involvement of microtubules in the regulation of neuronal growth cone morphologic remodeling

Involvement of Microtubules in the Regulation ofNeuronal Growth Cone Morphologic Remodeling

Gianluca Gallo*

Department of Biological Sciences, The University of Illinois at Chicago,845 West Taylor St., Chicago, Illinois 60607

Received 9 October 1997; accepted 12 December 1997

centrifugal displacement of lamellae in response toABSTRACT: The guidance of nerve fibers de-microtubule drugs. During microtubule drug-medi-pends on the constant protrusion, movement, and re-ated loss of growth cone lamellae, some filopodia weretraction (i.e., remodeling) of growth cone lamellae andobserved to elongate to greater than normal lengths.filopodia. We used drugs that interfere with the dy-Similarly, exposure to 20 nM vinblastine resulted innamics of microtubules to investigate the role of mi-an increase in filopodial length but not filopodial num-crotubules in the remodeling of larval amphibian spi-ber. As evidenced by DiOC6(3) staining, 8–20 nMnal cord neuronal growth cones. Vinblastine (8–100vinblastine altered the distribution of membranousnM) , taxol (10 nM) , and nocodazole (330 nM) alteredorganelles within growth cones, suggesting that themicrotubule distributions in growth cones and de-effects of microtubule drugs on growth cones may becreased the percentage of lamellar perimeter undergo-mediated in part by alterations in organelle localiza-ing remodeling, while not affecting the rates of lamel-tion. Our data show that microtubules are involvedlar protrusion and retraction. Also, 8–20 nM vinblas-in the maintenance and regulation of lamellar andtine caused temporary losses of the continuity of thefilopodial structures at the neuronal growth cone.originally fan-shaped lamella, resulting in two or moreThese findings have implications for the mechanismslamellae at the growth cone. At higher concentrationsby which growth cones are guided during develop-of microtubule drugs, the originally fan-shaped la-ment and regeneration. q 1998 John Wiley & Sons, Inc. Jmella broke up into separate smaller lamellae followedNeurobiol 35: 121–140, 1998by the centrifugal displacement from the base of theKeywords: microtubule; lamella; filopodium; organ-growth cone and eventual collapse of the resultantelles; motilitylamellae. Low doses of cytochalasin B prevented the

INTRODUCTION man, 1992). The C domain is the distal-most por-tion of the nerve fiber shaft and contains mainlymicrotubules and organelles, while the P domain isThe dynamic remodeling of the shape and underly-mostly organelle free and is characterized by motileing cytoskeleton of nerve growth cones is of funda-lamellae and filopodia, structures supported by anmental importance for both nerve fiber growth andactin cytoskeleton. Growth cone turning andguidance (Bentley and O’Connor, 1994; Black,branching is initiated by the restructuring of the P1994; Lin et al., 1994; Tanaka and Sabry, 1995;domain (Goldberg and Burmeister, 1986; OakleyLetourneau, 1996). Growth cones consist of a mo-and Tosney, 1993; Fan and Raper, 1995) and itstile peripheral (P) and a central (C) domain (Bridg-cytoskeleton (Sabry et al., 1991; O’Connor andBently, 1993; Lin and Forscher, 1994; Challacombe

* Present address: Department of Cell Biology and Neuro- et al., 1996); subsequently, the C domain advancesanatomy, 1-144 Jackson Hall, 321 Church St. S.E., Minneapolis, to complete the turn with a concomitant rearrange-MN 55455

ment of its microtubules (Williamson et al., 1996;Contract grant sponsor: University of Illinois at Chicagoq 1998 John Wiley & Sons, Inc. CCC 0022-3034/98/020121-20 Challacombe et al., 1997).

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122 Gallo

Microtubules have an important role in nerve fi- ranging from yeast to animal cells (Vega and Solo-mon, 1997). Experiments on the role of microtu-ber growth and may have additional roles in the

regulation of growth cone behavior. Advance of the bules in the regulation of cell morphology and mor-phologic remodeling of actin-based structures inC domain depends on both the polymerization of

new microtubules and the transport of preexisting nonneuronal cell types (Bershadsky et al., 1991;Ueda and Ogihara, 1994; Vasiliev, 1991) raise themicrotubules (Black, 1994; Letourneau, 1996; Yu

et al., 1996). Elongation of nerve fibers is believed possibility that microtubules are involved in the reg-ulation of the behavior of the P domain of the neu-to involve interactions between the components of

the P and C domains (Goldberg and Burmeister, ronal growth cone. In particular, Tanaka andKirschner (1995) reported that pharmacological sta-1986; Forscher and Smith, 1988; Lin et al., 1994;

Letourneau, 1996). Dynamic microtubules have bilization of microtubules results in a type of growthcone behavior they referred to as ‘‘wandering’’ andbeen observed to undergo changes in their distribu-

tion within growth cones and make excursions from a halt of nerve fiber growth in Xenopus embryonicneurons. Furthermore, Odde et al. (1996) reportedthe C domain into the P domain (Tanaka and

Kirschner, 1991). Furthermore, physical associa- the existence of coupling between growth cone andmicrotubule dynamics, further suggesting that mi-tions between microtubules and actin filaments in

the P domain have been reported (Letourneau and crotubules may regulate aspects of P-domain behav-ior. Owing to its relatively small size and abundantRessler, 1983), and microtubule associated proteins

have been implicated in the regulation of growth supply of microtubules, the neuronal growth coneprovides an advantageous model system to studycone morphology (DiTella et al., 1994). The cellu-

lar distribution of some organelles is dependent on the role of microtubules in the regulation of lamellarmotility.microtubules (Terasaki et al., 1984; Lee et al., 1989;

Terasaki and Reese, 1994; Allan, 1996; Lafont and To better appreciate the interactions between ele-ments of the C and P domains of the growth cone,Simons, 1996), and microtubule dynamics may reg-

ulate the structure and function of the growth cone we investigated the effects of drugs that affect mi-crotubule dynamics and stability on the behaviorcytoplasm by producing changes in organelle local-

ization (Dailey and Bridgman, 1989; Davenport et and morphology of the P domain of spinal nervefiber growth cones in vitro. We have previouslyal., 1996). Thus, an understanding of the role of

microtubules in the regulation of growth cone be- described the remodeling of larval Rana pipiens spi-nal cord growth cones (Gallo and Pollack, 1995,havior might provide insights into the cellular mech-

anisms underlying growth cone guidance. 1997) and found that these growth cones provide arelatively homogeneous population with character-The role of microtubule dynamics in nerve fiber

elongation has been studied using several drugs istic behaviors, thereby serving as a good modelsystem for the study of growth cone remodeling.which affect microtubule dynamics. Vinblastine at

nanomolar concentrations (2–50 nM) attenuates Our data show that microtubules are involved in themaintenance of the integrity of fan-shaped lamellaemicrotubule dynamics in vitro and in vivo in both

neuronal and nonneuronal cells (Zheng et al., 1991; at the growth cone, as well as the dynamics of lamel-lar remodeling and filopodial length, thereby reveal-Baas and Ahmad, 1993; Dhamodharan et al., 1995;

Tanaka and Kirschner, 1995; Challacombe et al., ing a new level of microtubule involvement in nervefiber growth.1997). Taxol both stabilizes growth cone microtu-

bules and may cause their polymerization (Letour-neau and Ressler, 1984; Letourneau et al., 1986) aswell as affect nerve fiber growth and aspects of MATERIALS AND METHODSgrowth cone morphology (Letourneau and Ressler,1984). Both taxol (Jordan et al., 1993) and nocoda- Tissue Culture Methodzole have been reported to attenuate microtubule

