Expression and extinction of glial properties in glioma X neuroblastoma cell hybrids

Rat glioma cells (clone C₆TK⁻) were hybridized with mouse neuroblastoma cells (clone NA), and 18 primary and secondary hybrid clones containing one chromosome set from each parent were isolated. The hybrids were assayed for the glial marker enzymes 2′,3′-cyclic nucleotide 3′-phosphohydrolase (CNP) and glycerol-3-phosphate dehydrogenase (GPDH). In many of the hybrid clones, the levels of CNP and GPDH were reduced to 5–20% of the activity of C₆TK⁻, as has been observed in other classes of glial X non-glial cell hybrids. In some hybrid clones, however, GPDH and CNP were expressed at high activity. Rat (glial) GPDH activity was not reduced in these clones, but mouse GPDH activity remained low, and was not “de-repressed” or “activated”. This suggests that the controls governing differentiation in neuroblastoma cells and extinction in hybrids may differ in some important details. There was a strong positive correlation between the specific activities of CNP and GPDH in the hybrid clones, suggesting that a mechanism regulates the activity of these two glial enzymes coordinately.     

Expression of A and B types of monoamine oxidase in neuroblastoma hybrid cells

Monoamine oxidase (MAO) activity towards kynuramine as substrate was measured in 6 hybrid cells derived by fusion of neuroblastoma and glioma, liver or brain cells, and was compared with that of parental or non-parental clones. Activities varied from the lowest level of less than 0.15 pmol/min/mg protein in a neuroblastoma clone NB2A to the highest level of 127 pmol/min/mg protein in NCB20 mouse neuroblastoma × Chinese hamster embryo brain hybrid cells. The relative proportions of A and B types of MAO activities were determined in homogenates of each cell line by inhibition curves with clorgyline and deprenyl. Although the A type activity was found in all cell lines measured, MAO A was predominant in 9 clones, except for NCB20 hybrid cells, N4G-B-a neuroblastoma × glioma hybrid cells, and G8-1 myoblast. The ratio of type A/type B activity in NCB20, N4G-B-a and G8-1 cells was 20/80, 75/25 and 95/5, respectively. The results suggest that NCB20 cells are highly enriched in MAO type B, and that the NCB20 cell is an excellent model for studying the type B activity found in the brain in vivo.     

Expression of biochemical properties of oligodendrocytes in oligodendrocyte x glioma cell hybrids proliferating in vitro

In order to generate cell lines that grow continuously in tissue culture and that express the biochemical properties of oligodendrocytes (the cells which produce myelin in the central nervous system), we isolated oligodendrocytes from calf brain and fused them with C₆ rat glioma cells. Of the 60 hybrid clones tested, several expressed oligodendroglial properties at levels comparable to isolated oligodendrocytes. In particular, hybrid clone CO-13-7 showed a high level of expression of all six oligodendroglial properties tested: 2′ : 3′-cyclic nucleotide 3′-phosphohydroiase, glycero-3-phosphate dehydrogenase, and induction of GPDH by hydrocortisone, all of which were also found in C₆ cells and in oligodendrocytes; and galactocerebroside, sulfatide, and myelin basic protein, which were found in normal oligodendrocytes but not in C₆ glioma cells. Therefore, the hybrids express a spectrum of oligodendrocyte biochemical properties that is not found in any other cell line that can be maintained continuously in tissue culture.     

Indirect immunofluorescence localization of beta-adrenergic receptors and G-proteins in human A431 cells.

Polyclonal antibodies directed against (i) rodent lung beta 2-adrenergic receptor, (ii) a synthetic fragment of an extracellular domain of the receptor, and (iii) human placenta G-protein beta-subunits, were used to localize these antigens in situ in intact and permeabilized human epidermoid carcinoma A431 cells. Antibodies directed against beta 2-adrenergic receptors showed a punctate immunofluorescence staining throughout the cell surface of fixed intact cells. Punctate staining was also observed in clones of Chinese hamster ovary cells transfected with an expression vector harbouring the gene for the hamster beta 2-adrenergic receptor. The immunofluorescence observed with anti-receptor antibodies paralleled the level of receptor expression. In contrast, the beta-subunits common to G-proteins were not stained in fixed intact cells, presumably reflecting their intracellular localization. In detergent-permeabilized fixed cells, strong punctate staining of G beta-subunits was observed throughout the cytoplasm. This is the first indirect immunofluorescence localization of beta-adrenergic receptors and G-proteins. Punctate immunofluorescence staining suggests that both antigens are distributed in clusters.     

