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Previous Article | Next Article
Volume 17, Number 13, Issue of July 1, 1997 pp. 4921-4932
Copyright ©1997 Society for NeuroscienceMicrotubule Organization and Stability in the Oligodendrocyte
Katharine F. Lunn1, 2, Peter W. Baas3, and Ian D. Duncan1 1 Department of Medical Sciences, 2 Neuroscience Training Program, and 3 Department of Anatomy, University of Wisconsin-Madison, Madison, Wisconsin 53706
ABSTRACT
The oligodendrocyte is the glial cell responsible for the formation and maintenance of CNS myelin. Because the development of neuronal morphology is known to depend on the presence of highly organized microtubule arrays, it may be hypothesized that the properties of microtubules influence the form and function of oligodendrocytes. The goals of the present study were to define the physical attributes of microtubules in oligodendrocytes maintained in vitro. The results of electron and confocal microscopy indicate that microtubules are present throughout the cell bodies and large and small processes of oligodendrocytes and are rarely associated with discrete microtubule-organizing centers. A modified "hooking" protocol demonstrated that the polarity orientation of microtubules is uniformly plus-end distal in small oligodendrocyte processes, compared with a nonuniform, predominantly plus-end distal orientation in large processes. Oligodendrocytes were exposed to the microtubule-depolymerizing drug nocodazole to examine microtubule stability in these cells. The results suggest that oligodendrocyte microtubules can be resolved into at least three distinct microtubule populations that differ in their kinetics of depolymerization in the presence of nocodazole. These findings suggest that the properties of the oligodendrocyte microtubule array reflect the functions of the different regions of this highly specialized cell.
Key words: oligodendrocyte; myelination; microtubule; cytoskeleton; microtubule polarity; microtubule stability; nocodazole
INTRODUCTION
The neurons and glial cells of the nervous system have complex morphologies that are intimately associated with their functions. The generation and maintenance of these morphologies require the establishment of highly organized arrays of microtubules within the cytoplasm of these cells. Microtubules are structural polymers that support the architecture of the cell and also provide a substrate for the active transport of cytoplasmic constituents. The role of microtubules in the differentiation of neurons has been examined in detail. Neurons extend two distinct types of process, axons and dendrites, that differ in their morphology and cytoplasmic composition. Many of these differences arise from the fact that microtubules in the axon are uniformly oriented with their plus-ends distal to the cell body, whereas microtubules in the dendrite have a nonuniform polarity orientation (Baas et al., 1988
, 1989
Although analyses of microtubules have provided significant insight into the cell biology of the neuron, relatively little is known about microtubule arrays within other cell types of the nervous system. One cell of particular importance is the oligodendrocyte, a glial cell that is specialized for the formation of myelin in the CNS. Oligodendrocytes are derived from mitotic progenitor cells that are initially monopolar and then become bipolar and migratory (Small et al., 1987
The present studies explore fundamental features of the oligodendrocyte microtubule array, using primary glial cultures as a model. The latter demonstrate many in vivo characteristics of oligodendrocytes, including the elaboration and maintenance of processes and membrane sheets, and the expression and compartmentalization of myelin-specific antigens (Knapp et al., 1987
MATERIALS AND METHODS
Cell culture Sprague Dawley rat pups were euthanized by an overdose of barbiturate at 10 d of age. The spinal cords were removed under sterile conditions and placed in Leibovitz-15 medium (L-15) (Life Technologies, Grand Island, NY) at 4°C, and the meninges and nerve roots were removed. The cords were transferred to Ca2+- and Mg2+-free Earle's balanced salt solution (EBSS) (Life Technologies) with 0.25% trypsin (Worthington, Freehold, NJ) and 0.05% DNase (Sigma, St. Louis, MO). The tissue was minced into 1 mm3 pieces and incubated in the enzyme solution in 5% CO2 at 37°C, on a rotating shaker at 70 rpm, for 60-90 min. The trypsin was inactivated by the addition of heat-inactivated fetal bovine serum (HIFBS) (Life Technologies) to a final concentration of 20%, and the tissue was recovered by centrifugation and resuspended in L-15 with 10% HIFBS. The final dissociation step involved trituration of the tissue at 4°C with flame-polished Pasteur pipettes of decreasing tip orifice size. The resulting cell suspension was diluted to 6.5 ml with L-15/10% HIFBS, mixed with 3 ml of 80% Percoll (Sigma) in 0.25 M sucrose buffered with 0.05 M sodium phosphate at pH 7.4, and centrifuged at 30,000 × g for 45 min. The oligodendrocyte-containing band was harvested from the bottom of the gradient, immediately above the red blood cell layer. The cells were suspended in L-15/10% HIFBS and recovered by centrifugation. The pellet was resuspended in culture medium, and a cell count was obtained from a 15 µl aliquot with use of a hemocytometer.
