Cytoskeletal regulation of normal rat glioblasts differentiated by glia maturation factor

Neurochem. Int. Vol. 16, No. 2, pp. 133-140, 1990 Printed in Great Britain. All rights reserved

0197-0186/90 $3.00 + 0.00 Copyright © 1990 Pergamon Press plc

CYTOSKELETAL R E G U L A T I O N OF N O R M A L RAT GLIOBLASTS D I F F E R E N T I A T E D BY GLIA M A T U R A T I O N FACTOR JIN-ICHI ITOl'*, TAIJI KATO2 and RYO TANAKAl tDepartment of Biochemistry and 2Department for Bioregulation Research, Nagoya City University Medical School, Mizuho-ku, Nagoya 467, Japan (Received 1 May 1989; accepted 5 September 1989)

Abstract--The morphological differentiation of normal rat glioblasts induced by glia maturation factor (GMF) was investigated with regard to the reorganization of the cytoskeletal structures by the use of indirect immunofluorescence staining methods. The microfilaments were reorganized from a stress fiber form to a plasmalemmal undercoat structure by GMF, and to a network-like structure by dBcAMP. The GMF- or dBcAMP-induced morphological differentiation was enhanced by cytochalasin D (over 2 # M) and conversely inhibited by colchicine (over l gM). Phalloidin, an F-actin stabilizer, inhibited more strongly the morphological differentiation induced by dBcAMP than that induced by GMF. Both gila filaments and microtubules existing as a network in control glioblasts assembled after GMF or dBcAMP stimulation to form thick filamentous structures, not only in the cell bodies but also in the processes. The bundle formation of glia filaments was inhibited by colchicine. These findings suggest that glial differentiation depends on both stress fiber disintegration and microtubule assembly, which are closely related to the bundle formation of glia filaments. The GMF- or dBcAMP-induced differentiation was promoted by 25/~M W-7 and inhibited by 1 mM CaCi 2 plus 4/~M A23187. At a higher concentration (50#M) W-7 promoted the differentiation even in the presence of CaC12 and A23187 at the same concentrations. These findings suggest that the suppression of the calmodulin function is important for morphological differentiation of glioblasts.

Glia maturation factor ( G M F ) , which is produced by normal glias, is a growth factor acting on glial cells themselves (Lim et al., 1987; Turriff and Lim, 1981). T u m o r o u s glial cells, whose proliferations are promoted by G M F , also produce G M F - l i k e growth factor(s) (Ito et al., 1986; Kato et al., 1984). G M F promotes both D N A synthesis and cell division of normal rat glioblasts earlier than 24 h after stimulation with G M F and induces continuous morphological differentiation later than 2 4 h after stimulation. G M F induces not only morphological differentiation but also biochemical differentiation, including increases in glial marker proteins such as glial fibrillary acidic protein ( G F A P ) and S-100 protein, d B c A M P and sialosyl cholesterol also provoke morphological differentiation, and the differentiation begins as early as 2 h after stimulation, unlike the case of G M F . G M F promotes D N A synthesis of glioblasts before the differentiation induction, suggesting that G M F induces the differentiation through more complex intracellular reactions

*To whom all correspondence should be addressed.

than d B c A M P or sialosyl cholesterol does (Ito et al., 1989; Kato et al., 1988). G o l d m a n and Chiu (1984) showed that astrocyte cytoskeletons consisted of mainly vimentin, G F A P and actin and that d B c A M P increased vimentin and G F A P levels and decreased the actin level (Goldman and Chiu, 1984), suggesting that alteration of cytoskeletal component levels and the cytoskeletal reorganization directly affected the morphological change of glioblasts. In this work we studied the reorganization of the cytoskeletal structure and involvement of calmodulin in its reorganization in the normal rat glioblasts stimulated with G M F . The results may facilitate the understanding of the alterations in the intracellular reactions, which take place during transition from the D N A synthesis phase to the morphological differentiation phase. EXPERIMENTAL PROCEDURES

