title: The actin nucleating Arp2/3 complex contributes to the formation of axonal filopodia and branches through the regulation of actin patch precursors to filopodia.
abstract: The emergence of axonal filopodia is the first step in the formation of axon collateral branches. In vitro, axonal filopodia emerge from precursor cytoskeletal structures termed actin patches. However, nothing is known about the cytoskeletal dynamics of the axon leading to the formation of filopodia in the relevant tissue environment. In this study we investigated the role of the actin nucleating Arp2/3 complex in the formation of sensory axon actin patches, filopodia, and branches. By combining in ovo chicken embryo electroporation mediated gene delivery with a novel acute ex vivo spinal cord preparation, we demonstrate that actin patches form along sensory axons and give rise to filopodia in situ. Inhibition of Arp2/3 complex function in vitro and in vivo decreases the number of axonal filopodia. In vitro, Arp2/3 complex subunits and upstream regulators localize to actin patches. Analysis of the organization of actin filaments in actin patches using platinum replica electron microscopy reveals that patches consist of networks of actin filaments, and filaments in axonal filopodia exhibit an organization consistent with the Arp2/3-based convergent elongation mechanism. Nerve growth factor (NGF) promotes formation of axonal filopodia and branches through phosphoinositide 3-kinase (PI3K). Inhibition of the Arp2/3 complex impairs NGF/PI3K-induced formation of axonal actin patches, filopodia, and the formation of collateral branches. Collectively, these data reveal that the Arp2/3 complex contributes to the formation of axon collateral branches through its involvement in the formation of actin patches leading to the emergence of axonal filopodia.
authors.affiliation: Department of Neurobiology and Anatomy, Drexel University College of Medicine, Philadelphia, Pennsylvania 19129, USA.
authors.forename: Mirela
authors.initials: M
authors.lastname: Spillane
authors.forename: Andrea
authors.initials: A
authors.lastname: Ketschek
authors.forename: Steven L
authors.initials: SL
authors.lastname: Jones
authors.forename: Farida
authors.initials: F
authors.lastname: Korobova
authors.forename: Bonnie
authors.initials: B
authors.lastname: Marsick
authors.forename: Lorene
authors.initials: L
authors.lastname: Lanier
authors.forename: Tatyana
authors.initials: T
authors.lastname: Svitkina
authors.forename: Gianluca
authors.initials: G
authors.lastname: Gallo
mesh_terms: D008841:Actin Cytoskeleton / Q000378:metabolism* / Q000502:physiology; D051377:Actin-Related Protein 2 / Q000037:antagonists & inhibitors / Q000502:physiology*; D051378:Actin-Related Protein 3 / Q000037:antagonists & inhibitors / Q000502:physiology*; D000818:Animals; D001369:Axons / Q000502:physiology*; D002454:Cell Differentiation / Q000502:physiology*; D002642:Chick Embryo; D002645:Chickens; D053595:Embryonic Stem Cells / Q000166:cytology / Q000378:metabolism / Q000502:physiology*; D020439:Growth Cones / Q000502:physiology*; D058953:Neural Stem Cells / Q000166:cytology / Q000378:metabolism / Q000502:physiology; D061251:Primary Cell Culture; D011554:Pseudopodia / Q000378:metabolism* / Q000502:physiology; D011984:Sensory Receptor Cells / Q000166:cytology / Q000502:physiology
chemical_list: D051377:Actin-Related Protein 2; D051378:Actin-Related Protein 3