Cells were plated onto poly-L-lysine (Sigma)-treated 12 mm glass coverslips at a density of 30,000 cells/coverslip for nocodazole treatment and indirect immunofluorescent antibody staining. Cultures for electron microscopy (EM) and the hooking protocol were plated onto poly-L-lysine-treated 35 mm plastic tissue culture dishes at a density of 50,000-60,000 cells/dish. The cells were maintained in medium consisting of DMEM with 1:1 F-12 nutrient mixture (DMEM/F-12) (Life Technologies) supplemented with 100 µM putrescine, 20 nM progesterone, 30 nM sodium selenite, 5 µg/ml insulin, 10 µg/ml rat transferrin (Jackson ImmunoResearch, West Grove, PA), 40 ng/ml L-thyroxine, 30 ng/ml 3,3 ,5-triiodo-L-thyronine, and 0.1 mg/ml bovine serum albumin (all components from Sigma, unless noted otherwise). The medium was supplemented further with 1% HIFBS and 0.1% gentamicin (Life Technologies).
Indirect immunofluorescence Glial cell cultures grown on glass coverslips were rinsed briefly with PHEM buffer (60 mM PIPES, 25 mM HEPES, 10 mM EGTA, 2 mM MgCl2, pH 6.9) followed by a 2 min extraction in 0.1% Triton X-100 in PHEM supplemented with 10 µM taxol (a gift from the National Cancer Institute). The cultures were fixed by the addition of an equal volume of PHEM containing 4% paraformaldehyde and 0.2% glutaraldehyde. After fixation for 15 min at room temperature, the cultures were rinsed with PBS, and autofluorescence was quenched with three 5 min incubations in 10 mg/ml NaBH4 in PBS. After they were rinsed in PBS, the cultures were postextracted in a graded series of ethanols and then incubated for 1 hr in a blocking solution containing 5% normal goat serum (Life Technologies) in PBS. The cultures were incubated in primary antibody for 15 hr at 4°C, rinsed three times in PBS, incubated in blocking solution for 1 hr, and then incubated in secondary antibody for 1 hr at 37°C. Finally, the coverslips were rinsed in PBS and mounted on slides in a glycerol/PBS mixture (Citifluor, UKC, Canterbury, UK) containing 1 mg/ml p-phenylenediamine, and the edges were sealed with nail polish. The primary antibodies were a monoclonal anti-
The cultures were maintained in a 5% CO2 environment at 37°C. The medium was changed 24 and 48 hr after plating, and the cells were used for additional studies after 4 d in vitro. -tubulin antibody (mouse IgG1 isotype; Sigma), used at 1:200, that recognizes all forms of tubulin, a rabbit polyclonal (WWTYR; provided by Dr. J. C. Bulinski, Columbia University) that recognizes tyrosinated
-tubulin, used at 1:500, and 6-11B-1, a monoclonal antibody that recognizes the acetylated form of
-specific) and a TRITC-conjugated goat anti-rabbit IgG (Jackson ImmunoResearch), both used at 1:150. All antibodies were diluted in PBS. To examine the distribution of microtubules (labeled by the anti-
) end is directed toward the observer. In the present study, cultures were rinsed once with Ca2+- and Mg2+-free EBSS and then treated at 37°C with 1.25% saponin in a microtubule assembly buffer (0.5 M PIPES, 1 mM EGTA, 0.1 mM EDTA, 1 mM MgCl2, 2.5% DMSO, 1 mM GTP) containing 1.2 mg/ml bovine brain microtubule protein. The cultures were exposed to the buffer for 5 min, before fixation by the addition of an equal volume of 4% glutaraldehyde in 0.1 M cacodylate buffer, followed by processing into epoxy resin as described above. Before they were sectioned, the embedded cells were visualized by staining with 1% alkaline toluidine blue, and oligodendrocyte processes were identified on phase-contrast microscopy and marked by scoring the resin. Ultrathin sections were taken perpendicular to the long axes of the processes, mounted on formvar-coated single-slot grids, and stained with uranyl acetate and lead citrate; we took care to ensure that the sections and grids were not inverted during handling. Photographs were taken of the processes, and the direction of the hooks was assessed from the vantage point of the distal end of the oligodendrocyte process, looking toward the cell body. For each oligodendrocyte process examined, hooked microtubules were scored as having clockwise hooks, counterclockwise hooks, ambiguous hooks, or no hooks. The ambiguous category includes microtubules