Stage V Rana pipiens larvae (Taylor and Kollros, 1946)dynamics at low concentrations (Jordan et al., 1992;were used as a source of lumbosacral spinal cord explants.Rochlin et al., 1996). Hence, these pharmacologicalTissues were cultured in Sykes–Moore chambers (Bellco

drugs are valuable tools which can be used to probe Glass, Vineland, NJ). Coverslips onto which tissue ex-the role of microtubules and their dynamics in the plants were cultured were coated overnight with 1-mg/regulation of cellular behaviors. mL poly-DL-lysine (Sigma; MW 30–70 kD) in borate

Understanding microtubule function in the gen- buffer solution (pH 8.4). The nutrient medium used foreration and maintenance of cell morphology is of culturing was Eagle’s minimum essential medium

(MEM) with Earle’s basic salts (without NaHCO3 andfundamental importance to the biology of cell types


Microtubules and Regulation of Neuronal Growth Cone Remodeling 123

glutamine) supplemented with 50 mg/mL gentamicin, Filopodial Length. The length of filopodia was mea-sured from the point of origin of the filopodium to its tip,200 mM L-glutamine (1 mL/100 mL), and 10 mM Hepes

buffer, pH 7.2. Prior to use, the medium was sterilized using the distance function.by microfiltration.

Tissue culture procedures were performed according Filopodial Number. The number of filopodia was deter-to previously established methods (Pollack and Koves, mined by visual inspection of the growth cone. Thin pro-1975; Muhlach and Pollack, 1978). Each spinal cord was jections longer than 1 mm were considered to be filopodia.used to prepare two lumbosacral cross-section explants(approximately 0.3–0.5 mm thick). Prepared chambers,

Nerve Fiber Width. A circle of 10 mm radius was drawneach containing one tissue explant, were stored in a darkon a transparent overlay which was placed on the monitorincubator at 197C.screen with its center positioned in the middle of the Cdomain of the growth cone. Using the distance function,nerve fiber width was measured at the intersection of theGrowth Cone Visualization,circle with the fiber.Data Collection, and Analysis

As detailed previously (Gallo and Pollack, 1997),measurements of lamellar area and perimeter were foundVideo microscopy was used to study the dynamics ofto be accurate within 2%, and measurements of filopodialgrowth cone remodeling and nerve fiber growth. Thislength were accurate within 1%. Nerve fiber width mea-allowed for visualization of growth cone morphology andsurements were accurate within 5%.the storage of data on high-grade videotapes. JAVA im-

age analysis software (Jandel, Inc.) was used to contrastenhance images, make measurements, and digitize im-

Determination of Gross Changes inages.Growth Cone Morphology andGrowth cones and nerve fibers were visualized on aNerve Fiber Growth RatesReicherdt inverted microscope using phase-contrast op-

tics. A final magnification of 148 (116 objective) wasThe maintenance or loss of fan-shaped lamellar morphol-used for observing nerve fibers and gross growth coneogy during a 1-h period was determined by digitizingmorphology, while a magnification of 1189 (163 objec-images of nerve fiber fields (148) and temporarily stor-tive) was used for the detailed study of growth cones.ing them in random access memory using the JAVA snap-An NEC camera (NC-15 CCD color camera) coupled toshot feature. Cultures were returned to the incubator forthe microscope sent the images to a Sony VCR connected1 h, after which the same nerve fiber fields were againin series with an IBM PS/2 computer. The VCR wasvisualized. The digitized snapshot allowed for a directused to make videotape records for later analysis. Thecomparison of growth cones before and after the incuba-video microscopy apparatus was kept at a temperature oftion. In this manner, the percentage of growth cones los-approximately 21–227C during periods of observation.ing fan-shaped lamellar morphology during a 1-h periodwas obtained.

Measurements of growth rates were performed byGrowth Cone Morphometricsstoring digitized images of nerve fiber fields (148) usingthe snapshot feature of JAVA and comparing these toTwo morphometric functions of the JAVA image analysisimages of the same nerve fiber fields 1 h later. Growthsoftware were used: area and distance. Area and distanceof the nerve fiber was determined by measuring the dis-measurements were calibrated using a micrometer cali-placement of the C domain during the 1-h period.brating slide. The following criteria were used to make

the measurements.

Measurements of GrowthLamellar Area. The phase-bright P domain was mea-Cone Remodelingsured from the point where it originated from the phase-

dark portion of the C domain, to where it could be seen Difference images (Kim and Wu, 1991; Gallo and Pol-to fade back into the C domain on the opposite side lack, 1995, 1997) were used to study the dynamics ofof the growth cone. Filopodia were excluded from this changes in lamellar expanse at the growth cone. A trans-measurement. parency sheet was affixed to the monitor screen and the

outline of the growth cone was traced from a contrastenhanced image. After 1 min, the procedure was repeatedLamellar Perimeter. JAVA automatically provides the

perimeter of a measured area. This value is the true perim- for the same growth cone, but with a different color. Theresult is the superimposition of images of the sameeter of the lamella plus the width of the nerve fiber.

Therefore, a measurement of the width of the nerve fiber, growth cone separated by a 1-min interval. The positionof particles located on the substratum was monitored toobtained with the distance function, was subtracted from

the original measurement to yield the true lamellar perim- make sure that stage drift had not occurred.Difference images allow the visualization and quanti-eter.


124 Gallo

fication of lamellar behavior. The lamellar perimeter ex- were digitized using a scanjet 3c/ t scanner (Hewlett-Packard) and prepared using Adobe Photoshop 2.5.1hibits regions undergoing protrusion or retraction, or re-

maining quiescent. The rate of lamellar protrusion, or (Adobe Systems).retraction, for each region can be determined by measur-ing the lamellar area which underwent protrusion/retrac-

Preparation and Handlingtion and dividing it by the length of lamellar perimeterfrom which the protrusion/retraction occurred. of Pharmacological Drugs

The percentage of lamellar perimeter undergoing re-All pharmacological drugs were purchased from Sigma.modeling at the growth cone is given byStock solutions of cytochalasin B, nocodazole, and taxolwere prepared in dimethyl sulfoxide (DMSO), aliquoted,% Perimeter undergoing remodeling Å (Pp / Pr) /Pand stored at 0207C, 0207C, and 2–47C, respectively.Vinblastine stock was prepared in MEM and stored at

where P is the total lamellar perimeter, and Pp and Pr2–47C. Final concentrations were prepared on the day of

are the total length of lamellar perimeter undergoing pro-use. To deliver drugs to cultured explants, 4 mL of drug-

trusion and retraction, respectively.containing medium was injected into the chamber at a

The percentage of lamellar perimeter undergoing pro-rate of approximately 1.3–1.5 mL/min. This volume was

trusion relative to total perimeter remodeling can be ob-used since it was greater than the volume required to

tained by dividing the total length of lamellar perimeterclear 1% Nile blue from the chamber, and hence expected

protruding or retracting by the length of perimeter under-to fully exchange the medium in the chamber.

going remodeling:

Relative % perimeter protruding Å Pp / (Pp / Pr) Fluorescent Visualization of OrganelleDistribution in Growth Cones

All growth cones were sampled on the fifth day invitro (DIV) unless otherwise noted. Only growth cones 3 *,3 *-Dihexyloxacarbocyanine iodide [DiOC6 ( 3 ) ] ;not contacting other cells or nerve fibers and that exhib- (Molecular Probes, Eugene, OR) was used to visualizeited a lamellar areaú80 mm2 at the beginning of observa- the organelles present in the C domain of growth conestion and a uniform fan-shaped lamella were used for mea- (Dailey and Bridgman, 1989). A concentration of 2.5surements of remodeling. mg/mL DiOC6(3) in PBS was prepared on the day of

use from a 0.5-mg/mL stock. Cells were fixed in PBScontaining 0.5% gluteraldehyde for 10 min. Following