Culture o f rat glioblasts Normal rat glioblasts were cultured as described by Ito et al. (1982) and Kato et al. (1981). Briefly, the cerebri of 17-day Wistar rat fetuses, from which blood and meninges had been removed, were minced with scissors and treated

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with 0.1% trypsin in Ca 2+, Mg2+-free Tyrode's solution. Tyrode's solution consisted of 137mM NaCI, 2.67mM KCI, 1.80 mM CaCI 2, 1.05 mM MgCI z, 0.32 mM NaH2PO4 HE0, 5.56mM glucose, and l l.9mM NaHCO 3. After removal of the trypsin solution by centrifugation (500 g, l0 min), glioblasts were cultured in F-IO medium (Gibco) supplemented with 20% fetal calf serum (FCS, Microbiological Associates) lbr 7 days at 37°C in a humidified atmosphere of 5% CO2/air. Subsequently the cells were collected by treatment with 0.1% trypsin, and secondary monolayer cultures were established in 12 well plates (Costar, 3512) using F- 10 medium containing 10% newborn bovine serum (NBS, Microbiological Associates). These cultures were maintained for 7 days.

GMF purification Partial purification of GMF was carried out as reported previously, except that DEAE-Sephacel was used in place of DEAE-Sephadex (Kato et al., 1981). One unit of GMF was defined as the amount causing morphological change to the extent equivalent to that produced by 1 mg protein in crude bovine brain extracts. A cell possessing at least one process longer than the diameter of the original cell soma was counted as a positive response and the results were expressed as percentage of morphologically changed cells with respect to the total cell population. Fluorescence staining of glial cytoskeletons Fluorescence staining of glial cytoskeletons were carried out according to the methods of Kato et al. (1988). After glioblasts cultured on tissue culture chamber/slide (Miles Scientific) had been washed with phosphate-buffered saline, pH 7.5, (PBS) for fluoresecence staining of microfilaments, they were fixed with 3.7% formaldehyde/PBS for 10 min. Acetone treatment was carried out at - 20°C for 5 rain after washing with PBS twice. 7-Nitrobenz-2-oxa-l,3-diazolephallacidin (NBD-phallacidin, Waco Chemicals) in PBS (33 ng/100/~l) was added and the slide was incubated for 20 min, followed by fluorescence microscopy. The indirect immunofluorescence staining of GFAP was carried out as follows. After monolayer glioblasts on slide glass had been washed three times with Hank's balanced salt solution (HBSS), they were fixed with formyl alcohol (mixtures of 95% ethanol and 37% formaldehyde (9:1) (4°C, 20 min) and then with 100% acetone (4°C, 30 min). Three percent Tween 80 treatment at room temperature for 1 min was followed by incubation with rabbit anti-human GFAP antiserum (Dako Co.) at 37°C for 30 min. After incubation with fluorescence isothiocyanate (FITC)-conjugated goat anti-rabbit IgG antibody (Cappel Laboratories) at 37°C for 30 min, the specimens were subjected to fluorescence microscopy. For fluorescence staining of microtubules, monolayer glioblasts on slide glass were fixed with 3.7% formaldebyde/PBS at 35°C for 20 min, followed by immersion in cold methanol ( - 20°C) and then in cold acetone (-20°C). After washing with PBS, the samples were incubated with rabbit anti-bovine tubulin antiserum for 30rain. Then a 30-min incubation was carried out in the presence of FITC-conjugated goat anti-rabbit IgG antibody. Phalloidin treatment of glioblasts The confluent glioblasts were treated with L-ct-lysophosphatidylcholine in Tyrode's solution (20#g/ml) for 2 min.

After washing with 2% NBS/F-10, the cells were treated with phalloidin in 2% NBS/F-10 (10/lg/ml) for 5 min and then washed three times with 2% NBS/F-10.

