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Previous Article | Next Article
Volume 17, Number 9, Issue of May 1, 1997 pp. 3085-3095
Copyright ©1997 Society for NeuroscienceDynamic Microtubule Ends Are Required for Growth Cone Turning to Avoid an Inhibitory Guidance Cue
Jean F. Challacombe, Diane M. Snow, and Paul C. Letourneau Department of Cell Biology and Neuroanatomy, The University of Minnesota, Minneapolis, Minnesota 55455
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 instability
INTRODUCTION
Growth 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
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
MTs are dynamic polymers, undergoing assembly and disassembly at their ends. These events are regulated by the gain and loss of a GTP cap from MT ends (Mitchison and Kirschner, 1984
Although these studies provide evidence that dynamic MTs are involved in neurite elongation, no one has examined their role in growth cone guidance. We used low concentrations of taxol and vinblastine to test the hypothesis that dynamic MT ends are required for the selective steering of MTs that is necessary for growth cone turning to avoid an inhibitory guidance cue, chondroitin sulfate proteoglycan (CSPG). Our results show that taxol and vinblastine partially reduced the rate of neurite elongation and completely inhibited growth cone turning. These findings indicate that splayed dynamic MT ends in the growth cone P-domain are necessary for the redirection of MTs and initiation of growth cone turning.
MATERIALS AND METHODS
Preparation of substrata. Heat-treated glass coverslips (24 × 30 mm) were mounted over 22 mm holes drilled into the bottom of 50 × 9 mm tissue culture dishes (Falcon Labware, Oxnard, CA) using aquarium sealant (Dow-Corning, Midland, MI). Coverslips were UV-sterilized for 30 min, coated with 0.1 mg/ml poly-L-lysine for 1 hr at 40°C, rinsed, and coated with nitrocellulose as described previously (Lagenaur and Lemmon, 1987
Cell culture. Dorsal root ganglia (DRG) were dissected from embryonic days 9-11 white Leghorn chicken embryos and cut into pieces to make explants. Explants were suspended in supplemented serum-free HEPES-buffered F14 medium (Letourneau et al., 1990
Videomicroscopy. After incubation, a culture dish was placed on the stage of an inverted microscope (Diaphot, Nikon, Garden City, NY) under an air curtain incubator (ASI 400, Carl Zeiss, Thornwood, NY) at 40°C. Growth cones were viewed by phase-contrast optics while rhodamine-labeled CSPG stripes were located under epi-illumination. Time-lapse phase-contrast images were acquired with a Newvicon video camera (Dage-MTI, Michigan City, IN) enhanced using Image 1 software (Universal Imaging, West Chester, PA) and recorded with an optical disk recorder (Panasonic TQ-2026F or TQ-3038F, Panasonic Industrial, Secaucus, NJ).
Images of a microscope field containing several growth cones approaching a CSPG stripe were recorded once per minute until the growth cones were 100 µm from the CSPG border. Then culture medium was replaced by medium containing either 7 nM taxol (Natural Products Branch, National Cancer Institute, Bethesda, MD) or 4 nM vinblastine (Sigma, St. Louis, MO) diluted from stock solutions dissolved in dimethyl sulfoxide (DMSO) at concentrations of DMSO that did not exceed 3 µl/ml. A similar volume of DMSO alone was added to control cultures. Growth rates were calculated by measuring the distance that growth cones migrated (in micrometers) in successive recorded image frames using Image 1 software.
