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 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 RNA-based mechanisms underlying axon guidance

2013 ◽  
Vol 202 (7) ◽  
pp. 991-999 ◽  
Author(s):  
Toshiaki Shigeoka ◽  
Bo Lu ◽  
Christine E. Holt
Keyword(s):  
Axon Guidance ◽  
Translational Control ◽  
Growth Cones ◽  
Receptor Activation ◽  
Neural Pathways ◽  
Neuronal Circuitry ◽  
Guidance Cues ◽  
Spatiotemporal Control

Axon guidance plays a key role in establishing neuronal circuitry. The motile tips of growing axons, the growth cones, navigate by responding directionally to guidance cues that pattern the embryonic neural pathways via receptor-mediated signaling. Evidence in vitro in the last decade supports the notion that RNA-based mechanisms contribute to cue-directed steering during axon guidance. Different cues trigger translation of distinct subsets of mRNAs and localized translation provides precise spatiotemporal control over the growth cone proteome in response to localized receptor activation. Recent evidence has now demonstrated a role for localized translational control in axon guidance decisions in vivo.

scholarly journals Robo recruitment of the Wave Regulatory Complex plays an essential and conserved role in midline repulsion

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Karina Chaudhari ◽  
Madhavi Gorla ◽  
Chao Chang ◽  
Artur Kania ◽  
Greg J Bashaw
Keyword(s):  
Axon Guidance ◽  
Actin Polymerization ◽  
Chick Embryos ◽  
Commissural Axons ◽  
Midline Crossing ◽  
Regulatory Complex ◽  
Axon Repulsion ◽  
Dorsal Spinal ◽  
Receptor Family

The Roundabout (Robo) guidance receptor family induces axon repulsion in response to its ligand Slit by inducing local cytoskeletal changes; however, the link to the cytoskeleton and the nature of these cytoskeletal changes are poorly understood. Here, we show that the heteropentameric Scar/Wave Regulatory Complex (WRC) which drives Arp2/3-induced branched actin polymerization, is a direct effector of Robo signaling. Biochemical evidence shows that Slit triggers WRC recruitment to the Robo receptor's WIRS motif. In Drosophila embryos, mutants of the WRC enhance Robo1-dependent midline crossing defects. Additionally, mutating Robo1's WIRS motif significantly reduces receptor activity in rescue assays in vivo, and CRISPR-Cas9 mutagenesis shows that the WIRS motif is essential for endogenous Robo1 function. Finally, axon guidance assays in mouse dorsal spinal commissural axons and gain-of-function experiments in chick embryos demonstrate that the WIRS motif is also required for Robo1 repulsion in mammals. Together, our data support an essential conserved role for the WIRS-WRC interaction in Robo1-mediated axon repulsion.

scholarly journals Robo recruitment of the Wave Regulatory Complex plays an essential and conserved role in midline repulsion

2020 ◽  
Author(s):  
Karina Chaudhari ◽  
Madhavi Gorla ◽  
Chao Chang ◽  
Artur Kania ◽  
Greg J. Bashaw
Keyword(s):  
Axon Guidance ◽  
Actin Polymerization ◽  
Biochemical Evidence ◽  
Commissural Axons ◽  
Midline Crossing ◽  
Regulatory Complex ◽  
Axon Repulsion ◽  
Dorsal Spinal ◽  
Receptor Family

SUMMARYThe Roundabout (Robo) guidance receptor family induces axon repulsion in response to its ligand Slit by inducing local cytoskeletal changes; however, the link to the cytoskeleton and the nature of these cytoskeletal changes are unclear. Here we show that the heteropentameric Scar/Wave Regulatory Complex (WRC) which drives Arp2/3-induced branched actin polymerization, is a direct effector of Robo signaling. Biochemical evidence shows that Slit triggers WRC recruitment to the Robo receptor’s WIRS motif. In Drosophila embryos, mutants of the WRC enhance Robol-dependent midline crossing defects. Additionally, mutating Robo1’s WIRS motif significantly reduces receptor activity in rescue assays in vivo, and CRISPR-Cas9 mutagenesis shows that the WIRS motif is essential for endogenous Robo1 function. Finally, axon guidance assays in mouse dorsal spinal commissural axons demonstrate that the WIRS motif is also required for Robo1 repulsion in mammals. Together, our data support an essential conserved role for the WRC in commissural axon repulsion.

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.

scholarly journals Gradients and Growth Cone Guidance of Grasshopper Neurons

2003 ◽  
Vol 51 (4) ◽  
pp. 445-454 ◽  
Author(s):  
Arthur T. Legg ◽  
Timothy P. O'Connor
Keyword(s):  
Nervous System ◽  
Long Range ◽  
Growth Cone ◽  
Growth Cones ◽  
Path Finding ◽  
Guidance Cues ◽  
Cone Function ◽  
The Absolute ◽  
Growth Cone Guidance

The generation of a functional nervous system is dependent on precise path-finding of axons during development. This pathfinding is directed by the distribution of local and long-range guidance cues, the latter of which are believed to be distributed in gradients. Gradients of guidance cues have been associated with growth cone function for over a hundred years. However, little is known about the mechanisms used by growth cones to respond to these gradients, in part owing to the lack of identifiable gradients in vivo. In the developing grasshopper limb, two gradients of the semaphorin Sema-2a are necessary for correct neuronal pathfinding in vivo. The gradients are found in regions where growth cones make critical steering decisions. Observations of different growth cone behaviors associated with these gradients have provided some insights into how growth cones respond to them. Growth cones appear to respond more faithfully to changes in concentration, rather than absolute levels, of Sema-2a expression, whereas the absolute levels may regulate growth cone size.

