title: Nerve growth factor promotes reorganization of the axonal microtubule array at sites of axon collateral branching.
abstract: The localized debundling of the axonal microtubule array and the entry of microtubules into axonal filopodia are two defining features of collateral branching. We report that nerve growth factor (NGF), a branch-inducing signal, increases the frequency of microtubule debundling along the axon shaft of chicken embryonic sensory neurons. Sites of debundling correlate strongly with the localized targeting of microtubules into filopodia. Platinum replica electron microscopy suggests physical interactions between debundled microtubules and axonal actin filaments. However, as evidenced by depolymerization of actin filaments and inhibition of myosin II, actomyosin force generation does not promote debundling. In contrast, loss of actin filaments or inhibition of myosin II activity promotes debundling, indicating that axonal actomyosin forces suppress debundling. MAP1B is a microtubule associated protein that represses axon branching. Following treatment with NGF, microtubules penetrating filopodia during the early stages of branching exhibited lower levels of associated MAP1B. NGF increased and decreased the levels of MAP1B phosphorylated at a GSK-3β site (pMAP1B) along the axon shaft and within axonal filopodia, respectively. The levels of MAP1B and pMAP1B were not altered at sites of debundling, relative to the rest of the axon. Unlike the previously determined effects of NGF on the axonal actin cytoskeleton, the effects of NGF on microtubule debundling were not affected by inhibition of protein synthesis. Collectively, these data indicate that NGF promotes localized axonal microtubule debundling, that actomyosin forces antagonize microtubule debundling, and that NGF regulates pMAP1B in axonal filopodia during the early stages of collateral branch formation.
authors.affiliation: Department of Anatomy and Cell Biology, Shriners Hospitals Pediatric Research Center, Temple University, Philadelphia, Pennsylvania, 19140.
authors.forename: Andrea
authors.initials: A
authors.lastname: Ketschek
authors.affiliation: Department of Biology, University of Pennsylvania, Philadelphia, Pennsylvania, 19104.
authors.forename: Steven
authors.initials: S
authors.lastname: Jones
authors.affiliation: Department of Anatomy and Cell Biology, Shriners Hospitals Pediatric Research Center, Temple University, Philadelphia, Pennsylvania, 19140.
authors.forename: Mirela
authors.initials: M
authors.lastname: Spillane
authors.affiliation: Department of Biology, University of Pennsylvania, Philadelphia, Pennsylvania, 19104.
authors.forename: Farida
authors.initials: F
authors.lastname: Korobova
authors.affiliation: Department of Biology, University of Pennsylvania, Philadelphia, Pennsylvania, 19104.
authors.forename: Tatyana
authors.initials: T
authors.lastname: Svitkina
authors.affiliation: Department of Anatomy and Cell Biology, Shriners Hospitals Pediatric Research Center, Temple University, Philadelphia, Pennsylvania, 19140.
authors.forename: Gianluca
authors.initials: G
authors.lastname: Gallo
mesh_terms: D000818:Animals; D030161:Avian Proteins / Q000378:metabolism*; D001369:Axons / Q000378:metabolism* / Q000648:ultrastructure; D002642:Chick Embryo; D005455:Fluorescent Antibody Technique; D038362:Glycogen Synthase Kinase 3 / Q000378:metabolism; D000071679:Glycogen Synthase Kinase 3 beta; D007150:Immunohistochemistry; D008854:Microscopy, Electron; D008869:Microtubule-Associated Proteins / Q000378:metabolism*; D008870:Microtubules / Q000378:metabolism* / Q000648:ultrastructure; D020932:Nerve Growth Factor / Q000378:metabolism*; D009828:Oleanolic Acid / Q000031:analogs & derivatives; D010766:Phosphorylation; D011554:Pseudopodia / Q000378:metabolism* / Q000648:ultrastructure; D012503:Saponins; D011984:Sensory Receptor Cells / Q000378:metabolism / Q000648:ultrastructure; D014162:Transfection
chemical_list: C116950:2,3,17,23-tetrahydroxy-28-norolean-12-en-16-one-3-O-arabinopyranosyl-1-2-arabinopyranosyl-1-6-glucopyranoside; D030161:Avian Proteins; D008869:Microtubule-Associated Proteins; D012503:Saponins; C085284:microtubule-associated protein 1B; D009828:Oleanolic Acid; D020932:Nerve Growth Factor; D000071679:Glycogen Synthase Kinase 3 beta; D038362:Glycogen Synthase Kinase 3
keywords: GSK-3β; bundling; consolidation; contractility; debundling; microtubule associated protein 1B; myosin II; splaying; sprout; sprouting; transport
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