'tSCa2+ uptake by glioblasts The confluent secondary culture glioblasts were stimulated with 1 unit of GMF at 24 h after exchange of medium with F-10 containing 2% NBS (2% NBS/F-10). After incubation with GMF for 24 h the glioblasts were washed three times with Tyrode's solution. The cells were incubated with 2/~ Ci/ml of 45Ca2+ (New England Nuclear) in Tyrode's solution for various periods of time, followed by washing three times with Tyrode's solution. The 45Ca2+ uptake by glioblasts was determined by scintillation counting. Chemicals Cytochalasin D, phalloidin, L-ct-lysophosphatidylcholine, colchicine and N6,2'-O-dibutyryladenosine 3': Y-cyclic monophosphate (dBcAMP) were purchased from Sigma Chemical Co. W-7 [N-(6-aminohexyl)-5-chloro-l-naphthalenesulfonamide hydrochloride] was purchased from Rikaken Co. Rabbit anti-bovine tubulin antiserum was kindly provided by Dr T. Sato, Department of Radiology, Aichi Cancer Center Research Institute. RESULTS

Effects o f cytoskeletal agents on G M F or d B c A M P induced morphological differentiation Rat glioblasts differentiated to astrocyte-like cells by 34-h stimulation of 1 unit of G M F as shown in Fig. 1. At 32 h after G M F stimulation, the addition of 50/~ M colchicine, a microtubule depolymerizer, to the culture system completely inhibited the G M F induced morphological differentiation of glioblasts. Conversely, cytochalasin D (1/~M), a microfilament depolymerizer, stimulated differentiation. The morphological differentiation induced by dBc A M P was similar to that induced by G M F (Fig. 2). However, whereas G M F induced differentiation later than 24 h, d B c A M P did that as early as 2 h after exposure. Moreover, a larger number of processes was extended after treatment with d B c A M P than with G M F , thus the morphology of glioblasts exposed with d B c A M P became more like that of astrocytes. Colchicine inhibited the dBcAMP-induced differentiation and cytochalasin D stimulated it. Phalloidin, an F-actin stabilizer, inhibited the dBcAMP-induced differentiation, but not the G M F induced differentiation (Figs 1 and 2). Although cytochalasin D stimulated dose-dependently the G M F - i n d u c e d differentiation, and the drug at concentrations < 1/~M inhibited the dBcAMP-induced differentiation as shown in Table 1. Colchicine at concentrations of 10-SM only inhibited differentiation of the glioblasts stimulated with dBcAMP.

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Fig. 1. Effects of cytoskeletal agents on GMF-induced morphological differentiation of glioblasts. After the culture media had been changed to fresh 2% NBS/F-10, rat glioblasts were cultured for 24 h. All of the glioblasts in 2% NBS/F-10 were stimulated with 1 unit of GMF, followed by incubation for 34 h (a). During the last 2 h of GMF-stimulation either 5/t M colchicine (b) or 2/~ M cytochalasin D (c) was added to the cultured glioblasts. The glioblasts which had been stimulated with G M F for 32 h were treated with 10 ~g/ml of phalloidin for 5 min as described in Experimental Procedures, followed by incubation for 2 h in the presence of G M F (d).

Reorganization of cytoskeleton to relate with morphological differentiation It was observed t h a t m a n y stress fibers existed in the same direction in the control glioblasts (Fig. 3). G M F stimulation for 42 h led to a reorganization of microfilaments to form a p l a s m a l e m m a l u n d e r c o a t a r r a n g e m e n t from a stress fiber a r r a n g e m e n t , dBc A M P stimulated the d i s a p p e a r a n c e of stress fibers as was seen with G M F , a n d the microfilaments were a r r a n g e d to form a n e t w o r k structure. Thus, the d i s a p p e a r a n c e o f stress fibers was closely related to the morphological differentiation o f the glia. Cytochalasin D p r o m o t e d the stress fiber disappearance, a n d also inhibited b o t h G M F a n d d B c A M P actions