Two independent mechanisms of induction of 2′:3′-cyclic-nucleotide 3′-phosphohydrolase in glioma cells by cyclic AMP and high cell density

Specific activity of the myelin enzyme, 2′:3′-cyclic-nucleotide 3′-phosphohydrolase (EC 3.1.4.37), increases 2- to 10-fold when sparsely inoculated cultures of C₆ rat glioma cells are allowed to grow to high cell density. Cyclic-nucleotide phosphohydrolase specific activity is also induced in C₆ cells and in oligodendrocytes by dibutyryl cyclic AMP or by agents that elevate intracellular cyclic AMP. In this report, we have compared the density-dependent induction of cyclic-nucleotide phosphohydrolase activity with the cyclic AMP-dependent induction. Dibutyryl cyclic AMP induced cyclic-nucleotide phosphohydrolase specific activity in both sparse and dense cultures which had very different density-dependent cyclic-nucleotide phosphohydrolase activities. Induction of both cyclic-nucleotide phosphohydrolase specific activity and intracellular cyclic AMP content by norepinephrine also occurred to a similar degree in sparse and dense cultures. Similar results were obtained for several clones of C₆ cells, and for a clone of oligodendrocyte x C₆ cell hybrids. Induction of cyclic-nucleotide phosphohydrolase by norepinephrine or dibutyryl cyclic AMP was not due to a change in cell density or rate of cell proliferation, nor did cell density have any appreciable effect on cyclic AMP content of the cells. These results show that regulation of cyclic-nucleotide phosphohydrolase activity in C₆ cells involves two distinct mechanisms.     

Flow cytometric analysis of tetanus toxin binding to neuroblastoma cells

A neuroblastoma cell line was assessed for its capacity to bind tetanus toxin (TT) by using immunofluorescence and flow cytometry to analyze cells on a single cell basis. A clone of Neuro 2a, N2AB-1, was shown to bind variable amounts of TT per cell and this binding could be saturated by increasing doses of the toxin. Toxin binding was specific for neuronal cells, as the non-neuronal cell line, C6 glioma, bound negligible amounts of toxin. Variability of immunofluorescence staining was due in part to the increase in size of N2AB-1 cells as they progress through the cell cycle as measured by cell surface densities of toxin binding and DNA levels by propidium iodide (PI) staining. When N2AB-1 cells were treated with exogenous gangliosides for 24 h, cells were induced to sprout neurites and cell growth was inhibited. Analysis of DNA histograms indicated that ganglioside treatment caused more cells to appear in G0G1 of the cell cycle than that seen for untreated controls. Upon cytometric analysis of TT binding to ganglioside treated cells, it was apparent that treatment stimulated all cells to bind TT in larger amounts per cell than that seen with untreated N2AB-1 cells. These data suggest that TT binding and, therefore, toxin receptors are constant in density throughout the cell cycle of these neuroblastoma cells and that exogenous gangliosides can cause differentiation followed by increased toxin binding.     

