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Bundling of Microtubules in the Growth Cone Induced by Laminin
Molecular and Cellular Neuroscience 15, 303–313 (2000) doi:10.1006/mcne.1999.0820, available online at http://www.idealibrary.com on
MCN
Bundling of Microtubules in the Growth Cone Induced by Laminin Dongjiang Tang and Daniel J. Goldberg Department of Pharmacology and Center for Neurobiology and Behavior, Columbia University, New York, New York 10032
Axons growing in the developing nervous system are guided by cues in the environment which act at the growth cone. So far, the initial cytoskeletal target of these cues has been found to be the network of actin filaments in the peripheral region of the growth cone. Laminins are constituents of the extracellular matrix which promote axonal growth. They exert effects on the actin network. Here, laminin 1 is shown to affect microtubules as well. Acute addition of laminin 1 to rat sympathetic neurons quickly caused the advance of microtubules and their bundling within the initial widely spread growth cone and then the outgrowth of thin, rapidly growing nascent axons. The bundling was pharmacologically separable from the advance of microtubules caused by laminin, as the former but not the latter was blocked by lithium. The bundling did not depend on the peripheral network of actin filaments, as it was unimpaired by the removal of this network with cytochalasin D. Thus, microtubules seem to be a direct cytoskeletal target for laminin 1 in the growth cone, with important consequences for axonal outgrowth.
INTRODUCTION The rate and direction of growth of neuronal processes are regulated by soluble and surface-bound environmental cues (Goodman, 1996). The growth cone, the specialized ending of a growing neurite, is the key structure for detecting and responding to these cues. How cues elicit the changes in growth cone morphology and behavior that lead to changes in growth is not well understood but, ultimately, the arrangement of the major cytoskeletal elements—actin filaments and microtubules—must be important. The peripheral region of the growth cone is richly invested with actin filaments, which drive the movements of its digitate filopodia and diaphanous veils and lamellipodia (Letourneau, 1983). These peripheral protrusions, especially the filopodia, 1044-7431/00 $35.00 Copyright r 2000 by Academic Press All rights of reproduction in any form reserved.
are important in detecting guidance cues; their absence gives rise to pathfinding errors (Bentley and ToroianRaymond, 1986). Microtubules project into the growth cone from the core of the axon and their entry into peripheral regions is probably important in transforming these into new lengths of axon (Goldberg and Burmeister, 1989). For those cues for which it has been examined, the peripheral actin filaments seem to be the primary cytoskeletal target. Nerve growth factor (NGF), a soluble growth promoter, rapidly induces actin-based motile activities such as filopodial protrusion (Seeley and Greene, 1983). Collapsin-1 (semaphorin III), a growth inhibitor, depletes the peripheral region of actin filaments (Fan et al., 1993). Surface-bound growth promoters link through their membrane receptors to the network of actin filaments undergoing a steady rearward flux in the peripheral region (Suter et al., 1998). This evidently permits the advance of other components of the growing axon, such as microtubules and membranebound organelles. Laminin 1 (mouse EHS laminin) is a widely studied cue that typically acts as a surface-bound molecule to promote axonal differentiation and growth (Lander, 1987; Lein and Higgins, 1989). Isoforms of laminin are major constituents of extracellular matrices (ECMs) and can also be expressed on the surfaces of Schwann and muscle cells (Lentz et al., 1997; Patton et al., 1997). Promotion of axonal growth by laminin 1 is mediated by integrin surface receptors (Bozyczko and Horwitz, 1986; Tomaselli et al., 1987; Reichardt and Tomaselli, 1991). Antibodies to integrin block growth of neurites on laminin 1 substrates in culture and regenerative growth of peripheral motor axons in situ. Laminin 1 elicits processes that not only grow fast, but also are thin, as is characteristic of axons (Rivas et al., 1992). It is possible that this streamlined shape facili-
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FIG. 1. Laminin causes bundling of microtubules in growth cones. Growth cones were fixed at various time points and stained for detection of microtubules by immunofluorescence microscopy. Phase-contrast images were taken before fixation. (A and B) A growth cone without exposure to laminin. (A) Phase-contrast image of a sympathetic neuron with a short thick axon and wide growth cone. (B) Immunofluorescent visualization of microtubules in this growth cone shows them to be confined to the central region. The brightness of the image has been reduced here to allow visualization of detail in the central region of the growth cone. (C–H) Two growth cones in laminin-treatment experiments. (C and F) The two growth cones before addition of laminin. (D) 45 min after the addition of laminin, material has advanced in the peripheral region as judged by the position of phase-dark material (denoted by arrowhead). (G) 90 min after the exposure of the other growth cone to laminin, thin neurites have sprouted from the edge of the peripheral region. (E and H) Fluorescence images of the growth cones of D and G. In E, two microtubule bundles
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tates the rapid growth. As might be expected, the thin processes and their small associated growth cones have more closely packed microtubules (Burden-Gulley and Lemmon, 1996). This, plus the fact that depletion of the microtubule-associated proteins MAP1b and tau reduces the acceleration of growth caused by laminin 1 (DiTella et al., 1996), suggests that the microtubules of the growth cone might also be a target for laminin 1. We show here that laminin 1 elicits bundling of microtubules in the growth cone of sympathetic neurons in culture and that this is not secondary to an effect on the peripheral network of actin filaments. The bundling precedes the appearance of the thin processes and thus is an early event in the differentiation of the neurite into an axon. Thus, microtubules may also serve as direct targets of action in the growth cone for environmental cues that regulate growth.
RESULTS
similar to axons in their thinness and rapid growth. A concentrating of the protein GAP-43 in the growth cone is the earliest biochemical marker of axonal identity when hippocampal cells begin to develop polarity in culture (Goslin and Banker, 1990). We thus examined quantitatively the distribution of GAP-43 in separate groups of neurons before and 2 and 4.5 h after the addition of laminin. GAP-43 was present in growth cones before and after the addition of laminin, but was much more concentrated in the small growth cones 2 h after the addition of laminin (Fig. 2). It became further concentrated by the later time point. Staining of the thin shaft of the neurite was dim. Two hours after the addition of laminin, the original widely spread growth cone often remained, with thin neurites projecting from its periphery. The GAP-43 staining intensity in the remaining wide growth cone was similar to that of the control condition (data not shown). Thus neurites, originally in an undifferentiated form, begin acquiring an axonal phenotype soon after exposure to laminin.
Thin Neurites Form after Addition of Laminin 1 We had previously shown that acute addition of laminin 1 to rat sympathetic neurons cultured on a polylysine-coated substrate causes a rapid increase in the neuritic growth rate (Rivas et al., 1992). Multiple thin processes were sometimes seen to sprout from large spread growth cones after the addition of laminin. We wanted to determine how common this rapid change in morphology was, so we observed neurons 4–8 h after plating in serum-free medium on polylysine/polyornithine. Numerous cells had a thick neurite ending in a growth cone with a large lamellipodium (Figs. 1A, 1C, and 1F). During the 1.5–2 h before adding laminin, these neurites elongated little. By 1.5 h after addition of laminin, 32 of 38 (84%) growth cones had developed thin neurites growing out from the original large growth cone (Fig. 1G).
Neuritic Outgrowth in Response to Laminin Is Axonal Laminin 1 has been shown to stimulate axonal growth specifically (Lein and Higgins, 1989). The neurites that grew out in response to laminin in our experiments are
Laminin Causes Bundling of Microtubules in the Growth Cone Before the addition of laminin, microtubules are largely restricted to the central region of the growth cone (Rivas et al., 1992, and Fig. 1B). Addition of laminin causes them to advance into the peripheral region within 10–20 min (Rivas et al., 1992, and Fig. 1E). We assessed the distribution of microtubules in growth cones fixed at various times 15–90 min after the addition of laminin or vehicle. Of the growth cones treated with laminin 59% had at least one discernible bundle of microtubules while only 12% without laminin did (Fig. 3). The 59% probably is an underestimate of the fraction of growth cones that would develop bundled microtubules in response to laminin, since some in our sample (those fixed at earlier times) probably did not have time to respond. Bundles in the thickened central region of the growth cone, to which microtubules are largely confined in the absence of laminin, are probably more difficult to detect than bundles in the very flat peripheral region. Therefore, the difference in bundling of microtubules with and without laminin might be more apparent
(arrows) formed within the growth cone before the emergence of thin neurites, while in H, bundles (arrow) run from the still spread growth cone into the thin neurites. The graph to the left of E shows the line scan of E (pixel brightness). The two peaks, which are both at least 30 units above the surrounding baseline (256-unit gray scale), represent the two bundles in E. The outlines of the growth cones, as traced from the phase-contrast micrographs, are drawn on the fluorescence micrographs. Bar, 10µm.
