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Role of microtubules in the cytoplasmic compartmentation of neurons. II. Endocytosis in the growth cone and neurite shaft
Brain Research, 450 (1988) 60-68 Elsevier
60 BRE 13553
Role of microtubules in the cytoplasmic compartmentation of neurons. II. Endocytosis in the growth cone and neurite shaft Gary I. Sinclair, Peter W. Baas and Steven R. Heidemann Department of Physiology, Michigan State University, E. Lansing, MI48824-1101 (U.S.A.) (Accepted 10 November 1987)
Key words: Microtubule; Neuron; Growth cone; Endocytosis; Taxol; Cell cortex
We investigated the role of microtubules in the compartmentation of motility and endocytosis in the neurite shaft and growth cone of cultured chick sensory ~.:urons. As reported previously by Letourneau and Ressler (J. Cell Biol., 98 (1984) 1355-1362), stimulating microtubule polymerization with taxol inhibits growth cone motility. In neurons that had grown for 18-30 h, taxol treatment caused growth cones to round up forming an obvious varicosity (taxol bulb) at the terminal. Removal of taxol allowed nearly immediate resumption of cortical motility in all 17 neurons observed by time-lapse videomicroscopy. However, only one of the 17 neurites was observed to elongate measurably even after 5 h of obs,~rvation. In 14 cases taxol was rinsed out and the concentration of nerve growth factor was increased 10x, 11/14 neurites retracted within the next hour. Endocytic activity was investigated by incubating control and taxol treated neurons in either cationized ferritin or horseradish peroxidase for 30 min. The number and area of label-containing vesicles was measured along with the total area of the growth cone or taxol bulb. We found that taxol treatment caused a 7-fold decrease in the ratio of the area of the labeled vesicles to the area of growth cone or taxol bulb. Conversely, in neurite shafts, normally relatively quiescent with respect to endocytosis, those regions devoid of microtubules in both control and nocodazole-treated cells contained a high concentration of label-containing vesicles. We conclude that the presence of microtubules plays a role in regulating endocytic activity by the overlying cell cortex. We speculate that this could be due either to transport activity mediated by microtubules or to cortical stabilization by microtubules.
INTRODUCTION Neurons in culture extend neurites that consist of two morphologically and functionally distinct compartments, the neurite shaft and the terminal growth cone. The neurite shaft is an elongated cylinder of cytoplasm with a dense paraxial array of microtubules and neurofilaments whose cortex does not engage in motile activity 5.25,27. The growth cone is a flat, palmate structure that lacks microtubules and neurofilaments, but has a cortex which is very active in filopodial and lamellipodia! extension and endocytosis5'22'25'27. Despite considerable attention given to describing these ultrastructural and functional differences between the neurite shaft and growth cone, little attention has been given to determining the mechanisms for this regionalization.
We reported previously that microtubules in the shaft of cultured chick sensory neurons play a role in the compartmentation of ribosomes to the cell bodies 1. The relative absence of microtubules in growth cones as compared to shafts suggests that microtubules may also play a role in the compartmentation of neurite shaft and growth cone. Support for this hypothesis comes from Letourneau and Ressler 14 who showed that adding taxol, a potent microtubulepolymerizing agent 21, to day-old cultures of dorsal root ganglion ( D R G ) neurons inhibits neuritic elongation and growth cone motility. We hypothesized that taxol may act by stimulating microtubule polymerization in the growth cone, thus eliminating its structural and functional specialization. We have extended the work of Letourneau and Ressler with particular emphasis on the effects of taxol-induced mi-
Correspondence: S. Heidemann, Department of Physiology, Michigan State University, E. Lansing, M148824-1101, U.S.A. 0006-8993/88/$03.50 c@.)i988 Elsevier Science Publishers B.V. (Biomedical Division)
61 crotubule assembly on the compartmentation of motility and endocytosis to the growth cone. We found that taxol treatment causes the growth cone to lose motility and endocytosis and to take on the quiescent characteristics of a neurite shaft. 1~IATERIALSAND METHODS
Materials Chemicals not otherwise noted were purchased from Sigma, St. Louis, MO. Cell culture Embryonic chick sensory neurons were cultured using a method similar to that described by Shaw and Bray 23. DRG were dissected from the lumbosacral regions of 10- to 12-day-old embryos, and placed in L-15 medium (Gibco, Grand Island, NY) supplemented with 0.6% glucose, 2 mM L-glutamine, 100 U/ml penicillin, and 100 mg/ml streptomycin (L15+). The ganglia were rinsed twice, then treated with 0.25% trypsin for 25 min at 37 °C. The trypsin was removed, L-15+ with 10% fetal calf serum (Hazleton Dutchland, Denver, PA) was added, and the ganglia were triturated with a pipette into a single cell dispersion. The cells were rinsed twice, and plated into medium with 10% fetal calf serum, 0.6% methyl cellulose (Metcel A4M, Dow Chemical, Midland, MI), and 100 ng/ml 7s nerve growth factor (NGF) isolated from mouse saliva 6. The cells were plated into 35-mm Falconware Primaria tissue culture dishes at a density low enough to render non-neuronal c,mtamination inconsequential. Cultures were incul:,oted at 37 °C for 18-30 h prior to experimentation. Experimental treatments Growth cones bearing filopodia and/or lamellipodia were selected for observation and circled with a diamond marker objective. Cells were treated with taxol (gift from the National Cancer Institute) by replacing the culture medium with fresh drug containing medium including L-15+, 10% FCS, 100 ng/ml NGF, and 5.7 x 10-8 M taxol. Taxol was removed from treated cultures by rinsing 3 times in fresh taxolfree medium after which the cells were allowed to incubaZe in L-15+, 10% fetal calf serum and 100 ng/ml NGF. In some experiments, taxol-treated cells were rinsed and replaced into taxol-free medium contain-
ing 1/~g/ml NGF, i.e. a 10x higher concentration of NGF. All cultures were constantly maintained at 37 °C prior to and during experimentation. Still photographs were taken with a Nikon FE2 camera through a Nikon phase contrast microscope on Technical Pan film developed in HC110 developer. Video recordings were made with a D A G E MTI 67 series video camera on an RCA time-lapse video recorder. Selected cells were assayed for endocytosis by adding either 500/lg/ml cationized ferritin (CF) or 1 mg/ml horseradish peroxidase (HRP) to the culture for a period of 30 min after the taxol had taken effect (see Results). In another set of experiments, cells were treated simultaneously with 1/~g/ml nocodazole (Sigma) and one of the endocytic markers (CF or HRP). After treatment with the endocytic marker, cultures were rinsed 3 times in serum-free medium prior to fixation. The HRP-treated cultures were exposed after fixation to a solution containing 0.5 mg/ml diaminobenzidine (HRP substrate) and 0.01% H20 2 in 0.05 M Tris, pH 7.6, for 10 min. The cultures were then prepared for electron microscopy as described below.
