Microtubule reorganization is obligatory for growth cone turning

Abstract

To examine the role of microtubules in growth cone turning, we have compared the microtubule organization in growth cones advancing on uniform laminin substrates with their organization in growth cones turning at a laminin-tenascin border. The majority (82%) of growth cones on laminin had a symmetrical microtubule organization, in which the microtubules entering the growth cone splay out toward the periphery of the growth cone. Growth cones at tenascin borders had symmetrically arranged microtubules in only 34% of cases, whereas in the majority of cases the microtubules were displaced toward one-half of the growth cone, presumably stabilizing in the direction of the turn along the tenascin border. These results suggest that reorganization of microtubules could underlie growth cone turning. Further evidence for the involvement of microtubule rearrangement in growth cone turning was provided by experiments in which growth cones approached tenascin borders in the presence of nanomolar concentrations of the microtubule stabilizing compound, Taxol. Taxol altered the organization of microtubules in growth cones growing on laminin by restricting their distribution to the proximal regions of the growth cone and increasing their bundling. Taxol did not stop growth cone advance on laminin. When growing in the presence of Taxol, growth cones at tenascin borders were not able to turn and grow along the laminin-tenascin border, and consequently stopped at the border. Growth cones were arrested at borders for as long as Taxol was present (up to 6 h) without showing any signs of drug toxicity. These effects of Taxol were reversible. Together, these results suggest that microtubule reorganization in growth cones is a necessary event in growth cone turning.

Axon Branching Requires Interactions between Dynamic Microtubules and Actin Filaments

“…We found that branching from the growth cone and the axon shaft is always preceded by splaying apart of looped or bundled microtubules which is accompanied by localized accumulation of F-actin. Dynamic microtubules colocalize with F-actin in transition regions of growth cones and axon branch points, consistent with observations in fixed growth cones (Bridgman and Dailey, 1989;Challacombe et al, 1996Challacombe et al, , 1997Williamson et al, 1996;Rochlin et al, 1999), whereas F-actin is excluded from regions of stable microtubules (Fig. 9).…”

“…Cultures were then mounted in 80% glycerol and PBS. To quantif y the distributions of tyrosinated and acetylated microtubules in relation to F-actin, cortical neuronal cultures were simultaneously extracted and fixed to preserve the majority of F-actin and microtubules but to extract cytoplasmic G-actin and tubulin (Challacombe et al, 1996;Williamson et al, 1996). This microtubule-F-actin fixative was composed of 4% paraformaldehyde, 0.25% glutaraldehyde (EM Sciences), 0.1% Triton X-100 (Sigma), 10 M taxol (Sigma), and 1.3 M phalloidin (Molecular Probes) in PH EM buffer (60 mM PI PES, 25 mM H EPES, 10 mM EGTA, and 2 mM MgC l 2 , pH 6.9).…”

“…Dynamic microtubules are capable of growth and shortening through cycles of polymerization and depolymerization (Mitchison and Kirschner, 1984;Desai and Mitchison, 1997) and previous studies have shown the importance of dynamic microtubules in growth cone turning at inhibitory boundaries Williamson et al, 1996;Challacombe et al, 1997). To determine whether F-actin-microtubule interaction is necessary for branch formation, we used drug treatments that selectively inhibited cytoskeletal dynamics.…”

“…Changes in the direction of axon outgrowth depend on reorganization of the microtubule and actin cytoskeleton (Lin and Forscher, 1993;Challacombe et al, 1996Challacombe et al, , 1997Williamson et al, 1996;Suter et al, 1998;Gallo and Letourneau, 1999). Microtubules in the central region of advancing growth cones are splayed apart but become bundled and form loops in slowly growing axons (Tsui et al, 1984;Sabry et al, 1991;Tanaka and Kirschner, 1991).…”

A Role for Epsin N-terminal Homology/AP180 N-terminal Homology (ENTH/ANTH) Domains in Tubulin Binding

“…This is consistent with the observation that PC12 cells require tubulin polymerization and reorganization of the microtubule cytoskeleton for neurite outgrowth to occur (47)(48)(49)(50)(51). To investigate whether ENTH domains might also be able to influence tubulin polymerization, we examined for alterations in neurite outgrowth in PC12 cells expressing ENTH domains.…”