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Previous Article | Next Article
Volume 16, Number 11, Issue of June 1, 1996 pp. 3601-3619
Copyright ©1996 Society for NeuroscienceTau Is Enriched on Dynamic Microtubules in the Distal Region of Growing Axons
Mark M. Black1, Theresa Slaughter1, Simon Moshiach1, Maria Obrocka2, and Itzhak Fischer2 1 Department of Anatomy and Cell Biology, Temple University School of Medicine, Philadelphia, Pennsylvania 19140, and 2 Department of Neurobiology and Anatomy, Medical College of Pennsylvania and Hahnemann University, Philadelphia, Pennsylvania 19129
ABSTRACT
It is widely held that tau determines the stability of microtubules in growing axons, although direct evidence supporting this hypothesis is lacking. Previous studies have shown that the microtubule polymer in the distal axon and growth cone is the most dynamic of growing axons; it turns over more rapidly and is more sensitive to microtubule depolymerizing drugs than the polymer situated proximally. We reasoned that if the stability of axonal microtubules is directly related to their content of tau, then the polymer in the distal axon should have less tau than the polymer in the proximal axon. We tested this proposition by measuring the relative tau content of microtubules along growing axons of cultured sympathetic neurons immunostained for tau and tubulin. Our results show that the tau content of microtubules varies along the axon, but in the opposite way predicted. Specifically, the relative tau content of microtubules increases progressively along the axon to reach a peak near the growth cone that is severalfold greater than that observed proximally. Thus, tau is most enriched on the most dynamic polymer of the axon. We also show that the gradient in tau content of microtubules does not generate corresponding gradients in the extent of tubulin assembly or in the sensitivity of axonal microtubules to nocodazole. On the basis of these findings, we propose that tau in growing axons has functions other than promoting microtubule assembly and stability and that key sites for these functions are the distal axon and growth cone.
Key words: microtubule-associated proteins; cytoskeleton; axon growth; quantitative digital image analysis; cultured sympathetic neurons
INTRODUCTION
Tau is a developmentally regulated microtubule-associated protein (MAP). Tau is encoded by a single gene, but because of alternative splicing and phosphorylation, it shows multiple isoforms (for review, see Wiche et al., 1991). During neuronal differentiation tau undergoes a transition from immature to mature forms that involves a dramatic increase in the number of isoforms (for review, see Schoenfeld and Obar, 1994
Although the importance of tau in axon growth is well established, its specific functions are unknown. Because tau binds MTs, its functions presumably involve, at least in part, binding to MTs. On the basis of its ability to promote MT assembly and stability in vitro, tau has been proposed to promote MT assembly and stabilization in growing axons (Brandt and Lee, 1993
Several recent observations have shown that the stability of MTs varies along the length of growing axons. Specifically, the polymer located distally near the growth cone turns over more rapidly and is more sensitive to treatments with MT depolymerizing drugs than the polymer situated elsewhere in the axon (Lim et al., 1989
MATERIALS AND METHODS
Materials. Culture media were obtained from Gibco (Grand Island, NY). Supplements for culture media were obtained from either Gibco or Sigma (St. Louis, MO), except for NGF, which was purified from mouse salivary glands according to Mobley et al. (1976). Nocodazole was obtained from Aldrich (Milwaukee, WI), and other reagents were obtained from Sigma unless otherwise indicated.Cell culture. All experiments reported here used rat sympathetic neurons dissociated from the SCGs of newborn pups as described by Black and Kurdyla (1983)
Preparation of polyclonal antibodies against tau. Two polyclonal antibodies against tau were prepared against fusion protein constructs containing discrete regions near the N terminus or C terminus of the protein (see Fig. 1A). Two cDNA clones encoding the N- and C-terminal ends of tau were prepared by RT-PCR using two sets of primers designed on the basis of the sequence of the high molecular weight isoform of rat tau (Goedert et al., 1992
end of the tau cDNA) spanned the N terminus at aa 2-113, whereas the 3TA and 3TB primers (sense and antisense sequences from the 3
Fig. 1. Characterization of polyclonal antibodies against tau. A shows a schematic of the tau cDNA, the primers used for RT-PCR, and the constructs used to prepare fusion proteins for use in antibody production. The sequences of the primers, from 5
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Fixation and extraction of neurons. Cultured neurons were processed for immunofluorescence localization of tubulin and tau according to one of the following four procedures. For procedure 1, cells were rinsed once with PBS, once with PHEM (60 mM PIPES, 25 mM HEPES, 10 mM EGTA, 2 mM MgCl2, pH 6.9; Schliwa and van Blerkom, 1981
For some experiments, cells were extracted before fixation using conditions that stabilize existing MTs and remove unassembled tubulin. Neurons were rinsed once with PBS, once with PHEM or PEM, and then extracted at room temperature for 2 min in PHEM or PEM containing 0.2% saponin and 10 µM taxol (a gift from Ms. Nancita Lomax of the National Cancer Institute); variations from this basic protocol are described in Results. Taxol was made as a 10 mM stock solution in DMSO, and appropriate volumes were added to the extraction buffer immediately before use. The extraction solution also contained a mixture of protease inhibitors (0.5 mM PMSF, 0.2 trypsin inhibitory units/ml aprotinin, and 10 µg/ml each leupeptin, chymostatin, and antipain). After extraction, the cells were fixed using procedure 1, 3, or 4 as described above.