Immunofluorescent Visualization fixation, cells were exposed to 0.1% sodium borohydrideof Microtubules in PBS for 10 min, and subsequently rinsed in PBS for

10 min and then stained with DiOC6(3) for a period ofThe staining procedure was adapted from that used by

2 min. Following staining, the preparations were rinsedTanaka and Kirschner (1995). Cultures were fixed for

in PBS for 10 min and the coverslips were then mounted10 min using cytoskeletal buffer (CB) (10 mM MES,

on glass slides using PBS as a mountant. VisualizationpH 6.0, 138 mM KCl, 3.0 mM MgCl2, and 2.0 mM

and documentation of DiOC6(3)-labeled preparationsEGTA) with 0.5% gulteraldehyde, and then extracted

were the same as for the immunofluorescent visualizationusing CB with 0.05% Triton X-100 for 10 min. Following

of microtubules.extraction, coverslips were placed in 0.1% sodium boro-hydride in phosphate-buffered saline (PBS) for 10 min.Cultures were subsequently washed for 15 min in PBSand then for 15 min in PBS with 3% bovine serum albu- RESULTSmin (BSA). Then, 300 mL of an antibody to b-tubulinwas applied for 2 h (Sigma; monoclonal clone DM1-a, General Observations on the Effects1:1000 dilution in PBS with 3% BSA), and coverslips of Microtubule Drugs on Growth Conewere washed for 15 min in PBS. The secondary antibody and Nerve Fiber Morphologywas applied for 1 h [Sigma; goat anti-mouse fluoresceinisothiocyanate (FITC)-conjugated immunoglobulin G Growth cones lost lamellar structures following a(IgG), 300 mL of 1:120 dilution in PBS per coverslip] . 1-h exposure to vinblastine in a dose-dependentCoverslips were then washed for 15 min in PBS, and manner (Table 1). Feeding cultures with MEM didmounted on glass slides using one drop of mountant (80%

not affect the frequency with which lamellar struc-glycerol, 10 mM tris hydrochloride, pH 7.8, with 20 nMtures were lost, compared to cultures not fed (Tablepropylgallate) . Slides were stored in slide boxes at1) . Neither growth cone lamellar area nor nerve0207C. A Zeiss Photomicroscope II research epifluore-fiber width was affected following a 24-h exposuresence microscope was used to visualize the FITC-taggedto 4–20 nM vinblastine (ANOVA, p ú 0.05) (Ta-microtubules. Photographs were taken using Fujicolor

film (400 ASA). For presentation purposes, photographs ble 1). Nerve fibers in cultures exposed to 4–20 nM



Table 1 Effects of Microtubule Drugs on Nerve Fiber and Growth Cone Morphology,and Nerve Fiber Growth Rate

% Growth ConesLosing Lamellar Lamellar Expanse Nerve Fiber Width Growth Rate

Treatment Morphology (mm2) (mm) (mm/h)

No treatment 3% (35) 76 { 4 (254) ND NDMEM feeding 5% (56) 82 { 6 (64) 0.9 { 0.05 (40) 20 { 1 (37)MEM / 1006 DMSO 4% (30) ND ND NDMEM / 1003 DMSO 4% (32) 73 { 6 (72) 0.9 { 0.05 (41) ND4 nM vinblastine 4% (28) 75 { 6 (51) 0.9 { 0.06 (38) 16 { 1 (31)8 nM vinblastine 17% (26) 72 { 4 (49) 0.9 { 0.05 (34) 12 { 1 (20)20 nM vinblastine 24% (21) 72 { 7 (53) 0.9 { 0.05 (36) 10 { 1 (15)50 nM vinblastine 75% (28) ND ND ND100 nM vinblastine 86% (29) ND ND ND1 mM vinblastine 90% (30) ND ND ND330 nM vinblastine 37% (27) ND ND ND10 nM taxol ND 63 { 6 (63) 1.4 { 0.1 (32)* ND

The percentage of growth cones losing lamellar morphology was determined 1 h after the initiation of the treatment. All othermeasurements were made at 24 h after the beginning of treatment. MEMÅmedium; DMSOÅ dimethyl sulfoxide; NDÅ not determined.Data are means { S.E.M. Values in parentheses represent the sample size.

* Different from control, Welch approximate t test, one-tailed; p õ 0.001.

vinblastine for 24 h continued to grow and exhibit to 10 nM taxol varied between cultures. In somecultures, up to 90% of growth cones lost fan-shapedgrowth cones with fan-shaped lamella [Fig. 1(A–

C)] without evident nerve fiber retraction or bead- lamellar morphology, while in others, only 10% un-derwent a similar change in morphology. However,ing. However, 20 nM vinblastine did result in the

formation of lamellar structures along nerve fiber by 4 h, all growth cones lost lamellar structures inresponse to 10 nM taxol (n Å 12 cultures) . Col-shafts [Fig. 1(C)] , a result similar to that of Bray

et al. (1978). Similar lamellar structures are very lapsed growth cones often exhibited a terminalswelling associated with the loss of most filopodiarare in untreated or MEM-treated cultures. It was

found that 4–20 nM vinblastine decreased nerve and all lamellae. In all cases, growth cone fan-shaped morphology was reestablished by 6–8 hfiber growth rate in a dose-dependent manner (AN-

OVA, p õ 0.05) (Table 1). Qualitative observa- after collapse. Therefore, although the temporal as-pect of the response to taxol was varied, growthtions of longer term (5–6 days post–drug addition)

cultures indicated that 4–20 nM vinblastine did not cones underwent an initial loss, with subsequentrecovery, of fan-shaped lamellar morphology. Noabolish nerve fiber growth, but prolonged exposure

to concentrations¢50 nM resulted in the disruption nerve fiber retraction or beading was noted. A simi-lar growth cone response, albeit much more rapid,of nerve fiber morphology and a block of fiber

growth. Cultures exposed to 50 nM to 1 mM vinblas- was documented by Letourneau and Ressler (1984)following treatment with 700 nM taxol. Morpho-tine for 24 h exhibited retraction and beading of

nerve fibers in conjunction with the absence of metric analysis of growth cones and nerve fibersfollowing a 24-h exposure to 10 nM taxol revealedgrowth cones with lamellar structures. Beading of

fibers was common after a 1-h exposure to concen- an increase in nerve fiber width and no effect ongrowth cone size (Table 1). In the prolonged pres-trations of vinblastine ¢100 nM .

One-hour exposure to 330 nM nocodazole re- ence of 10 nM taxol, growth cones exhibited P andC domains that were not distinguishable from con-sulted in the loss of lamellae in 37% of growth

cones (n Å 27), while treatment with MEM control growth cones [Fig. 1(D)] .taining DMSO resulted in a frequency of growthcone lamellar collapse equivalent to that in cultures Effects of Microtubule Drugs on Nervenot fed or fed with MEM alone (Table 1). Exposure Fiber and Growth Cone Microtubulesof cultures to 330 nM nocodazole for 24 h resultedin nerve fiber retraction, beading, and the absence As evidenced by immunofluorescent staining, mi-

crotubule arrays in nerve fibers are not disrupted byof growth cones with lamellar structures.The response of growth cones to a 1-h exposure a 24-h treatment with 4–20 nM vinblastine [Fig.


