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The Caenorhabditis elegans Eph receptor activates NCK and N-WASP, and inhibits Ena/VASP to regulate growth cone dynamics during axon guidance.

Mohamed AM, Boudreau JR, Yu FP, Liu J, Chin-Sang ID - PLoS Genet. (2012)

Bottom Line: We identified NCK-1 and WSP-1/N-WASP as downstream effectors of VAB-1.Furthermore, VAB-1, NCK-1, and WSP-1 can form a complex in vitro.We suggest that VAB-1/Eph RTK can stop axonal outgrowth by inhibiting filopodia formation at the growth cone by activating Arp2/3 through a VAB-1/NCK-1/WSP-1 complex and by inhibiting UNC-34/Ena activity.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, Queen's University, Kingston, Canada.

ABSTRACT
The Eph receptor tyrosine kinases (RTKs) are regulators of cell migration and axon guidance. However, our understanding of the molecular mechanisms by which Eph RTKs regulate these processes is still incomplete. To understand how Eph receptors regulate axon guidance in Caenorhabditis elegans, we screened for suppressors of axon guidance defects caused by a hyperactive VAB-1/Eph RTK. We identified NCK-1 and WSP-1/N-WASP as downstream effectors of VAB-1. Furthermore, VAB-1, NCK-1, and WSP-1 can form a complex in vitro. We also report that NCK-1 can physically bind UNC-34/Enabled (Ena), and suggest that VAB-1 inhibits the NCK-1/UNC-34 complex and negatively regulates UNC-34. Our results provide a model of the molecular events that allow the VAB-1 RTK to regulate actin dynamics for axon guidance. We suggest that VAB-1/Eph RTK can stop axonal outgrowth by inhibiting filopodia formation at the growth cone by activating Arp2/3 through a VAB-1/NCK-1/WSP-1 complex and by inhibiting UNC-34/Ena activity.

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Related in: MedlinePlus

VAB-1 activation affects PLM growth cone dynamics.(A–B) Series of time-lapse images of newly hatched L1 PLM growth cones as they migrate anteriorly. Scale bar represents 2 µm. (A) Wild-type PLM growth cones exhibit dynamic changes and display multiple filopodia protrusions. (B) Transgenic myr-vab-1 animals have PLM growth cones that are less dynamic and mostly void of any protrusions. (C) A model of how VAB-1 induces its termination effect during PLM axon guidance. (Left) In the absence of VAB-1 activation, UNC-34/Ena promotes axon extension through the polymerization of actin filaments and forming filopodia. UNC-34/Ena also physically binds to and inhibits NCK-1's role with WSP-1. It is possible that the UNC-34/NCK-1 heterodimer could work together in promoting actin polymerization downstream of other receptors and is indicated by question marks (?). (Right) Genetic and molecular data suggest that VAB-1 inhibits UNC-34 function. Activation of VAB-1 contributes to stopping axon outgrowth by binding to the NCK-1 SH2 domains, which disrupts the interaction between NCK-1 and UNC-34. VAB-1 over expression can reduce UNC-34 protein levels thereby preventing further actin filament polymerization. Furthermore, VAB-1, NCK-1 and WSP-1 can now form a complex and induce high levels of Arp2/3 activation to form an extensive network of short, branched actin filaments. The combination of inhibiting actin filament polymerization and increasing short, branched networks stop the formation of new filopodia. The net result is the termination of axon extension in response to VAB-1 signaling.
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pgen-1002513-g007: VAB-1 activation affects PLM growth cone dynamics.(A–B) Series of time-lapse images of newly hatched L1 PLM growth cones as they migrate anteriorly. Scale bar represents 2 µm. (A) Wild-type PLM growth cones exhibit dynamic changes and display multiple filopodia protrusions. (B) Transgenic myr-vab-1 animals have PLM growth cones that are less dynamic and mostly void of any protrusions. (C) A model of how VAB-1 induces its termination effect during PLM axon guidance. (Left) In the absence of VAB-1 activation, UNC-34/Ena promotes axon extension through the polymerization of actin filaments and forming filopodia. UNC-34/Ena also physically binds to and inhibits NCK-1's role with WSP-1. It is possible that the UNC-34/NCK-1 heterodimer could work together in promoting actin polymerization downstream of other receptors and is indicated by question marks (?). (Right) Genetic and molecular data suggest that VAB-1 inhibits UNC-34 function. Activation of VAB-1 contributes to stopping axon outgrowth by binding to the NCK-1 SH2 domains, which disrupts the interaction between NCK-1 and UNC-34. VAB-1 over expression can reduce UNC-34 protein levels thereby preventing further actin filament polymerization. Furthermore, VAB-1, NCK-1 and WSP-1 can now form a complex and induce high levels of Arp2/3 activation to form an extensive network of short, branched actin filaments. The combination of inhibiting actin filament polymerization and increasing short, branched networks stop the formation of new filopodia. The net result is the termination of axon extension in response to VAB-1 signaling.