scholarly journals Ephrin-A/EphA specific co-adaptation as a novel mechanism in topographic axon guidance

eLife ◽  
2017 ◽  
Vol 6 ◽  
Author(s):  
Felix Fiederling ◽  
Markus Weschenfelder ◽  
Martin Fritz ◽  
Anne von Philipsborn ◽  
Martin Bastmeyer ◽  
...  
Keyword(s):  
Axon Guidance ◽  
Surface Membrane ◽  
Growth Cones ◽  
Field Analysis ◽  
Membrane Microdomains ◽  
Recycling Endosomes ◽  
Guidance Cue ◽  
Retinal Axons ◽  
Retinal Growth ◽  
Neuronal Processing

Genetic hardwiring during brain development provides computational architectures for innate neuronal processing. Thus, the paradigmatic chick retinotectal projection, due to its neighborhood preserving, topographic organization, establishes millions of parallel channels for incremental visual field analysis. Retinal axons receive targeting information from quantitative guidance cue gradients. Surprisingly, novel adaptation assays demonstrate that retinal growth cones robustly adapt towards ephrin-A/EphA forward and reverse signals, which provide the major mapping cues. Computational modeling suggests that topographic accuracy and adaptability, though seemingly incompatible, could be reconciled by a novel mechanism of coupled adaptation of signaling channels. Experimentally, we find such ‘co-adaptation’ in retinal growth cones specifically for ephrin-A/EphA signaling. Co-adaptation involves trafficking of unliganded sensors between the surface membrane and recycling endosomes, and is presumably triggered by changes in the lipid composition of membrane microdomains. We propose that co-adaptative desensitization eventually relies on guidance sensor translocation into cis-signaling endosomes to outbalance repulsive trans-signaling.

scholarly journals Robo and Ror function in a common receptor complex to regulate Wnt-mediated neurite outgrowth in Caenorhabditis elegans

2018 ◽  
Vol 115 (10) ◽  
pp. E2254-E2263 ◽  
Author(s):  
Jiaming Wang ◽  
Mei Ding
Keyword(s):  
Caenorhabditis Elegans ◽  
Growth Cones ◽  
Asymmetric Distribution ◽  
Receptor Complex ◽  
Biological Processes ◽  
Membrane Recruitment ◽  
Guidance Cues ◽  
Downstream Effector ◽  
Common Receptor

Growing axons are exposed to various guidance cues en route to their targets, but the mechanisms that govern the response of growth cones to combinations of signals remain largely elusive. Here, we found that the sole Robo receptor, SAX-3, in Caenorhabditis elegans functions as a coreceptor for Wnt/CWN-2 molecules. SAX-3 binds to Wnt/CWN-2 and facilitates the membrane recruitment of CWN-2. SAX-3 forms a complex with the Ror/CAM-1 receptor and its downstream effector Dsh/DSH-1, promoting signal transduction from Wnt to Dsh. sax-3 functions in Wnt-responsive cells and the SAX-3 receptor is restricted to the side of the cell from which the neurite is extended. DSH-1 has a similar asymmetric distribution, which is disrupted by sax-3 mutation. Taking these results together, we propose that Robo receptor can function as a Wnt coreceptor to regulate Wnt-mediated biological processes in vivo.

scholarly journals Gradient-reading and mechano-effector machinery for netrin-1-induced axon guidance

eLife ◽  
2018 ◽  
Vol 7 ◽  
Author(s):  
Kentarou Baba ◽  
Wataru Yoshida ◽  
Michinori Toriyama ◽  
Tadayuki Shimada ◽  
Colleen F Manning ◽  
...  
Keyword(s):  
Cell Adhesion ◽  
Axon Guidance ◽  
Cell Adhesion Molecule ◽  
Growth Cones ◽  
Spatial Cues ◽  
Commissural Axons ◽  
Guidance Cues ◽  
Cell Adhesion Molecule L1 ◽  
Axon Guidance Molecule ◽  
Guidance Molecule

Growth cones navigate axonal projection in response to guidance cues. However, it is unclear how they can decide the migratory direction by transducing the local spatial cues into protrusive forces. Here we show that knockout mice of Shootin1 display abnormal projection of the forebrain commissural axons, a phenotype similar to that of the axon guidance molecule netrin-1. Shallow gradients of netrin-1 elicited highly polarized Pak1-mediated phosphorylation of shootin1 within growth cones. We demonstrate that netrin-1–elicited shootin1 phosphorylation increases shootin1 interaction with the cell adhesion molecule L1-CAM; this, in turn, promotes F-actin–adhesion coupling and concomitant generation of forces for growth cone migration. Moreover, the spatially regulated shootin1 phosphorylation within growth cones is required for axon turning induced by netrin-1 gradients. Our study defines a mechano-effector for netrin-1 signaling and demonstrates that shootin1 phosphorylation is a critical readout for netrin-1 gradients that results in a directional mechanoresponse for axon guidance.

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