title: Differential requirement of F-actin and microtubule cytoskeleton in cue-induced local protein synthesis in axonal growth cones.
abstract: BACKGROUND
  Local protein synthesis (LPS) via receptor-mediated signaling plays a role in the directional responses of axons to extrinsic cues. An intact cytoskeleton is critical to enact these responses, but it is not known whether the two major cytoskeletal elements, F-actin and microtubules, have any roles in regulating axonal protein synthesis.
  
  
  RESULTS
  Here, we show that pharmacological disruption of either microtubules or actin filaments in growth cones blocks netrin-1-induced de novo synthesis of proteins, as measured by metabolic incorporation of labeled amino acids, implicating both elements in axonal synthesis. However, comparative analysis of the activated translation initiation regulator, eIF4E-BP1, revealed a striking difference in the point of action of the two elements: actin disruption completely inhibited netrin-1-induced eIF4E-BP1 phosphorylation while microtubule disruption had no effect. An intact F-actin, but not microtubule, cytoskeleton was also required for netrin-1-induced activation of the PI3K/Akt/mTOR pathway, upstream of translation initiation. Downstream of translation initiation, microtubules were required for netrin-1-induced activation of eukaryotic elongation factor 2 kinase (eEF2K) and eEF2.
  
  
  CONCLUSIONS
  Taken together, our results show that while actin and microtubules are both crucial for cue-induced axonal protein synthesis, they serve distinct roles with F-actin being required for the initiation of translation and microtubules acting later at the elongation step.
authors.affiliation: Department of Physiology, Development and Neuroscience, University of Cambridge, Downing street, Cambridge, CB2 3DY, UK. m.piper@uq.edu.au.|Current address: The School of Biomedical Sciences and the Queensland Brain Institute, The University of Queensland, St Lucia, QLD, 4072, Australia. m.piper@uq.edu.au.
authors.forename: Michael
authors.initials: M
authors.lastname: Piper
authors.affiliation: Department of Physiology, Development and Neuroscience, University of Cambridge, Downing street, Cambridge, CB2 3DY, UK. orcalee@gmail.com.|Current address: Institute of Neuroscience, Chinese Academy of Sciences, Shanghai, 200031, China. orcalee@gmail.com.
authors.forename: Aih Cheun
authors.initials: AC
authors.lastname: Lee
authors.affiliation: Department of Physiology, Development and Neuroscience, University of Cambridge, Downing street, Cambridge, CB2 3DY, UK. francisca.wollerton@f-star.com.
authors.forename: Francisca P G
authors.initials: FP
authors.lastname: van Horck
authors.affiliation: Department of Physiology, Development and Neuroscience, University of Cambridge, Downing street, Cambridge, CB2 3DY, UK. heather.mcneilly@cantab.net.
authors.forename: Heather
authors.initials: H
authors.lastname: McNeilly
authors.affiliation: Department of Physiology, Development and Neuroscience, University of Cambridge, Downing street, Cambridge, CB2 3DY, UK. doggieadastra@gmail.com.
authors.forename: Trina Bo
authors.initials: TB
authors.lastname: Lu
authors.affiliation: Department of Physiology, Development and Neuroscience, University of Cambridge, Downing street, Cambridge, CB2 3DY, UK. wah20@cam.ac.uk.
authors.forename: William A
authors.initials: WA
authors.lastname: Harris
authors.affiliation: Department of Physiology, Development and Neuroscience, University of Cambridge, Downing street, Cambridge, CB2 3DY, UK. ceh33@cam.ac.uk.
authors.forename: Christine E
authors.initials: CE
authors.lastname: Holt
mesh_terms: D000199:Actins / Q000037:antagonists & inhibitors / Q000502:physiology*; D000818:Animals; D019086:Bridged Bicyclo Compounds, Heterocyclic / Q000494:pharmacology; D002352:Carrier Proteins / Q000378:metabolism; D003078:Colchicine / Q000494:pharmacology; D015638:Cytochalasin D / Q000494:pharmacology; D003599:Cytoskeleton / Q000187:drug effects / Q000648:ultrastructure; D054736:Elongation Factor 2 Kinase / Q000378:metabolism; D005786:Gene Expression Regulation; D020439:Growth Cones / Q000378:metabolism*; D008870:Microtubules / Q000502:physiology*; D009414:Nerve Growth Factors / Q000502:physiology; D009419:Nerve Tissue Proteins / Q000096:biosynthesis* / Q000235:genetics; D000075388:Netrin-1; D015739:Nocodazole / Q000494:pharmacology; D009865:Oocytes; D010441:Peptide Chain Elongation, Translational / Q000502:physiology*; D010442:Peptide Chain Initiation, Translational / Q000502:physiology*; D020652:Peptide Elongation Factor 2 / Q000378:metabolism; D019869:Phosphatidylinositol 3-Kinases / Q000502:physiology; D010766:Phosphorylation; D011499:Protein Processing, Post-Translational; D015398:Signal Transduction / Q000502:physiology; D053778:Thiazolidines / Q000494:pharmacology; D050257:Tubulin Modulators / Q000494:pharmacology; D025521:Tumor Suppressor Proteins / Q000502:physiology; D029867:Xenopus Proteins / Q000172:deficiency / Q000378:metabolism / Q000502:physiology*; D014982:Xenopus laevis
chemical_list: D000199:Actins; D019086:Bridged Bicyclo Compounds, Heterocyclic; D002352:Carrier Proteins; D009414:Nerve Growth Factors; D009419:Nerve Tissue Proteins; D020652:Peptide Elongation Factor 2; D053778:Thiazolidines; D050257:Tubulin Modulators; D025521:Tumor Suppressor Proteins; D029867:Xenopus Proteins; C000604142:eIF4E-BP1 protein, Xenopus; D000075388:Netrin-1; D015638:Cytochalasin D; D019869:Phosphatidylinositol 3-Kinases; D054736:Elongation Factor 2 Kinase; C037068:latrunculin B; D015739:Nocodazole; D003078:Colchicine