Dynamic Microtubule Ends Are Required for Growth Cone Turning to Avoid an Inhibitory Guidance Cue

Abstract

Growth cone turning is an important mechanism for changing the direction of neurite elongation during development of the nervous system. Our previous study indicated that actin filament bundles at the leading margin direct the distal microtubular cytoskeleton as growth cones turn to avoid substratum-bound chondroitin sulfate proteoglycan. Here, we investigated the role of microtubule dynamics in growth cone turning by using low doses of vinblastine and taxol, treatments that reduce dynamic growth and shrinkage of microtubule ends. We used time-lapse phase-contrast videomicroscopy to observe embryonic chick dorsal root ganglion neuronal growth cones as they encountered a border between fibronectin and chondroitin sulfate proteoglycan in the presence and absence of 4 nM vinblastine or 7 nM taxol. Growth cones were fixed and immunocytochemically labeled to identify actin filaments and microtubules containing tyrosinated and detyrosinated ␣-tubulin.Our results show that after contact with substratum-bound chondroitin sulfate proteoglycan, vinblastine-and taxol-treated growth cones did not turn, as did controls; instead, they stopped or sidestepped. Even before drug-treated growth cones contacted a chondroitin sulfate proteoglycan border, they were narrower than controls, and the distal tyrosinated microtubules were less splayed and were closer to the leading edges of the growth cones. We conclude that the splayed dynamic distal ends of microtubules play a key role in the actin filament-mediated steering of growth cone microtubules to produce growth cone turning. Key words: microtubule; growth cone; turning; actin filament; chondroitin sulfate proteoglycan; dynamic instabilityGrowth cones are the motile tips of elongating axons that guide growing axons to their targets during development of the nervous system. Growth cone navigation involves the detection and integration of extracellular signals, followed by a response that can include forward migration, retraction, branching, and turning. Detection of guidance cues is facilitated by protrusion and retraction of filopodia and lamellipodia from the peripheral region (P-domain) of the growth cone, which contains bundles and networks of actin filaments (AFs) (Letourneau and Ressler, 1983;Lewis and Bridgman, 1992). Axonal elongation depends on the advance of microtubules (MTs), which provide structural support and serve as tracks for axonal transport of membranous organelles. Stable MT bundles project from the axon into the central region (C-domain) of the growth cone, whereas dynamic MT ends splay apart and project into the actin-rich P-domain (Letourneau and Ressler, 1983;Gordon-Weeks, 1991;Challacombe et al., 1996).Recent studies indicate that the advance of MTs into specific growth cone regions initiates responses to guidance cues, such as advance toward a target (Lin and Forscher, 1993), turning toward a positive cue (Bentley and O'Connor, 1994), and turning away from an unfavorable substratum or inhibitory guidance cue (Challacombe et al., 1996). Interactions betw...

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).…”

“…Some studies have used live cell imaging to characterize dynamic changes in the microtubule array or in F-actin, but the approach has been to visualize one or the other cytoskeletal element in living neurons and then to determine accompanying changes in the other element by rapid fixation and staining (Challacombe et al, 1997;Suter et al, 1998;Rochlin et al, 1999;Kabir et al, 2001). However, associations between F-actin and microtubules are likely to be highly dynamic.…”