Fixation and immunocytochemistry. Cultures were fixed and extracted simultaneously for 10 min with PHEM buffer (60 mM PIPES, 25 mM HEPES, 10 mM EGTA, 2 mM MgCl2, pH 6.9) (Schliwa and van Blerkom, 1981
Confocal microscopy. Immunofluorescence images were acquired with either a BioRad MRC 1000 (Challacombe et al., 1996
Quantitation of MT organization in growth cones. To quantitatively examine the organization of MTs in taxol- and vinblastine-treated growth cones, images of tyrosinated and detyrosinated
RESULTS
Effects of taxol and vinblastine on growth cone migration rate
Low concentrations of taxol and vinblastine have recently been shown to be effective in reducing MT dynamics without dramatically affecting growth cone motility (Yu and Baas, 1995
Table 1. Rates of growth cone migration in the presence of low concentrations of taxol and vinblastine
Treatment Growth cone migration rate (no. of growth cones) DMSO control 45.1 ± 5.5 (18) Pre-taxol 39.9 ± 4.2 (26) 7 nM taxol 39.1 ± 3.2 (42) Taxol washout 40.8 ± 3.2 (16) Pre-vinblastine 43.9 ± 3.6 (30) 4 nM vinblastine 29.6 ± 1.5 (78)a Vinblastine washout 25.7 ± 2.9 (22)a Growth cone migration rates were calculated by measuring the distances individual growth cones migrated (in micrometers) over a 15-60 min period. Rates were expressed as µm/hr ± SEM. Differences in growth cone migration rates among all groups were assessed by the Kruskal-Wallis test with post hoc multiple comparison. a Significantly slower than pre-vinblastine rate (p < 0.05). Taxol and vinblastine prevent growth cone turning
To assess the role of MT dynamics in growth cone turning, we examined the behavior of growth cones in the presence and absence of vinblastine and taxol using our in vitro guidance assay, a substratum composed of alternating stripes of FN and CSPG (Snow et al., 1990
Table 2. Effects of taxol and vinblastine on growth cone behavior at FN/CSPG borders
Treatment Stopped Sidestepped Turned Combinations of stopped/ retracted/ sidestepped Misc.a DMSO controlb 0% 17% (4) 61% (14) 0% 22% (5)c Taxol (7 nM) 11.1% (2) 16.7% (3) 0% 66.7% (12) 5.5% (1)d Vinblastine (4 nM) 23.8% (5) 9.5% (2) 0% 57.2% (12) 9.5% (2)e Growth cone behaviors at FN/CSPG borders were categorized by reviewing video records of growth cones that approached, then contacted, a CSPG stripe. Growth cones were observed for several hours after first contact with CSPG. For each treatment, growth cone behaviors at FN/CSPG borders were noted. The number of growth cones exhibiting each behavior is given in parentheses. a Misc., Miscellaneous. b Data for DMSO controls from Challacombe et al. (1996) 
Fig. 1. Phase-contrast video sequences showing a control growth cone that turned to avoid a CSPG border compared with vinblastine- and taxol-treated growth cones that failed to turn. A-D, Turning control growth cone. E-H, Growth cone that stopped at the border in the presence of 4 nM vinblastine. I-L, 7 nM taxol-treated growth cone that sidestepped and stopped at the CSPG border. Arrowheads in I-L mark the position of the taxol-treated growth cone at the border and show that it sidestepped laterally along the border for a short distance. All three sequences include the first filopodial contact with CSPG (A, E, I) followed by C-domain advance to the FN/CSPG border (B, F, J), continued filopodial sampling of the CSPG stripe (C, G, K), and either turning (control; D) or continued interaction with the border (drug-treated; H, L). Drug-treated growth cones did not turn, even after interacting with the CSPG border for periods of 4-6 hr or overnight. For each sequence, the time interval after first filopodial contact is shown in the subsequent frames. Scale bar, 10 µm.
[View Larger Version of this Image (118K GIF file)]
Taxol and vinblastine alter MT organization
Because low concentrations of vinblastine and taxol prevented growth cone turning, we examined the organization of MTs and AFs in recorded growth cones that were migrating on homogeneous FN or at a CSPG border. To obtain fluorescent images of the cytoskeleton in the previously recorded growth cones, culture dishes were fixed and immunocytochemically labeled with fluorescent phalloidin and with antibodies directed against tyrosinated and detyrosinated
When untreated DRG growth cones migrate on homogeneous FN, the MT bundle extends throughout the C-domain, and individual Tyr-MTs splay into the P-domain where they overlap with AF bundles at the bases of filopodia. In contrast to controls, in vinblastine- and taxol-treated growth cones migrating on homogeneous FN, Tyr-MTs were less splayed throughout the growth cone (Fig. 2). In control growth cones migrating on FN, phalloidin-labeled AFs were present at the leading edge and extended as bundles within filopodia (Fig. 2A), whereas the ends of Tyr-MTs (Fig. 2B) projected into the P-domain, overlapping with AFs at the bases of filopodia. Detyr-MTs were confined to the C-domain where some of them formed loops (Fig. 2C). In growth cones treated with 4 nM vinblastine (Fig. 2D) and 7 nM taxol (Fig. 2G), phalloidin-labeled AFs were present at the leading edge and in filopodia. However, Tyr-MTs (Fig. 2E,H, arrows) were less splayed than in control growth cones as they extended forward into the AF-rich P-domain. Another difference between control and drug-treated growth cones was that in control growth cones, Detyr-MTs formed loops in the C-domain (Fig. 2C), whereas in vinblastine- (Fig. 2F) and taxol-treated (Fig. 2I) growth cones, such looped Detyr-MTs were less prevalent. In growth cones migrating on FN and exposed to vinblastine or taxol for 1-2 hr followed by washout of the drugs for 1-2 hr (Fig. 2J-L), the MTs were even more splayed than in controls. In a representative growth cone exposed to vinblastine, which was subsequently washed out, phalloidin-labeled AFs were present at the leading edge (Fig. 2J), the ends of Tyr-MTs were widely splayed throughout the P-domain (Fig. 2K), and Detyr-MTs formed loops in the C-domain (Fig. 2L). 