Electron microscopy After experimental treatments, cultures were fixed for 20-30 min with 2% glutaraldehyde in 0.1 M cacodylate, rinsed twice in 0.1 M cacodylate with 5% sucrose, postfixed for 5 min in 1% OsO4, dehydrated in ethanol series, and embedded in Polybed 812 (Polysciences, Warrington, PA). Thin sections were cut parallel to the substratum, stained with uranyl acetate and lead citrate, and observed with a Philips 300 transmission electron microscope. Measurement of areas Tissue areas were computed from tracings of scaled electron micrographs using numerical integration techniques. Areas were traced using a calibrated electromagnetic platten and handheld viewer coupled to an IBM XT computer for file storage. Integrations were made with a Sigma Scan (Jandel) computer program. RESULTS In addition to stopping the advance of preformed dorsal root ganglion neurons, as reported previous-
62. lyTM, taxol significantly changed the appearance of growth cones. Twenty-two well-spread growth cones were photographed before and 3 h after treatment with 5.7 x 10-8 M taxol. Fig. I shows a typical example of a cell's response to taxol. In all but one of the 22 cases the growth cones rounded into obvious varicosities at the neurite terminal (hereafter referred to as taxol bulbs) and displayed only a few filopodia and no lameUipodia. Time-lapse video observations revealed that the growth cones always assumed this shape within the first 35 min after taxol exposure and that the filopodia were not retracting and protruding as they do in normal growth cones. Letourneau and Ressler 14 reported that D R G neurons plated in the presence of taxol had the ability to recover and extend neurites after being rinsed free of taxol. We wished to examine the reversibility of taxol's effects on preformed neurites. We examined the effects of rinsing out taxol on 17 different taxol bulbs by making time-lapse video microscopy recordings of the bulbs for periods of 1 to 5 h following each rinseout. In all 17 cases, the taxol bulbs regained motility within minutes after taxol rinseout (Fig. 2), but only one of 17 bulbs recovered to the point where a new growth cone was formed and neurite elongation was measured. Peterson and Crain Is reported that relatively high concentrations of NGF attenuated the neurotoxic effects of taxol on cultures of fetal mouse D R G . The effects of 10x higher concentrations of N G F in drug-free medium was assessed by time lapse observation of 14 typical taxol bulbs. All 14 neurites in medium containing 1/~g/ml 7s NGF recovered motility within minutes, only one of 14 lengthened
Fig. 1. Morphological effect of taxol on pre-formed growth cones. Chick sensory neurites from 10-day-old embryo were treated with 5.7 x 10-8 M taxol, a: untreated neurites after 20 h of growth, b: same neurites 3 h after addition of taxol, x 260.
!
Fig. 2. Effect of removing taxol on pre-formed growth cones. Conditions of growth and taxol treatment were the same as in Fig. 1. a: prior to addition of taxol, b: 30 min after addition of taxol, just prior to removal of taxol, c: 10 min after removal of taxol from culture medium, d: 50 min after removal of taxol from culture medium, x580.
measurably. However, 11/14 neurites retracted within I h of being placed into the higher concentration of NGF. We speculate that this response is similar to the retraction response observed by Griffin :'nd Letourneau ~1. Although, except for a sudden increase in NGF concentrations, our conditions were rather different. Our transmission electron micrographs of thin sections confirmed the whole-mount observations of Letourneau and Ressler 14 on chick D R G and were entirely similar to observations from Horwitz's group on mouse 16 ~nd rat neurons 2°. The tips of taxol treated neurites contained a disorganized jumble of microtubules (Fig. 3). Our micrographs revealed that the microtubule density was not nearly as great as that typically found in a control neurite shaft but much greater than the extremely low density typical of growth
63
Fig. 3. Endocytic uptake of CF by taxol-treated growth cones. A transmission electron micrograph of a region of the distal terminal of a neuron treated with taxol (5.7 x 10-8) for 30 rain then exposed to 0.5 mg/ml CF for an additional 30 min. Thin arrows mark microtubules, stubby arrows mark ferritin-containing vesicles. Relative to growth cones without taxol (see Fig. 4) there is a large increase in number of microtubules and a concomitant decrease in labele~ivesicles, x25,200.
cones. Gordon-Weeks reported a similar taxol-induced stimulation of microtubule assembly in isolated growth cones from new-born rats 9. We wished to determine the effect of taxol treatment on endocytosis by the growth cone. In two experiments, CF was used as the label for endocytosis 8. In two other experiments, HRP was used 24. We measured the area of label-containing vesicles in randomly selected transmission electron micrographs from 4 different control growth cones (one from each experiment) and 7 different taxol bulbs (one or two from each experiment). Fig. 3 shows a taxol bulb and Fig. 4 a control growth cone assayed for endocytosis with CF as described in Materials and Methods. Despite the growth cone's smaller area, it contained 10.0 times as much !abel-containing, internalized membrane as the corresponding taxol bulb. Table I
summarizes our measurements. We found that the number and area of labeled vesicles was 3- to 5-fold greater in control growth cones than in taxol bulbs, despite the larger area of taxol bulbs surveyed. We used a ratio, area of endocytic vesicles/total area of growth cone or taxol bulb, as an estimate of endocytic activity. Using this measure, we found a 7.1-fold decrease in endocytic activity for taxol bulbs as compared to growth cones. A one-tailed t-test, adjusted for unequal variance and sample size, showed that this decrease in endocytic activity for the taxoltreated population was significant at a = 0.01. Since induced microtubule polymerization reduced endocytosis and motility in growth cones, we hypothesized that nocodazole-induced microtubule depolymerization would increase motility and endocytosis in neurite shafts. However, as previously re-
64
Fig. 4. Endocyticuptake of CF by control growth cones. A transmission electron micrograph of a region of a control growth cone incubated in 0.5 mg/mlCF in serum-free medium for 30 rain. Arrows mark ferritin-containing vesicles, x25,200.