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FIG. 2. GAP-43 concentrates in growth cones after exposure to laminin. Growth cones were fixed at three time points and stained for detection of GAP-43 by immunofluorescence microscopy. (A) Before addition of laminin (control), GAP-43 is evenly distributed in the widely spread growth cone, and its relative staining intensity is low. (B) GAP-43 staining was brighter in the small growth cones at the end of the thin neurites formed 2 h after addition of laminin. Bar, 10 µm. (C) Quantitation of the GAP-43 staining shows the increase of this protein in growth cones after addition of laminin. The ordinate shows the average pixel brightness (in arbitrary units, 0–4095). The data at each time point represent the mean value of 25 individual growth cones in three independent experiments. (Error bars show standard error of the mean.) Asterisks denote values that are significantly different from the control (0 h) (P , 0.0001), as determined by ANOVA followed by a t test using Bonferroni’s modification for multiple comparisons.
than real. Experiments with cytochalasin (described below) showed this not to be the case; even microtubules in the peripheral region could be seen to be largely unbundled in the absence of laminin. In those growth cones fixed after the emergence of thin neurites, the bundles of microtubules in the spread original growth cone typically ran into the thin neurites (Fig. 1H). Thus, it seemed possible that microtubules were being zippered together by constriction of the plasma membrane to form the thin neurites. This appears not to be the case. Many growth cones were fixed at a time at which microtubules had advanced from the central region deep into the peripheral region in response to laminin, but thin neurites had not yet emerged (Fig. 1E). [There is often a delay between those two stages in the response to laminin (Rivas et al., 1992).] Of
these growth cones 46% showed some bundling of microtubules (Fig. 3); this, also, should be viewed as a minimum estimate. The advance and subsequent bundling of microtubules caused by laminin were pharmacologically separable events. Lithium (20 µM, with 20 µM myoinositol to prevent depletion of inositol in the cell), which can affect the packing of microtubules in neurites, did not by itself cause microtubules to advance or to bundle (Figs. 4F and 5) and had no evident effect on F-actin in the peripheral region of the growth cone (Fig. 4E). Addition of laminin in the presence of lithium elicited advancement of microtubules and membrane-bound organelles into the peripheral region, as typically seen (Fig. 5). However, only 32% of these growth cones displayed bundling of microtubules and only 19% produced thin
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FIG. 3. Laminin (LN) causes bundling of microtubules (MTs) which is not prevented by cytochalasin D (CD). 59% of the growth cones investigated (including growth cones of the types shown in Figs. 1D, 1E, 1G, and 1H) exhibited bundling of microtubules after addition of laminin (left black bar), as opposed to 12% without laminin (left white bar). Bundled microtubules were seen in 46% of those growth cones (like the one shown in Figs. 1D and 1E) from which thin neurites had not yet emerged though microtubules had advanced into the peripheral region (hatched bar). The percentage of growth cones showing bundling of microtubules after addition of laminin was only slightly reduced by preexposure to CD (middle and right pairs of bars; gray bars represent cells exposed only to CD). Asterisks denote those percentages that are significantly different from their controls (P , 0.002), as determined by the approximate test for the comparison of two proportions. For each condition above, at least 30 growth cones from four independent experiments were counted.
neurites. In addition, the acceleration of elongation typically caused by laminin was blocked by lithium (Fig. 6).
The Peripheral Network of Actin Filaments Is Not Needed for Bundling of Microtubules nor Are Neurofilaments Although constriction of the plasma membrane was not needed for the bundling of microtubules, we thought they might be forced into bundles by actions of the rich internal peripheral network of actin filaments. We used the fungal metabolite cytochalasin D (CD) to eliminate this network. The network rapidly withdraws from the growth cone in the presence of CD, allowing microtubules and membrane-bound organelles to advance deep into the peripheral region (Forscher and Smith, 1988). One hour of exposure to 1 µM CD (without laminin) left the peripheral region of the growth cone, which is
normally richly invested with F-actin, largely devoid of F-actin (Fig. 4C). Scattered clumps of F-actin sometimes remained at the edge, but not colocalized with microtubules. Of the observed large growth cones 77% had microtubules and membrane-bound organelles filling much or all of the peripheral region (Fig. 4D), much as was seen with laminin (without CD). Yet, only 14% of the growth cones displayed bundling of microtubules (Fig. 3), demonstrating that advance of microtubules into the peripheral region is not sufficient for bundling to occur or for us to be able to detect it. In contrast, when laminin was added 20 min after the addition of CD, 45% of the growth cones subsequently showed bundling of microtubules, only slightly less than was seen without CD (Fig. 3). Increasing the concentration of CD 10-fold had no further effect. Many fewer thin neurites formed in response to laminin when CD was present (Fig. 7), as expected because the peripheral actin network facilitates neuritic elongation (Letourneau et al., 1987). However, 28% of the observed cells produced thin neurites, suggesting that the advance and bundling of microtubules could be sufficient to produce this type of process. Neurofilaments are the other major elements of the cytoskeleton of the axon. We used antibodies monospecific for the three subunits of the neurofilament. Antibodies against the 200-kDa subunit (Fig. 8) and the 68-kDa subunit (not shown) stained the neurite more brightly by immunofluorescence than when primary antibody was omitted, but neither stained the growth cone strongly and neither stained fibers within the growth cone, either before laminin had been added or when microtubules had projected deep into the peripheral region of the growth cone after the addition of laminin. We detected no specific staining with antibody directed against the 160-kDa subunit (not shown). Therefore, neurofilaments do not appear to be present in appreciable numbers in these growth cones.
DISCUSSION These studies demonstrate that the axonal growthpromoting molecule laminin 1 causes microtubules in the growth cone to bundle. This effect is not mediated by the network of actin filaments, with which laminin interacts, nor by neurofilaments, indicating that the microtubules are probably a direct cytoskeletal target for laminin. The bundling of microtubules may contribute to fast growth and to differentiating a process into an axon.
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FIG. 4. The impact of cytochalasin D (CD) and lithium on the actin network and microtubules (MTs) in growth cones. Growth cones were fixed and double stained for detection of actin and microtubules by immunofluorescence microscopy. (A, C, and E) Actin staining of three growth cones. (B, D, and F) MT staining in the same growth cones. (A) F-actin network heavily invests the peripheral region of a growth cone in the control condition. (B) MTs in the growth cone were confined to the central region. (C) 1 h after addition of 1 µM CD, the F-actin network was eliminated, with some staining remaining at edges. (D) MTs advanced to the peripheral region of the growth cone, yet no MT bundles formed. (E and F) 1 h after addition of lithium and myoinositol, the patterns of F-actin and MT staining were the same as without them, indicating that these chemicals do not overtly affect those networks. Bar, 10µm.
Laminin Induces Bundling of Microtubules
FIG. 5. Lithium blocks the bundling of microtubules (MTs) and the formation of thin neurites, but not the advance of microtubules, caused by laminin (LN). LN was added for 90 min either in the absence of lithium (bars 2) or after 30 min of lithium preincubation (bars 3). Control experiments involved addition of vehicle alone (bars 1) or lithium alone for 90 min (bars 4). Asterisks denote those ‘‘LN 1 Lithium’’ percentages that are significantly different from their corresponding ‘‘LN only’’ percentages (P , 0.002). At least 40 growth cones from four independent experiments were counted for each condition.
