Resul ts Low Concentrations of Vinblastine Inhibit Neurite Outgrowth

A question of broad importance in cellular neurobiology has been, how is microtubule cytoskeleton of the axon organized? It is of particular interest because of the history of conflicting results concerning the form in which tubulin is transported in the axon. While many studies indicate a stationary nature of axonal microtubules, a recent series of experiments reports that microtubules are recruited into axons of neurons grown in the presence of a microtubule-inhibitor, vinblastine (Baas, P.W., and F.J. Ahmad. 1993. J. Cell Biol. 120:1427-1437; Ahmad F.J., and P.W. Baas. 1995. J. Cell Sci. 108:2761-2769; Sharp, D.J., W. Yu, and P.W. Baas. 1995. J. Cell Biol. 130:93-103; Yu, W., and P.W. Baas. 1995. J. Neurosci. 15:6827--6833.). Since vinblastine stabilizes bulk microtubule-dynamics in vitro, it was concluded that preformed microtubules moved into newly grown axons. By visualizing the polymerization of injected fluorescent tubulin, we show that substantial microtubule polymerization occurs in neurons grown at reported vinblastine concentrations. Vinblastine inhibits, in a concentrat ion-dependent manner, both neurite outgrowth and microtubule assembly. More importantly, the neuron growth conditions of low vinblastine concentration allowed us to visualize the footprints of the tubulin wave as it polymerized and depolymerized during its slow axonal transport. In contrast, depolymerization resistant fluorescent microtubules did not move when injected in neurons. We show that tubulin subunits, not microtubules, are the primary form of tubulin transport in neurons. ECAUSE microtubules serve as tracks for fast transport (400 mm/d) of many cellular components into and down the axon, an axonal microtubule array must be elaborated and maintained. To determine the mechanisms by which cytoskeletal components move from their place of synthesis in the cell body to their place of use in the axon, which lacks protein synthetic machinery, Hoffman and Lasek (1975) injected radiolabeled amino acids into neuronal cell bodies in the spinal cord and then followed the progression of the newly synthesized radiolabeled tubulin down the cat sciatic nerve. They observed tubulin movement down the axon as a discrete, but slow (N1-4 mm/d), wave and concluded that polymers may assemble in the cell body and move slowly into and down the axon. Hoffman and Lasek's conclusion was first challenged by Bamburg et al. (1986). They locally applied microtubuledisrupting drugs to the growing tip of the axon or to the cell body and then monitored neurite outgrowth. They found axonal elongation stopped when drug (<0.1 Ixg/ml) was applied to the growing tip, but axonal elongation conPlease address all correspondence to H.C. Joshi, Department of Anatomy and Cell Biology, Emory University School of Medicine, Atlanta, GA 30322. Tel. : (404) 727-0445. Fax: (404) 727-6256. E-mail: Joshi@anatomy.emory. edu 1. Abbreviat ion used in this paper: EGS, ethylene glycol-bis-succinimidylsuccinate. tinued when the same concentration of the drug was applied to the cell body. They concluded that microtubule polymerization at the growth cone was required for neurite outgrowth. These results have since been confirmed and greatly enlarged upon in several studies (Brown et al., 1992; Li and Black, 1996; Okabe and Hirokawa, 1988). Nonetheless, the hypothesis of polymer transport gained support from subsequent experiments where neuronal microtubules were rendered fluorescent by growing them with injected fluorescent tubulin, and then creating a discrete marked zone in the axon by photobleaching. Although the initial study of this type reported the photobleached zone moved down the neurite at the rate of slow axonal transport (Keith, 1987), later experiments using an identical technique showed that the photobleached zone remained stationary (Lim et al., 1990; Okabe and Hirokawa, 1990). The reasons for these differing results are not clear. It is possible that fluorescent microtubules and their associated movement mechanisms may be sensitive to radiation energy used for photobleaching (Vigers et al., 1988). To address this issue, Reinch et al. (1991) used the technique of photoactivation. Photoactivation involves shining a brief pulse of laser light onto tubulins conjugated with a caged fluorophore (Mitchison, 1989). After illumination, the caged fluorophore becomes fluorescent. This technique is less damaging than photobleaching because it requires much lower radiation energy to photoactivate caged tubulin than it takes to photobleach fluorescent tu© The Rockefeller University Press, 0021-9525/96/06/1355/12 $2.00 The Journal of Cell Biology, Volume 133, Number 6, June 1996 1355-1366 1355 on F ebuary 8, 2007 w w w . jc.org D ow nladed fom bulin. To monitor the movement of a photoactivated zone in Xenopus axons, embryos were injected with caged fluorescent tubulin and the embryos were allowed to develop a neural tube. The caged tubulin in the neurites of neurons from the neural tube was then photoactivated, and the position of the photoactivated region was monitored over time. Based upon these experiments, Reinch et al. (1991) reported that the photoactivated mark moved down the axons of Xenopus neurons. Subsequently, Okabe and Hirokawa (1992, 1993) reported that while both photobleaching and photoactivation procedures can show movement in Xenopus axons, these techniques reveal an absence of tubulin transport in mammalian axons. However, the movement of photobleached/photoactivated zone in the Xenopus axons were believed not to be due to slow axonal transport (Okabe and Hirokawa, 1992; Sabre et al., 1995), but rather as a consequence of passive pulling of the axon by the growth cone (Okabe and Hirokawa, 1992; Takeda et al., 1995; Popov et al., 1993). The stationary nature of the axonal microtubules has also been recently reported in vivo in grasshopper and zebra fish neurons (Takeda et al., 1995; Sabry et al., 1995). An alternative approach to the study of microtubule transport has recently been taken by Baas and Ahmad (1993), Ahmad and Baas (1995), Sharp et al. (1995), and Yu and Baas (1995). They report that the relative mass of microtubules in the cell body is less than in the neurites when neurons are grown in nanomolar concentrations of vinblastine. Because low concentrations of vinblastine have been shown to inhibit both polymerization and depolymerization of microtubules in vitro (Toso et al., 1993), Baas and Ahmad (1993) concluded that microtubules that were pre-formed within the cell body must be transported into the newly grown axon. One critical limitation of these studies is that microtubule polymerization rates were not measured under the low drug concentrations used. Thus, the conclusions of Baas and Ahmad (1993), Ahmad and Baas (1995), Sharp et al. (1995), and Yu and Baas (1995) were based upon the assumption that vinblastine stopped microtubule polymerization and depolymerization in cultured neurons. To draw firm conclusions from those observations, it is important to determine quantitatively the effects of vinblastine on microtubule dynamics in neurons. In this paper, we show substantial polymerization of microtubules throughout neurons in the presence of vinblastine concentrations used in previously published reports (Baas and Ahmad, 1993; Ahmad and Baas, 1995; Sharp et al., 1995; Yu and Baas, 1995). A quantitative analysis of microtubule polymerization reveals that injected tubulin moves from the cell body into the axon as a discrete wave of tubulin subunits, and fluorescent microtubules stabilized against depolymerization remain stationary within the neuronal cell bodies. Materials and Methods