Cytoskeletal Dynamics Underlying Collateral Membrane Protrusions Induced by Neurotrophins in Cultured Xenopus Embryonic Neurons Jean Gibney, James Q. Zheng Department of Neuroscience and Cell Biology, University of Medicine and Dentistry of New Jersey, Robert Wood Johnson Medical School, 675 Hoes Lane, Piscataway, New Jersey 08854 Received 19 June 2002; accepted 31 July 2002 ABSTRACT: The establishment and refinement of neuronal connections depend on dynamic modification of the morphology and physiology of developing axons in response to extrinsic factors. In embryonic cultures of Xenopus spinal neurons, acute application of brain-de- rived neurotrophic factor (BDNF) induced rapid collat- eral protrusion of filopodium-like microspikes and la- mellipodia along the neurite processes, leading to a morphologic alternation of the neuron. Both types of membrane protrusions contained high concentrations of actin filaments and depended on the polymerization of the actin cytoskeleton. Immunofluorescent staining, however, revealed the presence of microtubules (MTs) in lamellipodia induced by BDNF. These MTs appeared to have arisen from debundling of MTs in the neurite shaft at the protrusion sites, splaying and extending in the rapidly protruding lamellipodia. Inhibition of microtu- bule polymerization by nocodazole largely abolished the formation of lamellipodia but not of microspikes. Taken together, our results suggest that collateral sprouting of microspikes and lamellipodia involve distinctly different cytoskeletal mechanisms. Although the actin cytoskele- ton is solely responsible for microspike formation, coop- erative efforts by microtubules and actin filaments are essential for lamellipodial protrusion in response to ex- trinsic factors. © 2002 Wiley Periodicals, Inc. J Neurobiol 54: 393– 405, 2003 Keywords: microtubule; actin; sprouting; lamellipodia; microspikes; membrane ruffling INTRODUCTION Filopodia and lamellipodia are two motile structures at the nerve growth cone that are crucial to the growth and guidance of nerve fibers during development (Lockerbie, 1987; Bray and Hollenbeck, 1988; Smith, 1988; Goldberg and Burmeister, 1989). Filopodia and lamellipodia also play an important part in the struc- tural plasticity of neuronal connections. The sprouting of new motile filopodia and lamellipodia from previ- ously quiescent regions of a neuron is fundamental for the development of collateral branches (Bastmeyer et al., 1998) as well as for the dynamic morphologic changes involved in the refinement of neuronal con- nections (Robbins and Polak, 1988; Ziv and Smith, 1996; Fiala et al., 1998; Maletic-Savatic et al., 1999). There is ample evidence that the core structural com- ponent in motile filopodia and lamellipodia is the actin cytoskeleton, and its dynamic reorganization underlies the motile activity of filopodia and lamelli- podia (Smith, 1988; Okabe and Hirokawa, 1991; Lin et al., 1994). Microtubules (MTs), on the other hand, are generally absent from these motile structures in non-neuronal (Heath and Holifield, 1991a,b) as well as neuronal (Smith, 1988) cells. It is believed that the actin-based motility in lamellipodia drives the for- ward movement of the cell or the growth cone fol- Correspondence to: J.Q. Zheng (zhengjq@umdnj.edu). Contract grant sponsor: American Heart Association and Na- tional Institutes of Health. © 2002 Wiley Periodicals, Inc. DOI 10.1002/neu.10149 393