Bundling of Microtubules Caused by Laminin The present work is in agreement with previous work that microtubules in the growth cone assume a more closely packed arrangement in response to laminin 1 (Burden-Gulley and Lemmon, 1996). However, in those
FIG. 6. Lithium blocks the acceleration of neuritic growth elicited by laminin (LN). Phase-contrast images were taken for the same group of neurons at three time points and the lengths of the neurites were measured. Asterisks denote those values that are significantly different from their controls (P , 0.002). The data represent the average of 50 individual neurites from three independent experiments for each condition.
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FIG. 7. Cytochalasin D (CD) reduces the formation of thin neurites elicited by treatment with laminin (LN). Growth cones were assessed 90 min after the addition of laminin. CD was added 20 min before the addition of laminin. Asterisks denote those percentages that are significantly different from the control (laminin only) (P , 0.002). For each condition above, at least 40 growth cones from four independent experiments were counted.
studies of chick retinal ganglion cells, laminin also caused actin filaments to fill more of the growth cone and to assemble into cables running into the area containing the microtubules. The network of actin filaments affects the distribution of microtubules in the growth cone. It can retard the entry of microtubules into the peripheral region, as evidenced by the rapid entry of microtubules when the actin network is eliminated by cytochalasin (Forscher and Smith, 1988, and the present results). It can also facilitate the advance of microtubules by exerting tension on them (Suter et al., 1998). The actin filaments are also needed to reorient microtubules (perhaps by pulling them) either toward or away from external cues during growth cone turning (Lin and Forscher, 1993; Challacombe et al., 1996). Thus, the bundling of microtubules might be mediated by the network of actin filaments. The fact that the bundling occurred in our experiments even after this network had been eliminated from the growth cone by CD demonstrates that this is not the case. Neurofilaments, the other major component of the cytoskeleton, do not project into these growth cones in appreciable numbers. Our evidence, therefore, points to the microtubule as a primary cytoskeletal target for laminin 1 in the growth cone. Laminin 1 thus appears to have two actions on the growth cone which are distinguishable temporally, pharmacologically, and, probably, mechanistically. Laminin 1 initially causes an advance of microtubules and cytoplasm into the peripheral region of the growth cone in
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FIG. 8. Neurofilaments are not present in the widely spread growth cones. Growth cones were fixed and stained for detection of neurofilaments by immunofluorescence microscopy. (A) Staining of a growth cone to visualize microtubules. (B) Staining of the same growth cone with a different secondary antibody but no primary antibody, showing the level of nonspecific staining for comparison to C and D. (C and D) mAb (NE14) against the 200-kDa subunit of neurofilaments was used as the primary antibody. Bright staining appeared only in the neurite. The faint signal in the growth cone is diffuse rather than fibrillar. (C) A growth cone without exposure to laminin. (D) 30 min after addition of laminin, when microtubules have advanced. Bar, 10µm.
our experiments, followed by bundling of microtubules and the appearance of thin neurites growing rapidly (Rivas et al., 1992). Only the latter events were blocked by lithium in the present experiments. Kuhn et al. (1995, 1998) showed that when a filopodium touches a bead coated with laminin 1, there is a rapid entry of cytoplasm into the filopodium, followed at some delay by growth at an accelerated rate. The advance of microtubules and cytoplasm into the peripheral region of the growth cone is probably caused by physical linking of laminin on the substrate to the network of actin filaments in the peripheral region of the growth cone and is probably a general mechanism for substrate-bound growth promoters (Felsenfeld et al., 1996; Grabham and Goldberg, 1997; Suter et al., 1998). We show here that the later effect is independent of actin filaments but is associated with bundling of microtubules. Our identification of the microtubule as a primary cytoskeletal target is a novel twist for ECM molecules and for other molecules that affect the growth cone to regulate neuritic growth. Binding of ECM molecules like laminin 1 to their integrin receptors [b1 integrin mediates the effects of laminin on the growth cone in our preparation (Rivas et al., 1992)] had so far been
shown to target the actin cytoskeleton to cause cell spreading (Hynes and Destree, 1978; Mooney et al., 1995), adhesion, and migration (Burridge et al., 1988; Felsenfeld et al., 1996). As for the growth cone, those external cues studied so far, including laminin 1, have been found to target the peripheral network of actin filaments (Seeley and Greene, 1983; Fan et al., 1993; Grabham and Goldberg, 1997; Suter et al., 1998; and see Introduction). Effects on the synthesis or phosphorylation of microtubules and MAPs have been described, though not specifically related to growth cone functioning (Black et al., 1986; Aletta et al., 1988).
Roles for Microtubule Bundling Microtubule bundling has been shown to precede constriction of the plasma membrane during neuritic elongation and turning (Tanaka and Kirschner, 1991, 1995). We also often detected bundles in growth cones from which thin neurites had not yet emerged. Thin neurites emerged from some growth cones even after peripheral F-actin had been eliminated with CD, suggesting that the effects of laminin on the microtubules are sufficient in some cases for the formation of thin neu-
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rites. Thus, it seems likely that the bundling induced here is important in causing the formation of the thin neurites. The bundling of microtubules may contribute to accelerating neuritic elongation and to the conversion of an undifferentiated neurite into an axon. Bundling might accelerate elongation by reducing lateral excursions of the growth cone (Aigner and Caroni, 1993; Tanaka et al., 1995). It could also allow more efficient delivery of the transported materials upon which growth depends. Observations of axons growing in vivo show that rapid elongation is typically associated with small, narrow growth cones (LoPresti et al., 1973; Kaethner and Stuermer, 1992), as would be associated with compact microtubules. The thin processes that form in response to laminin are nascent axons (our results and Lein and Higgins, 1989). The bundling we observe here is one of the earliest changes that have been observed in the transformation of undifferentiated neurite into axon, as it precedes the emergence of the fast growing nascent axon. Within an hour of the acceleration of growth that marks the conversion of neurite to axon in hippocampal neurons (Goslin and Banker, 1989), the number of microtubules in the growing process has greatly increased and they have become more closely packed and aligned in parallel (Yu and Baas, 1994). Even before the nascent hippocampal axon accelerates its growth (i.e., becomes recognizable as an axon), it displays a greatly increased transport of organelles (Bradke and Dotti, 1997). Since organelles are transported along microtubules, it is reasonable to suggest that a key early event in axonal differentiation is a change in the organization or properties of the microtubules, perhaps bundling. This could permit the increased delivery of materials needed to support the faster growth of the axon.
(Eugene, OR). Monoclonal antibody against GAP-43 (7b10/c3) was generously provided by Dr. K. Meiri. Polyclonal antibodies against a-tubulin in Glu and Tyr forms (SG and W2) were generously provided by Dr. J. C. Bulinski.
Tissue Culture Sympathetic superior cervical ganglion and/or sympathetic chains from the thoracic region of the spinal cord were dissected from postnatal day 1–4 rats and placed in Leibowitz’s L-15 medium (GIBCO). Ganglia were incubated in 0.25% trypsin in Ca21/Mg21-free Hanks’ balanced salt solution for 30 min at 37°C and were dissociated by gentle trituration with fire-polished Pasteur pipettes. All coverslips, glued under a hole in the bottom of a 60-mm plastic dish, were coated with a mixture of 100 µg/ml poly-D-lysine and polyornithine for 2 h at room temperature or overnight at 4°C, then rinsed twice with double-distilled water. Defined culture medium was L-15 supplemented with NGF (100 ng/ml), bovine insulin (5 µg/ml), human transferrin (10 µg/ml), sodium selenite (5 ng/ml), sodium pyruvate (1 mM), and bovine serum albumin (0.5 mg/ml). Two protocols were used to obtain growth cones suitable for study. In one, cells were cultured for 4–8 h before observation. In the other, cells were maintained for 16–24 h and then replated to induce new short thick neurites ending in a spread growth cone. For all experiments, laminin 1 was added to the culture medium at a final concentration of 25 µg/ml to assess its effect. In some experiments, 20 mM lithium chloride and 20 mM myoinositol were added to the medium 30 min before the addition of laminin 1.