ported ~, the response of DRG microtubules to microtubule poisons proved highly variable. Some regions of our nocodazole-treated neurites were devoid of microtubules while other regions appeared to contain normal microtubule arrays. Measurement of endocytic .~ctlvi~y as in Table I was therefore not ':teaslu~e. "-' .... However, in neurites treated with nocodazole, as well as in control neurites, we found that areas along the shafts that were devoid of microtubules often contained an accumulation of endocytic vesicles. Fig. 5 shows a bundle of neurites treated with both nocodazole and CF. The two outer neurites were devoid of microtubules and contained an accumulation of CF labeled vesicles while the inner neurite had an intact array of microtubules and was devoid of label. As shown in this figure, labeled vesicles in nocodazole-treated neurites frequently appeared abnormally large and empty. Fig. 6 shows a microtubulesparse region of a control neurite. The absence of mi-
crotubules is associated with the formation of a 'preterminal growth cone' characterized by filopodia and the generally splayed appearance of the cytoplasm. This sample had been treated with HRP, and the high density of labeled vesicles in the vicinity of the microtubule-sparse area should be noted. We never observed a similar concentration of endocytic vesicles in control or drug-treated neurites in areas of the shaft containing a dense array of microtubules. DISCUSSION Our results confirm Letourneau and Ressler's ~4report that taxol causes growth cones to lose their motility and take on the quiescent characteristic of neurite shafts. We showed that rinsing out taxol from such cultures (which presumably allows for some microtubule depolymerization) causes the distal terminals of neurites to regain cortical motility, and we
65 TABLE I Effect o f taxol treatment on endocytosis at neurite terminals Cell
Number o f vesicles*
Area o f vesicles*'**
1 control 2 control 3 control 4 control Control subtotal
Treatment
16 28 30 9 74
0.839 3.193 2.427 0.597 7.056
20.303 41.292 32.965 13.869 108.456
Total area**
Ratio o f vesicle to total area
0.041 0.077 0.043 0.043 0.065
5 taxol 6 taxol 7 taxol 8 taxol 9 taxol 10 taxol 11 taxol Taxol subtotals
3 6 1 0 1 2 10 23
0.090 0.561 0.355 0.000 0.095 0.070 0.354 1.525
22.107 30.507 25.049 18.430 9.842 16.241 49.103 171.279
0.004 0.018 0.014 0.000 0.010 0.004 0.007 0.009
* Only vesicles labeled with HRP or CF. ** Area in/~m2.
found that taxol bulbs, unlike g r o w t h cones, were similar to n e u r i t e shafts with respect to their low content of internalized vesicles. F u r t h e r , we f o u n d that microtubule-free regions of both control and nocodazole-treated n e u r i t e shafts often c o n t a i n e d endocytic
vesicles and occasionally formed motile structures (Fig. 6). W e thus propose t h a t microtubule polymerization plays an i m p o r t a n t role in the c o m p a r t m e n t a tion of neurites into shafts and growth cones. This int e r p r e t a t i o n is consistent with the observations of
Fig. 5. Relationship of microtubules to endocytosis in the neurite shaft. A transmission electron micrograph of a region of neurite shafts from a small bundle of neurites exposed to 1 mg/ml nocodazole and 0.5 mg/mi CF for 25 rain. Note the absence of microtubules in both outer neurites and the presence of label-containing vesicles while the inner neurite contains microtubules but lacks labeled vesicles, x22,700.
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Fig. 6. Relationshipof microtubules to endocytosisin the neurite shaft. A region of a neurite not treated with any microtubule drug but exposed to 1 mg/ml HRP for 30 rain. This area of normal neurite shaft was sparse in microtubules, apparently had a 'pre-terminal growth cone"which engaged in endocytosis. No equivalent uptake of label was observed in control neurites in regions containing a normal microtubulearray, x 11,200.
Bray et al. 4 and of Tosney and Wessels 25. Bray et al. reported that they routinely found 'preterminal' growth cones on DRG neurites exposed to drugs which depolymerize microtubules. From this and other observations, they concluded that the capacity for growth cone formation is distributed throughout the neurite and seems to be repressed by microtubules. Tosney and Wessels found that stable areas of the growth cone (the palm, non-motile veils) contained microtubules while motile portions did not. Apparently, the absence of microtubules permits motility and endocytosis, while the presence of microtubules inhibits motility and endocytosis in the overlying cell cortex. The mechanism of microtubule-associated compartmentation is unknown but two broad categories
of explanation suggest themselves. One explanation for the changes induced by taxol invokes the role of microtubules in vesicular transport 1°. It is possible that taxol bulbs are capable of endocytosis but lack vesicles because, unlike growth cones, they contain sufficient numbers of microtubules to rapidly transport the internalized vesicles back to the cell body. The absence of motility in taxol bulbs might also be explained by changes in transport. Lloyd et al. 15 proposed that directed fibroblast motility requires the channelling of membrane precursors towards a preferred edge by a linear array of microtubules. The disorganized jumble of microtubules found in taxol bulbs may be incapable of such membrane channelling and thus incapable of supporting growth cone motility.