After fixation and permeabilization, the dishes were rinsed with PBS, treated with sodium borohydride (10 mg/ml in PBS for 7 min), incubated with 0.1 M glycine in PBS for 20 min, rinsed with PBS again, and then incubated with blocking solution for 30 min. Blocking solution consisted of either 4% normal donkey serum in PBS or 1% nonfat dry milk + 0.1% Tween-20 in PBS. The cells were then double-stained to reveal tau and tubulin (see below). All antibodies were diluted in blocking solution and then clarified before use by centrifugation at 200,000 × g for 10 min in a Beckman TL-100 ultracentrifuge (Beckman Instruments, Palo Alto, CA). After incubation with secondary antibody, cells were rinsed extensively with PBS and then mounted in 80% (w/v) glycerol in PBS containing 10 mg/ml n-propyl gallate.
Immunofluorescence procedures. Cells were double-stained for tubulin and tau using one of the following conditions. Staining condition 1: cells were incubated first with a mouse monoclonal anti-
-tubulin antibody [purchased from Amersham (Arlington Heights, IL; Blose et al., 1984
-tubulin (diluted 1:400, generously provided by Dr. Chloe Bulinski, Columbia University School of Medicine, New York, NY) (Gundersen et al., 1984). Cells were first incubated with the anti-tubulin antibody for 45 min at 37°C, rinsed with blocking solution, and then incubated with a cocktail of mouse monoclonal antibodies against tau consisting of tau1, tau14, and tau60 for 2 hr at room temperature (tau14 and tau60 were generously provided by Dr. Virginia Lee, Department of Pathology, University of Pennsylvania, Philadelphia, PA; tau1 was generously provided by Dr. Lester Binder, Northwestern University Medical School, Chicago, IL). After extensive rinsing and reblocking, the cells were incubated simultaneously with FITC-conjugated donkey anti-rabbit antibody and lissamine-conjugated donkey anti-mouse antibody, both diluted 1:100.
For some experiments, the rabbit polyclonal antibody against the N-terminal region of tau (tau-5
Image acquisition and analysis. Neurons were observed by epifluorescence microscopy using a Zeiss Axiovert 135 inverted microscope (Zeiss, Thornwood, NY), and images were obtained with a CH250 cooled CCD camera (Photometrics Ltd., Tucson, AZ) equipped with a Thompson 7883 CCD chip. The details of the imaging system have been described previously (Brown et al., 1992
To evaluate the overall distribution of tubulin and tau in growing axons, cells were imaged using a 25×/0.8 N.A. plan neofluar oil-immersion objective to capture the entire length of the axons in single images. To examine MT staining for tubulin and tau at higher resolution, cells were imaged with a 100×/1.3 N.A. plan neofluar oil-immersion objective. We used the segmented mask procedure to quantify the intensity of tubulin and tau staining along the length of individual axons (Brown et al., 1992
Fig. 6. Quantitative analyses of the relative amounts of total tubulin and total tau along the axon. Cells fixed and then permeabilized (according to procedure 4) were double-stained for
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Fig. 11. Quantitative analyses of the relative amounts of assembled tubulin and assembled tau along the axon. Cells processed by combined fixation and extraction (according to procedure 3) were double-stained for
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Measurements of axonal volume. Measurements of volume along the length of axons were based on
Fig. 7. Quantitative analyses of the volume densities of tubulin and tau along the axon. Cells fixed and then permeabilized (according to procedure 4) were double-stained for
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Preparation of extracts of cultured neurons for biochemical analyses. Total tau as well as tau in cytoskeletal and soluble fractions of cultured neurons were prepared for analysis by immunoblotting. For total tau, whole-cell SDS extracts were prepared by scraping cultured neurons into 1% SDS plus a cocktail of protease inhibitors that included 0.2 trypsin inhibitory units/ml of aprotinin and 10 µg/ml each leupeptin, chymostatin, and antipain, and 0.5 mM PMSF. The extracts were incubated at 100°C for 5 min and then clarified by centrifugation in the TL100.2 rotor (Beckman Instruments) at 75,000 rpm for 15 min at 22°C.
