rac GTPases

2017 ◽  
Author(s):  
Mahekta Gujar ◽  
Aubrie M. Stricker ◽  
Erik A. Lundquist
Keyword(s):  
Axon Guidance ◽  
Growth Cone ◽  
Molecular Mechanisms ◽  
Direct Oxidation ◽  
Rac Gtpase ◽  
C Elegans ◽  
Growth Cone Collapse ◽  
Rac Gtpases ◽  
Inhibition Of Growth

AbstractThe 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 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 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.Author SummaryMolecular mechanisms of axon repulsion mediated by UNC-6/Netrin are not well understood. Inhibition of growth cone lamellipodial and filopodial protrusion is critical to repulsive axon guidance. Previous work identified a novel pathway involving Rac GTPases and the cytoskeletal interacting molecule UNC-33/CRMP required for UNC-6/Netrin-mediated inhibition of growth cone protrusion. In other systems, CRMP mediates growth cone collapse in response to semaphorin. Here we demonstrate a novel role of flavoprotein monooxygenases (FMOs) in repulsive axon guidance and inhibition of growth cone protrusion downstream of UNC-6/Netrin signaling and Rac GTPases. In Drosophila and vertebrates, the multidomain MICAL FMO mediates semaphorin-dependent growth cone collapse by direct oxidation and depolymerization of F-actin. The C. elegans genome does not encode a multidomain MICAL-like molecule, and we speculate that the C. elegans FMOs might have an equivalent role downstream of UNC-6/Netrin signaling. Indeed, we show that EHBP-1, similar to the non-FMO portion of MICAL, also controls repulsive axon guidance and growth cone inhibition, suggesting that in C. elegans, the functions of the multidomain MICAL molecule might be distributed across different molecules. In sum, we show conservation of function of molecules involved in semaphorin growth cone collapse with inhibition of growth cone protrusion downstream of UNC-6/Netrin signaling.

scholarly journals Frazzled promotes growth cone attachment at the source of a Netrin gradient in the Drosophila visual system

eLife ◽  
2016 ◽  
Vol 5 ◽  
Author(s):  
Orkun Akin ◽  
S Lawrence Zipursky
Keyword(s):  
Colorectal Cancer ◽  
Visual System ◽  
Axon Guidance ◽  
Growth Cone ◽  
Short Range ◽  
Growth Cones ◽  
Live Imaging ◽  
Deleted In Colorectal Cancer ◽  
Binding Partner

Axon guidance is proposed to act through a combination of long- and short-range attractive and repulsive cues. The ligand-receptor pair, Netrin (Net) and Frazzled (Fra) (DCC, Deleted in Colorectal Cancer, in vertebrates), is recognized as the prototypical effector of chemoattraction, with roles in both long- and short-range guidance. In the Drosophila visual system, R8 photoreceptor growth cones were shown to require Net-Fra to reach their target, the peak of a Net gradient. Using live imaging, we show, however, that R8 growth cones reach and recognize their target without Net, Fra, or Trim9, a conserved binding partner of Fra, but do not remain attached to it. Thus, despite the graded ligand distribution along the guidance path, Net-Fra is not used for chemoattraction. Based on findings in other systems, we propose that adhesion to substrate-bound Net underlies both long- and short-range Net-Fra-dependent guidance in vivo, thereby eroding the distinction between them.

scholarly journals Endoglycan plays a role in axon guidance by modulating cell adhesion

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Thomas Baeriswyl ◽  
Alexandre Dumoulin ◽  
Martina Schaettin ◽  
Georgia Tsapara ◽  
Vera Niederkofler ◽  
...  
Keyword(s):  
Cell Adhesion ◽  
Axon Guidance ◽  
Negative Regulator ◽  
Fine Tuning ◽  
In Vivo Studies ◽  
Commissural Axons ◽  
Guidance Cue ◽  
Cell Cell

Axon navigation depends on the interactions between guidance molecules along the trajectory and specific receptors on the growth cone. However, our in vitro and in vivo studies on the role of Endoglycan demonstrate that in addition to specific guidance cue – receptor interactions, axon guidance depends on fine-tuning of cell-cell adhesion. Endoglycan, a sialomucin, plays a role in axon guidance in the central nervous system of chicken embryos, but it is neither an axon guidance cue nor a receptor. Rather, Endoglycan acts as a negative regulator of molecular interactions based on evidence from in vitro experiments demonstrating reduced adhesion of growth cones. In the absence of Endoglycan, commissural axons fail to properly navigate the midline of the spinal cord. Taken together, our in vivo and in vitro results support the hypothesis that Endoglycan acts as a negative regulator of cell-cell adhesion in commissural axon guidance.