67 The other explanation is that microtubules play a role in regulating the ability of the cortex to change shape, e.g. regulating the surface tension of neurons. Bray and C h a p m a n 3 speculated that the increasing quiescence found in going from the tip of the growth cone back to the neurite may reflect an increasing gradient of surface tension. Similarly, the work of Tosne~ and Wessels 25 suggests that the increasing gradient of quiescence is accompanied by an increase in the density of the microtubule array. O u r observations of drug-treated neurons are uniformly consistent with the hypothesis that the density of the microtubule array directly correlates with the quiescence of the neuronal surface. In taxol-treated neurons, decreased motility and endocytosis at the growth cone, as well as the characteristic 'rounded up' shape of taxol bulbs, could be explained by an increase in surface tension. Conversely, nocodazole-induced increases in cortical motility and endocytosis and the fragmentation of neurites into separate beads of cytoplasm in response to lengthy nocodazole treatments I could be caused by a decrease in surface tension. Other workers have proposed that microtubules have the ability to regulate the quiescence of the cellular cortex. A number of studies have proREFERENCES 1 Baas, P.W., Sinclair, G.I. and Heidemann, S.R., Role of microtubules in the cytoplasmic compartmentation of neurons, Brain Research, in press. 2 Bamburg, J.R., Bray, D. and Chairman. K.. Assembly of microtubules at the tips of growing axons. Nature (Lond.), 321 (1986) 788-800. 3 Bray, D. and Chapman, K., Analysis of microspike movements on the neuronal growth cone, J. Neurosci., 5 (1985) 3204-3213. 4 Bray, D., Thomas, C. and Shaw, G., Growth cone formation in cultures of sensory neurons, Proc. Natl. Acad. Sci. U.S.A., 75 (1978) 5226-5229. 5 Bunge, M.B.~ Initial cndocytGsi~ of peroxidase or ferritin by growth cones of cultured nerve cells, J. Neurocytol.. 6 (1977) 407-439. 6 Burton, L.E., Wilson, W.H. and Shooter, E.M., Nerve growth factor from mouse saliva, J. Biol. Chem., 253 (1978) 7807-7812. 7 Caron, J.M., and Berlin, R.D., Dynamic interactions between microtubules and artificial membranes, Biochemistry, 26 (1987) 3681-3688. 8 Chan, K.Y., Bunt, A.H. and Haschke, R.H., Endocytosis and compartmentation of peroxidases and cationized ferritin in neuroblastoma cells, J. Neurocytol., 9 (1980) 381-403. 9 Gordon-Weeks, P.R., The cytoskeleton of isolated, neuronal growth cones, Neuroscience, 21 (1987) 977-989.
vided evidence for a class of microtubules that have the ability to cross-link to membrane-associated cytoskeletal elements and thus potentially stabilize the cell cortex 12.13-17-19. Further, Vassilev et al. 26 showed that increasing tubulin in a lipid monolayer decreases its surface tension. Caron and Berlin 7 found that stimulated microtubule polymerization can deplete artificial membranes of adsorbed tubulin. Stimulating microtubule polymerization within a cell by taxol may deplete the membrane of tubulir, thus causing an increase in surface tension and, consequently, a decrease in cortical motility. ACKNOWLEDGEMENTS We thank R.E. Buxbaum for many stimulating discussions and for reading and commenting on the manuscript. We are indebted to H. Stuart Pankratz for his invaluable assistance and the Department of Microbiology for the use of their electron microscope facility. We also thank Thomas Adams for access to and help with the morphometric measurements. This work was supported by Grant BNS 8706741 from the National Science Foundation and Grant G M 36894 from the NIH. 10 Grafstein, B. and Forman, D.S., Intracellular transport in neurons, Physiol. Rev., 60 (1980) 1167-1283. 11 Griffin, C.G. and Letourneau, P., Rapid retraction of neurites of sensory neurons in response to increased concentrations of nerve growth factor, J. Cell Biol., 86 (1980) 156-164. 12 Hirokawa, N., Cross linker system between neurofilaments, microtubules, and membranous organelles revealed by the quick freeze, deep etching method, J. Cell. Biol., 94 (1982) 129-142. 13 Joshi, H.C., Baas, P., Chu, D.T. and Heidemann, S.R., The cytoskeleton of neurites after microtubule depolymerization, Exp. Cell. Res., 163 (1986) 233-245. 14 Letourneau, P.C. and Ressler, A.H., Inhibition of neurite initiation and growth by taxol, J. Cell Biol., 98 (1984) 1355-1362. 15 Lloyd, C.W., Smith, C.G., Woods, A. and Rees, O.A., Mechanisms of cellular adhesion. II. The interplay between adhesion, the cytoskeleton and morphology in substrate attached cells. Exp. Cell Res., 110 (1977) 427-437. 16 Masurovsky, E.B., Peterson, E.R., Crain, S.M. and !"~rwitz, S.B., Morphological alteration in dorsal root ganglion neurons and supporting cells of organotypic mouse spinal cord-ganglion cultures exposed to taxol, Neuroscience, 10 (1983) 491-509. 17 Miller, R.H., Lasek. R.J. and Katz, M.J., Preferred microtubules for vesicle transport in lobster axons, Science, 235 (1987) 220-222. 18 Peterson, E.R, and Crain, S.M., Nerve growth factor at-
68 tenuates neurotoxic effects of taxol on spinal cord-ganglion explants from fetal mouse, Science, 217 (1982) 377-379. 19 Pollard, T.D., Selden, C.S. and Maupin, P., Interaction of actin filaments with microtubules, J. Cell Biol., 99 (1984) 33s-37s. 20 Roytta, M., Horwitz, S.B. and Crain, C.S., Taxol-induced neuropathy: short term effects of local injection, J. Neurocytol., 13 (1984) 685-701. 21 Schiff, P.B., Fant, J. and Horwitz, S.B., Promotion of microtubule assembly in vitro by taxol., Nature (Lond.), 277 (1979) 665-667. 22 Schnapp, BJ. and Reese, T.S., Cytoplasmic structure in rapid frozen axons, J. Cell. Biol., 94 (1982) 667-679. 23 Shaw, G. and Bray, D., Movement and extension of isola-
ted growth cones, Exp. Cell Res., 104 (1977) 55-62. 24 Steinman. R.M., Silver, J.M. and Cohn, Z.A., Pinocytosis in fibrobh :~, J. Cell Biol., 63 (1974) 949-969. 25 Tosney, K.W. and Wessels, N.K., Neuronal motility: the ultrastructure of veils and microspikes correlates with their motile activities, J. Cell Sci., 61 (1983) 389-411. 26 Vassilev, P.M., Tanneva, S., Pannaiotov, I. and Georgiev, G., Dilational viscoelastic properties of tubulin and mixed tubulin-lipid monolayers, J. Colloid Interface Sci., 84 (1981) 169-174. 27 Yamada, K.M., Spooner, B.S. and Wessels, N.K., Axon growth: role of microfilaments and microtubules, Proc. Natl. Acad. $ci. U.S.A., 66 (1970) 1206-1212.