To prepare soluble and cytoskeletal fractions from cultures, cultures were rinsed once with PBS, once with PHEM or PEM, and then scraped into PHEM or PEM supplemented with 0.2% Triton X-100 or 0.2% saponin, respectively, 10 µM taxol, and protease inhibitors (all buffers were at room temperature). Soluble and cytoskeletal fractions were obtained by centrifugation of cell extracts at 75,000 rpm for 10 min at 22°C in the TL100.2 rotor. The soluble fraction was removed and its protein precipitated by the addition of ~8 volumes of cold methanol. The resulting methanol-precipitated material was collected by centrifugation, air-dried, dissolved in 1% SDS, and then incubated at 100°C for 5 min. The cytoskeletal pellet was rinsed once with PHEM or PEM + 10 µM taxol and then dissolved directly in 1% SDS, and then incubated at 100°C for 5 min. The resulting materials were then analyzed by SDS-PAGE and immunoblotting.
SDS-PAGE and immunoblotting. Protein samples were resolved in 4-10% SDS-PAGE gels and then transferred onto nitrocellulose membranes (1000 mA for 3 hr at 4°C). The transfers were then incubated with primary antibodies followed by HRP-conjugated secondary antibodies and detection by chemiluminescence using ECL reagents (Amersham). The antibodies against the recombinant tau fragments (tau-3
RESULTS
The present experiments examine the tau content of MTs in axons and growth cones of cultured sympathetic neurons, and the effects of tau on MT stability and the extent of MT assembly in growing axons of these neurons. In a previous study, we used quantitative immunofluorescence procedures to evaluate the MAP1b content of MTs along the length of growing axons (Black et al., 1994Characterization of antibody reagents against tau
The present studies used two polyclonal antibodies raised against distinct regions of the tau molecule. One antibody (tau-5We used the polyclonal antibodies (tau-3
The association of tau with MTs is very sensitive to conditions of fixation and extraction
In our previous studies (Black et al., 199499% of the tau was Triton-soluble whereas, by comparison, only ~54% of MAP1b was Triton-soluble (Fig. 2). Many possible explanations can account for the lack of tau association with MTs under these conditions. As we document below, tau binding to MTs in these neurons is very labile to a variety of conditions of fixation and extraction that preserve MTs and MAP1b association with MTs.
Fig. 2. Immunoblot analyses of tau partitioning during extraction with an MT-stabilizing buffer containing Triton X-100. Cells were extracted with PHEM + 0.2% Triton X-100 + 10 µM taxol as described in Materials and Methods to obtain Triton-soluble and Triton-insoluble fractions, which contain unassembled and assembled MT proteins, respectively. The entire Triton-soluble and Triton-insoluble fractions were resolved on 4-10% gradient gels and then transferred to nitrocellulose. The transfer was first probed with the tau monoclonal cocktail and then, after obtaining the necessary exposures, the transfer was stripped and reprobed with the anti-MAP1b polyclonal antibody. Shown are portions of the resulting exposures showing the partitioning of tau and MAP1b between the Triton-soluble (S) and Triton-insoluble (I) fractions. Note that the middle portion of the immunoblot is shown for tau, whereas the top portion is shown for MAP1b.
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We tested several different procedures to visualize tau in cultured neurons using immunofluorescence procedures. Figure 3A-F shows representative results obtained with a modified combined fixation and extraction procedure in which 0.2% saponin was used in place of 0.5% Triton X-100 (procedure 1); comparable results were also obtained when cells were fixed in the absence of detergent and then permeabilized using procedure 2 or the PBS/sucrose fixative described by Mandell and Bankler (1995) (data not shown). All of these procedures resulted in strong staining for tubulin and tau in the cell body and all along the axon, and the tau staining could be completely blocked by preincubating the anti-tau antibody with a boiled MAP fraction prepared from neonatal rat brain (data not shown). However, in spread regions where MTs were clearly visible by tubulin staining, tau staining was diffuse and did not colocalize with the MTs (see Fig. 3C-F). This lack of colocalization was observed wherever MTs could be seen, including in lamellapodial expansions of the cell body, in spread regions that occurred occasionally along the axon shaft, and in the growth cone. Using this same combined fixation and extraction procedure, MAP1b clearly and unambiguously colocalized with axonal MTs (see Fig. 3G-J), as did MAP2 in immature axons (Fig. 4C,D); tau did not localize to MTs in such immature axons (data not shown).