Immunocytochemistry
EXPERIMENTAL METHODS Materials Cytochalasin D, lithium chloride, myoinositol, bovine insulin, human transferrin, sodium selenite, poly-Dlysine, polyornithine, osmium tetroxide, monoclonal antibody against b-tubulin (JDR.38B), and monoclonal antibodies against neurofilament subunits (NE14, NR4, and NN18) were purchased from Sigma Chemical (St. Louis, MO). Sodium pyruvate was from GIBCO (Grand Island, NY). NGF and laminin 1 (from mouse EHS sarcoma) were from Boehringer Mannheim (Indianapolis, IN). Texas red-conjugated goat anti-mouse antibody, FITC-conjugated goat anti-rabbit antibody, and Texas red–phalloidin were obtained from Molecular Probes
Immunofluorescence procedures to visualize microtubules were as previously described by Black et al. (1994), with fixation procedure 2 used most often. Briefly, cells were extracted for 2 min in buffered medium containing 0.2% saponin and 10µM Taxol. After extraction, the cells were fixed for 10 min in medium containing 2% paraformaldehyde and 0.05% glutaraldehyde. To visualize GAP-43 and neurofilaments, cells were fixed in PBS containing 4% paraformaldehyde, 0.1% glutaraldehyde, and 400 mM sucrose for 5 min at 37°C, followed by one rinse and three 5-min washes with 0.15% Triton X-100 in PBS. Nonspecific binding was blocked by incubation in 10% normal goat serum (NGS) for 30 min at room temperature. The cells were incubated overnight at 4°C with primary antibodies against b-tubulin or neurofila-
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ment subunits at a 1:100 dilution or against GAP-43 at a 1:5 dilution. The antibodies were diluted in PBS/4% NGS. Cells were then washed and incubated for 1 h with 1:100 secondary antibody conjugated to Texas red followed by mounting in 20 mg/ml propyl gallate in 90% glycerol/10%PBS. Immunofluorescence procedures for double staining of F-actin and another antigen were essentially as previously described by Lewis and Bridgman (1996). After the F-actin staining was recorded, cells were processed further for staining to visualize microtubules. Cells were incubated with a mixture of primary antibodies (SG and W2) against tubulin at a 1:100 dilution at 4°C overnight. The cells were then washed and incubated for 1 h with 1:100 fluorescein-conjugated secondary antibody.
that met our criteria. Because of variation in the time course of the response, growth cones at different time points from 0.5 to 1.5 h were pooled in the analysis. A growth cone was scored as having had an advance of microtubules if the distance between the neck of the growth cone and the front edge of the envelope of microtubules was more than 85% of the total distance from the neck to the distal edge (not including filopodia) of the growth cone. Control growth cones never displayed this; the average percentage of the length occupied by microtubules in control growth cones was 65%. The area measurement function of the Metamorph system was used to quantitate GAP-43 staining by measuring average pixel brightness for the entire growth cone.
Image Acquisition and Analysis
ACKNOWLEDGMENTS
Phase-contrast microscopy was done with 20 and 633 objectives. An automated stage was used to relocate multiple growth cones. Images were recorded at times before and after the addition of laminin, cytochalasin D, and/or lithium chloride. The preparation was then fixed and processed for immunofluorescent visualization of microtubules in the same growth cones observed with phase-contrast microscopy. Microtubules were visualized with a 403, 1.3 NA objective lens to optimize horizontal and vertical resolution and images were captured with a cooled CCD camera (Princeton Instruments, Trenton, NJ) to allow accurate quantitation of fluorescence intensity. The following procedure was used to identify a bundle. First, we inspected the whole growth cone we were analyzing to find potential bundles of microtubules. For each potential bundle, we measured its pixel brightness along a line drawn across the growth cone perpendicular to the long axis of the potential bundle using the line scan function of the Metamorph image analysis system (Universal Imaging, West Chester, PA). We also measured the thickness and length of the potential bundle using the measure distance function. A bundle was scored as such if it met all of the following criteria: (1) a peak in brightness at least 30 units above the surrounding baseline (256-unit gray scale), (2) an apparent thickness of at least 1.44 µm, and (3) a length of at least 6.67 µm. This somewhat conservative definition selected fibers that were clearly different from single microtubules. However, it probably excluded some small bundles. It would rule out bright spots because they would not be long enough. A growth cone was scored as having bundling if it had at least one bundle
We are grateful to Drs. J. Chloe Bulinski and Karina Meiri for generous gifts of antibodies. We are grateful to Dr. Peter W. Grabham for assistance with some procedures and for comments on an earlier version of the manuscript and to Ms. Anuli Umeojiako for excellent technical assistance. This work was supported by NIH Grants NS25161 and GM32099.
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Laminin Induces Bundling of Microtubules
Challacombe, J. F., Snow, D. M., and Letourneau, P. C. (1996). Actin filament bundles are required for microtubule reorientation during growth cone turning to avoid an inhibitory guidance cue. J. Cell Sci. 109: 2031–2040. DiTella, M. C., Feiguin, F., Carri, N., Kosik, K. S., and Ca´ceras, A. (1996). MAP-1B/tau functional redundancy during lamininenhanced axonal growth. J. Cell Sci. 109: 467–477. Fan, J., Mansfield, S. G., Redmond, T., Gordon-Weeks, P. R., and Raper, J. (1993). The organization of F-actin and microtubules in growth cones exposed to a brain-derived collapsing factor. J. Cell Biol. 121: 867–878. Felsenfeld, D. P., Choquet, D., and Sheetz, M. P. (1996). Ligand binding regulates the directed movement of b1 integrins on fibroblasts. Nature 383: 438–440. Forscher, P., and Smith, S. J. (1988). Actions of cytochalasins on the organization of actin filaments and microtubules in a neuronal growth cone. J. Cell Biol. 107: 1505–1516. Goldberg, D. J., and Burmeister, D. W. (1989). Looking into growth cones. Trends Neurosci. 12: 503–506. Goodman, C. S. (1996). Mechanisms and molecules that control growth cone guidance. Annu. Rev. Neurosci. 19: 341–377. Goslin, K., and Banker, G. (1989). Experimental observations on the development of polarity by hippocampal neurons in culture. J. Cell Biol. 108: 1507–1516. Goslin, K., and Banker, G. (1990). Rapid changes in the distribution of GAP-43 correlate with the expression of neuronal polarity during normal development and under experimental conditions. J. Cell Biol. 110: 1319–1331. Grabham, P. W., and Goldberg, D. J. (1997). Nerve growth factor stimulates the accumulation of b1 integrin at the tips of filopodia in the growth cones of sympathetic neurons. J. Neurosci. 17: 5455–5465. Hynes, R. O., and Destree, A. T. (1978). Relationships between fibronectin (LETS protein) and actin. Cell 15: 875–886. Kaethner, R. J., and Stuermer, C. A. O. (1992). Dynamics of terminal arbor formation and target approach of retinotectal axons in living zebrafish embryos: A time-lapse study of single axons. J. Neurosci. 12: 3257–3271. Kuhn, T. B., Schmidt, M. F., and Kater, S. B. (1995). Laminin and fibronectin guideposts signal sustained but opposite effects to passing growth cones. Neuron 14: 275–285. Kuhn, T. B., Williams, C. V., Dou, P., and Kater, S. B. (1998). Laminin directs growth cone navigation via two temporally and functionally distinct calcium signals. J. Neurosci. 18: 184–194. Lander, A. (1987). Molecules that make axons grow. Mol. Neurobiol. 1: 213–245. Lein, P. J., and Higgins, D. (1989). Laminin and a basement membrane extract have different effects on axonal and dendritic outgrowth from embryonic rat sympathetic neurons in vitro. Dev. Biol. 136: 330–345. Lentz, S. I., Miner, J. H., Sanes, J. R., and Snider, W. D. (1997). Distribution of the ten known laminin chains in the pathways and targets of developing sensory axons. J. Comp. Neurol. 378: 547–561.