with hooks of both direction and those with hooks that were too short to interpret. For each process, the percentage of interpretable hooks that were clockwise or counterclockwise was determined, and mean + SD was calculated. Effects of nocodazole on oligodendrocyte microtubules Nocodazole treatment. To assess the stability of oligodendrocyte microtubules, glial cultures grown on coverslips were exposed to the microtubule depolymerizing drug nocodazole (Aldrich Chemical, Milwaukee, WI) for 15, 30, 60, 120, and 360 min. The nocodazole was prepared as a 10 mg/ml stock in DMSO that was diluted to a final concentration of 10 µg/ml in warmed culture medium, before being added to the cells. After exposure to nocodazole, the cells were fixed, permeabilized, and prepared for indirect immunofluorescence as described above. Additional control coverslips that had not been exposed to nocodazole were prepared in the same way. Quantitation of the indirect immunofluorescent labeling of the cells with the mouse monoclonal anti-
Analysis of the kinetics of nocodazole-induced microtubule depolymerization. A modeling approach was used to investigate the kinetics of nocodazole-induced microtubule depolymerization in oligodendrocytes. On the basis of the results of previous investigations in neurons (Baas et al., 1991 Bt) + (Ce
In this model the expression Ae
For the rapidly depolymerizing and slowly depolymerizing forms of polymer, the values of the parameters A and C, respectively, give the amount of each type of microtubule (expressed in AFUs) present at time 0. These values were used to calculate the relative amounts of each type of microtubule in the different regions of the oligodendrocytes. The values of B and D were used to estimate half times for depolymerization of each subpopulation of microtubules.
RESULTS
Indirect immunofluorescence
When the glial cultures were examined by phase microscopy, oligodendrocytes were easily recognized on the basis of their morphology. These cells have a small soma of ~10 µm in diameter. A number of large processes emerge from the cell body and branch numerous times, giving rise to progressively smaller processes that finally terminate in a fringe of fine processes around the periphery of the cell. Figure 1 illustrates the distribution of microtubules, labeled with the anti-
Fig. 1. Distribution of microtubules in the cell body and processes of a cultured oligodendrocyte. Optical sections of ~0.6 µm axial resolution through an oligodendrocyte labeled with an anti-
[View Larger Version of this Image (127K GIF file)]
Oligodendrocytes labeled with the anti-tyrosinated Electron microscopy
EM examination of cultured oligodendrocytes confirmed the results of the indirect immunofluorescent labeling. Thus microtubules were found throughout the cell bodies and processes of these cells. In the cell bodies the microtubules were scattered through the cytoplasm and were seen in both transverse and longitudinal section (Fig. 2A). Centrioles occasionally were found in the oligodendrocyte cell body, located close to the nucleus, and in proximity to the Golgi apparatus (Fig. 2). The centrioles that were identified were sometimes associated with a small number of microtubules radiating from the pericentriolar region (Fig. 2A), whereas others showed no association with microtubules (Fig. 2B). In the largest oligodendrocyte processes (Fig. 3), which emerged directly from the cell body, microtubules were prominent, with variable spacing between them, and little evidence of tight bundling (Fig. 4A). These processes also contained ribosomes, both single and clustered (Figs. 3, 4A), and mitochondria. Granular structures, ~0.5 µm in diameter, were also noted in oligodendrocyte cell bodies and processes (Figs. 2A, 4A). These had the appearance of dense accumulations of ribosomes. As the processes divided, microtubules continued into the daughter branches and were consistently present as further process subdivision occurred (Fig. 3). The finest processes at the periphery of the oligodendrocyte were frequently seen to contain one or two microtubules and the fine granular material typical of the oligodendrocyte cytoplasm, bounded by plasma membrane (Figs. 4B,C).
