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Cellular and Molecular Life Sciences

, Volume 75, Issue 22, pp 4207–4222 | Cite as

Neuronal GAP-Porf-2 transduces EphB1 signaling to brake axon growth

  • Guo-Hui Huang
  • Lin Guo
  • Liang Zhu
  • Xian-Dong Liu
  • Zhao-Liang Sun
  • Hong-Jiang Li
  • Nan-Jie Xu
  • Dong-Fu Feng
Original Article

Abstract

Axonal outgrowth and guidance require numerous extracellular cues and intracellular mediators that transduce signals in the growth cone to regulate cytoskeletal dynamics. However, the way in which cytoskeletal effectors respond to these signals remains elusive. Here, we demonstrate that Porf-2, a neuron-expressed RhoGTPase-activating protein, plays an essential role in the inhibition of initial axon growth by restricting the expansion of the growth cone in a cell-autonomous manner. Furthermore, the EphB1 receptor is identified as an upstream controller that binds and regulates Porf-2 specifically upon extracellular ephrin-B stimulation. The activated EphB forward signal deactivates Rac1 through the GAP domain of Porf-2, which inhibits growth cone formation and brakes axon growth. Our results therefore provide a novel GAP that regulates axon growth and braking sequentially through Eph receptor-independent and Eph receptor-dependent pathways.

Keywords

Axon growth Porf-2 EphB GAP Vilse 

Abbreviations

GAPs

GTPase-activating proteins

GEFs

Guanine nucleotide exchange factors

Porf-2

Preoptic regulatory factor-2

MS

Mass spectrometry

WT

Wild type

Robo

Roundabout

Co-IP

Co-immunoprecipitation

shRNA

Short hairpin RNA

cAMP

Cyclic adenosine monophosphate

PDL

Poly-d-lysine

NSC

Neural stem cell

PFA

Paraformaldehyde

CNK2

Connector enhancer of KSR-2

GFP

Green fluorescent protein

FITC

Fluorescein isothiocyanate

BSA

Bovine serum albumin

GST

Glutathione S-transferase

PBD

p21-binding domain

Notes

Acknowledgements

We thank Prof. Jialin C. Zheng for providing the PDMS stamp model used in the stripe assay.

Author contributions

G-HH and LG designed and performed the experiments and wrote the manuscript. LZ, X-DL, Z-LS and H-JL performed the data and statistical analysis. N-JX and D-FF designed the experiments and revised the manuscript critically. All authors agree that all the questions related to the accuracy or integrity of the paper have been appropriately investigated and resolved and have given the final approval of the version to be published.

Funding

This study was supported by the National Natural Science Foundation of China (81772059 to D.-F.F, 31671062 and 31371097 to N.-J.X.), National Basic Research Program of China (Program 973 Grant 2014CB965002), 1000-Talents Program, Grants of Shanghai Brain-Intelligence Project from STCSM (16JC1420501), and the Program for Professor of Special Appointment (Eastern Scholar) at Shanghai Institutions of Higher Learning (No. 2013-25) to N.-J.X.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

18_2018_2858_MOESM1_ESM.docx (371 kb)
Supplementary material 1 (DOCX 371 kb)