313 Letourneau, P. C. (1983). Differences in the organization of actin in the growth cones compared with the neurites of cultured neurons from chick embryos. J. Cell Biol. 97: 963–973. Letourneau, P. C., Shattuck, T. A., and Ressler, A. H. (1987). ‘‘Pull’’ and ‘‘push’’ in neurite elongation: Observations on the effects of different concentrations of cytochalasin B and Taxol. Cell Motil. Cytoskeleton 8: 193–209. Lewis, A. K., and Bridgman, P. C. (1996). Mammalian myosin Ia is concentrated near the plasma membrane in nerve growth cones. Cell Motil. Cytoskeleton 33: 130–150. Lin, C.-H., and Forscher, P. (1993). Cytoskeletal remodeling during growth cone-target interactions. J. Cell Biol. 121: 1369–1383. LoPresti, V., Macagno, E. R., and Levinthal, C. (1973). Structure and development of neuronal connections in isogenic organisms: Cellular interactions in the development of the optic lamina of Daphnia. Proc. Natl. Acad. Sci. USA 70: 433–437. Mooney, D. J., Langer, R., and Ingber, D. E. (1995). Cytoskeletal filament assembly and the control of cell spreading and function by extracellular matrix. J. Cell Sci. 108: 2311–2320. Patton, B. L., Miner, J. H., Chiu, A. Y., and Sanes, J. R. (1997). Distribution and function of laminins in the neuromuscular system of developing, adult, and mutant mice. J. Cell Biol. 139: 1507–1521. Reichardt, L. F., and Tomaselli, K. J. (1991). Extracellular matrix molecules and their receptors: Functions in neural development. Annu. Rev. Neurosci. 14: 531–570. Rivas, R. J., Burmeister, D. W., and Goldberg, D. J. (1992). Rapid effects of laminin on the growth cone. Neuron 8: 107–115. Seeley, P. J., and Greene, L. A. (1983). Short-latency local actions of nerve growth factor at the growth cone. Proc Natl. Acad. Sci. USA 80: 2789–2793. Suter, D. M., Errante, L. D., Belotserkovsky, V., and Forscher, P. (1998). The Ig superfamily cell adhesion molecule, apCAM, mediates growth cone steering by substrate–cytoskeletal coupling. J. Cell Biol. 141: 227–240. Tanaka, E. M., Ho, T., and Kirschner, M. W. (1995). The role of microtubule dynamics in growth cone motility and axonal growth. J. Cell Biol. 128: 139–155. Tanaka, E. M., and Kirschner, M. W. (1991). Microtubule behavior in the growth cones of living neurons during axon elongation. J. Cell Biol. 115: 345–363. Tanaka, E. M., and Kirschner, M. W. (1995). The role of microtubules in growth cone turning at substrate boundaries. J. Cell Biol. 128: 127–137. Tomaselli, K. J., Damsky, C. H., and Reichardt, L. F. (1987). Interactions of a neuronal cell line (PC12) with laminin, collagen IV, and fibronectin: Identification of integrin-related glycoproteins involved in attachment and process outgrowth. J. Cell Biol. 105: 2347–2358. Yu, W., and Baas, P. W. (1994). Changes in microtubule number and length during axon differentiation. J. Neurosci. 14: 2818–2829. Received July 16, 1999 Revised November 1, 1999 Accepted November 12, 1999
MCN
Bundling of Microtubules in the Growth Cone Induced by Laminin Dongjiang Tang and Daniel J. Goldberg Department of Pharmacology and Center for Neurobiology and Behavior, Columbia University, New York, New York 10032
Axons growing in the developing nervous system are guided by cues in the environment which act at the growth cone. So far, the initial cytoskeletal target of these cues has been found to be the network of actin filaments in the peripheral region of the growth cone. Laminins are constituents of the extracellular matrix which promote axonal growth. They exert effects on the actin network. Here, laminin 1 is shown to affect microtubules as well. Acute addition of laminin 1 to rat sympathetic neurons quickly caused the advance of microtubules and their bundling within the initial widely spread growth cone and then the outgrowth of thin, rapidly growing nascent axons. The bundling was pharmacologically separable from the advance of microtubules caused by laminin, as the former but not the latter was blocked by lithium. The bundling did not depend on the peripheral network of actin filaments, as it was unimpaired by the removal of this network with cytochalasin D. Thus, microtubules seem to be a direct cytoskeletal target for laminin 1 in the growth cone, with important consequences for axonal outgrowth.
INTRODUCTION The rate and direction of growth of neuronal processes are regulated by soluble and surface-bound environmental cues (Goodman, 1996). The growth cone, the specialized ending of a growing neurite, is the key structure for detecting and responding to these cues. How cues elicit the changes in growth cone morphology and behavior that lead to changes in growth is not well understood but, ultimately, the arrangement of the major cytoskeletal elements—actin filaments and microtubules—must be important. The peripheral region of the growth cone is richly invested with actin filaments, which drive the movements of its digitate filopodia and diaphanous veils and lamellipodia (Letourneau, 1983). These peripheral protrusions, especially the filopodia, 1044-7431/00 $35.00 Copyright r 2000 by Academic Press All rights of reproduction in any form reserved.
are important in detecting guidance cues; their absence gives rise to pathfinding errors (Bentley and ToroianRaymond, 1986). Microtubules project into the growth cone from the core of the axon and their entry into peripheral regions is probably important in transforming these into new lengths of axon (Goldberg and Burmeister, 1989). For those cues for which it has been examined, the peripheral actin filaments seem to be the primary cytoskeletal target. Nerve growth factor (NGF), a soluble growth promoter, rapidly induces actin-based motile activities such as filopodial protrusion (Seeley and Greene, 1983). Collapsin-1 (semaphorin III), a growth inhibitor, depletes the peripheral region of actin filaments (Fan et al., 1993). Surface-bound growth promoters link through their membrane receptors to the network of actin filaments undergoing a steady rearward flux in the peripheral region (Suter et al., 1998). This evidently permits the advance of other components of the growing axon, such as microtubules and membranebound organelles. Laminin 1 (mouse EHS laminin) is a widely studied cue that typically acts as a surface-bound molecule to promote axonal differentiation and growth (Lander, 1987; Lein and Higgins, 1989). Isoforms of laminin are major constituents of extracellular matrices (ECMs) and can also be expressed on the surfaces of Schwann and muscle cells (Lentz et al., 1997; Patton et al., 1997). Promotion of axonal growth by laminin 1 is mediated by integrin surface receptors (Bozyczko and Horwitz, 1986; Tomaselli et al., 1987; Reichardt and Tomaselli, 1991). Antibodies to integrin block growth of neurites on laminin 1 substrates in culture and regenerative growth of peripheral motor axons in situ. Laminin 1 elicits processes that not only grow fast, but also are thin, as is characteristic of axons (Rivas et al., 1992). It is possible that this streamlined shape facili-
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FIG. 1. Laminin causes bundling of microtubules in growth cones. Growth cones were fixed at various time points and stained for detection of microtubules by immunofluorescence microscopy. Phase-contrast images were taken before fixation. (A and B) A growth cone without exposure to laminin. (A) Phase-contrast image of a sympathetic neuron with a short thick axon and wide growth cone. (B) Immunofluorescent visualization of microtubules in this growth cone shows them to be confined to the central region. The brightness of the image has been reduced here to allow visualization of detail in the central region of the growth cone. (C–H) Two growth cones in laminin-treatment experiments. (C and F) The two growth cones before addition of laminin. (D) 45 min after the addition of laminin, material has advanced in the peripheral region as judged by the position of phase-dark material (denoted by arrowhead). (G) 90 min after the exposure of the other growth cone to laminin, thin neurites have sprouted from the edge of the peripheral region. (E and H) Fluorescence images of the growth cones of D and G. In E, two microtubule bundles
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tates the rapid growth. As might be expected, the thin processes and their small associated growth cones have more closely packed microtubules (Burden-Gulley and Lemmon, 1996). This, plus the fact that depletion of the microtubule-associated proteins MAP1b and tau reduces the acceleration of growth caused by laminin 1 (DiTella et al., 1996), suggests that the microtubules of the growth cone might also be a target for laminin 1. We show here that laminin 1 elicits bundling of microtubules in the growth cone of sympathetic neurons in culture and that this is not secondary to an effect on the peripheral network of actin filaments. The bundling precedes the appearance of the thin processes and thus is an early event in the differentiation of the neurite into an axon. Thus, microtubules may also serve as direct targets of action in the growth cone for environmental cues that regulate growth.