Fig. 3. Double-staining of neurons for tubulin and either tau (A-F) or MAP1b (G-J). The cells were processed according to procedure 1 and then stained using staining condition 1. A, C, E, G, and I show tubulin staining; B, D, and F show tau staining; and H and J show MAP1b staining. A and B show low-magnification images (scale bar, 56 µm) depicting the overall distribution of tubulin and tau in the neurons, whereas the remaining panels show higher-magnification views that reveal details of MT staining for tubulin, tau, and MAP1b under these fixation and staining conditions. Note that tubulin and tau are distributed throughout the axon but that tau does not colocalize with MTs in the growth cone, whereas MAP1b clearly decorates MTs in the growth cone to or near their tips (see also Black et al., 1994
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Fig. 4. Double-staining of neurons for
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One possible interpretation for the lack of tau localization to MTs is that little or none of the tau associates with MTs. It is also possible that tau associates with MTs but that the conditions of fixation and extraction, while preserving tau in the cells, did not preserve its association with MTs. Because tau is a known MT binding protein (for review, see Wiche et al., 1991
Fig. 5. Double-staining of neurons for tubulin (A, C, E, G) and tau (B, D, F, H) in cells processed according to staining condition 2. A-D show low- and high-magnification views of cells processed using the combined fixation and extraction procedure of Lee and Rook (1992)
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Fig. 8. Tau association with MTs in the growth cone. High-magnification views of growth cones were processed by combined fixation and extraction (according to procedure 3) and then double-stained for
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The above results were obtained with the rabbit antibody against the N-terminal region of tau (tau-5
The procedures that resulted in strong tau staining without MT colocalization used PHEM + saponin as the basic buffer and a mixture of glutaraldehyde and paraformaldehyde (0.1 and 4%, respectively) for fixation, whereas the procedures that resulted in both strong staining and clear colocalization of tau with MT used PEM + NP40 as the buffer and only glutaraldehyde, at 0.3%, as the fixative. We tested different combinations of these buffers and fixatives to determine how they influence tau staining. Using the combination of glutaraldehyde and paraformaldehyde always resulted in diffuse tau staining without MT colocalization, regardless of the buffer used, and increasing the glutaraldehyde concentration to 0.3 or 0.5% did not change this result. Using 0.3% glutaraldehyde in PEM or PHEM resulted in clear tau colocalization to MTs. Although we did not pursue these experiments in detail, the available information indicates that paraformaldehyde interferes with the preservation of tau in cultured sympathetic neurons, even in the presence of more potent fixatives such as glutaraldehyde, such that the association of tau with MTs is lost even though tau is fixed in the cells.
The above findings show that the specific staining patterns obtained for tau depend on the specific conditions of fixation and extraction and that conditions that preserve MTs and reveal MAP1b and MAP2 localization to MTs do not necessarily reveal tau or tau binding to MTs. Indeed, we have identified conditions that result in clear MAP1b localization to MTs and either no tau staining whatsoever, or strong tau staining but no apparent colocalization with MTs. It is only with procedures 3 and 4 that we have been able to visualize tau associated with MTs. Whereas these results are primarily technical in nature, they also indicate that the interaction of tau with MTs differs from that of MAP1b and MAP2. Such differences are not unexpected for tau and MAP1b because they have very different MT binding domains (for review, see Wiche et al., 1991
Given the sensitivity of tau localization patterns to fixation conditions, it is not possible to determine how accurately any given procedure reflects the true localization within the cell. We assume that of all the methods tested, the picture of tau localization provided by procedures 3 and 4 provides the most reliable picture of tau localization in growing axons because of its ability to consistently reveal tau association with MTs. Accordingly, we have used these procedures to examine in detail the localization and MT association of tau in growing axons.
Tau localization in growing axons
We quantified several parameters of tubulin and tau distribution in growing axons based on low-magnification images of neurons fixed and then permeabilized (procedure 4). Representative images of cells processed in this way are shown in Figure 5E-H. In the vast majority of neurons, the staining intensity of both tubulin and tau varied along the length of the axon (Figs. 5, 6, Table 1). Staining for both tubulin and tau was highest in the cell body. Moving out from the cell body into the axon, staining intensity declined rapidly, over a distance of 30-50 µm, to a relatively low value that remained more or less constant until the distal one-half to one-third of the axon, where the staining intensity for both tubulin and tau began to increase, reaching a peak that was, on average, 20-50 µm from the axon tip. As a measure of the magnitude of the proximal-to-distal increase in the amount of tubulin and tau in the axon, we expressed the peak values in the distal axon relative to the average values in the proximal axon. Tubulin levels increased ~6-fold, whereas tau increased ~11-fold (see Table 1). These results reveal a proximal-to-distal gradient in the amount of tau and tubulin within the axon, with the amount tau and tubulin rising steadily in the distal part of the axon to reach a peak near the growth cone.