on the reorganization of microfilament structures as m e n t i o n e d above. Colchicine inhibited G M F - or d B c A M P - i n d u c e d morphological differentiation of glioblasts, a n d inhibited b o t h microfilament reorganization to p l a s m a l e m m a l u n d e r c o a t induced by G M F a n d t h a t to network structure induced by d B c A M P . Indirect immunofluorescence staining using antiG F A P a n t i s e r u m showed the n e t w o r k structure of glia filaments in glioblast cell bodies (Fig. 4). After differentiation by G M F or d B c A M P , glia filaments assembled to form thick bundles which were also distributed in the processes. Cytochalasin D enh a n c e d b o t h G M F a n d d B c A M P actions to form glia filament bundles, whereas colchicine inhibited these actions.

Fig. 2. Effects of cytoskeletal agents on dBcAMP-induced morphological differentiation. The glioblasts cultured in 2% NBS/F-10 for 24 h were stimulated with 2 mM dBcAMP for 2 h without cytoskeletal agents (a), with 2 ~M cytochalasin D (b), or with 50/~M colchicine (c). After pretreatment with 10 pg/ml of phalloidin for 5 min, glioblasts in 2% NBS/F-10 were stimulated with 2 mM dBcAMP (d).

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Table 1. Effects of cytochalasin D and colchicine on morphological differentiation of glioblasts stimulated with GMF or dBcAMP Colchicine (M) 0 (%) 10 9 10 ~ 10 7

GMF (1 unit)

dBcAMP (2raM)

Cytochalasin D (/~M)

GMF (1 unit)

dBcAMP (2raM)

90 90 90 60

40 40 5 5

0 (%) 0.4 1.0 2.0

35 70 90 100

35 10 20 60

After the culture media were replaced with fresh 2% NBS/F-10, the confluent glioblasts were stimulated with 1 unit of GMF. Then either cytochalasin D or colchicine at 40 h after GMF stimulation was added to the cultured glioblasts, and glial morphological changes were observed after 2-h incubation. Percentage of cell number of differentiated glioblasts to total cell number is shown. The glioblasts which had been cultured in 2% NBS/F-10 for 24 h were stimulated with 2 mM dBcAMP simultaneously with either cytochalasin D or colchicine. After 2 h, morphological differentiation of glioblasts were observed as case of GMF stimulation. All the data are the means of duplicate experiments.

[]

[] Fig. 3. R e o r g a n i z a t i o n o f m i c r o f i l a m e n t s in glioblasts s t i m u l a t e d with G M F or d B c A M P . The glioblasts c u l t u r e d in 2 % N B S / F - 1 0 for 24 h (control, a) were s t i m u l a t e d with G M F for 40 h, followed by i n c u b a t i o n for 2 h w i t h o u t c y t o s k e l e t a l agents (b), or with 2 # M c y t o c h a l a s i n D (d), or with 5 0 / a M colchicine (e). The cells in 2 % N B S / F - 1 0 were s t i m u l a t e d with 2 m M d B c A M P w i t h (f) or w i t h o u t (c) 50 # M colchicine for 2 h. N B D - p h a l l a c i d i n s t a i n i n g was carried out as described in E x p e r i m e n t a l Procedures.

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3

Fig. 4. Reorganization of glia filaments in glioblasts stimulated with GMF or dBcAMP. Glioblasts cultured in 2% NBS/F-10 for 24h were treated as described in Fig. 3 and subsequently indirect immunofluorescencestaining was carried out using anti-human GFAP antiserum. (a) Control glioblasts, (b) 1 unit of GMF, (c) 2mM dBcAMP, (d) GMF plus 2/IM cytochalasin D, (e) GMF plus 50#M colchicine, (f) dBcAMP plus colchicine. The microtubule structure of glioblasts stimulated with G M F was investigated by indirect immunofluorescence staining by the use of anti-bovine tubulin antibody. Microtubules formed networks in control glioblasts (Fig. 5). Either G M F or dBcAMP stimulation made the network highly dense, and especially microtubules stretched strongly to form bundles in the processes.