126 Gallo

Figure 1 Effects of microtubule drugs of growth cone morphology and microtubules. Allimages were obtained from cultures 24 h after treatment. (A) Representative growth cone froma culture fed with medium. Growth cones fed with medium or not fed usually maintained thefan-shaped lamellar morphology for a period of at least 1 h (Table 1) (Gallo and Pollack,1997). (B–D) Examples of growth cones morphology after treatment with 8 and 20 nMvinblastine, and 10 nM taxol, respectively. Note that although growth cone morphology follow-ing treatment with these drugs was not obviously altered, in the presence of 20 nM vinblastinenerve fiber shafts often protruded lamellae (C, arrow). (E–G) Microtubule arrays of nervefibers treated with medium, 8 and 20 nM , respectively. Note that there is no obvious disruptionof microtubule arrays in the vinblastine-treated cultures. (H–J) Examples of the three growthcone microtubule distribution types observed in our system. These distribution types are hereaf-ter referred to as deep (H), intermediate (I) , and neck (J) . In some cases, individual microtu-bules could be seen to extend into filopodia (H, arrow). (K) Example of microtubule bundles(arrows) in a growth cone in the presence of 10 nM taxol. The white dots delineate the borderof the growth cone lamellae, as determined by the faint background fluorescence. For eachrow, the bar in the leftmost panel Å 10 mm.

1(E–G)]. However, the distribution of microtu- centage of growth cones exhibited microtubuleswhich splayed out in the P domain but did not ex-bules within fan-shaped growth cones was altered

by a 24-h exposure to 8–20 nM vinblastine. In tend far into the P domain [Fig. 1(I) and Table 2].Following a 24-h exposure to 8–20 nM vinblastine,growth cones treated with MEM for 24 h, the major-

ity of growth cones had microtubules which splayed microtubules tended to terminate in the C domainand did not extend further [Fig. 1(J) and Table 2].out as they extended from the C domain into the P

domain, often almost reaching the leading edge of These types of microtubule distributions have beeninterpreted to be the result of the attenuation oflamellae [Fig. 1(H) and Table 2]. A smaller per-



Table 2 Effects of Vinblastine on Growth Cone growth cones sampled from an equal number ofMicrotubule Distribution separate cultures): 50 nM vinblastine (n Å 8), 100

nM vinblastine (n Å 7), 1 mM vinblastine (n Å 6),% Growth Cones withand 330 nM nocodazole (n Å 8). Overall, growthMicrotubule Distributioncone disruption in response to vinblastine and noco-

Treatment Type (n) Deep Intermediate Neck dazole was similar; therefore, the data are presentedcollectively. In 30% of growth cones (n Å 29), aMEM 24 h (48) 82 16 2

8 nM VB 24 h (25) 40 32 28 loss of lamella at the leading edge occurred during20 nM VB 24 h (25) 28 21 51 the first 20 min following delivery of the drug (Fig.

2) , resulting in a bifurcated appearance. On occa-For the definition of microtubule distribution types, see Fig-sion, one of the resultant lamellae was lost, leavingure 1(H–J). MEM Å medium; VB Å vinblastine.the growth cone asymmetrical. A more generalizedresponse was the centrifugal migration of lamellarstructures away from the C domain following themicrotubule dynamics in growth cones (Tanaka and

Kirschner, 1995; Rochlin et al., 1996). F-actin breakup of the originally fan-shaped lamella intomultiple smaller lamellae. Individual lamellae werestaining did not reveal obvious differences in

growth cone actin filament meshworks following observed to undergo centrifugal migration from theC domain [Fig. 2(A), min 11–22]. In these cases,vinblastine treatment (data not shown), consistent

with other reports of minor (Tanaka and Kirschner, continuity with the nerve fiber was maintained bythin membranous connections. Immunofluorescent1995) or no effects (Challacombe et al., 1997) of

vinblastine on growth cone actin filament organiza- visualization of microtubules in growth cones ex-hibiting lamellae undergoing centripetal separationtion.

A 24-h exposure to 10 nM taxol resulted in rela- from the C domain failed to reveal any microtubulesconnecting the displaced lamellae and the C domaintively unsplayed distributions of microtubules

within growth cones, with most of the microtubules (data not shown). Microtubule distributions werevisualized in 13 growth cones exhibiting lamellaeappearing bundled into one or more fascicles [Fig.

1(K) (four cultures)] . Twenty-four hours of treat- that, following an exposure of 15–30 min to 1 mMvinblastine (five cultures) , appeared separate fromment of cultures with MEM containing 1003 DMSO

did not change microtubule distributions from those the C domain, as well as in eight growth conesexposed to 100 nM vinblastine (five cultures) forobserved in MEM-fed cultures (one culture; data

not shown). 30 min.Frequently, starting as soon as 10–15 min fol-Visualization of microtubules in the presence of

doses of microtubule drugs which result in the loss lowing exposure to microtubule drugs, individualfilopodia underwent a change in morphology whichof fan-shaped lamellae at the growth cone (50 nM

to 1 mM vinblastine and 330 nM nocodazole) re- was termed ‘‘filopodial spreading’’ [Fig. 3(A)] .This consisted of lamellae forming either at the tipvealed that some nerve fibers retain relatively nor-

mal nerve fiber microtubules arrays even after 1 h of or along the shaft of the filopodium. Lamellaeformed by filopodial spreading on occasion devel-exposure to microtubule drugs (not shown); these

nerve fibers were the ones which still exhibited oped filopodia along their perimeter [Fig. 3(B)]and could move both centripetally and centrifugallygrowth cones with fan-shaped lamellae after 1 h in

the presence of microtubule drugs. However, al- on the filopodial shaft. Filopodial spreading is nota common feature of spinal cord growth cones inthough nerve fiber microtubules were still evident,

only a few growth cones retained some microtu- our culturing system (Gallo and Pollack, 1997).Another consistent feature of the response to vin-bules within the P domain. These likely represent

growth cones with relatively more stable microtu- blastine and nocodazole was the growth of 1–3 fi-lopodia/growth cone to lengths reaching 30 mm,bules at the time of drug delivery.starting as early as 15 min following drug addition[Fig. 3(C)] . This is in contrast to the finding thatEffects of Microtubule-Disrupting Drugsonly 3% and 1% of the filopodia of spinal cordon Maintenance of Fan-Shapedgrowth cones fed with MEM grew to lengths ú16Lamellae at the Growth Coneand 20 mm, respectively (n Å 199; maximum ob-served length was 24 mm) (see Table 5) during theThe immediate response of growth cone morphol-

ogy to microtubule drugs was investigated at the 1-h period following feeding. The loss of lamellararea with time, in conjunction with the unabated,following drug concentrations (n Å number of


128 Gallo

Figure 2 Effects of microtubule disruption on growth cone morphology. In both panels, theminutes before (õ0) and after (¢0) addition of the drug are indicated in the lower right-handside of each image. (A) Example of growth cone behavior in response to 100 nM vinblastine.Note that an initial loss of lamellar surface occurred at the leading edge of the growth cone(9 min, arrow), directly ahead of the nerve fiber shaft. This resulted in the formation of twoindependent lamellae which subsequently migrated centrifugally from the neck of the growthcone (11–22 min) (*fixed substratum point) and eventually underwent further breaking upinto smaller lamellae (41 min). (B) Example of growth cone behavior after 330 nM nocodazole.In this case, the breaking up of the main lamella occurred both to the left of the growth coneneck (0 min, arrow) and at the leading edge (5 min, arrow). Although the centrifugal migrationof lamellae from the growth cone neck was not as striking as in (A), lamellae still underwentcentrifugal migration (15 min, arrow). Bar Å 10 mm.