Table 1. Distribution of total
Profile parameters Average ± SD (minimum/maximum) Tau Proportion of axons showing a distal rise in staining intensity 93% 100% Length of distal region (µm)a 117 ± 59 162 ± 57 (7.5/212) (71/245) Distance from axon tip of distal segment containing peak ADU (µm) 19 ± 13 24 ± 14 (0/47) (0/55) (Peak ADU distal axon)/(mean ADU proximal axon) 6.3 ± 4.1 10.9 ± 6.9 (2.6/18.8) (4.1/26.6) (Peak ADU distal axon)/(median ADU entire axon)b 5.1 ± 2.4 7.1 ± 4.0 (2.4/11.4) (2.5/15.4) (Amount in distal axon)/(total amount in axon) 0.41 ± 0.24 0.61 ± 0.18 (0.06/0.82) (0.33/0.87) The data were obtained from 14 axons fixed in the absence of detergent (using procedure 4), and then double-stained with the mouse monoclonal antibody against a This refers to the length of the distal axon over which the staining intensity for tubulin and tau increased from the proximal plateau region. b Note: we presented the peak value in distal axon relative to the median in the axon as a whole because the value of the median is much less influenced by the extremes than the mean. Comparison of tubulin and tau in terms of the ratio of the peak staining intensity in the distal axon to the mean in the axon shaft reveals that the magnitude of the distal increase in tau staining exceeds that for tubulin. This point is made more dramatically in plots of the ratio of tau fluorescence to tubulin fluorescence along the length of the axon (see Fig. 6C,F). In all but one axon analyzed quantitatively (13 of 14 axons), these plots showed a progressive proximal-to-distal increase in the amount of tau relative to tubulin along the length of the axon. As a measure of the magnitude of this increase, the peak ratio in the distal axon was, on average, 3.7 + 1.9 µm (mean ± SD, range = 1.7-8.6) times the average in the plateau region of the axon shaft. Because the method of fixation followed by permeabilization (procedure 4) preserves all of the tubulin and all of the tau in the cells, these data show that the amount of total tau relative to total tubulin increases progressively along the length of the axon.
The proximal-to-distal increase in the amount of tau reflects a corresponding increase in tau concentration. This was determined by normalizing the tau fluorescence along the axon to axon volume in cells fixed and then permeabilized (Fig. 7). In 12 of 14 cells, the volume density of tau increased in a proximal-to-distal manner to reach a peak near the growth cone that was, on average, 3.5 ± 1 (range = 2.2-5) times greater than the average in the axon shaft. In the two axons that did not show a distal increase in the volume density of tau, this parameter was more or less uniform along the length of the axon. Thus, the proximal-to-distal increase in the amount of tau typically reflects an increase in its concentration.
With regard to tubulin, 10 of 14 neurons showed either no change or a slight decline in the volume density of tubulin along the axon. In the remaining four neurons, the volume density of tubulin increased in the distal part of the axon, reaching a peak that was on average ~2 times that observed more proximally. In axons that showed a distal increase in the volume density of both tubulin and tau, the magnitude of the increase for tau always exceeded that for tubulin.
Tau binding to MTs in the growth cone
High-magnification images of growth cones double-stained for tubulin and tau reveal that tau is present on MTs in the growth cone. In approximately half of the well spread growth cones observed, tau staining of MTs extended to or near their tips (Fig. 8; see also DiTella et al., 1994Behavior of tau during detergent extraction under MT stabilizing conditions: immunofluorescence and biochemical analyses
We attempted to use extracted cell models prepared under MT stabilizing conditions to evaluate the relative content of tau on MTs along the length of the axon. Initially, we used extraction conditions (PHEM or PEM + 0.2% saponin + 10 µM taxol; see Materials and Methods) that preserve MTs as well as MAP1b binding to MTs in extracted cells (Black et al., 1994One possible explanation for the lack of tau staining in these cytoskeletal preparations is that the tau dissociates from MTs during extraction and is removed from the cells. This interpretation is also consistent with the finding that tau binding to MTs is lost under a variety of conditions of combined fixation and extraction (see Fig. 3 and above). Previously we showed that a portion of the tau in 10- to 14-d-old cultures of sympthetic neurons partions with MTs during extraction under MT stabilizing conditions (Peng et al., 1985
Fig. 9. Immunoblot analyses of tau partitioning during extraction with an MT-stabilizing buffer containing saponin. Cells were extracted with PEM + 0.2% saponin + 10 µM taxol as described in Materials and Methods to obtain saponin-soluble and saponin-insoluble fractions, which contain unassembled and assembled MT proteins, respectively. The entire amount of each fraction was resolved on 4-10% gradient gels, transferred to nitrocellulose, and then probed with the anti-tau polyclonal (tau-3
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Tau is enriched on MTs in the distal part of the axon