Localization of kinesin in cultured cells

Kinesin was isolated from bovine brain and used to elicit polyclonal antibodies in rabbits. The specificities of the resulting antibodies were evaluated by immunoblotting. Antibodies purified from these sera by their affinity for brain kinesin react with a polypeptide of approximately 120 kD in extracts from bovine brain, PtK1 cells, and mouse neuroblastoma cells. They bind to a pair of polypeptides of approximately 120 kD present in crude kinesin prepared from Xenopus eggs and with a single polypeptide of approximately 115 kD in extracts from Drosophila embryos. Antibodies raised against kinesin prepared from fruit fly embryos (by W. M. Saxton, Indiana University, Bloomington, IN) and from neural tissues of the squid (by M. P. Sheetz, Washington University, St. Louis, MO) cross react with the mammalian, the fly, and the frog polypeptides. Kinesin antigen was localized in cultured cells by indirect immunofluorescence. PtK1 cells in interphase showed dim background staining of cytoplasmic membranous components and bright staining of a small, fibrous, juxtanuclear structure. Double staining with antibodies to microtubules showed that the fibrous object was usually located near the centrosome. On the basis of shape, size, and location, we identify the kinesin-positive structure as a primary cilium. PtK1 cells in mitosis are stained at their poles during all stages of division. The structure stained is approximately spherical, but wisps of faint fluorescence also extend into the body of the spindle. Antibodies to squid or fruit fly kinesin produce identical patterns in PtK1 cells. Controls with preimmune and preabsorbed sera show that the centrosome staining is not due simply to the common tendency of rabbit antisera to stain this structure. Similar centrosome and spindle pole staining was visible when antibodies to bovine brain or squid kinesin were applied to the A6 cell line (kidney epithelial cells from Xenopus laevis). Some possible functions of kinesin localized at the spindle poles are discussed.     

Clonal characterization of mouse mammary luminal epithelial and myoepithelial cells separated by fluorescence-activated cell sorting

Summary Lineage analysis in vitro of heterogeneous tissues such as mammary epithelium requires the separation of constituent cell types and their growth as clones. The separation of virgin mouse mammary luminal epithelial and myoepithelial cells by fluorescence-activated cell-sorting, their growth at clonal density, and the phenotyping of the clones obtained with cell-type specific markers are described in this paper. Epithelial cells were isolated by collagenase digestion followed by trypsinization, and the luminal and myoepithelial cells were flow-sorted with the rat monoclonal antibodies 33A10 and JB6, respectively. Sorted cells were cloned under, using low oxygen conditions (<5% vol/vol), in medium containing cholera toxin and insulin, with an irradiated feeder layer of 3T3-L1 cells. Clones were characterized morphologically, and antigenically by multiple immunofluorescence with a panel of antibodies to cytoskeletal antigens specific to either luminal epithelial or myoepithelial cells in situ. Whereas sorted myoepithelial cells gave a single clone type, sorted luminal cells gave three morphological clone types, two of which grew rapidly. All myoepithelially derived clones showed a limited proliferative capacity in vitro, in contrast to their rat and human counterparts, as shown in previous studies. The present results with sorted mouse cells have also allowed the stability of the differentiated phenotype in mouse, rat, and human mammary luminal epithelial and myoepithelial cells in primary clonal culture to be compared. They show that the mouse mammary cells are the least stable in terms of expression of differentiation-specific cytoskeletal markers in vitro.     

Extinction of differentiated glial functions in somatic ce-l hybrids is incomplete.

Rat glioma cells of clone C₆ were hybridized in vitro with mouse L cells of clone A9 or with freshly isolated mouse macrophages, and the hybrids were assayed for glial cell functions. C₆ cells expressed high levels of 2′,3′-cyclic nucleotide 3′-phosphohydrolase (CNP; EC 3.1.4.37), β-hydroxybutyrate dehydrogenase (HBDH; EC 1.1.1.30), glycerol-3-phosphate dehydrogenase (GPDH; EC 1.1.1.8), and inducibility of GPDH by hydrocortisone (HC). A9 cells and macrophages had very low activities of these functions. Hybrids between C₆ and A9 or between C₆ and macrophages had greatly reduced activities of these functions, but the hybrids expressed significantly higher activities than the non-glial parent. This incomplete extinction was not due to fusion of two glioma cells with one L cell or macrophage. The difference in GPDH activity in the hybrids as compared with the non-glial parent was due to incomplete shut-off of GPDH of the glial parent, and not to an increase in GPDH production by the non-glial genome.     