scholarly journals A mathematical model explains saturating axon guidance responses to molecular gradients

eLife ◽  
2016 ◽  
Vol 5 ◽  
Author(s):  
Huyen Nguyen ◽  
Peter Dayan ◽  
Zac Pujic ◽  
Justin Cooper-White ◽  
Geoffrey J Goodhill
Keyword(s):  
Mathematical Model ◽  
Nervous System ◽  
Axon Guidance ◽  
Predictive Model ◽  
Growth Cone ◽  
Growth Cones ◽  
Mathematical Explanation ◽  
In Vitro Growth

Correct wiring is crucial for the proper functioning of the nervous system. Molecular gradients provide critical signals to guide growth cones, which are the motile tips of developing axons, to their targets. However, in vitro, growth cones trace highly stochastic trajectories, and exactly how molecular gradients bias their movement is unclear. Here, we introduce a mathematical model based on persistence, bias, and noise to describe this behaviour, constrained directly by measurements of the detailed statistics of growth cone movements in both attractive and repulsive gradients in a microfluidic device. This model provides a mathematical explanation for why average axon turning angles in gradients in vitro saturate very rapidly with time at relatively small values. This work introduces the most accurate predictive model of growth cone trajectories to date, and deepens our understanding of axon guidance events both in vitro and in vivo.

The metalloproteinase ADAM10 is required for retinal ganglion cell axon guidance in the developing visual systems

2007 ◽  
Vol 30 (4) ◽  
pp. 77
Author(s):  
Y. Y. Chen ◽  
C. L. Hehr ◽  
K. Atkinson-Leadbeater ◽  
J. C. Hocking ◽  
S. McFarlane
Keyword(s):  
Ganglion Cell ◽  
Axon Guidance ◽  
Retinal Ganglion Cell ◽  
Growth Cone ◽  
Expression Patterns ◽  
Optic Tract ◽  
Axon Growth ◽  
Proteolytic Processing ◽  
Retinal Ganglion

Background: The growth cone interprets cues in its environment in order to reach its target. We want to identify molecules that regulate growth cone behaviour in the developing embryo. We investigated the role of A disintegrin and metalloproteinase 10 (ADAM10) in axon guidance in the developing visual system of African frog, Xenopus laevis. Methods: We first examined the expression patterns of adam10 mRNA by in situ hybridization. We then exposed the developing optic tract to an ADAM10 inhibitor, GI254023X, in vivo. Lastly, we inhibited ADAM10 function in diencephalic neuroepithelial cells (through which retinal ganglion cell (RGC) axons extend) or RGCs by electroporating or transfecting an ADAM10 dominant negative (dn-adam10). Results: We show that adam10 mRNA is expressed in the dorsal neuroepithelium over the time RGC axons extend towards their target, the optic tectum. Second, pharmacological inhibition of ADAM10 in an in vivo exposed brain preparation causes the failure of RGC axons to recognize their target at low concentrations (0.5, 1 μM), and the failure of the axons to make a caudal turn in the mid-diencephalon at higher concentration (5 μM). Thus, ADAM10 function is required for RGC axon guidance at two key guidance decisions. Finally, molecular inhibition of ADAM10 function by electroporating dn-adam10 in the brain neuroepithelium causes defects in RGC axon target recognition (57%) and/or defects in caudal turn (12%), as seen with the pharmacological inhibitor. In contrast, molecular inhibition of ADAM10 within the RGC axons has no effect. Conclusions: These data argue strongly that ADAM10 acts cell non-autonomously within the neuroepithelium to regulate the guidance of RGC axons. This study shows for the first time that a metalloproteinase acts in a cell non-autonomous fashion to direct vertebrate axon growth. It will provide important insights into candidate molecules that could be used to reform nerve connections if destroyed because of injury or disease. References Hattori M, Osterfield M, Flanagan JG. Regulated cleavage of a contact-mediated axon repellent. Science 2000; 289(5483):1360-5. Janes PW, Saha N, Barton WA, Kolev MV, Wimmer-Kleikamp SH, Nievergall E, Blobel CP, Himanen JP, Lackmann M, Nikolov DB. Adam meets Eph: an ADAM substrate recognition module acts as a molecular switch for ephrin cleavage in trans. Cell 2005; 123(2):291-304. Pan D, Rubin GM. Kuzbanian controls proteolytic processing of Notch and mediates lateral inhibition during Drosophila and vertebrate neurogenesis. Cell 1997; 90(2):271-80.