Although we could not evaluate tau assembled into MTs using extracted cell models, the following results indicate that in cells processed by combined fixation and extraction (procedure 3), the staining for tau and tubulin is attributable principally to tau and tubulin assembled into MTs. First, the amount of diffuse staining for tubulin and tau observed in spread regions of cells processed by combined fixation and extraction is much less than that seen in comparable regions of cells fixed in the absence of detergent (see Fig. 5, compare C and D with G and H). Assuming that the diffuse staining corresponds to unassembled tubulin and tau, then the relative lack of diffuse staining in cells processed by combined fixation and extraction indicates that unassembled tubulin and tau are removed from the cells and that the remaining staining corresponds to assembled forms of these proteins.To directly test this possibility, cells were treated with 5 µg/ml nocodazole for 30 min to depolymerize their MTs and then processed by combined fixation and extraction. If unassembled tubulin and tau are not extracted, then the intensity of tubulin and tau staining will resemble that observed in control cells. On the other hand, if unassembled tubulin and tau are extracted during the combined fixation and extraction procedure, then tubulin and tau staining will be substantially diminished relative to controls. As shown in Figure 10, staining for tubulin and tau is dramatically reduced in drug-treated neurons compared to control neurons (compare A and B with C and D). Quantitative analyses reveal that the drug treatment reduced the intensity of axonal staining for tubulin and tau by 90% and 95%, respectively (based on analyses of three control axons and four drug-treated axons). More detailed inspection reveals that staining for both tubulin and tau was diminished throughout the axons of drug-treated cells, including in the distal region, where tubulin and tau staining are normally relatively high. Also, gaps in tubulin and tau staining occurred sporadically along drug-treated axons (see double-headed arrow in Fig. 9G,H), and in some axons both tubulin and tau staining were at background levels in the distal axon and growth cone, situations never observed in control axons. When drug-treated cells were fixed in the absence of detergent (using procedure 4), staining for tubulin and tau remained strong throughout the axon (Fig. 10E,F), indicating that drug treatment did not lead to the wholesale proteolysis of tubulin or tau over the time course of these experiments (see also Drubin et al., 1988
Fig. 10. Effects of nocodazole on the intensity of axonal staining for tubulin and tau. Cells were treated with or without nocodazole (5 µg/ml) for 30 min, and then processed by combined extraction and fixation (procedure 3, A-D, G, H) or by fixation without extraction (procedure 4, E, F), and then double-stained for tubulin (A, C, E, G) and tau (B, D, F, H) using staining condition 1. A and B show a control cell demonstrating typical staining patterns for tubulin and tau, respectively. The intensity of staining in drug-treated cells is dependent on the conditions of fixation. With combined fixation and extraction (C, D), tubulin and tau staining is reduced to negligible levels compared to controls, whereas with fixation without extraction, tubulin and tau staining is strong. The cell shown in C and D is shown again in G and H, but scaled to better reveal the dim staining remaining in this cell. Entirely similar results were also obtained using a modified procedure 3 in which the Triton X-100 treatment after fixation was omitted. Note that staining for tubulin and tau is at or near background in the distal 30-40 µm of the axon (see C, D, G, H). The arrowheads identify non-neuronal cells (non-neuronal cells processed by combined fixation and extraction and then viewed at higher magnification contain a few wavy MTs; data not shown), the single-headed arrows identify the axon tip in drug-treated cells, and the double-headed arrows identify a gap in staining along the length of a drug-treated axon.
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We used the combined extraction and fixation procedure (procedure 3) to quantify the relative amounts of assembled tubulin and assembled tau along the length of growing axons (Fig. 11, Table 2). The overall staining patterns of assembled tubulin and assembled tau resemble those for total tubulin and total tau. Specifically, the relative amounts of assembled tubulin and assembled tau increase progressively in the distal one-half to one-third of the axon, to reach a peak at variable, though relatively short, distances from the growth cone; on average, the peak amounts of assembled tubulin and assembled tau occurred within 30-50 µm from the axon tip. The proximal-to-distal gradient in assembled tau was steeper than that for assembled tubulin. As one measure of this, we expressed the peak staining intensity for assembled tubulin and assembled tau in the distal axon relative to the average values in the proximal axon. The relative levels of assembled tubulin increased ~4-fold, whereas the relative amount of assembled tau increased ~13-fold (see Table 2). As another measure of this, we calculated the ratio of staining caused by assembled tau to that of assembled tubulin along the length of the axon (Fig. 11C,F). This ratio was relatively low and constant in the proximal half of the axon. However, within the distal half, the amount of assembled tau relative to assembled tubulin increased progressively to reach a peak near the growth cone; the peak ratio in the distal axon was, on average, 6.2 ± 2.7 µm (range = 2.8-12.1) times the average in the proximal axon. Thus, the tau content of MTs (i.e., the amount of tau per unit amount of MT polymer) increases progressively in a proximal-to-distal direction along the length of growing axons.