RESULTS
similar to axons in their thinness and rapid growth. A concentrating of the protein GAP-43 in the growth cone is the earliest biochemical marker of axonal identity when hippocampal cells begin to develop polarity in culture (Goslin and Banker, 1990). We thus examined quantitatively the distribution of GAP-43 in separate groups of neurons before and 2 and 4.5 h after the addition of laminin. GAP-43 was present in growth cones before and after the addition of laminin, but was much more concentrated in the small growth cones 2 h after the addition of laminin (Fig. 2). It became further concentrated by the later time point. Staining of the thin shaft of the neurite was dim. Two hours after the addition of laminin, the original widely spread growth cone often remained, with thin neurites projecting from its periphery. The GAP-43 staining intensity in the remaining wide growth cone was similar to that of the control condition (data not shown). Thus neurites, originally in an undifferentiated form, begin acquiring an axonal phenotype soon after exposure to laminin.
Thin Neurites Form after Addition of Laminin 1 We had previously shown that acute addition of laminin 1 to rat sympathetic neurons cultured on a polylysine-coated substrate causes a rapid increase in the neuritic growth rate (Rivas et al., 1992). Multiple thin processes were sometimes seen to sprout from large spread growth cones after the addition of laminin. We wanted to determine how common this rapid change in morphology was, so we observed neurons 4–8 h after plating in serum-free medium on polylysine/polyornithine. Numerous cells had a thick neurite ending in a growth cone with a large lamellipodium (Figs. 1A, 1C, and 1F). During the 1.5–2 h before adding laminin, these neurites elongated little. By 1.5 h after addition of laminin, 32 of 38 (84%) growth cones had developed thin neurites growing out from the original large growth cone (Fig. 1G).
Neuritic Outgrowth in Response to Laminin Is Axonal Laminin 1 has been shown to stimulate axonal growth specifically (Lein and Higgins, 1989). The neurites that grew out in response to laminin in our experiments are
Laminin Causes Bundling of Microtubules in the Growth Cone Before the addition of laminin, microtubules are largely restricted to the central region of the growth cone (Rivas et al., 1992, and Fig. 1B). Addition of laminin causes them to advance into the peripheral region within 10–20 min (Rivas et al., 1992, and Fig. 1E). We assessed the distribution of microtubules in growth cones fixed at various times 15–90 min after the addition of laminin or vehicle. Of the growth cones treated with laminin 59% had at least one discernible bundle of microtubules while only 12% without laminin did (Fig. 3). The 59% probably is an underestimate of the fraction of growth cones that would develop bundled microtubules in response to laminin, since some in our sample (those fixed at earlier times) probably did not have time to respond. Bundles in the thickened central region of the growth cone, to which microtubules are largely confined in the absence of laminin, are probably more difficult to detect than bundles in the very flat peripheral region. Therefore, the difference in bundling of microtubules with and without laminin might be more apparent
(arrows) formed within the growth cone before the emergence of thin neurites, while in H, bundles (arrow) run from the still spread growth cone into the thin neurites. The graph to the left of E shows the line scan of E (pixel brightness). The two peaks, which are both at least 30 units above the surrounding baseline (256-unit gray scale), represent the two bundles in E. The outlines of the growth cones, as traced from the phase-contrast micrographs, are drawn on the fluorescence micrographs. Bar, 10µm.
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FIG. 2. GAP-43 concentrates in growth cones after exposure to laminin. Growth cones were fixed at three time points and stained for detection of GAP-43 by immunofluorescence microscopy. (A) Before addition of laminin (control), GAP-43 is evenly distributed in the widely spread growth cone, and its relative staining intensity is low. (B) GAP-43 staining was brighter in the small growth cones at the end of the thin neurites formed 2 h after addition of laminin. Bar, 10 µm. (C) Quantitation of the GAP-43 staining shows the increase of this protein in growth cones after addition of laminin. The ordinate shows the average pixel brightness (in arbitrary units, 0–4095). The data at each time point represent the mean value of 25 individual growth cones in three independent experiments. (Error bars show standard error of the mean.) Asterisks denote values that are significantly different from the control (0 h) (P , 0.0001), as determined by ANOVA followed by a t test using Bonferroni’s modification for multiple comparisons.
than real. Experiments with cytochalasin (described below) showed this not to be the case; even microtubules in the peripheral region could be seen to be largely unbundled in the absence of laminin. In those growth cones fixed after the emergence of thin neurites, the bundles of microtubules in the spread original growth cone typically ran into the thin neurites (Fig. 1H). Thus, it seemed possible that microtubules were being zippered together by constriction of the plasma membrane to form the thin neurites. This appears not to be the case. Many growth cones were fixed at a time at which microtubules had advanced from the central region deep into the peripheral region in response to laminin, but thin neurites had not yet emerged (Fig. 1E). [There is often a delay between those two stages in the response to laminin (Rivas et al., 1992).] Of
these growth cones 46% showed some bundling of microtubules (Fig. 3); this, also, should be viewed as a minimum estimate. The advance and subsequent bundling of microtubules caused by laminin were pharmacologically separable events. Lithium (20 µM, with 20 µM myoinositol to prevent depletion of inositol in the cell), which can affect the packing of microtubules in neurites, did not by itself cause microtubules to advance or to bundle (Figs. 4F and 5) and had no evident effect on F-actin in the peripheral region of the growth cone (Fig. 4E). Addition of laminin in the presence of lithium elicited advancement of microtubules and membrane-bound organelles into the peripheral region, as typically seen (Fig. 5). However, only 32% of these growth cones displayed bundling of microtubules and only 19% produced thin
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FIG. 3. Laminin (LN) causes bundling of microtubules (MTs) which is not prevented by cytochalasin D (CD). 59% of the growth cones investigated (including growth cones of the types shown in Figs. 1D, 1E, 1G, and 1H) exhibited bundling of microtubules after addition of laminin (left black bar), as opposed to 12% without laminin (left white bar). Bundled microtubules were seen in 46% of those growth cones (like the one shown in Figs. 1D and 1E) from which thin neurites had not yet emerged though microtubules had advanced into the peripheral region (hatched bar). The percentage of growth cones showing bundling of microtubules after addition of laminin was only slightly reduced by preexposure to CD (middle and right pairs of bars; gray bars represent cells exposed only to CD). Asterisks denote those percentages that are significantly different from their controls (P , 0.002), as determined by the approximate test for the comparison of two proportions. For each condition above, at least 30 growth cones from four independent experiments were counted.
neurites. In addition, the acceleration of elongation typically caused by laminin was blocked by lithium (Fig. 6).