Cellular Acetylcholine Content and Neuronal Differentiation

Abstract: N18TG2 neuroblastoma clone is defective for biosynthetic neurotransmitter enzymes; its inability to establish functional synapses is overcome in the neuroblastoma × glioma 108CC15, where acetylcholine synthesis is also activated. These observations suggest a possible relation between the ability to produce acetylcholine and the capability to advance in the differentiation program and achieve a fully differentiated state. Here, we report the characterization of several clones after transfection of N18TG2 cells with a construct containing a cDNA for rat choline acetyltransferase (ChAT). The ability of these clones to synthesize acetylcholine is demonstrated by HPLC determination on cellular extracts. In the transfected clones, northern blot analysis shows increased expression of mRNAs for a specific neuronal protein associated with synaptic vesicles, synapsin I. Fiber outgrowth of transfected clones is also evaluated to establish whether there is any relation between ChAT levels and morphological differentiation. This analysis shows that the transfected clone 1/2, not expressing ChAT activity, displays a very immature morphology, and its ability to extend fibers also remains rather poor in the presence of "differentiation" agents such as retinoic acid. In contrast, clones 2/4, 3/1, and 3/2, exhibiting high ChAT levels, display higher fiber outgrowth compared with clone 1/2 in both the absence and the presence of differentiating agents.     

Tubulin constancy during morphological differentiation of mouse neuroblastoma cells

Clonal cell lines N18 and N103 of the mouse neuroblastoma C1300 possess an undifferentiated neuroblast morphology under optimal growth conditions; however, when deprived of serum, N18 can be induced to extend long neurites. Although initial neurite outgrowth is rapid, very long fibers are found only after several days. Both initial outgrowths and established neurites contain microtubules; however, the number and density of these polymerized tubules increase markedly during this time. Optimum conditions have been established for assessing the colchicine-binding activity of neuroblastoma sonicates. A time-decay colchicine-binding assay was used to make a comparative study of the tubulin content of both undifferentiated and differentiated N18 as well as the nondifferentiating N103 and the rat glioma C6. Both morphologies of clone N18 possessed similar concentrations of tubulin (130-140 pmol/10(6) cells). Although cells of clone N103 contain 20% less tubulin than N18 cells, this is considerably more tubulin than is present in the glioma C6 (30 pmol/10(6) cells) which has a similar generation time. Quantitative densitometry of neuroblastoma extracts electrophoresed on SDS-polyacrylamide gels confirmed the constancy of tubulin. Radiolabeled proteins from neuroblastoma cells subjected to both growth conditions show that neurite outgrowth does not create a disproportionate demand for tubulin synthesis. Thus, the morphological differentiation of neuroblastoma cells probably reflects the regulation of tubulin storage and microtubule polymerization.     

低氧对NG108－15细胞生长及分化的影响

神经母细胞瘤和神经胶质瘤细胞融合的克隆细胞系ＮＧ１０８－１５细胞在含分化剂双丁基环化单磷酸腺苷（ｄＢｆｃＡＭＰ）的培养液培养后分化,成为具神经细胞特征的细胞。本实验利用四唑盐（ＭＴＴ）微量比色法,并结合焦油紫染色,测定和观察细胞的生长及分化状况,研究了低氧（２％Ｏ２＋９３％Ｎ２＋５／ＣＯ２）对未分化的,分化中的和已分化完成的ＮＧ１０８－１５细胞的影响。获得的主要结果是;低氧明显降低未分化细胞增殖和存活率,使分化完成的细胞大量死亡;低氧影响ＮＧ１０８－１５细胞的分化,使细胞在分化中出现体积膨大,突起短等异常特征,经焦油紫染色,胞质中无尼氏体（即不着色）的细胞增多。低氧是否可能使未分化ＮＧ１０８－１５细胞向更多地表达胶质细胞特征的方向分化？将是一个十分有趣的问题。     

Extinction of differentiated glial functions in somatic cell hybrids is incomplete

Rat glioma cells of clone C₆ were hybridized in vitro with mouse L cells of clone A9 or with freshly isolated mouse macrophages, and the hybrids were assayed for glial cell functions. C₆ cells expressed high levels of 2′,3′-cyclic nucleotide 3′-phosphohydrolase (CNP; EC 3.1.4.37), β-hydroxybutyrate dehydrogenase (HBDH; EC 1.1.1.30), glycerol-3-phosphate dehydrogenase (GPDH; EC 1.1.1.8), and inducibility of GPDH by hydrocortisone (HC). A9 cells and macrophages had very low activities of these functions. Hybrids between C₆ and A9 or between C₆ and macrophages had greatly reduced activities of these functions, but the hybrids expressed significantly higher activities than the non-glial parent. This incomplete extinction was not due to fusion of two glioma cells with one L cell or macrophage. The difference in GPDH activity in the hybrids as compared with the non-glial parent was due to incomplete shut-off of GPDH of the glial parent, and not to an increase in GPDH production by the non-glial genome.     