scholarly journals Regulation of axon repulsion by MAX-1 SUMOylation and AP-3

2018 ◽  
Vol 115 (35) ◽  
pp. E8236-E8245
Author(s):  
Shih-Yu Chen ◽  
Chun-Ta Ho ◽  
Wei-Wen Liu ◽  
Mark Lucanic ◽  
Hsiu-Ming Shih ◽  
...  
Keyword(s):  
Axon Guidance ◽  
Neural Development ◽  
Biochemical Analysis ◽  
Genetic Interaction ◽  
Motor Axons ◽  
Surface Receptors ◽  
C Elegans ◽  
Guidance Receptors ◽  
Axon Repulsion

During neural development, growing axons express specific surface receptors in response to various environmental guidance cues. These axon guidance receptors are regulated through intracellular trafficking and degradation to enable navigating axons to reach their targets. In Caenorhabditis elegans, the UNC-5 receptor is necessary for dorsal migration of developing motor axons. We previously found that MAX-1 is required for UNC-5–mediated axon repulsion, but its mechanism of action remained unclear. Here, we demonstrate that UNC-5–mediated axon repulsion in C. elegans motor axons requires both max-1 SUMOylation and the AP-3 complex β subunit gene, apb-3. Genetic interaction studies show that max-1 is SUMOylated by gei-17/PIAS1 and acts upstream of apb-3. Biochemical analysis suggests that constitutive interaction of MAX-1 and UNC-5 receptor is weakened by MAX-1 SUMOylation and by the presence of APB-3, a competitive interactor with UNC-5. Overexpression of APB-3 reroutes the trafficking of UNC-5 receptor into the lysosome for protein degradation. In vivo fluorescence recovery after photobleaching experiments shows that MAX-1 SUMOylation and APB-3 are required for proper trafficking of UNC-5 receptor in the axon. Our results demonstrate that SUMOylation of MAX-1 plays an important role in regulating AP-3–mediated trafficking and degradation of UNC-5 receptors during axon guidance.

scholarly journals A pair of E3 ubiquitin ligases compete to regulate filopodial dynamics and axon guidance

2019 ◽  
Vol 219 (1) ◽  
Author(s):  
Nicholas P. Boyer ◽  
Laura E. McCormick ◽  
Shalini Menon ◽  
Fabio L. Urbina ◽  
Stephanie L. Gupton
Keyword(s):  
Axon Guidance ◽  
Growth Cone ◽  
Ubiquitin Ligases ◽  
Critical Element ◽  
E3 Ubiquitin Ligases ◽  
Related Protein ◽  
Guidance Cues ◽  
Guidance Cue ◽  
Neuronal Connections ◽  
Novel Model

Appropriate axon guidance is necessary to form accurate neuronal connections. Axon guidance cues that stimulate cytoskeletal reorganization within the growth cone direct axon navigation. Filopodia at the growth cone periphery have long been considered sensors for axon guidance cues, yet how they respond to extracellular cues remains ill defined. Our previous work found that the filopodial actin polymerase VASP and consequently filopodial stability are negatively regulated via nondegradative TRIM9-dependent ubiquitination. Appropriate VASP ubiquitination and deubiquitination are required for axon turning in response to the guidance cue netrin-1. Here we show that the TRIM9-related protein TRIM67 outcompetes TRIM9 for interacting with VASP and antagonizes TRIM9-dependent VASP ubiquitination. The surprising antagonistic roles of two closely related E3 ubiquitin ligases are required for netrin-1–dependent filopodial responses, axon turning and branching, and fiber tract formation. We suggest a novel model in which coordinated regulation of VASP ubiquitination by a pair of interfering ligases is a critical element of VASP dynamics, filopodial stability, and axon guidance.

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