Fig. 2. Comparison of AF and MT organization in chick DRG growth cones migrating on FN in the presence and absence of 4 nM vinblastine and 7 nM taxol. A, D, G, J, Phalloidin-labeled AFs. B, E, H, K, Tyrosinated
[View Larger Version of this Image (69K GIF file)]
Figure 3 compares the organization of AFs and MTs in a control growth cone turning at a CSPG border with vinblastine- and taxol-treated growth cones that were either stopped or sidestepping at a CSPG border. In the turning control growth cone (Fig. 3A-C), the ends of Tyr-MTs (Fig. 3B) and Detyr-MTs (Fig. 3C) are bundled and turned to be aligned along the border, with Tyr-MT ends projecting into the bases of AF-rich filopodia at the growth cone tip (see also Fig. 4D). The arrangement of MTs and AFs in vinblastine- and taxol-treated growth cones at a CSPG border is shown in Figure 3D-I. The organization of Tyr-MTs (Fig. 3E,H) and Detyr-MTs (Fig. 3F,I) within the growth cones is similar to the MT organization in the drug-treated growth cones migrating on FN (Fig. 2E,F,H,I). 
Fig. 3. Comparison of AF and MT organization in chick DRG growth cones at a CSPG border in the presence and absence of 4 nM vinblastine and 7 nM taxol. A, D, G, Phalloidin-labeled AFs. B, E, H, Tyrosinated
[View Larger Version of this Image (144K GIF file)]
Fig. 4. Merged three-color images showing the organization of AFs and MTs in growth cones migrating on FN and at CSPG borders in the presence and absence of vinblastine and taxol. A-C correspond to Figure 2, and D-F correspond to Figure 3. In F, the CSPG border was digitally enhanced to make it more visible. In all panels, AF labeling is blue, tyrosinated
[View Larger Version of this Image (111K GIF file)]
A more direct comparison of the MT distribution between vinblastine- and taxol-treated growth cones and controls can be made by examining the three-color images in Figure 4. A-C correspond to Figure 2, and D-F correspond to Figure 3. The splaying and close proximity of Tyr-MT ends (red) to AFs (blue) are apparent in the control growth cones (Fig. 4A,D), as is the reduced splaying of Tyr-MT ends (red) in the vinblastine- (Fig. 4B) and taxol-treated (Fig. 4C) growth cones migrating on FN. Figure 4 also illustrates the bundling and turning of the MTs in the control growth cone that was turning at a CSPG border (Fig. 4D), whereas the configuration of Tyr-MTs in the vinblastine- (Fig. 4E) and taxol-treated (Fig. 4F) growth cones that were not turning is similar to that of drug-treated growth cones migrating on FN. Quantitative analysis of MT organization in vinblastine- and taxol-treated growth cones
In a previous study, we quantitatively analyzed the organization of Tyr-MTs, Detyr-MTs, and AFs in turning growth cones and in growth cones that failed to turn after treatment with cytochalasin B (Challacombe et al., 1996
Fig. 5. Schematic diagram of a growth cone illustrating how measurements of MT organization were made. Detyr-MTs are shown in black, and Tyr-MT ends are gray. Measurements from the leading edge (excluding filopodia) to the distal extents of Tyr-MTs and Detyr-MTs are depicted by the thin black double-headed arrows marked a and b, respectively. The width of the Tyr-MT ends is represented by a thicker black double-headed arrow (c), and the width of Detyr-MT staining is illustrated by a white double-headed arrow (d). The width of the growth cone (not marked) was from side to side at the widest point, excluding filopodia.