Fig. 2. Electron micrographs of the perinuclear region of cultured oligodendrocytes. Both cells contain scattered microtubules, and a centriole can be seen in each (arrowheads). In A, a number of microtubules appear to radiate from the pericentriolar region, whereas no microtubules appear to be associated with the centriole in B. Granules are present in the cytoplasm of the oligodendrocyte in A (arrows). These appear to consist of accumulations of ribosomes. Scale bars, 0.5 µm.
[View Larger Version of this Image (196K GIF file)]
Fig. 3. Electron micrograph demonstrating the branching of oligodendrocyte processes and the presence of microtubules in the branches. In the widest process the microtubules are generally parallel to the long axis of the process, although they also appear to curve. Ribosomes are prominent in the wide processes but infrequent in the narrow process (arrow). Scale bar, 0.5 µm.
[View Larger Version of this Image (155K GIF file)]
Fig. 4. Microtubules are present in oligodendrocyte processes of different sizes. These electron micrographs demonstrate that microtubules are present in both large (A) and small (B, C) oligodendrocyte processes. In A, the spacing between the microtubules is irregular. Ribosomes are also present and appear to be clumped together (arrow) as described in Figure 2A. In B, microtubules appear to curve around the branch point of a small process (arrow). The process in C was found at the periphery of an oligodendrocyte, and although only ~0.15 µm in width, it contains two microtubules in this view. Scale bars: A, B, 0.5 µm; C, 0.1 µm.
[View Larger Version of this Image (135K GIF file)]
Microtubule polarity analysis
Table 1 summarizes the results of the analysis of the polarity orientation of microtubules in oligodendrocyte processes, using a modified hooking procedure. These results clearly show that the orientation of microtubules is uniform in small oligodendrocyte processes and nonuniform in larger processes. The percentage of hooked microtubules was found to be high (Table 1, Fig. 5). A total of 26 oligodendrocyte processes were examined, and of these 13 were defined as "large" (4-10 µm in diameter) and 13 were designated as "small" processes (<1.5 µm). All of the large processes and three of the small processes were of known orientation. Thus in each of these it was known that the hooks on the microtubules were being viewed from the vantage point of the end of the process, looking toward the cell body. The remaining 10 small processes were sectioned incidentally while the identified processes were examined; thus they were of unknown orientation. For these processes, the direction of the hooks was designated as being the "majority" or "minority" direction. The results in Table 1 show that in the large oligodendrocyte processes, 80.5% (±16.4%) of microtubules are oriented with their plus ends distal to the cell body. The percentage of clockwise hooks in the large processes ranged from 50 to 100%. Four processes within this group had >90% clockwise hooks, and if these were not included in the final calculation, the percentage of microtubules with plus ends distal to the cell body fell to 72.4 (±12.8%) in the large oligodendrocyte processes. Figure 5 illustrates the typical appearance of hooked microtubules in small oligodendrocyte processes. In the small processes of known orientation, the microtubules were uniformly oriented, with plus ends distal to the cell body in 95.8% (±7.2%). For the 10 small oligodendrocyte processes of unknown orientation, 97.6% (±5.2%) of the microtubules had hooks in the majority direction, indicating that microtubule polarity was also uniform in these processes. Given that the small processes of known orientation had uniformly plus-end distal microtubules, this was also assumed to be true for the processes of unknown orientation. Thus the percentage of microtubules with plus ends distal to the cell body was calculated to be 97.2% (±5.5%) in the small processes.
Table 1. Direction of microtubule hooks in oligodendrocyte processes
Process size Number % CW (±SD) % CCW (±SD) % Hooked Large 13 80.5 (±16.4) 19.5 (±16.4) 86.3 (±8.4) Small 3 95.8 (±7.2) 4.2 (±7.2) 93.3 (±11.5) Small 13a 97.2 (±5.5) 2.8 (±5.5) 91.0 (±9.0) The values for clockwise (CW) and counterclockwise (CCW) hooks are means of the values from each process, expressed as a percentage of the total number of interpretable hooks. The hooking percentage is expressed as a mean of the percentage of the total number of microtubules that were hooked in each process. The 13 large and 3 small processes were viewed from the vantage point of the end of the process, looking toward the cell body. a The 13 small processes include the 3 processes of known orientation and the 10 processes of unknown orientation. The latter are described separately in Results. The two data sets were combined after assuming that all the small processes contain microtubules with a uniformly plus-end distal orientation. 