Effect of Ca-calmodulin on GMF-induced morphological differentiation The glioblasts to which W-7, a calmodulin antagonist, had been added at 24 h after G M F stimulation were cultured for a further 24 h in the presence of GMF. Approximately 70% glioblasts of the total

cell population differentiated morphologically after G M F stimulation without W-7. W-7 promoted dosedependently the differentiation induced by GMF, and most of the cells differentiated at 50 #M W-7 (Table 2). The number of glioblasts differentiated by G M F diminished to 5% when 4 # M A23187 and 1 mM CaC12 were added in the culture medium. The simultaneous addition of W-7 (50/~M) with A23187 plus Ca 2+, reversed the inhibition of morphological differentiation. DISCUSSION

This paper investigated the GMF-induced morphological differentiation of normal rat glioblasts

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Fig. 5. Reorganization of microtubules in glioblasts stimulated with GMF or dBcAMP. Glioblasts in 2% NBS/F-10 (a) were stimulated with 1 unit of GMF for 42 h (b), or 2 mM dBcAMP for 2 h (c). Indirect immunofluorescence staining was carried out using rabbit anti-bovine tubulin antiserum.

with regard to the alteration of the cytoskeletal structure using mainly the indirect immunofluorescence staining method, and compared it with the dBcAMP-induced differentiation. G M F from bovine brain is a growth factor of Mr 19,500 and p14.75. The factor possesses two distinct functions: it stimulates both DNA synthesis and proliferation of glioblasts and subsequently induces their morphological differentiation (Kato et al., 1981). The induced differentiation is a process independent-from DNA synthesis, since G M F evokes differentiation even in the presence of glial growth inhibitory factor (GGIF), which Table 2. Effect o f W-7 with or without Ca :+ and A23187 on morphological differentiation o f glioblasts stimulated with G M F or dBcAMP Concentration o f W-7 (,uM) Stimulant

0

10

25

50

GMF

70 5 40 15

80 10 60 30

90 20 70 60

100 65 70 55

+ Ca z+ A23187 ( % ) dBcAMP + C a :+ A23187 ( % )

The glioblasts were stimulated with 1 unit of G M F at 2 4 h after replacement o f culture media with 2 % NBS/F-10, followed by incubation for 40 h. The glioblasts were treated with various a m o u n t s o f W-7 with or without 1 m M CaCI 2 and 4/1M A23187 and were incubated further for 2 h . T h e glioblasts in 2 % NBS/F-10 were stimulated with 2 m M o f d B c A M P and various a m o u n t s o f W-7 with or without 1 m M CaCI 2 and 4 / t M A23187 for 2 h. Percentage o f cell n u m b e r o f differentiated glioblasts to total cell n u m b e r were shown. All the data are the means o f duplicate experiments.

suppresses GMF-stimulated DNA synthesis (Ito et al., 1987; Kato et al., 1987). There was a difference between G M F and dBcAMP with regard to the ability to induce morphological changes. The glioblasts differentiated by dBcAMP possessed a larger number of processes than those differentiated by G M F to attain a morphology more closely resembling that of astrocytes. The induction of morphological differentiation by both G M F and dBcAMP was inhibited by 10 6M colchicine and stimulated by cytochalasin D at concentrations over 2 It M, suggesting that the differentiation depends on the microtubule assembly and the stress fiber disappearance. Since the GMF-induced differentiation was inhibited by phalloidin, an F-actin stabilizer (Barak et al., 1980), to a smaller extent than the dBcAMP-induced differentiation, GMF-induced differentiation may implicate the disappearance of a small amount of stress fibers. Fluorescence staining of F-actin revealed that microfilaments were rearranged to form plasmalemmal undercoats after stimulation with GMF, and to make network structures after stimulation with dBcAMP. The difference between morphological changes induced by G M F and dBcAMP may be caused, at least partly, by a different alteration of actin filament structure. The morphological differentiation induced by either G M F or dBcAMP converted gila filaments from fine networks to thick bundles. Similarly to glia