References

  1. 1.
    O’Donnell M, Chance RK, Bashaw GJ (2009) Axon growth and guidance: receptor regulation and signal transduction. Annu Rev Neurosci 32:383–412CrossRefGoogle Scholar
  2. 2.
    Cheng PL, Poo MM (2012) Early events in axon/dendrite polarization. Annu Rev Neurosci 35:181–201CrossRefGoogle Scholar
  3. 3.
    Heng JI, Chariot A, Nguyen L (2010) Molecular layers underlying cytoskeletal remodelling during cortical development. Trends Neurosci 33:38–47CrossRefGoogle Scholar
  4. 4.
    Bilimoria PM, Bonni A (2013) Molecular control of axon branching. Neuroscientist 19:16–24CrossRefGoogle Scholar
  5. 5.
    Heasman SJ, Ridley AJ (2008) Mammalian Rho GTPases: new insights into their functions from in vivo studies. Nat Rev Mol Cell Biol 9:690–701CrossRefGoogle Scholar
  6. 6.
    Miller MB, Yan Y, Eipper BA, Mains RE (2013) Neuronal Rho GEFs in synaptic physiology and behavior. Neuroscientist 19:255–273CrossRefGoogle Scholar
  7. 7.
    Moon SY, Zheng Y (2003) Rho GTPase-activating proteins in cell regulation. Trends Cell Biol 13:13–22CrossRefGoogle Scholar
  8. 8.
    Huang GH, Sun ZL, Li HJ, Feng DF (2017) Rho GTPase-activating proteins: regulators of Rho GTPase activity in neuronal development and CNS diseases. Mol Cell Neurosci 80:18–31CrossRefGoogle Scholar
  9. 9.
    Hu H, Li M, Labrador JP, McEwen J, Lai EC, Goodman CS, Bashaw GJ (2005) Cross GTPase-activating protein (CrossGAP)/Vilse links the Roundabout receptor to Rac to regulate midline repulsion. Proc Natl Acad Sci USA 102:4613–4618CrossRefGoogle Scholar
  10. 10.
    Lundstrom A, Gallio M, Englund C, Steneberg P, Hemphala J, Aspenstrom P, Keleman K, Falileeva L, Dickson BJ, Samakovlis C (2004) Vilse, a conserved Rac/Cdc42 GAP mediating Robo repulsion in tracheal cells and axons. Genes Dev 18:2161–2171CrossRefGoogle Scholar
  11. 11.
    Lim J, Ritt DA, Zhou M, Morrison DK (2014) The CNK2 scaffold interacts with vilse and modulates Rac cycling during spine morphogenesis in hippocampal neurons. Curr Biol 24:786–792CrossRefGoogle Scholar
  12. 12.
    Lee JY, Lee LJ, Fan CC, Chang HC, Shih HA, Min MY, Chang MS (2017) Important roles of Vilse in dendritic architecture and synaptic plasticity. Sci Rep 7:45646CrossRefGoogle Scholar
  13. 13.
    Xu NJ, Henkemeyer M (2009) Ephrin-B3 reverse signaling through Grb4 and cytoskeletal regulators mediates axon pruning. Nat Neurosci 12:268–276CrossRefGoogle Scholar
  14. 14.
    Zhu XN, Liu XD, Zhuang H, Henkemeyer M, Yang JY, Xu NJ (2016) Amygdala EphB2 signaling regulates glutamatergic neuron maturation and innate fear. J Neurosci 36:10151–10162CrossRefGoogle Scholar
  15. 15.
    Huang GH, Yang XT, Chen K, Xing J, Guo L, Zhu L, Li HJ, Li XC, Zhang SY, Feng DF (2016) Porf-2 inhibits neural stem cell proliferation through Wnt/beta-catenin pathway by its GAP domain. Front Cell Neurosci 10:85PubMedPubMedCentralGoogle Scholar
  16. 16.
    Yue X, Son AI, Zhou R (2013) Growth cone collapse assay. Methods Mol Biol 1018:221–227CrossRefGoogle Scholar
  17. 17.
    Meyer LA, Kaselis A, Satkauskas S, Bagnard D (2017) Analysis of semaphorin-induced growth cone collapse and axon growth inhibition. Methods Mol Biol 1493:171–183CrossRefGoogle Scholar
  18. 18.
    Zhang M, Song A, Lai S, Qiu L, Huang Y, Chen Q, Zhu B, Xu D, Zheng JC (2015) Applications of stripe assay in the study of CXCL12-mediated neural progenitor cell migration and polarization. Biomaterials 72:163–171CrossRefGoogle Scholar
  19. 19.
    Diez-Roux G, Banfi S, Sultan M, Geffers L, Anand S, Rozado D, Magen A, Canidio E, Pagani M, Peluso I et al (2011) A high-resolution anatomical atlas of the transcriptome in the mouse embryo. PLoS Biol 9:e1000582CrossRefGoogle Scholar
  20. 20.
    Huber AB, Kolodkin AL, Ginty DD, Cloutier JF (2003) Signaling at the growth cone: ligand–receptor complexes and the control of axon growth and guidance. Annu Rev Neurosci 26:509–563CrossRefGoogle Scholar
  21. 21.
    Dent EW, Gupton SL, Gertler FB (2011) The growth cone cytoskeleton in axon outgrowth and guidance. Cold Spring Harb Perspect Biol 3:a001727CrossRefGoogle Scholar
  22. 22.
    Robichaux MA, Chenaux G, Ho HY, Soskis MJ, Dravis C, Kwan KY, Sestan N, Greenberg ME, Henkemeyer M, Cowan CW (2014) EphB receptor forward signaling regulates area-specific reciprocal thalamic and cortical axon pathfinding. Proc Natl Acad Sci USA 111:2188–2193CrossRefGoogle Scholar
  23. 23.
    Egea J, Klein R (2007) Bidirectional Eph-ephrin signaling during axon guidance. Trends Cell Biol 17:230–238CrossRefGoogle Scholar
  24. 24.
    Nowak FV (2014) Preoptic regulatory factor-2, a Rhogap domain protein that modifies cell cycle progression and apoptosis in the CNS. In: Hayat MA (ed) Stem cells and cancer stem cells, vol 12: Therapeutic applications in disease and injury. Springer, Dordrecht, pp 219–230CrossRefGoogle Scholar
  25. 25.
    Ma S, Nowak FV (2011) The RhoGAP domain-containing protein, Porf-2, inhibits proliferation and enhances apoptosis in neural stem cells. Mol Cell Neurosci 46:573–582CrossRefGoogle Scholar
  26. 26.
    Yang XT, Huang GH, Li HJ, Sun ZL, Xu NJ, Feng DF (2017) Rac1 guides Porf-2 to Wnt pathway to mediate neural stem cell proliferation. Front Mol Neurosci 10:172CrossRefGoogle Scholar
  27. 27.
    Kerber ML, Cheney RE (2011) Myosin-X: a MyTH-FERM myosin at the tips of filopodia. J Cell Sci 124:3733–3741CrossRefGoogle Scholar
  28. 28.
    Lu Q, Li J, Zhang M (2014) Cargo recognition and cargo-mediated regulation of unconventional myosins. Acc Chem Res 47:3061–3070CrossRefGoogle Scholar
  29. 29.
    Weck ML, Grega-Larson NE, Tyska MJ (2017) MyTH4-FERM myosins in the assembly and maintenance of actin-based protrusions. Curr Opin Cell Biol 44:68–78CrossRefGoogle Scholar
  30. 30.
    Koh CG (2006) Rho GTPases and their regulators in neuronal functions and development. Neurosignals 15:228–237CrossRefGoogle Scholar
  31. 31.
    Flanagan JG, Vanderhaeghen P (1998) The ephrins and Eph receptors in neural development. Annu Rev Neurosci 21:309–345CrossRefGoogle Scholar
  32. 32.
    Chen ZY, Sun C, Reuhl K, Bergemann A, Henkemeyer M, Zhou R (2004) Abnormal hippocampal axon bundling in EphB receptor mutant mice. J Neurosci 24:2366–2374CrossRefGoogle Scholar
  33. 33.
    Marler KJ, Becker-Barroso E, Martinez A, Llovera M, Wentzel C, Poopalasundaram S, Hindges R, Soriano E, Comella J, Drescher U (2008) A TrkB/EphrinA interaction controls retinal axon branching and synaptogenesis. J Neurosci 28:12700–12712CrossRefGoogle Scholar
  34. 34.
    Xu NJ, Sun S, Gibson JR, Henkemeyer M (2011) A dual shaping mechanism for postsynaptic ephrin-B3 as a receptor that sculpts dendrites and synapses. Nat Neurosci 14:1421–1429CrossRefGoogle Scholar
  35. 35.
    Penzes P, Beeser A, Chernoff J, Schiller MR, Eipper BA, Mains RE, Huganir RL (2003) Rapid induction of dendritic spine morphogenesis by trans-synaptic ephrinB–EphB receptor activation of the Rho-GEF kalirin. Neuron 37:263–274CrossRefGoogle Scholar
  36. 36.
    Um K, Niu S, Duman JG, Cheng JX, Tu YK, Schwechter B, Liu F, Hiles L, Narayanan AS, Ash RT et al (2014) Dynamic control of excitatory synapse development by a Rac1 GEF/GAP regulatory complex. Dev Cell 29:701–715CrossRefGoogle Scholar