60 BRE 13553
Role of microtubules in the cytoplasmic compartmentation of neurons. II. Endocytosis in the growth cone and neurite shaft Gary I. Sinclair, Peter W. Baas and Steven R. Heidemann Department of Physiology, Michigan State University, E. Lansing, MI48824-1101 (U.S.A.) (Accepted 10 November 1987)
Key words: Microtubule; Neuron; Growth cone; Endocytosis; Taxol; Cell cortex
We investigated the role of microtubules in the compartmentation of motility and endocytosis in the neurite shaft and growth cone of cultured chick sensory ~.:urons. As reported previously by Letourneau and Ressler (J. Cell Biol., 98 (1984) 1355-1362), stimulating microtubule polymerization with taxol inhibits growth cone motility. In neurons that had grown for 18-30 h, taxol treatment caused growth cones to round up forming an obvious varicosity (taxol bulb) at the terminal. Removal of taxol allowed nearly immediate resumption of cortical motility in all 17 neurons observed by time-lapse videomicroscopy. However, only one of the 17 neurites was observed to elongate measurably even after 5 h of obs,~rvation. In 14 cases taxol was rinsed out and the concentration of nerve growth factor was increased 10x, 11/14 neurites retracted within the next hour. Endocytic activity was investigated by incubating control and taxol treated neurons in either cationized ferritin or horseradish peroxidase for 30 min. The number and area of label-containing vesicles was measured along with the total area of the growth cone or taxol bulb. We found that taxol treatment caused a 7-fold decrease in the ratio of the area of the labeled vesicles to the area of growth cone or taxol bulb. Conversely, in neurite shafts, normally relatively quiescent with respect to endocytosis, those regions devoid of microtubules in both control and nocodazole-treated cells contained a high concentration of label-containing vesicles. We conclude that the presence of microtubules plays a role in regulating endocytic activity by the overlying cell cortex. We speculate that this could be due either to transport activity mediated by microtubules or to cortical stabilization by microtubules.
INTRODUCTION Neurons in culture extend neurites that consist of two morphologically and functionally distinct compartments, the neurite shaft and the terminal growth cone. The neurite shaft is an elongated cylinder of cytoplasm with a dense paraxial array of microtubules and neurofilaments whose cortex does not engage in motile activity 5.25,27. The growth cone is a flat, palmate structure that lacks microtubules and neurofilaments, but has a cortex which is very active in filopodial and lamellipodia! extension and endocytosis5'22'25'27. Despite considerable attention given to describing these ultrastructural and functional differences between the neurite shaft and growth cone, little attention has been given to determining the mechanisms for this regionalization.
We reported previously that microtubules in the shaft of cultured chick sensory neurons play a role in the compartmentation of ribosomes to the cell bodies 1. The relative absence of microtubules in growth cones as compared to shafts suggests that microtubules may also play a role in the compartmentation of neurite shaft and growth cone. Support for this hypothesis comes from Letourneau and Ressler 14 who showed that adding taxol, a potent microtubulepolymerizing agent 21, to day-old cultures of dorsal root ganglion ( D R G ) neurons inhibits neuritic elongation and growth cone motility. We hypothesized that taxol may act by stimulating microtubule polymerization in the growth cone, thus eliminating its structural and functional specialization. We have extended the work of Letourneau and Ressler with particular emphasis on the effects of taxol-induced mi-
Correspondence: S. Heidemann, Department of Physiology, Michigan State University, E. Lansing, M148824-1101, U.S.A. 0006-8993/88/$03.50 c@.)i988 Elsevier Science Publishers B.V. (Biomedical Division)
61 crotubule assembly on the compartmentation of motility and endocytosis to the growth cone. We found that taxol treatment causes the growth cone to lose motility and endocytosis and to take on the quiescent characteristics of a neurite shaft. 1~IATERIALSAND METHODS
Materials Chemicals not otherwise noted were purchased from Sigma, St. Louis, MO. Cell culture Embryonic chick sensory neurons were cultured using a method similar to that described by Shaw and Bray 23. DRG were dissected from the lumbosacral regions of 10- to 12-day-old embryos, and placed in L-15 medium (Gibco, Grand Island, NY) supplemented with 0.6% glucose, 2 mM L-glutamine, 100 U/ml penicillin, and 100 mg/ml streptomycin (L15+). The ganglia were rinsed twice, then treated with 0.25% trypsin for 25 min at 37 °C. The trypsin was removed, L-15+ with 10% fetal calf serum (Hazleton Dutchland, Denver, PA) was added, and the ganglia were triturated with a pipette into a single cell dispersion. The cells were rinsed twice, and plated into medium with 10% fetal calf serum, 0.6% methyl cellulose (Metcel A4M, Dow Chemical, Midland, MI), and 100 ng/ml 7s nerve growth factor (NGF) isolated from mouse saliva 6. The cells were plated into 35-mm Falconware Primaria tissue culture dishes at a density low enough to render non-neuronal c,mtamination inconsequential. Cultures were incul:,oted at 37 °C for 18-30 h prior to experimentation. Experimental treatments Growth cones bearing filopodia and/or lamellipodia were selected for observation and circled with a diamond marker objective. Cells were treated with taxol (gift from the National Cancer Institute) by replacing the culture medium with fresh drug containing medium including L-15+, 10% FCS, 100 ng/ml NGF, and 5.7 x 10-8 M taxol. Taxol was removed from treated cultures by rinsing 3 times in fresh taxolfree medium after which the cells were allowed to incubaZe in L-15+, 10% fetal calf serum and 100 ng/ml NGF. In some experiments, taxol-treated cells were rinsed and replaced into taxol-free medium contain-
ing 1/~g/ml NGF, i.e. a 10x higher concentration of NGF. All cultures were constantly maintained at 37 °C prior to and during experimentation. Still photographs were taken with a Nikon FE2 camera through a Nikon phase contrast microscope on Technical Pan film developed in HC110 developer. Video recordings were made with a D A G E MTI 67 series video camera on an RCA time-lapse video recorder. Selected cells were assayed for endocytosis by adding either 500/lg/ml cationized ferritin (CF) or 1 mg/ml horseradish peroxidase (HRP) to the culture for a period of 30 min after the taxol had taken effect (see Results). In another set of experiments, cells were treated simultaneously with 1/~g/ml nocodazole (Sigma) and one of the endocytic markers (CF or HRP). After treatment with the endocytic marker, cultures were rinsed 3 times in serum-free medium prior to fixation. The HRP-treated cultures were exposed after fixation to a solution containing 0.5 mg/ml diaminobenzidine (HRP substrate) and 0.01% H20 2 in 0.05 M Tris, pH 7.6, for 10 min. The cultures were then prepared for electron microscopy as described below.