Cytoskeletal regulation of rat glial differentiation filaments, microtubules also formed thick bundles in the cell bodies as well as the processes after stimulation with G M F or dBcAMP. The actions of G M F and dBcAMP with regard to reorganization of both glia filaments and microtubules were essentially the same. This finding supports the contention that the fundamental difference between the morphological differentiation induced by G M F and that by dBcAMP is in the reorganization of microfilaments. Since colchicine, a specific microtubule depolymerizer, affected not only microtubule arrangement but also glia filaments and microfilaments in the G M F or dBcAMP-stimulated cells, the reorganization of microfilaments and glia filaments may be regulated by microtubules. Goldman and Chiu (1984) have reported that the level of cytoskeleton-associated actin decreased approx. 35% and conversely G F A P increased 2-3 times in cultured astrocytes which had been stimulated with dBcAMP for a week. The cytoskeleton-associated actin and GFAP levels, however, changed little in glioblasts after such a short time of stimulation with G M F as 48 h (data not shown). Therefore, morphological differentiation appears to involve the bundling~iebundling cycle of cytoskeletal filaments rather than the polymerization~lepolymerization cycle. What kind of second messenger, then, regulates reorganization of the cytoskeleton after G M F stimulation? It was suggested that a suppression of calmodulin function participated in morphological differentiation after G M F stimulation, inasmuch as the differentiation induced by either G M F or dBcAMP was inhibited by Ca 2+ plus A23187, and the inhibition was reversed by the addition of W-7 (a calmodulin antagonist) at 50/~M, where the inhibition is known to suppress calmodulin specifically (Morimoto et al., 1982). The finding that the 45Ca2+ uptake by GMF-stimulated glioblasts decreased by 25-35% (data not shown) suggested importance of the intracellular Ca 2+ for the cytoskeleton reorganization through calmodulin. The effect of time of W-7 addition on the morphological differentiation is of interest with regard to understanding of the relationship between differentiation and proliferation. The ability of G M F to induce differentiation was enhanced by the addition of W-7 12 h or longer after G M F stimulation, but was suppressed by the addition before 12 h (data not shown). Glioblasts were induced to enter the DNA synthesis phase (S phase) at 10-12h after G M F stimulation, suggesting that glial morphological differentiation needs suppression of calmodulin func-

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tion after the S phase. It has previously been reported that Ca-calmodulin plays an important role in the process of DNA synthesis (Okumura et al., 1982; Okumura-Noji et al., 1983). Therefore, G M F may be instrumental in the promotion of DNA synthesis through calmodulin-mediated reactions, and may stimulate Ca-ATPase to cause a decrease in the intracellular Ca 2+ level and in turn a suppression of calmodulin function. There have been many reports that Ca2+-calmodulin regulates the organization of the cytoskeletal structure through flil~flop interaction with actin-associated proteins and/or with microtubule-associated proteins (Kumagai et al.. 1982; Sobue et al., 1981, 1983), and also through phosphorylation of cytoskeletal components by Ca 2+, calmodulin-dependent protein kinases (Jameson et al., 1980; Sobue et al., 1982; Yamamoto et al., 1983; Yamaguchi and Fujisawa, 1982; Yen and Fields, 1981). These facts suggested that the suppression of the Ca2+-calmodulin function after G M F stimulation promotes polymerization and/or bundle formation of microtubules. The bundle formation may in turn stimulate that of glia filaments, and hence morphological differentiation may be accelerated. The intracellular Ca 2+ mobilization prior to morphological differentiation and the detailed correlation of Cacalmodulin with reorganization of microfilaments, or microtubules are under investigation.

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