Table 2. Distribution of assembled
Profile parameters Average ± SD (minimum/maximum) Tau Proportion of axons showing a distal rise in staining intensity 69% 94% Length of distal region (µm)a 189 ± 80 198 ± 71 (55/358) (90/345) Distance from axon tip of distal segment containing peak ADU (µm) 51 ± 65 29 ± 19 (13/251) (8/74) (Peak ADU distal axon)/(mean ADU proximal axon) 4.0 ± 2.6 13.4 ± 9.7 (1.4/10.2) (2.7/32.4) (Peak ADU distal axon)/(median ADU entire axon) 3.5 ± 2.2 10.1 ± 8.8 (1.2/8.3) (1.8/29.0) (Amount in distal axon)/(total amount in axon) 0.48 ± 0.2 0.66 ± 0.15 (0.1/0.75) (0.32/0.85) The data were obtained from 16 axons processed by combined extraction and fixation (according to procedure 3), double-stained with the mouse monoclonal antibody against To facilitate the comparison of the distribution of total tubulin and total tau along the axon (Table 1) with that of assembled tubulin and assembled tau (Table 2), the quantitative data shown in both tables were obtained from a single experiment using sister cultures. The staining procedures used on all cells were identical and were performed in parallel using the same reagents. Also, the images of the cells on which the quantitative analyses were based were obtained using the same exposure times. Thus, we have controlled for as many variables as possible so that the specific quantitative results obtained with the two fixation procedures can be reliably compared with each other. a This refers to the length of the distal axon over which the staining intensity for tubulin and tau increased from the proximal plateau region. Tau and the stability of MTs in growing axons
Tau influences the assembly and stability of MTs in vitro and in intact cells. For example, in the test tube, tau promotes tubulin assembly into MTs and enhances MT stability principally by reducing the probability that MTs will undergo depolymerization (Pryer et al., 1992To determine whether the sensitivity of axonal MTs to treatment with MT depolymerizing drugs varies as a function of their content of tau, neurons treated with 5 µg/ml nocodazole for 0, 10, or 20 min were fixed (according to procedure 3) and then immunostained to reveal assembled tubulin and assembled tau. We then used the segmented mask procedure to quantify the staining intensity attributable to assembled tubulin and assembled tau in the entire axon and in two discrete ~100-µm-long segments of the axon, one situated proximally and one situated distally. The distal segment included the most distal ~100 µm of the axon. The proximal segment was situated between 50 and 150 µm from the cell body. The MT polymer in this proximal region has the lowest average content of tau of the axon, whereas the polymer in the distal region has a higher tau content, and includes the axonal polymer with the highest average content of tau. Thus, if the differences in tau content of the polymer in these regions are sufficient to influence their sensitivity to MT depolymerizing drugs, then the polymer in these two regions will decline at distinctly different rates in the presence of such drugs. However, as shown in Figure 12, the amount of polymer in the entire axon as well as in proximal and distal regions decline in the presence of drug, and the time course of the decline in the distal axon is indistinguishable from that in the proximal axon. Thus, in spite of the severalfold difference in tau content, the MT polymer in the proximal and distal axon exhibit very similar sensitivities to nocodazole.
Fig. 12. Quantitative analyses of the time course of nocodazole effects on the amount of MT polymer in the axon. Cultures were treated with 5 µg/ml nocodazole for 0, 10, or 20 min and processed by combined extraction and fixation using procedure 3. The cells were then double-stained for tubulin and tau using staining condition 1. Images were obtained from the resulting cells and subjected to the segmented mask procedure to quantify tubulin and tau staining along the length of the axon. We then computed the total staining intensity of tubulin and tau for the entire axon, and for a 100 µm segment located proximally, and a 100 µm segment located distally (10 or 11 axons were measured for each drug treatment condition). The proximal segment was situated between 50 and 150 µm from the cell body and contains the polymer that has the lowest average content of tau in the axon. The distal segment included the most distal 100 µm of the axon and contains polymer that has a much higher tau content than the polymer in the proximal segment, and includes the axonal polymer with the highest average content of tau. The staining intensity for tubulin and tau of drug-treated axons is expressed as a percentage of the intensity in control axons. These analyses show that the time course of polymer loss is the same in the proximal and distal axonal regions in spite of the severalfold difference in tau content of the polymer in these regions.
[View Larger Version of this Image (32K GIF file)]
Drug treatment also did not detectably affect the ratio of tau fluorescence to tubulin fluorescence in axonal MTs. We evaluated this by measuring the average tau-to-tubulin ratio and the maximum tau-to-tubulin ratio for the entire axon, and for the proximal and distal regions described above. In control axons, the average values for this ratio for the entire axon and for the proximal and distal regions described above were 0.43 ± 0.20 (mean ± SD), 0.25 ± 0.13, and 0.82 ± 0.42, respectively, whereas the maximum values were 1.26 ± 0.64, 0.36 ± 0.19, and 1.26 ± 0.64, respectively. In axons treated with nocodazole for 20 min, the corresponding average values were 0.54 ± 0.22, 0.28 ± 0.12, and 0.83 ± 0.53, whereas the maximum values were 1.37 ± 0.64, 0.48 ± 0.17, and 1.13 ± 0.65. If the tau content of axonal MTs influences the time course of polymer loss in the presence of drug, then this parameter, as measured by the tau-to-tubulin ratio, should change as a function of time in drug. For example, if MT polymer with a relatively high tau content depolymerized slower than polymer with a relatively low content, then the average and maximum values for the tau-to-tubulin ratio would increase with time in drug. Clearly, this is not the case over the time course of the present experiments. This result further reinforces the conclusion that the naturally occurring variation in the tau content of the MT polymer in growing axons does not influence the sensitivity of this polymer to nocodazole-induced depolymerization.