Mentions: The VAB-1 RTK effectors NCK-1 and WSP-1 are known actin regulators and therefore implicate VAB-1 signaling in regulating actin cytoskeleton for axon guidance. To confirm this, we monitored the PLM growth cone of wild-type and myr-vab-1 transgenic animals at the time of hatching. In wild-type animals, most of the PLM growth cones exhibited dynamic changes and had many filopodia protrusions (70%; N = 20 movies) (Figure 7A, Video S1). Transgenic myr-vab-1 animals, on the other hand, had growth cones that were less dynamic and were usually void of filopodia like structures with only 25% (N = 16 movies) showing some filopodia structures (Figure 7B, Video S2). Since our molecular and genetic data suggest that VAB-1 inhibits UNC-34/Ena function we also observed the growth cones of unc-34(e566) animals. We found that unc-34(e566) mutants, like myr-vab-1 animals, had growth cones void of filopodia structures with only 25% displaying filopodia structures (N = 12 movies; not shown). Our results show that activated VAB-1 can affect the PLM growth cone morphology by inhibiting filopodia formation.


The Caenorhabditis elegans Eph receptor activates NCK and N-WASP, and inhibits Ena/VASP to regulate growth cone dynamics during axon guidance.

Mohamed AM, Boudreau JR, Yu FP, Liu J, Chin-Sang ID - PLoS Genet. (2012)

VAB-1 activation affects PLM growth cone dynamics.(A–B) Series of time-lapse images of newly hatched L1 PLM growth cones as they migrate anteriorly. Scale bar represents 2 µm. (A) Wild-type PLM growth cones exhibit dynamic changes and display multiple filopodia protrusions. (B) Transgenic myr-vab-1 animals have PLM growth cones that are less dynamic and mostly void of any protrusions. (C) A model of how VAB-1 induces its termination effect during PLM axon guidance. (Left) In the absence of VAB-1 activation, UNC-34/Ena promotes axon extension through the polymerization of actin filaments and forming filopodia. UNC-34/Ena also physically binds to and inhibits NCK-1's role with WSP-1. It is possible that the UNC-34/NCK-1 heterodimer could work together in promoting actin polymerization downstream of other receptors and is indicated by question marks (?). (Right) Genetic and molecular data suggest that VAB-1 inhibits UNC-34 function. Activation of VAB-1 contributes to stopping axon outgrowth by binding to the NCK-1 SH2 domains, which disrupts the interaction between NCK-1 and UNC-34. VAB-1 over expression can reduce UNC-34 protein levels thereby preventing further actin filament polymerization. Furthermore, VAB-1, NCK-1 and WSP-1 can now form a complex and induce high levels of Arp2/3 activation to form an extensive network of short, branched actin filaments. The combination of inhibiting actin filament polymerization and increasing short, branched networks stop the formation of new filopodia. The net result is the termination of axon extension in response to VAB-1 signaling.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3285579&req=5