Figure 3 Effects of microtubule drugs on filopodial behavior. (A) In the presence of vinblas-tine (8 nM in this case) , filopodia often underwent swelling (long arrow) and lateral spreading(short arrow). The two images are 2 min apart. Example of filopodial spreading (B) andelongation (C) in response to concentrations of microtubule drugs which resulted in the lossof growth cone morphology [both (B) and (C) were obtained from the same growth cone, at40 and 25 min after the addition of 1 mM vinblastine, respectively] . (B) Filopodium initiallyunderwent swelling (left image, arrow), which spread into a small lamella ( the extend ofwhich is delineated by the thin arrows in the right image) and two individual filopodial shafts(right image, thick arrows). During growth cone disruption, individual filopodia hyperextended(C). Note the relationship of the filopodial tip (arrow) in relation to the fixed substratum point(X) in both images (5 min apart) . Bars Å 3 mm for (A) and 10 mm for (B,C).

apparently promoted growth of some filopodia re- microtubules was investigated. We have previouslysulted in a filopodial morphology by the time all determined that low concentrations of cytochalasinlamellar structures had disappeared. Eventually, as B (0.002–0.2 ng/mL) and D (0.002–0.02 ng/mL)the nerve fiber underwent beading and retraction diminish lamellar remodeling by decreasing the ratecommenced, the filopodia were also withdrawn into of lamellar protrusion and the percentage of lamellarthe nerve fiber tip. perimeter remodeling, without causing the loss of

Feeding cultures with MEM (n Å 10) or MEM lamellar structures (Kapur et al., 1995). Therefore,with 1003 DMSO (n Å 8) had no apparent effects cytochalasins were used to inhibit but not abolishon growth cone remodeling. Growth cones main- actin-dependent growth cone lamellar remodeling.tained a fan-shaped lamellar morphology through- Eight growth cones from eight separate culturesout the 1-h observation period in a manner not dis- were exposed to MEM containing 1 mM vinblastinetinguishable from that of growth cones from nonfed and 0.2 ng/mL cytochalasin B and observed for acultures (Gallo and Pollack, 1995, 1997). 1-h period. These concentrations of cytoskeletal

drugs were chosen since 1 mM vinblastine resultedInvestigation of Role of Actin in a consistent morphological response and 0.2 ng/Cytoskeleton in Lamellar Response mL cytochalasin B maximally inhibits lamellar re-to Microtubule Disruption modeling without causing decreases in the expanse

of lamellae. In response to this dual treatment, sevenThe role of the actin cytoskeleton in the behaviorof lamellae following pharmacological disruption of of eight growth cones did not undergo the character-


130 Gallo

istic loss of fan-shaped morphology associated with The response of eight growth cones, each froma separate culture, to 20 nM vinblastine was docu-1 mM vinblastine (Fig. 4) . Fan-shaped morphology

was maintained, as was overall lamellar size. In mented. Two growth cones underwent a transitionto a filopodial morphology similar to that occurringonly one case was the behavior of the growth cone

reminiscent of that in response to 1 mM vinblastine. at higher concentrations. One growth cone under-went branching, as suggested by the fact that theIn this case, one filopodium underwent spreading

and two filopodia reached lengths of approximately phase-dark nerve fiber shaft appeared to have split,resulting in the presence of two C and P domains.30 mm. Immunofluorescent visualization of micro-

tubules in the combined presence of 1 mM vinblas- In one instance, growth cone lamellae were lostwithin 15 min of drug addition. Filopodial spreadingtine and 0.2 ng/mL cytochalasin B revealed that

microtubules were affected in a manner similar to was observed, but no breaking up of the lamellaor filopodial growth occurred. The remaining fourthat occurring in response to vinblastine alone (data

not shown). growth cones underwent repeated instances of la-mellar fragmentation similar to that described forgrowth cones in 8 nM vinblastine.Effects of 4–20 nM Vinblastine

on Growth Cone RemodelingEffects of Microtubule Drugs

Growth cone behavior in response to concentrations on Lamellar Perimeter Dynamicsof vinblastine (4–20 nM) that do not disrupt nervefiber microtubule arrays but alter the distribution of Difference images were used to study remodeling

of the lamellar perimeter in the presence of microtu-microtubules within the growth cone was investi-gated. The response to 4 nM vinblastine varied bule drugs. Growth cones were sampled 1 and 24

h after the addition of 4–20 nM vinblastine, MEMgreatly among growth cones (n Å 8, eight separatecultures) . One growth cone underwent a loss of fan- (medium) feeding, and MEM with 1003 and 1006

DMSO, and 24 h after 10 nM taxol. For each experi-shaped morphology similar to that observed withgreater vinblastine concentrations, followed by re- mental condition, a minimum of five growth cones

from two or more separate cultures were each sam-traction of the nerve fiber. In this case, two filopodiagrew to lengths ú20 mm. Five growth cones exhib- pled for three to five difference images. Since a

subpopulation of fan-shaped growth cones wasited behavior indistinguishable from that of MEM-fed growth cones. The remaining two growth cones noted to persist after 1 h in 330 nM nocodazole and

50–100 nM vinblastine, different images were alsounderwent a transformation from having a singlefan-shaped lamella to having multiple smaller la- obtained from these growth cones. The nerve fibers

of these growth cones did not exhibit beading fol-mellae. In these cases, the fan-shaped lamella brokeup into two or more smaller lamellae. These small lowing 1 h in the presence of microtubule drugs.

Growth cones sampled after 1 h in 330 nM nocoda-lamellae often shrank as others formed elsewhere atthe growth cone, often through filopodial spreading. zole and 50–100 nM vinblastine satisfied the mor-

phological criteria for sampling (see Methods) .The response of 10 growth cones from 10 sepa-rate cultures to 8 nM vinblastine was observed. One The percentage of lamellar perimeter undergoing

remodeling decreased with increasing concentrationgrowth cone underwent a loss of lamellar structuresexhibiting features associated with the effects of of vinblastine [Figs. 5(C) and 6(A)]. However,

the percentage of active lamellar perimeter ( i.e.,higher concentrations of vinblastine. Three growthcones retained a fan-shaped morphology but under- either protruding or retracting) undergoing protru-

sion was not affected by vinblastine treatments (Ta-went minimal remodeling, giving them the appear-ance of being frozen [Fig. 5(A)] . The remaining ble 3). Similar effects on lamellar perimeter remod-

eling were noted in growth cones exposed to 330growth cones underwent repeated instances of theloss of a fan-shaped lamellar morphology, resulting nM nocodazole, 50–100 nM vinblastine, or 10 nM

taxol [Fig. 6(B)] , ruling out the possibility that thein the presence of multiple smaller lamellae, fol-lowed by reestablishment of the fan-shaped mor- effects of vinblastine were drug specific.

Whether microtubule drugs affected the rates ofphology [Fig. 5(B)] . In contrast to the response ofgrowth cone lamellae to higher concentrations of lamellar protrusion or retraction was investigated.

Spinal cord growth cones undergo naturally oc-microtubule drugs, although lamellae broke up, theydid not separate from the C domain and undergo curring lamellar size fluctuations (Gallo and Pol-

lack, 1995), termed ‘‘lamellar cycles.’’ A lamellarcentrifugal migration. Filopodial spreading oc-curred in most growth cones at 8 nM vinblastine. cycle is described as consisting of a phase of lamel-



Figure 4 Cytochalasin B prevents growth cone disruption in response to microtubule agents.Concurrent exposure to 0.2 ng/mL cytochalasin B and 1 mM vinblastine prevented the disruptionof growth cone morphology associated with exposure to microtubule drugs. Note that breakupand centripetal migration of the fan-shaped lamella did not occur. Bar Å 10 mm.