Phosphatase activity in cultured glioma and neuroblastoma cells

Acid phosphatase activity in human glioma cells (138 MG) and mouse neuroblastoma cells (C 1300) was associated with structures accumulating neutral red and acridine orange. Only neuroblastoma cells gave a significant positive histochemical reaction for alkaline phosphatase. Glioma and neuroblastoma cell homogenates exhibited maximal phosphatase activity at pH 5 as measured by spectrophotometer. The specific activity; μmoles phosphate released per hour/mg protein was 1.1 in glioma and 0.9 in neuroblastoma. At pH 8, glioma cells lacked activity whereas neuroblastoma cells showed another maximum. The acid phosphatase activity of both cell types was strongly inhibited by CuCl₂ (0.3 mM) and NaF (10 mM) and moderately by -tartaric acid (10 mM). cGMP (1 mM) stimulated the phosphatase activity of both cell lines. db-cAMP, in serum-free medium, induced characteristic morphological changes of the cells studied. This process was unaffected by CuCl₂, c-GMP and -tartaric acid. db-cAMP (1 mM) inhibited proliferation in both glioma and neuroblastoma cells during a 48 h incubation in serum-containing medium. This growth inhibition was associated with an increase in acid phosphatase activity of the glioma but not of the neuroblastoma cells.     

CD8+ suppressor T cell clone capable of inhibiting the antigen- and anti-T cell receptor-induced proliferation of Th clones without cytolytic activity

A CD8+ Ts clone 13G2 was established from lymph node cells of bovine alpha s1-casein-primed C57BL/6 mice by in vitro antigenic stimulation followed by maintenance with IL-2-containing medium. The clone suppressed the Ag-induced proliferative responses of CD4+ Th cell clones without detectable cytotoxicity for both APC and responding T cells. The clone was able to suppress the in vitro proliferative response and antibody formation of Ag-primed lymph node cells. The suppression was Ag-nonspecific and not restricted to the MHC. The clone was able to suppress the proliferation of Th clones induced by an immobilized anti-TCR antibody in which APC was absent. The clone was, however, unable to suppress the proliferation of Th clones induced by anti-CD3 or IL-2. Thus, the mechanism of suppression by 13G2 was found to be due to a direct action on Th by inhibiting a consequence of signal transduction initiated through the TCR.     

Only Pyrimidinoceptors Are Functionally Expressed in Mouse Neuroblastoma Cell Lines

The ability of UTP, UDP, ATP, and ADP to influence inositol phospholipid hydrolysis in neuroblastoma origin cell lines was assessed. The mouse neuroblastoma lines N1E 115, Neuro 2a, and NB4 1A3 and the rat glioma/mouse neuroblastoma hybrid line NG108-15 gave robust responses to both UTP and UDP, which were essentially equipotent. Thus a range of cell lines of mouse neuroblastoma origin express a pyrimidine-selective P2Y receptor. The NG108-15 cells were the only cell type tested at which ATP and ADP displayed activity with EC₅₀ values of greater than 100 μM, compared with values of 0.58 and 1.25 μM for UTP and UDP, respectively. In contrast to the cell lines derived from mouse neuroblastoma, the human neuroblastoma lines SH-SY5Y and SK-N-SH did not respond to any nucleotides, although both responded well to carbachol.     

Localization of the coding region for an Epstein-Barr virus early antigen and inducible expression of this 60-kilodalton nuclear protein in transfected fibroblast cell lines.