The proximal-to-distal gradient in the amount of tau relative to tubulin does not generate a corresponding gradient in the tubulin monomer-to-polymer ratio
To obtain information on the possible effects of tau on the assembly of MTs in growing axons, we evaluated whether the proportion of tubulin in polymer increases along the axon in parallel with the proximal-to-distal increase in the tau-to-tubulin ratio. We used an indirect analysis based on the following considerations. Quantitative analyses of tubulin staining indicates that the amounts of total tubulin (Fig. 6, Table 1) and of assembled tubulin (Fig. 11, Table 2) increase in a proximal-to-distal manner for most axons. If the proportion of the total tubulin pool that is assembled increases along the length of the axon, then the magnitude of the proximal-to-distal increase in staining for assembled tubulin (seen in cells processed by combined fixation and extraction according to procedure 3) will be greater than that observed for total tubulin (seen in cells fixed and then permeabilized according to procedure 4). To evaluate this, we calculated two parameters, the ratio of the peak staining for tubulin in the distal axon to the mean staining in the proximal axon and the ratio of the peak staining for tubulin in the distal axon to the median staining for the entire axon (see Tables 1 and 2). For total tubulin, these ratios were 6.3 ± 4.1 and 5.1 ± 2.4, respectively, whereas for assembled tubulin they were 4 ± 2.6 and 3.5 ± 2.2, respectively. Whereas considerable cell-to-cell variation exists in the values obtained for these parameters, there is no indication that the values for the assembled tubulin pool are, overall, greater than (or less than) those for the total tubulin pool. Thus, the tubulin monomer-to-polymer ratio does not appear to vary systematically along the axon in relation to the tau-to-tubulin ratio. This in turn indicates that the proximal-to-distal increase in the tau-to-tubulin ratio (see Fig. 6 and above) is not reflected in the extent of tubulin assembly within these axons.
DISCUSSION
Tau has a nonuniform distribution in axons of cultured sympathetic neurons. Its levels are relatively low and constant in the proximal half of the axon. However, within the distal half, the amount of total tau and assembled tau undergoes a progressive proximal-to-distal increase, reaching a peak near the growth cone that is ~10-fold greater than that observed proximally. The volume of the axon also increases proximodistally. However, the proximodistal increase in total tau exceeds by severalfold that of axon volume, indicating that the concentration of tau increases progressively along the axon. The amounts of total tubulin and assembled tubulin also exhibit a proximodistal increase. However, the gradients of total tau and assembled tau are steeper than those of tubulin. Thus, the ratio of total tau to total tubulin and the tau content of MTs increases progressively between the proximal axon and the axon tip (Figs. 6, 11).We have taken advantage of the naturally occurring gradient in the tau content of MTs and in the ratio of total tau to total tubulin to evaluate whether tau promotes MT assembly and stability in growing axons. Functional studies of tau in the test tube and in non-neuronal cells transfected with tau have revealed several activities of tau, most notably the ability to promote the assembly of tubulin into MTs and to enhance the stability of MTs (for review, see Hirokawa, 1994
Other observations also question whether tau promotes MT assembly and stability in growing axons. First, if tau stabilizes axonal MTs, then it should have a reasonably stable interaction with these MTs (Trinczek et al., 1995
Given that tau promotes MT assembly and stability in vitro (for review, see Hirokawa, 1994
The issues concerning whether tau promotes MT assembly and stability in growing axons also apply to other MAPs. Tau is only one of several MAPs in growing axons (Black and Smith, 1988
What are the functional specializations of tau and MAP1b in growing axons? The answer still remains a mystery. Given that both MAP1b and tau are MT binding proteins, it seems reasonable that their functions in axon growth are MT dependent and involve at least in part binding to MTs. In addition, both tau and MAP1b have sidearm domains that project away from the surface of the MT. Through their sidearm domains, tau and MAP1b influence the packing density of MTs (Bloom et al., 1985
FOOTNOTES
Received Feb. 8, 1996; revised March 5, 1996; accepted March 6, 1996.
This work was supported by National Institutes of Health Grants NS17681 (M.M.B.) and NS24275 and NS24707 (I.F.). We thank Jonathan Fischer for his skilled technical assistance with computer analyses of gel images.
Correspondence should be addressed to Dr. Mark M. Black, Department of Anatomy and Cell Biology, Temple University School of Medicine, 3400 North Broad Street, Philadelphia, PA 19140.
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