[View Larger Version of this Image (17K GIF file)]
Results of this analysis (Table 3) showed that, for growth cones migrating on homogeneous FN, the mean widths of Tyr-MT staining in vinblastine- and taxol-treated growth cones were significantly decreased compared with control growth cones. In addition, the mean growth cone widths also decreased. These results indicate that under the influence of vinblastine and taxol, the distal Tyr-MT ends are significantly less splayed in the P-domain, and the growth cones are narrower than control growth cones. Vinblastine and taxol treatment also resulted in a significant decrease in the distance from the leading edge to the distal extents of Tyr-MTs but not Detyr-MTs, indicating that the distance between the distal extents of Tyr- and Detyr-MTs was greater than in controls. However, the distal limit of detyrosination may not indicate the limit of MT stability in the presence of these drugs, because the Tyr-MT ends may become stabilized before Tyr-tubulin is accessible to the enzyme responsible for detyrosination. A significant observation was that when taxol and vinblastine were washed out, growth cones became significantly wider than even the control growth cones, and the splayed width of Tyr-MT ends was also increased significantly beyond controls. This observation supports a link between the dynamic growth and shrinkage of the distal MT ends and a splayed MT distribution in the P-domain of growth cones.
Table 3. Quantitation of MT organization in growth cones migrating on FN and interacting with a CSPG border in the presence and absence of 4 nM vinblastine and 7 nM taxol
Substratum Treatment Leading edge to Tyr-MTs Leading edge to Detyr-MTs Width Tyr-MTs Width Detyr-MTs Width growth cone Homogeneous Control 5.63 ± 0.34 9.10 ± 0.40 7.85 ± 0.89 4.01 ± 0.58 16.44 ± 1.50 FN (178) (150) (45) (44) (43) 4 nM 3.04 ± 0.54* 9.3 ± 0.78 3.85 ± 0.54* 2.43 ± 0.23 7.47 ± 1.03* vinblastine (20) (20) (15) (15) (15) 7 nM taxol 2.57 ± 0.50* 8.72 ± 0.77 2.88 ± 0.44* 3.14 ± 0.60 7.83 ± 1.01* (16) (16) (15) (17) (15) Vinblastine 3.77 ± 0.69 7.80 ± 0.96 13.64 ± 2.90** 8.84 ± 2.31** 22.94 ± 3.06** washout (23) (22) (11) (11) (11) Taxol 4.28 ± 0.54 9.37 ± 0.67 13.73 ± 2.38** 7.37 ± 1.24** 26.86 ± 2.51** washout (27) (25) (10) (10) (10) CSPG border Control 4.66 ± 0.38 7.94 ± 0.49 4.39 ± 0.46 2.17 ± 0.20 9.25 ± 0.97 turning (107) (92) (41) (41) (41) 4 nM 1.76 ± 0.50* 6.80 ± 0.65 3.80 ± 0.59 2.46 ± 0.29 8.27 ± 1.10 vinblastine (20) (20) (21) (21) (19) 7 nM taxol 1.72 ± 0.65* 6.22 ± 0.75 5.01 ± 1.59 3.61 ± 1.17 7.63 ± 0.91 (20) (20) (15) (15) (13) The measurement categories represent mean ± SEM distances in micrometers. For each substratum, the Kruskal-Wallis test with post hoc multiple comparison was used to assess the significance of differences between the distributions of measurements in each category. * p < 0.05, significantly different from control. ** p < 0.05, significantly different from all other groups.
Table 3 also shows a similar analysis of these MT parameters in growth cones at a CSPG border. Controls were turning, and vinblastine- and taxol-treated growth cones were either stopped at a border or sidestepping along it before fixation. Results of this analysis show that the altered MT organization induced by vinblastine and taxol treatment persists in growth cones interacting with a CSPG border. The distance from the leading edge to Tyr-MTs is significantly smaller than in controls, and the distance from the leading edge to Detyr-MTs is similar to controls. Furthermore, the widths of Tyr- and Detyr-MT staining, as well as the widths of drug-treated growth cones, are similar to controls. One of the key changes in growth cone morphology and MT organization that occurs during normal turning behavior is the bundling of the MT ends and narrowing of the growth cone (Tanaka and Kirschner, 1995
DISCUSSION
The goal of this study was to examine the role of dynamic MTs in growth cone turning by using low concentrations of vinblastine and taxol. Vinblastine and taxol are unrelated plant metabolites that suppress MT dynamics at low substoichiometric concentrations, although they bind to different sites on MTs (Jordan et al., 1991
When control growth cones migrate to and contact a CSPG border, they turn or stop to avoid crossing the border (Challacombe et al., 1996
Previous studies used low concentrations of MT-specific drugs to investigate the role of dynamic MTs in neurite elongation. Low doses of vinblastine and nocodazole inhibited axonal elongation from Xenopus neural tube (Tanaka et al., 1995
Our results do show that dynamic MTs in the growth cone are needed for responses to guidance cues, such as turning away from CSPG. In support of this idea, observations of MT ends in living cells indicate that MT polymerization is responsible, at least in part, for advancing MTs from the growth cone C-domain into the P-domain (Tanaka et al., 1995
Our data indicate that vinblastine- and taxol-treated growth cones fail to turn because their MT organization is altered such that MTs are not steered to one side. How does the reduction in MT dynamics by vinblastine and taxol interfere with the MT rearrangements that are crucial for growth cone turning? One possibility is that, because the MTs of vinblastine- and taxol-treated growth cones are less splayed in the P-domain, the MT ends may not interact to a sufficient degree with AF bundles, and are therefore not steered to one side to initiate a growth cone turn (Fig. 6). 