132 Gallo



Figure 6 Effects of microtubule drugs on the percentage of lamellar perimeter remodeling.(A) Feeding cultures with medium (MEM) had no effect on the percentage of lamellar perime-ter undergoing remodeling at either 1 or 24 h after feeding (CNT Å growth cones not treated;ANOVA, p ú 0.05). It was found that 8 and 20 nM vinblastine (VB) decreased the percentageof lamellar perimeter remodeling at both 1 and 24 h following feeding (ANOVA with Bonfer-roni multiple comparisons tests; *p° 0.05 and **p° 0.001). Since the percentage of lamellarperimeter remodeling following 1 h washout of 20 nM VB returned levels characteristic ofMEM-fed cultures (Welch’s approximate t test; pú 0.05), the effects of vinblastine on lamellarperimeter remodeling were shown to be reversible. (B) Nocodazole (NOC) and taxol (TAX)also decreased the percentage of lamellar perimeter remodeling. Nocodazole data were collectedfrom growth cones which, following 30 min to 1 h exposure to 330 nM NOC, still exhibiteda fan-shaped morphology. The percentage of lamellar perimeter undergoing remodeling wasdecreased relative to cultures fed with dimethylsulfoxide (DMSO) (Welch’s approximate ttest, one-tailed, p õ 0.0001). Taxol data were collected 24 h following the addition of taxolto the culture owing to the instability of the response at earlier time intervals (see Results) .The percentage of lamellar perimeter undergoing remodeling was decreased by TAX (Welch’sapproximate t test, one-tailed; p õ 0.0006) relative to cultures fed with DMSO for 24 h. Dataare means { S.E.M. There were 21–37 difference images measured in each group.

lar size increase (A/) followed by one of decrease (see Methods) were compared. It was found that4–20 nM vinblastine did not have a significant ef-(A0) . Since lamellar protrusion rate is greatest dur-

ing A/ (Gallo and Pollack, 1997), and that of re- fect on lamellar rates of protrusion or retraction ateither 1 or 24 h after drug addition (Table 4). Simi-traction is invariant across cycle phases, only rates

obtained from difference images classified as A/ larly, rates were not affected by a 1 h exposure to

Figure 5 Effects of vinblastine on growth cone remodeling. (A) Example of a growth coneundergoing minimal remodeling following the administration of 8 nM vinblastine. The imageswere obtained 15 and 25 min after treatment, respectively. This example is an extreme casedemonstrating that in the presence of vinblastine, the whole lamella can become immotilewithout undergoing retraction. (B) Example of repeated loss of growth cone fan-shaped mor-phology in the presence of 8 nM vinblastine. Fan-shaped lamellar morphology was lost by 3min following the introduction of vinblastine and was regained by 20 min. Similar break upof fan-shaped lamella occurred again at 35–41 min. (C) Examples of difference images (seeMethods) obtained from growth cones treated with medium (MEM) of vinblastine (VB). Whiteareas denote lamellar retraction; black areas indicate lamellar protrusion. Portions of the perimeterwhich were quiescent are represented by a line. Note the increase in regions of quiescent lamellarperimeter with increasing vinblastine concentration. All difference images were obtained duringthe A/ phase of lamellar cycles (Gallo and Pollack, 1997). Bars Å 10 mm.


134 Gallo

Table 3 Effects of Vinblastine on Relative Lamellar Perimeter Undergoing Protrusion

Relative Perimeter Protruding [% (n)]

Treatment A/ A0 A0

MEM 57 { 2 (29) 46 { 2 (15) 33 { 2 (14)4 nM VB 47 { 3 (8) 40 { 5 (8) 25 { 6 (5)8 nM VB 57 { 3 (13) 49 { 3 (9) 34 { 4 (4)20 nM VB 57 { 2 (10) 47 { 2 (15) 35 { 3 (4)

Data were collected at 24 h following treatment. MEM Å medium; VB Å vinblastine; Data are means { S.E.M.

330 nM nocodazole (Table 4). Exposure to 10 nM filopodial length was greater in vinblastine than inMEM-treated cultures. Growth cones exposed to 20taxol slightly decreased lamellar retraction rate but

had no effect on protrusion rate (Table 4). nM vinblastine for 24 h exhibited a mean filopodiallength of 8.3 { 0.25 mm [mean { standard errorof the mean (S.E.M.)] , while those treated withEffects of 20 nM Vinblastine onmedium had a mean length of 6.8 { 0.15 mm (meanGrowth Cone Filopodia { S.E.M.) (n ú 267 filopodia measured from ú30growth cones in each group; Welch t test; pThe observation that some filopodia underwent ex-

tensive elongation during the loss of growth cone õ 0.0001). The increase in mean filopodial lengthwas not due to the elongation of filopodia to abnor-morphology in response to microtubule drugs

prompted us to investigate whether lower concentra- mal lengths, as is the case during microtubule drug-induced growth cone collapse, but rather, resultedtions of vinblastine also affected filopodia. Cultures

were fed with either MEM or 20 nM vinblastine, from an increase in the percentage of filopodiareaching intermediate lengths (6–16 mm) comparedand filopodial length and number were measured

24 h later. As a positive control for the effects of to MEM-fed control cultures (Table 5).vinblastine, the percentage of lamellar perimeter inthese experiments was measured and found to be Distribution of Membranous Organellesdecreased (61 { 4; n Å 10; Welch approximate t within Growth Cones in the Presencetest, one-tailed, p õ 0.05), relative to MEM-fed of Vinblastinecontrols. The number of filopodia per 10 mm oflamellar perimeter was not affected by vinblastine Cultures were fed with MEM or 8–20 nM vinblas-

tine and stained with DiOC6(3), an organelle stain(ANOVA; p ú 0.05; data not shown). However,

Table 4 Effects of Microtubule Drugs on Rates of Lamellar Protrusion and Retraction

Lamellar Protrusion Rate Lamellar Retraction RateTreatment (mm/min) (n) (mm/min) (n)

1 h MEM 1.51 { 0.20 (26) 0.96 { 0.05 (56)24 h MEM 1.35 { 0.10 (77) 0.94 { 0.04 (62)1 h 1 1 1003 DMSO 1.15 { 0.10 (50) 0.86 { 0.04 (43)24 h 1 1 1003 DMSO 1.36 { 0.10 (45) 1.02 { 0.07 (37)1 h 4 nM VB 1.23 { 0.10 (30) 0.86 { 0.07 (25)1 h 8 nM VB 1.16 { 0.10 (32) 0.92 { 0.07 (28)1 h 20 nM VB 1.17 { 0.10 (32) 0.93 { 0.05 (45)24 h 4 nM VB 1.47 { 0.10 (27) 1.12 { 0.10 (34)24 h 8 nM VB 1.31 { 0.10 (60) 0.99 { 0.06 (49)24 h 20 nM VB 1.20 { 0.10 (46) 0.94 { 0.05 (39)1 h 330 nM NOC 1.34 { 0.10 (56) 0.98 { 0.06 (37)24 h 10 nM TAX 1.25 { 0.10 (40) 0.84 { 0.05 (34)*

Data were collected only during the A/ phases, since the rate of lamellar protrusion is greatest during A/ and that of retractiondoes not vary during lamellar cycles. MEM Å medium; DMSO Å dimethylsulfoxide; VB Å vinblastine; NOC Å nocodazole; TAX Åtaxol. Data means { S.E.M.