Electron microscopy After experimental treatments, cultures were fixed for 20-30 min with 2% glutaraldehyde in 0.1 M cacodylate, rinsed twice in 0.1 M cacodylate with 5% sucrose, postfixed for 5 min in 1% OsO4, dehydrated in ethanol series, and embedded in Polybed 812 (Polysciences, Warrington, PA). Thin sections were cut parallel to the substratum, stained with uranyl acetate and lead citrate, and observed with a Philips 300 transmission electron microscope. Measurement of areas Tissue areas were computed from tracings of scaled electron micrographs using numerical integration techniques. Areas were traced using a calibrated electromagnetic platten and handheld viewer coupled to an IBM XT computer for file storage. Integrations were made with a Sigma Scan (Jandel) computer program. RESULTS In addition to stopping the advance of preformed dorsal root ganglion neurons, as reported previous-
62. lyTM, taxol significantly changed the appearance of growth cones. Twenty-two well-spread growth cones were photographed before and 3 h after treatment with 5.7 x 10-8 M taxol. Fig. I shows a typical example of a cell's response to taxol. In all but one of the 22 cases the growth cones rounded into obvious varicosities at the neurite terminal (hereafter referred to as taxol bulbs) and displayed only a few filopodia and no lameUipodia. Time-lapse video observations revealed that the growth cones always assumed this shape within the first 35 min after taxol exposure and that the filopodia were not retracting and protruding as they do in normal growth cones. Letourneau and Ressler 14 reported that D R G neurons plated in the presence of taxol had the ability to recover and extend neurites after being rinsed free of taxol. We wished to examine the reversibility of taxol's effects on preformed neurites. We examined the effects of rinsing out taxol on 17 different taxol bulbs by making time-lapse video microscopy recordings of the bulbs for periods of 1 to 5 h following each rinseout. In all 17 cases, the taxol bulbs regained motility within minutes after taxol rinseout (Fig. 2), but only one of 17 bulbs recovered to the point where a new growth cone was formed and neurite elongation was measured. Peterson and Crain Is reported that relatively high concentrations of NGF attenuated the neurotoxic effects of taxol on cultures of fetal mouse D R G . The effects of 10x higher concentrations of N G F in drug-free medium was assessed by time lapse observation of 14 typical taxol bulbs. All 14 neurites in medium containing 1/~g/ml 7s NGF recovered motility within minutes, only one of 14 lengthened
Fig. 1. Morphological effect of taxol on pre-formed growth cones. Chick sensory neurites from 10-day-old embryo were treated with 5.7 x 10-8 M taxol, a: untreated neurites after 20 h of growth, b: same neurites 3 h after addition of taxol, x 260.
!
Fig. 2. Effect of removing taxol on pre-formed growth cones. Conditions of growth and taxol treatment were the same as in Fig. 1. a: prior to addition of taxol, b: 30 min after addition of taxol, just prior to removal of taxol, c: 10 min after removal of taxol from culture medium, d: 50 min after removal of taxol from culture medium, x580.
measurably. However, 11/14 neurites retracted within I h of being placed into the higher concentration of NGF. We speculate that this response is similar to the retraction response observed by Griffin :'nd Letourneau ~1. Although, except for a sudden increase in NGF concentrations, our conditions were rather different. Our transmission electron micrographs of thin sections confirmed the whole-mount observations of Letourneau and Ressler 14 on chick D R G and were entirely similar to observations from Horwitz's group on mouse 16 ~nd rat neurons 2°. The tips of taxol treated neurites contained a disorganized jumble of microtubules (Fig. 3). Our micrographs revealed that the microtubule density was not nearly as great as that typically found in a control neurite shaft but much greater than the extremely low density typical of growth
63
Fig. 3. Endocytic uptake of CF by taxol-treated growth cones. A transmission electron micrograph of a region of the distal terminal of a neuron treated with taxol (5.7 x 10-8) for 30 rain then exposed to 0.5 mg/ml CF for an additional 30 min. Thin arrows mark microtubules, stubby arrows mark ferritin-containing vesicles. Relative to growth cones without taxol (see Fig. 4) there is a large increase in number of microtubules and a concomitant decrease in labele~ivesicles, x25,200.
cones. Gordon-Weeks reported a similar taxol-induced stimulation of microtubule assembly in isolated growth cones from new-born rats 9. We wished to determine the effect of taxol treatment on endocytosis by the growth cone. In two experiments, CF was used as the label for endocytosis 8. In two other experiments, HRP was used 24. We measured the area of label-containing vesicles in randomly selected transmission electron micrographs from 4 different control growth cones (one from each experiment) and 7 different taxol bulbs (one or two from each experiment). Fig. 3 shows a taxol bulb and Fig. 4 a control growth cone assayed for endocytosis with CF as described in Materials and Methods. Despite the growth cone's smaller area, it contained 10.0 times as much !abel-containing, internalized membrane as the corresponding taxol bulb. Table I
summarizes our measurements. We found that the number and area of labeled vesicles was 3- to 5-fold greater in control growth cones than in taxol bulbs, despite the larger area of taxol bulbs surveyed. We used a ratio, area of endocytic vesicles/total area of growth cone or taxol bulb, as an estimate of endocytic activity. Using this measure, we found a 7.1-fold decrease in endocytic activity for taxol bulbs as compared to growth cones. A one-tailed t-test, adjusted for unequal variance and sample size, showed that this decrease in endocytic activity for the taxoltreated population was significant at a = 0.01. Since induced microtubule polymerization reduced endocytosis and motility in growth cones, we hypothesized that nocodazole-induced microtubule depolymerization would increase motility and endocytosis in neurite shafts. However, as previously re-
64
Fig. 4. Endocyticuptake of CF by control growth cones. A transmission electron micrograph of a region of a control growth cone incubated in 0.5 mg/mlCF in serum-free medium for 30 rain. Arrows mark ferritin-containing vesicles, x25,200.