The Peripheral Network of Actin Filaments Is Not Needed for Bundling of Microtubules nor Are Neurofilaments Although constriction of the plasma membrane was not needed for the bundling of microtubules, we thought they might be forced into bundles by actions of the rich internal peripheral network of actin filaments. We used the fungal metabolite cytochalasin D (CD) to eliminate this network. The network rapidly withdraws from the growth cone in the presence of CD, allowing microtubules and membrane-bound organelles to advance deep into the peripheral region (Forscher and Smith, 1988). One hour of exposure to 1 µM CD (without laminin) left the peripheral region of the growth cone, which is
normally richly invested with F-actin, largely devoid of F-actin (Fig. 4C). Scattered clumps of F-actin sometimes remained at the edge, but not colocalized with microtubules. Of the observed large growth cones 77% had microtubules and membrane-bound organelles filling much or all of the peripheral region (Fig. 4D), much as was seen with laminin (without CD). Yet, only 14% of the growth cones displayed bundling of microtubules (Fig. 3), demonstrating that advance of microtubules into the peripheral region is not sufficient for bundling to occur or for us to be able to detect it. In contrast, when laminin was added 20 min after the addition of CD, 45% of the growth cones subsequently showed bundling of microtubules, only slightly less than was seen without CD (Fig. 3). Increasing the concentration of CD 10-fold had no further effect. Many fewer thin neurites formed in response to laminin when CD was present (Fig. 7), as expected because the peripheral actin network facilitates neuritic elongation (Letourneau et al., 1987). However, 28% of the observed cells produced thin neurites, suggesting that the advance and bundling of microtubules could be sufficient to produce this type of process. Neurofilaments are the other major elements of the cytoskeleton of the axon. We used antibodies monospecific for the three subunits of the neurofilament. Antibodies against the 200-kDa subunit (Fig. 8) and the 68-kDa subunit (not shown) stained the neurite more brightly by immunofluorescence than when primary antibody was omitted, but neither stained the growth cone strongly and neither stained fibers within the growth cone, either before laminin had been added or when microtubules had projected deep into the peripheral region of the growth cone after the addition of laminin. We detected no specific staining with antibody directed against the 160-kDa subunit (not shown). Therefore, neurofilaments do not appear to be present in appreciable numbers in these growth cones.
DISCUSSION These studies demonstrate that the axonal growthpromoting molecule laminin 1 causes microtubules in the growth cone to bundle. This effect is not mediated by the network of actin filaments, with which laminin interacts, nor by neurofilaments, indicating that the microtubules are probably a direct cytoskeletal target for laminin. The bundling of microtubules may contribute to fast growth and to differentiating a process into an axon.
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FIG. 4. The impact of cytochalasin D (CD) and lithium on the actin network and microtubules (MTs) in growth cones. Growth cones were fixed and double stained for detection of actin and microtubules by immunofluorescence microscopy. (A, C, and E) Actin staining of three growth cones. (B, D, and F) MT staining in the same growth cones. (A) F-actin network heavily invests the peripheral region of a growth cone in the control condition. (B) MTs in the growth cone were confined to the central region. (C) 1 h after addition of 1 µM CD, the F-actin network was eliminated, with some staining remaining at edges. (D) MTs advanced to the peripheral region of the growth cone, yet no MT bundles formed. (E and F) 1 h after addition of lithium and myoinositol, the patterns of F-actin and MT staining were the same as without them, indicating that these chemicals do not overtly affect those networks. Bar, 10µm.
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FIG. 5. Lithium blocks the bundling of microtubules (MTs) and the formation of thin neurites, but not the advance of microtubules, caused by laminin (LN). LN was added for 90 min either in the absence of lithium (bars 2) or after 30 min of lithium preincubation (bars 3). Control experiments involved addition of vehicle alone (bars 1) or lithium alone for 90 min (bars 4). Asterisks denote those ‘‘LN 1 Lithium’’ percentages that are significantly different from their corresponding ‘‘LN only’’ percentages (P , 0.002). At least 40 growth cones from four independent experiments were counted for each condition.
Bundling of Microtubules Caused by Laminin The present work is in agreement with previous work that microtubules in the growth cone assume a more closely packed arrangement in response to laminin 1 (Burden-Gulley and Lemmon, 1996). However, in those
FIG. 6. Lithium blocks the acceleration of neuritic growth elicited by laminin (LN). Phase-contrast images were taken for the same group of neurons at three time points and the lengths of the neurites were measured. Asterisks denote those values that are significantly different from their controls (P , 0.002). The data represent the average of 50 individual neurites from three independent experiments for each condition.
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FIG. 7. Cytochalasin D (CD) reduces the formation of thin neurites elicited by treatment with laminin (LN). Growth cones were assessed 90 min after the addition of laminin. CD was added 20 min before the addition of laminin. Asterisks denote those percentages that are significantly different from the control (laminin only) (P , 0.002). For each condition above, at least 40 growth cones from four independent experiments were counted.
studies of chick retinal ganglion cells, laminin also caused actin filaments to fill more of the growth cone and to assemble into cables running into the area containing the microtubules. The network of actin filaments affects the distribution of microtubules in the growth cone. It can retard the entry of microtubules into the peripheral region, as evidenced by the rapid entry of microtubules when the actin network is eliminated by cytochalasin (Forscher and Smith, 1988, and the present results). It can also facilitate the advance of microtubules by exerting tension on them (Suter et al., 1998). The actin filaments are also needed to reorient microtubules (perhaps by pulling them) either toward or away from external cues during growth cone turning (Lin and Forscher, 1993; Challacombe et al., 1996). Thus, the bundling of microtubules might be mediated by the network of actin filaments. The fact that the bundling occurred in our experiments even after this network had been eliminated from the growth cone by CD demonstrates that this is not the case. Neurofilaments, the other major component of the cytoskeleton, do not project into these growth cones in appreciable numbers. Our evidence, therefore, points to the microtubule as a primary cytoskeletal target for laminin 1 in the growth cone. Laminin 1 thus appears to have two actions on the growth cone which are distinguishable temporally, pharmacologically, and, probably, mechanistically. Laminin 1 initially causes an advance of microtubules and cytoplasm into the peripheral region of the growth cone in
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FIG. 8. Neurofilaments are not present in the widely spread growth cones. Growth cones were fixed and stained for detection of neurofilaments by immunofluorescence microscopy. (A) Staining of a growth cone to visualize microtubules. (B) Staining of the same growth cone with a different secondary antibody but no primary antibody, showing the level of nonspecific staining for comparison to C and D. (C and D) mAb (NE14) against the 200-kDa subunit of neurofilaments was used as the primary antibody. Bright staining appeared only in the neurite. The faint signal in the growth cone is diffuse rather than fibrillar. (C) A growth cone without exposure to laminin. (D) 30 min after addition of laminin, when microtubules have advanced. Bar, 10µm.
our experiments, followed by bundling of microtubules and the appearance of thin neurites growing rapidly (Rivas et al., 1992). Only the latter events were blocked by lithium in the present experiments. Kuhn et al. (1995, 1998) showed that when a filopodium touches a bead coated with laminin 1, there is a rapid entry of cytoplasm into the filopodium, followed at some delay by growth at an accelerated rate. The advance of microtubules and cytoplasm into the peripheral region of the growth cone is probably caused by physical linking of laminin on the substrate to the network of actin filaments in the peripheral region of the growth cone and is probably a general mechanism for substrate-bound growth promoters (Felsenfeld et al., 1996; Grabham and Goldberg, 1997; Suter et al., 1998). We show here that the later effect is independent of actin filaments but is associated with bundling of microtubules. Our identification of the microtubule as a primary cytoskeletal target is a novel twist for ECM molecules and for other molecules that affect the growth cone to regulate neuritic growth. Binding of ECM molecules like laminin 1 to their integrin receptors [b1 integrin mediates the effects of laminin on the growth cone in our preparation (Rivas et al., 1992)] had so far been
shown to target the actin cytoskeleton to cause cell spreading (Hynes and Destree, 1978; Mooney et al., 1995), adhesion, and migration (Burridge et al., 1988; Felsenfeld et al., 1996). As for the growth cone, those external cues studied so far, including laminin 1, have been found to target the peripheral network of actin filaments (Seeley and Greene, 1983; Fan et al., 1993; Grabham and Goldberg, 1997; Suter et al., 1998; and see Introduction). Effects on the synthesis or phosphorylation of microtubules and MAPs have been described, though not specifically related to growth cone functioning (Black et al., 1986; Aletta et al., 1988).