* Different from DMSO control, Welch approximate t test, two-tailed; p Å 0.04.



Table 5 Effects of Vinblastine on Distribution of Growth Cone Filopodial Lengths

% Filopodia with Length in Given Range (mm)

Treatment 1–5.9 6–10.9 10–15.9 16–20.0 ú21

MEM (199) 52 34 10 3 120 nM VB (150) 32 37 23 5 3

All data were collected 24 h after treatment. MEM Å medium; VB Å vinblastine.

(Dailey and Bridgman, 1989; Lee et al., 1989; Tera- fed growth cones (Fisher’s exact test, one-tailed;p õ 0.05). The distribution of DiOC6(3)-stainedsaki et al., 1984; Terasaki and Reese, 1992) 24 h

later. In MEM-fed cultures (n Å 7), staining ap- organelles in growth cones exposed to 8 nM vin-blastine for 24 h was similar to that observed follow-peared bright along the extent of all nerve fibers

having lamellar growth cones, and dimmer along ing a 24-h exposure to 20 nM vinblastine. In thepresence of 8 nM vinblastine, 46% of growth conesthose not having lamellae. A fluorescent organelle

mass was located in the C domain and often had (n Å 28, five cultures) had finger-like projectionsextending from the fluorescent organelle mass, sig-finger-like projections extending into the P domain,

similar to those described by Dailey and Bridgman nificantly less than growth cones fed with MEM(Fisher’s exact test, one-tailed; p õ 0.01). A 1-h(1989) [Fig. 7(A)] . Finger-like projections were

present in 85% of growth cones (n Å 34) treated exposure to 20 nM vinblastine also decreased thepercentage of growth cones exhibiting finger-likewith MEM for 24 h. The distribution of membra-

nous organelles was studied in cultures exposed to protrusions from the fluorescent organelle mass(50%, n Å 10, four cultures; Fisher’s exact test,8 and 20 nM vinblastine for 24 h (seven cultures

for both concentrations) . In vinblastine-treated cul- one-tailed; p õ 0.05). Nonneuronal cells that mi-grated out of the spinal cord explants stained simi-tures, growth cones often exhibited staining which

was localized to a swelling, in some cases pro- larly in both MEM- and vinblastine-fed cultures(data not shown).nounced, of the neck of the growth cone [Fig.

7(B)] , just behind where the C-domain organellemass is usually located in growth cones in MEM-fed cultures. In the presence of 20 nM vinblastine, DISCUSSION50% of growth cones (n Å 24) exhibited finger-like projections of the fluorescent organelle mass To further the understanding of the mechanisms un-into the P domain. The percentage of finger-like derlying nerve fiber growth and guidance, we inves-projections in growth cones exposed to 20 nM vin- tigated the role of microtubules in growth cone re-blastine was significantly reduced relative to MEM- modeling using drugs that interfere with micro-

tubule dynamics. Vinblastine at low nanomolarconcentrations has been shown to attenuate microtu-bule dynamics by interfering with tubulin polymer-ization (Jordan and Wilson, 1990; Dhamodharan etal., 1995). Although we did not directly measuremicrotubule dynamics, the findings that microtubulearrays within nerve fiber shafts were not disruptedby 8–20 nM vinblastine, nerve fiber width was notaffected, nerve fiber growth rate decreased in adose-dependent manner, and, most important, mi-

Figure 7 Examples of organelle distribution in growth crotubule distribution within growth cones was af-cones. Growth cones were stained with DiOC6(3) to visu- fected are consistent with the attenuation of micro-alize membranous organelles, as detailed in Methods. (A)

tubule dynamic instability in spinal cord growthIn MEM-treated or untreated growth cones, organellecones by 8–20 nM vinblastine. Similar concentra-finger-like projections were often found in the P domaintions of vinblastine have been reported to stabilize(arrowheads) . (B) In the presence of vinblastine (8 nMmicrotubules in other neuronal cell types (Baas andin this case) , organelles relocated to the neck of theAhmad, 1993; Tanaka and Kirschner, 1995; Challa-growth cone (arrows). Note the absence of finger-like

projections into the P domain. Bar Å 10 mm. combe et al., 1997) as well as nonneuronal cells


136 Gallo

(Dhamodharan et al., 1995). The concentrations of ated proteins or organelles) are responsible for themaintenance of continuity between the P and C do-vinblastine at which microtubule depolymerization

became prominent were also similar to those pre- mains. Lamellae that underwent centrifugal separa-tion from the C domain eventually collapsed, evenviously reported to have such effects (Dhamodharan

et al., 1995; Bass and Ahmad, 1993). The possibil- though no nerve fiber retraction had yet com-menced, indicating a requirement of C-domain ele-ity that vinblastine interfered with transport mecha-

nisms within the axon is unlikely, since Horie et al. ments for the long-term maintenance of lamellarstructures. Experiments using cytochalasin B to at-(1983) did not find any effects of the drug on axonal

particle movement at doses lower than 1007 M. . tenuate but not abolish lamellar remodeling showedthat actin-dependent remodeling of lamellae was re-Further, even after 24 h in the presence of the drug,

axons still grew and did not exhibit beading, and quired for the centrifugal separation of the P domainfrom the C domain.the size of lamellar growth cones did not differ

from that of controls (data summarized in Table 1). Neuronal growth cones pull (Lamoureux et al.,1989), a phenomenon dependent on the actin cy-Furthermore, organelle distribution along axons 24

h after 20 nM vinblastine treatment did not appear toskeleton (Letourneau et al., 1987; Lafont et al.,1993). Under normal in vitro conditions, the P do-different from controls (data not shown).

Spinal cord growth cones responded differently main generates tension by pulling on the nerve fibershaft, a force possibly transduced by associationsto concentrations of microtubule drugs resulting in

the attenuation of microtubule dynamics (i.e., between P-domain actin filaments with microtu-bules or of microtubules with the plasmalemma and8–20 nM vinblastine and 10 nM taxol) than to

microtubule disruption (i.e., ¢100 mM vinblastine the cortical actin cytoskeleton (Chalfie and Thomp-son, 1979; Letourneau and Ressler, 1983; Sasakiand 330 nM nocodazole) . Doses of vinblastine and

taxol that altered growth cone microtubule distribu- et al., 1983; Jacobs and Stevens, 1986). If suchconnections were to be lost (e.g., in response totions decreased the extent of growth cone lamellar

remodeling and resulted in the occasional loss of microtubule disruption), the pull generated by la-mellar structures might then be able to separate thethe integrity of fan-shaped lamellae. Also, filopodial

length was increased by 20 nM vinblastine, but no P domain from the C domain. Consistent with thisidea is the finding that if lamellar remodeling iseffects were seen on the number of filopodia at

the growth cone. The disruption of microtubules inhibited by concentrations of cytochalasin B thataffect lamellar protrusion rate and the percentageresulted in the loss of the growth cone fan-shaped

morphology characterized by an initial transition to of lamellar perimeter undergoing remodeling (Ka-pur et al., 1995), lamellar centrifugal separation ina type of morphology having multiple lamellae and

decreased lamellar expanse, and resulting in the ac- response to microtubule drugs does not occur. Thebehavior of lamellar structures following microtu-quisition of a morphology exhibiting only filopodia

by the time when all lamellar expanse had been lost. bule disruption may be due to a loss of the compres-sive support that microtubules provide for the ten-In such cases, filopodia had grown to lengths not

usually observed in spinal cord growth cones. We sion produced by the growth cone and nerve fiberactin network (Joshi et al., 1985). Normally, growththerefore conclude that microtubules have a role

in the maintenance and regulation of growth cone cone lamellar pull may be restrained by microtu-bules that support a fraction of the tension producedremodeling.