ported ~, the response of DRG microtubules to microtubule poisons proved highly variable. Some regions of our nocodazole-treated neurites were devoid of microtubules while other regions appeared to contain normal microtubule arrays. Measurement of endocytic .~ctlvi~y as in Table I was therefore not ':teaslu~e. "-' .... However, in neurites treated with nocodazole, as well as in control neurites, we found that areas along the shafts that were devoid of microtubules often contained an accumulation of endocytic vesicles. Fig. 5 shows a bundle of neurites treated with both nocodazole and CF. The two outer neurites were devoid of microtubules and contained an accumulation of CF labeled vesicles while the inner neurite had an intact array of microtubules and was devoid of label. As shown in this figure, labeled vesicles in nocodazole-treated neurites frequently appeared abnormally large and empty. Fig. 6 shows a microtubulesparse region of a control neurite. The absence of mi-
crotubules is associated with the formation of a 'preterminal growth cone' characterized by filopodia and the generally splayed appearance of the cytoplasm. This sample had been treated with HRP, and the high density of labeled vesicles in the vicinity of the microtubule-sparse area should be noted. We never observed a similar concentration of endocytic vesicles in control or drug-treated neurites in areas of the shaft containing a dense array of microtubules. DISCUSSION Our results confirm Letourneau and Ressler's ~4report that taxol causes growth cones to lose their motility and take on the quiescent characteristic of neurite shafts. We showed that rinsing out taxol from such cultures (which presumably allows for some microtubule depolymerization) causes the distal terminals of neurites to regain cortical motility, and we
65 TABLE I Effect o f taxol treatment on endocytosis at neurite terminals Cell
Number o f vesicles*
Area o f vesicles*'**
1 control 2 control 3 control 4 control Control subtotal
Treatment
16 28 30 9 74
0.839 3.193 2.427 0.597 7.056
20.303 41.292 32.965 13.869 108.456
Total area**
Ratio o f vesicle to total area
0.041 0.077 0.043 0.043 0.065
5 taxol 6 taxol 7 taxol 8 taxol 9 taxol 10 taxol 11 taxol Taxol subtotals
3 6 1 0 1 2 10 23
0.090 0.561 0.355 0.000 0.095 0.070 0.354 1.525
22.107 30.507 25.049 18.430 9.842 16.241 49.103 171.279
0.004 0.018 0.014 0.000 0.010 0.004 0.007 0.009
* Only vesicles labeled with HRP or CF. ** Area in/~m2.
found that taxol bulbs, unlike g r o w t h cones, were similar to n e u r i t e shafts with respect to their low content of internalized vesicles. F u r t h e r , we f o u n d that microtubule-free regions of both control and nocodazole-treated n e u r i t e shafts often c o n t a i n e d endocytic
vesicles and occasionally formed motile structures (Fig. 6). W e thus propose t h a t microtubule polymerization plays an i m p o r t a n t role in the c o m p a r t m e n t a tion of neurites into shafts and growth cones. This int e r p r e t a t i o n is consistent with the observations of
Fig. 5. Relationship of microtubules to endocytosis in the neurite shaft. A transmission electron micrograph of a region of neurite shafts from a small bundle of neurites exposed to 1 mg/ml nocodazole and 0.5 mg/mi CF for 25 rain. Note the absence of microtubules in both outer neurites and the presence of label-containing vesicles while the inner neurite contains microtubules but lacks labeled vesicles, x22,700.
66 ,~!~!.~.
"
:~.~,~.~ .
.
.
.
2
.,
.
o
Fig. 6. Relationshipof microtubules to endocytosisin the neurite shaft. A region of a neurite not treated with any microtubule drug but exposed to 1 mg/ml HRP for 30 rain. This area of normal neurite shaft was sparse in microtubules, apparently had a 'pre-terminal growth cone"which engaged in endocytosis. No equivalent uptake of label was observed in control neurites in regions containing a normal microtubulearray, x 11,200.
Bray et al. 4 and of Tosney and Wessels 25. Bray et al. reported that they routinely found 'preterminal' growth cones on DRG neurites exposed to drugs which depolymerize microtubules. From this and other observations, they concluded that the capacity for growth cone formation is distributed throughout the neurite and seems to be repressed by microtubules. Tosney and Wessels found that stable areas of the growth cone (the palm, non-motile veils) contained microtubules while motile portions did not. Apparently, the absence of microtubules permits motility and endocytosis, while the presence of microtubules inhibits motility and endocytosis in the overlying cell cortex. The mechanism of microtubule-associated compartmentation is unknown but two broad categories
of explanation suggest themselves. One explanation for the changes induced by taxol invokes the role of microtubules in vesicular transport 1°. It is possible that taxol bulbs are capable of endocytosis but lack vesicles because, unlike growth cones, they contain sufficient numbers of microtubules to rapidly transport the internalized vesicles back to the cell body. The absence of motility in taxol bulbs might also be explained by changes in transport. Lloyd et al. 15 proposed that directed fibroblast motility requires the channelling of membrane precursors towards a preferred edge by a linear array of microtubules. The disorganized jumble of microtubules found in taxol bulbs may be incapable of such membrane channelling and thus incapable of supporting growth cone motility.