Fig. 5. Electron micrograph of "hooked" microtubules in oligodendrocyte processes. The orientation of these two small processes is unknown, but almost all the interpretable hooks are counterclockwise in this view. Scale bar, 0.2 µm.
[View Larger Version of this Image (112K GIF file)]
Effects of nocodazole on oligodendrocyte microtubules
Appearance of cells exposed to nocodazole When viewed by confocal microscopy and epifluorescence, the control oligodendrocytes showed immunofluorescence of the cell body and large and small processes, reflecting the distribution of microtubules in these cells (Fig. 6A). After 15 min of exposure to nocodazole, however, there was an obvious reduction in fluorescence intensity throughout the whole cell, with a marked decrease in the small processes (Fig. 6B). With further exposure to nocodazole, there was continued loss of fluorescence intensity from the processes, although the cell body showed little change with time (Fig. 6C,D). The loss of intensity from the processes appeared more marked in those of small diameter, and after 6 hr exposure to nocodazole most of the labeled microtubules in the oligodendrocytes appeared to be in the cell body and large processes (Fig. 6D). When the labeled cells were examined with epifluorescence and phase optics, it was found that oligodendrocytes retained their small processes after exposure to nocodazole, confirming that the loss of fluorescence intensity was attributable to loss of microtubules rather than loss of the processes themselves.
Fig. 6. The effect of nocodazole on oligodendrocyte microtubules. Confocal images of a control oligodendrocyte (A) and cells exposed to nocodazole for 15 min (B), 1 hr (C), and 6 hr (D). The cells were indirectly immunofluorescently labeled for
[View Larger Version of this Image (110K GIF file)]
Kinetics of nocodazole-induced microtubule depolymerization Three separate nonlinear regression analyses were performed to describe the kinetics of the rapidly depolymerizing and slowly depolymerizing microtubules in oligodendrocytes. Initially the data were summarized by a single two-compartment model to describe the whole cell; however, the images of the fluorescently labeled cells suggested that the microtubules in the oligodendrocyte cell bodies and processes depolymerized with different kinetics in the presence of nocodazole. Therefore two additional models were proposed to describe these two regions separately. Figure 7 shows the theoretical curves predicted for each model, together with the experimental data from the whole cell, the oligodendrocyte cell body, and the processes. Table 2 summarizes the model used to describe each region and the values of the parameters A, B, C, and D obtained from the nonlinear regression analyses. These results show that of the total tubulin in oligodendrocytes, 73% is present in the processes.
Fig. 7. Theoretical curves predicted from three separate models describing the kinetics of nocodazole-induced microtubule depolymerization in oligodendrocytes. The nonlinear regression analyses suggest the presence of rapidly depolymerizing and slowly depolymerizing microtubules in the whole oligodendrocyte (a), the processes (b), and the cell body (c). The observed data values are plotted on the same axes as the theoretical curves, with n = 12 at each time point. The vertical axis represents the total amount of tubulin expressed in arbitrary fluorescence units (AFU).
[View Larger Version of this Image (18K GIF file)]
Table 2. Values of the parameters A, B, C, and D from the nonlinear regression analysis of nocodazole-induced microtubule depolymerization in the whole oligodendrocyte, the cell body, and the processes
Region Model Parameter estimate (±SE) A B C D Whole cell Ae 2,085,000 (±274,000) 0.1659 (±0.0492) 1,085,000 (±73,500) 0.0011 (±0.0003) Processes Ae 1,510,000 (±211,000) 0.1776 (±0.0600) 777,000 (±55,500) 0.0018 (±0.0004) Cell body Ae 555,000 (±88,000) 0.1398 (±0.0413) 306,000 (±16,500) (see Results) See Results for further details.