The lamellar component of the P domain of by the actin cytoskeleton. If the microtubules wereto be removed, the lamellae might then become ablegrowth cones of spinal cord nerve fibers in vitro

normally remained continuous with the C domain to exert their whole pull unto the substratum, asdiscussed by Dennerll et al. (1988), and therebyduring a 1-h observation period (Gallo and Pollack,

1995, 1997; this report) . In the presence of microtu- pull enough to migrate away from the C domain.Therefore, while growth cone–produced tensionbule drugs, at concentrations resulting in the loss

of growth cone lamellae and eventual nerve fiber may regulate aspects of microtubule assembly (De-nnerll et al., 1988; Zheng et al., 1991), microtubulesretraction, the P domain (lamellae) separated from

the C domain leaving only thin, membranous con- may in turn regulate the shape and behavior of theP domain, perhaps by modulating its ability to exertnections between the two domains. The lamellae

displaced centrifugally relative to the C domain, a pulling force on the substratum and advance.The decrease in lamellar perimeter undergoingwhich underwent no further movement. These ob-

servations indicate that microtubules, or microtu- remodeling in the presence of vinblastine may un-derlie the mechanism by which growth cone fan-bule-associated elements (e.g., microtubule-associ-



shaped lamellae break up and migrate away from found in filopodia. In this case, as dynamic microtu-bules are abolished, F-actin filament organizationthe C domain. While the percentage of lamellar pe-

rimeter actively remodeling was decreased by vin- may tend toward bundling instead of meshworking,resulting in filopodial growth. In favor of this hy-blastine and nocodazole, the rate of lamellar protru-

sion and the percentage of lamellar perimeter under- pothesis, it has been shown that the localizationof members of the ERM family of actin-associatedgoing protrusion relative to the total amount of

lamellar perimeter remodeling were not affected, proteins is dependent upon microtubules (Goslin etal., 1989). Further, tubulin binds to Rac1 (Best etindicating that the initiation of lamellar protrusion

domains is affected by microtubule drugs but not al., 1996), a member of the family of Ras-relatedGTP-binding proteins which has been shown to bethe process of protrusion once it is commenced.

In the presence of microtubule drugs, domains of involved in the regulation of lamellar behavior (Ta-pon and Hall, 1997). In turn, Rac1 has been demon-lamellar protrusion became separated from one an-

other by portions of lamellar perimeter that re- strated to bind IQGAP1, a protein which crosslinksactin filaments into gellike meshworks (Bashour etmained quiescent. If the quiescent lamellar perime-

ter does not undergo further protrusion, it may begin al., 1997), perhaps similar to those which supportlamellar structures. Another GTP-binding protein,to retract in the absence of protrusive activity. Re-

traction of the lamellar perimeter between two pro- Rho, has been shown to be involved in mediatingthe effects of microtubule agents on the formationtrusion domains would physically separate the two

protrusive domains, resulting in the breaking up of of actin stress fibers and focal adhesions in 3T3cells (Enomoto, 1996). Therefore, dynamic micro-growth cone fan-shaped lamellae. The leading edge

of the lamella appears to be particularly susceptible tubules may regulate lamellar and filopodial behav-ior through interactions with molecules which regu-to microtubule drugs. Taxol also decreased the per-

centage of lamellar perimeter actively remodeling, late lamellar actin filament reorganization.The behavior of filopodia and lamellae in re-but did not result in the breaking up of lamellae

during the loss of fan-shaped lamellar morphology. sponse to microtubule drugs may result fromchanges in the organization of the growth cone cyto-This may be due to the fact that taxol does not result

in the loss of microtubules within growth cones, but plasm following microtubule disruption or stabiliza-tion. Our experiments visualizing membranous or-does affect their splaying into the P domain.

The observation that elongation of filopodia is ganelle distribution following exposure to 8–20 nMvinblastine have revealed changes in organelle dis-promoted during microtubule drug mediated growth

cone collapse, and that in the presence of 20 nM tribution in growth cones. In particular, the fre-quency of finger-like projections from the growthvinblastine filopodia have greater mean lengths than

controls, shows that microtubules are also involved cone neck into the P domain was decreased by vin-blastine. These experiments provide support for thein the regulation of filopodial morphology at the

neuronal growth cone. Colocalization of microtu- hypothesis that microtubule dynamics may have arole in determining the localization of finger-likebules and the actin filament bundles supporting fi-

lopodial structures have been reported (Gordon- membranous projections into the P domain (Daileyand Bridgman, 1989). Although the present data doWeeks, 1991). Possibly, microtubules that contact

the actin bundles of filopodia may prevent the filo- not allow us to determine the role of organelles inthe regulation of growth cone lamellar remodeling,podia from growing beyond a certain length. When

microtubules are no longer present, filopodia may the correlated alteration in organelle and microtu-bule distribution in response to microtubule drugthen elongate to greater than normal lengths. In sup-

port of a role for microtubules in regulating filopod- treatments suggests their involvement. In supportfor a role of organelles in the regulation of lamellarial morphology, taxol also has been reported to af-

fect aspects of filopodial morphology in dorsal root motility, Rodionov et al. (1993) found that inhibi-tion of kinesin, an organelle motor protein, resultedganglion growth cones (Letourneau and Ressler,

1984; Williamson et al., 1996). Interactions be- in an inhibition of nonneuronal lamellar motility in amanner similar to treatment with microtubule drugstween microtubules and filopodia, possibly via the

filopodial actin bundles, may have a significant role (Bershadsky et al., 1991). Further work will berequired to determine the exact role of C-domainin the regulation of growth cone remodeling and

play an important role in nerve fiber guidance. organelles in the regulation of growth cone lamellarbehavior.Alternatively, the activity of dynamic microtu-

bules may promote the generation of F-actin mesh- The alterations in growth cone morphology oc-curring as a result of concentrations of microtubulesworks, as found in lamellae, instead of bundles, as


138 Gallo

drugs that cause a halt, or attenuation, of nerve fiber placement of the microtubule tips themselves or al-terations in the localization of microtubule associ-growth may hold significance for growth cone guid-

ance. When growth cones arrive at ‘‘decision re- ated cellular constituents (e.g., organelles) . There-fore, although the present data are suggestive ofgions’’ in vivo, they often balk and adopt a morphol-

ogy characterized by the extension of P-domain a relationship between microtubule dynamics andlamellar motility, alternative hypothesis cannot atstructures (Godement et al., 1994; Halloran and

Kalil, 1994; Holt, 1989; Kaethner and Stuermer, present be ruled out.1992). The spinal cord growth cones studied herehave been reported to undergo frequent transitions The author thanks Dr. E. D. Pollack (University ofto a filopodial morphology in the presence of cocul- Illinois) for providing the laboratory facilities and supporttured target tissue in vitro (Gallo and Pollack, 1995, required to complete this research, Dr. P. C. Letourneau1997), to which they exhibit a tropic response (Pol- (University of Minnesota) for commenting on the manu-lack et al., 1981). Interestingly, the changes in la- script, and A. Zartaysky for technical assistance. This

research was supported in part by the Campus Researchmellar and filopodial morphology that spinal cordBoard of the University of Illinois at Chicago.growth cones exhibit in the presence of target tissue

are very similar to those observed in the presenceof microtubule drugs (Gallo and Pollack, 1997).Since filopodia (Kater et al., 1994) and possibly

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