Fig. 6. A, A model for growth cone turning. In a growth cone migrating on homogeneous FN, stable MTs (black) are bundled in the neurite by the activity of MAPs (striped rectangles) and extend into the C-domain, whereas dynamic MT ends (gray) splay throughout the P-domain. Some dynamic MT ends are linked to AF bundles (thin black lines) at the bases of filopodia by an unknown linkage molecule (filled circles). When the growth cone contacts a CSPG border, filopodia sample the CSPG, but these are unstable contacts. Some filopodia remain on FN, forming more stable contacts with the substratum. When the AF bundles of these filopodia are linked to dynamic MT ends, the AF bundles steer the dynamic MT ends asymmetrically along the border to initiate the turn. During turning, the growth cone MTs become bundled and aligned along the border. B, A growth cone that fails to turn in the presence of low concentrations of taxol or vinblastine. In a growth cone treated with taxol or vinblastine, the MT ends in the P-domain are less dynamic and become less splayed while the growth cone is migrating on FN. When the growth cone contacts a CSPG border, filopodia sample the CSPG but the distal MTs remain less splayed in the P-domain, the MT ends are not steered by AFs, and the growth cone fails to turn. This may be attributable to a premature stabilization and stiffening of the MT ends in the P-domain, resulting either directly from drug-induced changes in MT properties or from increased binding of MAPs to the less dynamic MTs. The failure to turn also may be because of an inability of the linkage molecule to connect AFs to the less dynamic MT ends.
[View Larger Version of this Image (53K GIF file)]
The reduced dynamics of these MT ends may lead to a premature stabilization and stiffening of the MT ends in the P-domain, so they assume the bundled configuration that is seen in the C-domain and more proximal growth cone (Fig. 6B). This reduced splaying of the MT ends may result either directly from drug-induced changes in MT properties (Mickey and Howard, 1995
Based on results from other studies of the effects of MT drugs on cell motility, we suggest that another contribution to the inhibition of growth cone turning by these drugs is that dynamic MT ends are necessary for active remodeling of AF networks in the growth cone P-domain. Bershadsky et al. (1991)
We noted that when taxol and vinblastine were removed from the culture medium of growth cones migrating on FN, the distal Tyr-MT ends recovered and became widely splayed in the P-domain, and growth cones were wider than control growth cones. This result supports our hypothesis that the splayed configuration of MT ends in the P-domain of growth cones on FN requires dynamic MT ends (but not AF bundles, because cytochalasin B-treated growth cones contain widely splayed MTs) (Challacombe et al., 1996
In summary, we found that low concentrations of taxol and vinblastine, which reduce MT dynamics, prevent DRG growth cone turning. These results support the hypothesis proposed by Tanaka et al. (1995)
FOOTNOTES
Received Nov. 7, 1996; revised Feb. 5, 1997; accepted Feb. 10, 1997.
This work was supported by National Institutes of Health Grants HD19950 (P.C.L.), F32-NS09971 (J.F.C.), and EY10545 (D.M.S.), and the Minnesota Medical Foundation. We thank Dr. G. Gallo for suggestions on this manuscript, Drs. A. I. Caplan and D. A. Carrino for their generous gift of chick limb bud CSPG, and Drs. J. C. Bulinski and G. G. Gundersen for providing antiserum against detyrosinated
Correspondence should be addressed to Jean F. Challacombe, Department of Cell Biology and Neuroanatomy, The University of Minnesota, 4-144 Jackson Hall, 321 Church Street SE, Minneapolis, MN 55455.
Dr. Snow's present address: Department of Anatomy and Neurobiology, The University of Kentucky, Lexington, KY 40536.
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