For the whole oligodendrocyte (Fig. 7a), the amount of tubulin, measured in AFUs, shows an initial rapid decline in the presence of nocodazole (described by the Ae
The regression analysis of the data for the oligodendrocyte cell bodies (Fig. 7c) showed that the value of the component D was not significantly different from zero. Therefore the model used to describe this region of the cell was of the form y = Ae
DISCUSSION
The primary function of the oligodendrocyte is the myelination of axons in the CNS. Knowledge of the spatial organization, polarity orientation, and stability properties of oligodendrocyte microtubules is central to understanding how this cell achieves and maintains its specialized morphology and how it is able to spatially regulate and coordinate the transport of lipids, proteins, and mRNAs essential for myelin synthesis and maintenance. The present studies were performed on oligodendrocytes grown in the absence of neurons, and future experiments should address the influence of axons on the microtubule arrays of these cells. Axons have been shown to regulate the distribution of microtubules in Schwann cells (Kidd et al., 1996The organization of oligodendrocyte microtubules
The presence of centrioles and microtubules in oligodendrocytes in vivo and in vitro has been described by many authors (Mori and Leblond, 1970Polarity orientation of oligodendrocyte microtubules
Microtubules are intrinsically polar structures, with the plus end of the microtubule favored for assembly over the minus end (Bergen and Borisy, 1980
Microtubule polarity orientation in neurons has been studied in some detail, showing conclusively that axonal microtubules have a uniformly plus-end distal orientation (Burton and Paige, 1981 The stability properties of oligodendrocyte microtubules
The results of the nonlinear regression analysis suggest that oligodendrocyte microtubules can be resolved into at least three distinct subpopulations that differ in their kinetics of depolymerization in the presence of nocodazole. Because the values for B were very close in the separate models for the cell body and processes, the labile microtubules in each region may be part of the same population, accounting for ~65% of the total and depolymerizing with a combined estimated half time of 4.2 min. The processes contain a second subpopulation of microtubules that depolymerize slowly, with an estimated half time of 389 min. Within the oligodendrocyte cell body there is a third subpopulation of microtubules that are extremely stable and do not depolymerize in the presence of nocodazole within the time frame of the present study. Therefore, oligodendrocyte microtubule kinetics in the presence of nocodazole may be described by a three-compartment model, although more data are required to test this hypothesis. In neurons, microtubules can be resolved into two subpopulations: one that is labile and one that is relatively stable in the presence of nocodazole (Baas and Black, 1990The role of microtubules in the biology of the oligodendrocyte
The cytoskeletal elements present in oligodendrocytes comprise microtubules and microfilaments (Wilson and Brophy, 1989
The distribution of labile and stable microtubules in the oligodendrocyte reflects the functions of the different regions of this cell. In cultured oligodendrocytes, the small processes, which rapidly lose fluorescence intensity in the presence of nocodazole, are likely to be analogous to the processes that in vivo contact and spiral around axons to form the myelin sheath. The presence of a dynamic microtubule array is likely to be central to this early phase of myelin formation. The relatively stable microtubules, also detected in oligodendrocyte processes, may help to maintain the highly branched morphology of the cell, and they serve as a substrate for the transport of organelles and macromolecules. The highly stable microtubules in the cell body of the oligodendrocyte may be similar to a stable population of microtubules described in chick sensory neurons in culture (Letourneau and Wire, 1995
It has been proposed that neurons maintain a polarized morphology by organizing their cytoplasmic constituents through interactions between microtubules and microtubule motors (Baas et al., 1988
In the present EM studies, ribosomes were detected in large and medium-sized oligodendrocyte processes. This is in contrast to axons in which it is suggested that ribosomes are excluded by the absence of minus-end distal microtubules (Baas et al., 1988
FOOTNOTES
Received Jan. 27, 1997; revised April 9, 1997; accepted April 11, 1997.
This work was supported by National Institutes of Health Grant NS32361. Excellent technical assistance was provided by the personnel of the laboratories of Drs. Duncan and Baas. We are also grateful to M. K. Clayton for help with the statistical analysis.
Correspondence should be addressed to Dr. Ian D. Duncan, Department of Medical Sciences, School of Veterinary Medicine, University of Wisconsin-Madison, 2015 Linden Drive West, Madison, WI 53706.
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