Copyright information

© Springer International Publishing AG, part of Springer Nature 2018

Authors and Affiliations

  • Guo-Hui Huang
    • 1
    • 2
    • 3
  • Lin Guo
    • 4
    • 5
  • Liang Zhu
    • 1
  • Xian-Dong Liu
    • 4
    • 5
  • Zhao-Liang Sun
    • 1
  • Hong-Jiang Li
    • 1
  • Nan-Jie Xu
    • 4
    • 5
    • 6
  • Dong-Fu Feng
    • 1
    • 3
  1. 1.Department of Neurosurgery, Shanghai Ninth People’s HospitalShanghai Jiao Tong University School of MedicineShanghaiChina
  2. 2.Department of Neurosurgery, Shanghai Tenth People’s HospitalTongji University School of MedicineShanghaiChina
  3. 3.Institute of Traumatic MedicineShanghai Jiao Tong University School of MedicineShanghaiChina
  4. 4.Center for Brain Science Research, Department of Anatomy and PhysiologyShanghai Jiao Tong University School of MedicineShanghaiChina
  5. 5.Department of Biochemistry and Molecular Cell Biology, Shanghai Key Laboratory for Tumor Microenvironment and InflammationShanghai Jiao Tong University School of MedicineShanghaiChina
  6. 6.Key Laboratory of Cell Differentiation and Apoptosis of the Chinese Ministry of EducationShanghai Jiao Tong University School of MedicineShanghaiChina

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