pgen-1002513-g007: VAB-1 activation affects PLM growth cone dynamics.(A–B) Series of time-lapse images of newly hatched L1 PLM growth cones as they migrate anteriorly. Scale bar represents 2 µm. (A) Wild-type PLM growth cones exhibit dynamic changes and display multiple filopodia protrusions. (B) Transgenic myr-vab-1 animals have PLM growth cones that are less dynamic and mostly void of any protrusions. (C) A model of how VAB-1 induces its termination effect during PLM axon guidance. (Left) In the absence of VAB-1 activation, UNC-34/Ena promotes axon extension through the polymerization of actin filaments and forming filopodia. UNC-34/Ena also physically binds to and inhibits NCK-1's role with WSP-1. It is possible that the UNC-34/NCK-1 heterodimer could work together in promoting actin polymerization downstream of other receptors and is indicated by question marks (?). (Right) Genetic and molecular data suggest that VAB-1 inhibits UNC-34 function. Activation of VAB-1 contributes to stopping axon outgrowth by binding to the NCK-1 SH2 domains, which disrupts the interaction between NCK-1 and UNC-34. VAB-1 over expression can reduce UNC-34 protein levels thereby preventing further actin filament polymerization. Furthermore, VAB-1, NCK-1 and WSP-1 can now form a complex and induce high levels of Arp2/3 activation to form an extensive network of short, branched actin filaments. The combination of inhibiting actin filament polymerization and increasing short, branched networks stop the formation of new filopodia. The net result is the termination of axon extension in response to VAB-1 signaling.
Mentions: The VAB-1 RTK effectors NCK-1 and WSP-1 are known actin regulators and therefore implicate VAB-1 signaling in regulating actin cytoskeleton for axon guidance. To confirm this, we monitored the PLM growth cone of wild-type and myr-vab-1 transgenic animals at the time of hatching. In wild-type animals, most of the PLM growth cones exhibited dynamic changes and had many filopodia protrusions (70%; N = 20 movies) (Figure 7A, Video S1). Transgenic myr-vab-1 animals, on the other hand, had growth cones that were less dynamic and were usually void of filopodia like structures with only 25% (N = 16 movies) showing some filopodia structures (Figure 7B, Video S2). Since our molecular and genetic data suggest that VAB-1 inhibits UNC-34/Ena function we also observed the growth cones of unc-34(e566) animals. We found that unc-34(e566) mutants, like myr-vab-1 animals, had growth cones void of filopodia structures with only 25% displaying filopodia structures (N = 12 movies; not shown). Our results show that activated VAB-1 can affect the PLM growth cone morphology by inhibiting filopodia formation.

Bottom Line: We identified NCK-1 and WSP-1/N-WASP as downstream effectors of VAB-1.Furthermore, VAB-1, NCK-1, and WSP-1 can form a complex in vitro.We suggest that VAB-1/Eph RTK can stop axonal outgrowth by inhibiting filopodia formation at the growth cone by activating Arp2/3 through a VAB-1/NCK-1/WSP-1 complex and by inhibiting UNC-34/Ena activity.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, Queen's University, Kingston, Canada.

ABSTRACT
The Eph receptor tyrosine kinases (RTKs) are regulators of cell migration and axon guidance. However, our understanding of the molecular mechanisms by which Eph RTKs regulate these processes is still incomplete. To understand how Eph receptors regulate axon guidance in Caenorhabditis elegans, we screened for suppressors of axon guidance defects caused by a hyperactive VAB-1/Eph RTK. We identified NCK-1 and WSP-1/N-WASP as downstream effectors of VAB-1. Furthermore, VAB-1, NCK-1, and WSP-1 can form a complex in vitro. We also report that NCK-1 can physically bind UNC-34/Enabled (Ena), and suggest that VAB-1 inhibits the NCK-1/UNC-34 complex and negatively regulates UNC-34. Our results provide a model of the molecular events that allow the VAB-1 RTK to regulate actin dynamics for axon guidance. We suggest that VAB-1/Eph RTK can stop axonal outgrowth by inhibiting filopodia formation at the growth cone by activating Arp2/3 through a VAB-1/NCK-1/WSP-1 complex and by inhibiting UNC-34/Ena activity.

Show MeSH
Related in: MedlinePlus