Roles for Microtubule Bundling Microtubule bundling has been shown to precede constriction of the plasma membrane during neuritic elongation and turning (Tanaka and Kirschner, 1991, 1995). We also often detected bundles in growth cones from which thin neurites had not yet emerged. Thin neurites emerged from some growth cones even after peripheral F-actin had been eliminated with CD, suggesting that the effects of laminin on the microtubules are sufficient in some cases for the formation of thin neu-
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rites. Thus, it seems likely that the bundling induced here is important in causing the formation of the thin neurites. The bundling of microtubules may contribute to accelerating neuritic elongation and to the conversion of an undifferentiated neurite into an axon. Bundling might accelerate elongation by reducing lateral excursions of the growth cone (Aigner and Caroni, 1993; Tanaka et al., 1995). It could also allow more efficient delivery of the transported materials upon which growth depends. Observations of axons growing in vivo show that rapid elongation is typically associated with small, narrow growth cones (LoPresti et al., 1973; Kaethner and Stuermer, 1992), as would be associated with compact microtubules. The thin processes that form in response to laminin are nascent axons (our results and Lein and Higgins, 1989). The bundling we observe here is one of the earliest changes that have been observed in the transformation of undifferentiated neurite into axon, as it precedes the emergence of the fast growing nascent axon. Within an hour of the acceleration of growth that marks the conversion of neurite to axon in hippocampal neurons (Goslin and Banker, 1989), the number of microtubules in the growing process has greatly increased and they have become more closely packed and aligned in parallel (Yu and Baas, 1994). Even before the nascent hippocampal axon accelerates its growth (i.e., becomes recognizable as an axon), it displays a greatly increased transport of organelles (Bradke and Dotti, 1997). Since organelles are transported along microtubules, it is reasonable to suggest that a key early event in axonal differentiation is a change in the organization or properties of the microtubules, perhaps bundling. This could permit the increased delivery of materials needed to support the faster growth of the axon.
(Eugene, OR). Monoclonal antibody against GAP-43 (7b10/c3) was generously provided by Dr. K. Meiri. Polyclonal antibodies against a-tubulin in Glu and Tyr forms (SG and W2) were generously provided by Dr. J. C. Bulinski.
Tissue Culture Sympathetic superior cervical ganglion and/or sympathetic chains from the thoracic region of the spinal cord were dissected from postnatal day 1–4 rats and placed in Leibowitz’s L-15 medium (GIBCO). Ganglia were incubated in 0.25% trypsin in Ca21/Mg21-free Hanks’ balanced salt solution for 30 min at 37°C and were dissociated by gentle trituration with fire-polished Pasteur pipettes. All coverslips, glued under a hole in the bottom of a 60-mm plastic dish, were coated with a mixture of 100 µg/ml poly-D-lysine and polyornithine for 2 h at room temperature or overnight at 4°C, then rinsed twice with double-distilled water. Defined culture medium was L-15 supplemented with NGF (100 ng/ml), bovine insulin (5 µg/ml), human transferrin (10 µg/ml), sodium selenite (5 ng/ml), sodium pyruvate (1 mM), and bovine serum albumin (0.5 mg/ml). Two protocols were used to obtain growth cones suitable for study. In one, cells were cultured for 4–8 h before observation. In the other, cells were maintained for 16–24 h and then replated to induce new short thick neurites ending in a spread growth cone. For all experiments, laminin 1 was added to the culture medium at a final concentration of 25 µg/ml to assess its effect. In some experiments, 20 mM lithium chloride and 20 mM myoinositol were added to the medium 30 min before the addition of laminin 1.
Immunocytochemistry
EXPERIMENTAL METHODS Materials Cytochalasin D, lithium chloride, myoinositol, bovine insulin, human transferrin, sodium selenite, poly-Dlysine, polyornithine, osmium tetroxide, monoclonal antibody against b-tubulin (JDR.38B), and monoclonal antibodies against neurofilament subunits (NE14, NR4, and NN18) were purchased from Sigma Chemical (St. Louis, MO). Sodium pyruvate was from GIBCO (Grand Island, NY). NGF and laminin 1 (from mouse EHS sarcoma) were from Boehringer Mannheim (Indianapolis, IN). Texas red-conjugated goat anti-mouse antibody, FITC-conjugated goat anti-rabbit antibody, and Texas red–phalloidin were obtained from Molecular Probes
Immunofluorescence procedures to visualize microtubules were as previously described by Black et al. (1994), with fixation procedure 2 used most often. Briefly, cells were extracted for 2 min in buffered medium containing 0.2% saponin and 10µM Taxol. After extraction, the cells were fixed for 10 min in medium containing 2% paraformaldehyde and 0.05% glutaraldehyde. To visualize GAP-43 and neurofilaments, cells were fixed in PBS containing 4% paraformaldehyde, 0.1% glutaraldehyde, and 400 mM sucrose for 5 min at 37°C, followed by one rinse and three 5-min washes with 0.15% Triton X-100 in PBS. Nonspecific binding was blocked by incubation in 10% normal goat serum (NGS) for 30 min at room temperature. The cells were incubated overnight at 4°C with primary antibodies against b-tubulin or neurofila-
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ment subunits at a 1:100 dilution or against GAP-43 at a 1:5 dilution. The antibodies were diluted in PBS/4% NGS. Cells were then washed and incubated for 1 h with 1:100 secondary antibody conjugated to Texas red followed by mounting in 20 mg/ml propyl gallate in 90% glycerol/10%PBS. Immunofluorescence procedures for double staining of F-actin and another antigen were essentially as previously described by Lewis and Bridgman (1996). After the F-actin staining was recorded, cells were processed further for staining to visualize microtubules. Cells were incubated with a mixture of primary antibodies (SG and W2) against tubulin at a 1:100 dilution at 4°C overnight. The cells were then washed and incubated for 1 h with 1:100 fluorescein-conjugated secondary antibody.
that met our criteria. Because of variation in the time course of the response, growth cones at different time points from 0.5 to 1.5 h were pooled in the analysis. A growth cone was scored as having had an advance of microtubules if the distance between the neck of the growth cone and the front edge of the envelope of microtubules was more than 85% of the total distance from the neck to the distal edge (not including filopodia) of the growth cone. Control growth cones never displayed this; the average percentage of the length occupied by microtubules in control growth cones was 65%. The area measurement function of the Metamorph system was used to quantitate GAP-43 staining by measuring average pixel brightness for the entire growth cone.
Image Acquisition and Analysis
ACKNOWLEDGMENTS
Phase-contrast microscopy was done with 20 and 633 objectives. An automated stage was used to relocate multiple growth cones. Images were recorded at times before and after the addition of laminin, cytochalasin D, and/or lithium chloride. The preparation was then fixed and processed for immunofluorescent visualization of microtubules in the same growth cones observed with phase-contrast microscopy. Microtubules were visualized with a 403, 1.3 NA objective lens to optimize horizontal and vertical resolution and images were captured with a cooled CCD camera (Princeton Instruments, Trenton, NJ) to allow accurate quantitation of fluorescence intensity. The following procedure was used to identify a bundle. First, we inspected the whole growth cone we were analyzing to find potential bundles of microtubules. For each potential bundle, we measured its pixel brightness along a line drawn across the growth cone perpendicular to the long axis of the potential bundle using the line scan function of the Metamorph image analysis system (Universal Imaging, West Chester, PA). We also measured the thickness and length of the potential bundle using the measure distance function. A bundle was scored as such if it met all of the following criteria: (1) a peak in brightness at least 30 units above the surrounding baseline (256-unit gray scale), (2) an apparent thickness of at least 1.44 µm, and (3) a length of at least 6.67 µm. This somewhat conservative definition selected fibers that were clearly different from single microtubules. However, it probably excluded some small bundles. It would rule out bright spots because they would not be long enough. A growth cone was scored as having bundling if it had at least one bundle
We are grateful to Drs. J. Chloe Bulinski and Karina Meiri for generous gifts of antibodies. We are grateful to Dr. Peter W. Grabham for assistance with some procedures and for comments on an earlier version of the manuscript and to Ms. Anuli Umeojiako for excellent technical assistance. This work was supported by NIH Grants NS25161 and GM32099.
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