67 The other explanation is that microtubules play a role in regulating the ability of the cortex to change shape, e.g. regulating the surface tension of neurons. Bray and C h a p m a n 3 speculated that the increasing quiescence found in going from the tip of the growth cone back to the neurite may reflect an increasing gradient of surface tension. Similarly, the work of Tosne~ and Wessels 25 suggests that the increasing gradient of quiescence is accompanied by an increase in the density of the microtubule array. O u r observations of drug-treated neurons are uniformly consistent with the hypothesis that the density of the microtubule array directly correlates with the quiescence of the neuronal surface. In taxol-treated neurons, decreased motility and endocytosis at the growth cone, as well as the characteristic 'rounded up' shape of taxol bulbs, could be explained by an increase in surface tension. Conversely, nocodazole-induced increases in cortical motility and endocytosis and the fragmentation of neurites into separate beads of cytoplasm in response to lengthy nocodazole treatments I could be caused by a decrease in surface tension. Other workers have proposed that microtubules have the ability to regulate the quiescence of the cellular cortex. A number of studies have proREFERENCES 1 Baas, P.W., Sinclair, G.I. and Heidemann, S.R., Role of microtubules in the cytoplasmic compartmentation of neurons, Brain Research, in press. 2 Bamburg, J.R., Bray, D. and Chairman. K.. Assembly of microtubules at the tips of growing axons. Nature (Lond.), 321 (1986) 788-800. 3 Bray, D. and Chapman, K., Analysis of microspike movements on the neuronal growth cone, J. Neurosci., 5 (1985) 3204-3213. 4 Bray, D., Thomas, C. and Shaw, G., Growth cone formation in cultures of sensory neurons, Proc. Natl. Acad. Sci. U.S.A., 75 (1978) 5226-5229. 5 Bunge, M.B.~ Initial cndocytGsi~ of peroxidase or ferritin by growth cones of cultured nerve cells, J. Neurocytol.. 6 (1977) 407-439. 6 Burton, L.E., Wilson, W.H. and Shooter, E.M., Nerve growth factor from mouse saliva, J. Biol. Chem., 253 (1978) 7807-7812. 7 Caron, J.M., and Berlin, R.D., Dynamic interactions between microtubules and artificial membranes, Biochemistry, 26 (1987) 3681-3688. 8 Chan, K.Y., Bunt, A.H. and Haschke, R.H., Endocytosis and compartmentation of peroxidases and cationized ferritin in neuroblastoma cells, J. Neurocytol., 9 (1980) 381-403. 9 Gordon-Weeks, P.R., The cytoskeleton of isolated, neuronal growth cones, Neuroscience, 21 (1987) 977-989.
vided evidence for a class of microtubules that have the ability to cross-link to membrane-associated cytoskeletal elements and thus potentially stabilize the cell cortex 12.13-17-19. Further, Vassilev et al. 26 showed that increasing tubulin in a lipid monolayer decreases its surface tension. Caron and Berlin 7 found that stimulated microtubule polymerization can deplete artificial membranes of adsorbed tubulin. Stimulating microtubule polymerization within a cell by taxol may deplete the membrane of tubulir, thus causing an increase in surface tension and, consequently, a decrease in cortical motility. ACKNOWLEDGEMENTS We thank R.E. Buxbaum for many stimulating discussions and for reading and commenting on the manuscript. We are indebted to H. Stuart Pankratz for his invaluable assistance and the Department of Microbiology for the use of their electron microscope facility. We also thank Thomas Adams for access to and help with the morphometric measurements. This work was supported by Grant BNS 8706741 from the National Science Foundation and Grant G M 36894 from the NIH. 10 Grafstein, B. and Forman, D.S., Intracellular transport in neurons, Physiol. Rev., 60 (1980) 1167-1283. 11 Griffin, C.G. and Letourneau, P., Rapid retraction of neurites of sensory neurons in response to increased concentrations of nerve growth factor, J. Cell Biol., 86 (1980) 156-164. 12 Hirokawa, N., Cross linker system between neurofilaments, microtubules, and membranous organelles revealed by the quick freeze, deep etching method, J. Cell. Biol., 94 (1982) 129-142. 13 Joshi, H.C., Baas, P., Chu, D.T. and Heidemann, S.R., The cytoskeleton of neurites after microtubule depolymerization, Exp. Cell. Res., 163 (1986) 233-245. 14 Letourneau, P.C. and Ressler, A.H., Inhibition of neurite initiation and growth by taxol, J. Cell Biol., 98 (1984) 1355-1362. 15 Lloyd, C.W., Smith, C.G., Woods, A. and Rees, O.A., Mechanisms of cellular adhesion. II. The interplay between adhesion, the cytoskeleton and morphology in substrate attached cells. Exp. Cell Res., 110 (1977) 427-437. 16 Masurovsky, E.B., Peterson, E.R., Crain, S.M. and !"~rwitz, S.B., Morphological alteration in dorsal root ganglion neurons and supporting cells of organotypic mouse spinal cord-ganglion cultures exposed to taxol, Neuroscience, 10 (1983) 491-509. 17 Miller, R.H., Lasek. R.J. and Katz, M.J., Preferred microtubules for vesicle transport in lobster axons, Science, 235 (1987) 220-222. 18 Peterson, E.R, and Crain, S.M., Nerve growth factor at-
68 tenuates neurotoxic effects of taxol on spinal cord-ganglion explants from fetal mouse, Science, 217 (1982) 377-379. 19 Pollard, T.D., Selden, C.S. and Maupin, P., Interaction of actin filaments with microtubules, J. Cell Biol., 99 (1984) 33s-37s. 20 Roytta, M., Horwitz, S.B. and Crain, C.S., Taxol-induced neuropathy: short term effects of local injection, J. Neurocytol., 13 (1984) 685-701. 21 Schiff, P.B., Fant, J. and Horwitz, S.B., Promotion of microtubule assembly in vitro by taxol., Nature (Lond.), 277 (1979) 665-667. 22 Schnapp, BJ. and Reese, T.S., Cytoplasmic structure in rapid frozen axons, J. Cell. Biol., 94 (1982) 667-679. 23 Shaw, G. and Bray, D., Movement and extension of isola-
ted growth cones, Exp. Cell Res., 104 (1977) 55-62. 24 Steinman. R.M., Silver, J.M. and Cohn, Z.A., Pinocytosis in fibrobh :~, J. Cell Biol., 63 (1974) 949-969. 25 Tosney, K.W. and Wessels, N.K., Neuronal motility: the ultrastructure of veils and microspikes correlates with their motile activities, J. Cell Sci., 61 (1983) 389-411. 26 Vassilev, P.M., Tanneva, S., Pannaiotov, I. and Georgiev, G., Dilational viscoelastic properties of tubulin and mixed tubulin-lipid monolayers, J. Colloid Interface Sci., 84 (1981) 169-174. 27 Yamada, K.M., Spooner, B.S. and Wessels, N.K., Axon growth: role of microfilaments and microtubules, Proc. Natl. Acad. $ci. U.S.A., 66 (1970) 1206-1212.