Expression of a component of the Epstein-Barr virus early antigen (EA) complex has been studied in fibroblast cells transfected with both wild-type and P3HR-1 defective DNA fragments covering the BamHI-M-S region of the Epstein-Barr virus genome. Baby hamster kidney (BHK) cells transfected with the BglII-J fragment and stained with human serum that was positive for the diffuse component of EA [EA(D)] in an indirect immunofluorescence assay exhibited positive nuclear staining in 5% of the cell population. Cleavage of BglII-J before transfection with the restriction enzyme BglII, StuI, HindIII, or PvuII did not affect EA expression, whereas prior cleavage with BamHI or EcoRI reduced or eliminated synthesis of EA. These observations were confirmed by using individual cloned subfragments. A Bal 31 deletion clone (pTS1) in which the HindIII and StuI sites were eliminated retained activity, whereas a clone (pTS5) in which the deletion extended closer to the EcoRI site had greatly reduced activity. Transfection of the individual BamHI-M or BamHI-S fragments, which span BglII-J, also resulted in little or no EA expression. The 2.1-kilobase biologically active region defined by these experiments corresponds precisely to the BMLF1 open reading frame. Immunoblot analyses of BHK cells transfected with either P3HR-1 defective DNA clones or the BglII-J wild-type fragment identified the product of this EA(D) coding region as a family of polypeptides consisting of a major 60-kilodalton product and minor 45- and 50-kilodalton species. In latently Epstein-Barr virus-infected lymphocytes these early antigens are not expressed, but can be induced by treatment of the cultures with sodium butyrate or phorbol esters. Using the BglII-J and pTS6 clones that were positive in transient assays, we also established Neor coselected BHK and Vero cell lines which showed similar regulated expression of the 60-kilodalton EA(D) protein. In these cell lines constitutive expression of EA(D) was limited (0.1% positive by indirect immunofluorescence and undetectable by immunoblot analysis). However, expression of EA(D) could be induced by treatment with sodium butyrate. In the induced cultures, up to 30% of the cells were EA(D) positive by immunofluorescence, and there was a concomitant appearance of the 60-kilodalton EA(D) polypeptide.     

Characterization of human and rat immortalized clones of parotid acinar cells with respect to specific proteins and their mRNAs,and receptor-linked adenylate cyclase

Summary This study reports the isolation and characterization of a rat nontumorigenic parotid acinar cell clone (2RSG), a human nontumorigenic parotid acinar cell clone (2HPC8), and a human tumorigenic acinar clone (2HP₁G). The levels ofα-amylase mRNAs detected when usingα-amylase cDNA of 1176 and 702 bp for hybridization were higher in 2RSG and 2HPC8 cells than their respective whole parotid glands. The level of these mRNAs decreased in 2HP₁G cells. In contrast toα-amylase mRNAs levels, theα-amylase activity in cultured acinar cells was extremely low in comparison to whole glands, irrespective of species or cell status. The levels of proline-rich protein (PRP) mRNA and parotid secretory protein (PSP) mRNA detected when using PRP cDNA of 600 bp and PSP cDNA of 805 bp for hybridization were higher in 2RSG cells than those in rat parotid glands; the reverse was observed in 2HPC8 cells and human parotid glands. The levels of PRP mRNA and PSP mRNA in 2HPC8 and 2HP₁G acinar cells were similar. The level of mRNA was not detectable in murine neuroblastoma cells (NBP2) using the sameα-amylase cDNA, PRP cDNA and PSP cDNA for hybridization. The PSP level in rat parotid gland was lower than that found in 2RSG cells; the reverse was observed in 2HPC8 cells and human parotid glands. The level of PSP in 2HP₁G cells was higher than that found in 2HPC8 cells. Isoproterenol increased the cAMP level in 2RSG, 2HPC8, and 2HP₁G clones, being most effective in 2RSG cells, and least effective in 2HPG cells. Prostaglandin E₁ (PGE₁) also increased cAMP level, being most effective in 2HPC8 cells and ineffective in 2HP₁G cells, suggesting that the PGE₁ receptor-linked adenylate cyclase becomes inactive upon transformation. These results suggest that the three clonal acinar cells from rat and human parotid glands reported here can be useful in comparative studies on regulation of growth, differentiation, and transformation.     

Differential distribution of microtubule-associated proteins MAP-1 and MAP-2 in neurons of rat brain and association of MAP-1 with microtubules of neuroblastoma cells (clone N2A).

To study the individual location of the microtubule proteins MAP-1 and MAP-2 in neuronal tissues and cells, antisera to electrophoretically purified MAP-1 and MAP-2 components were raised in rabbits. When frozen sections through rat brain were examined by immunofluorescence microscopy the antibodies to MAP-1 strongly stained a variety of nerve cells including dendrites and myelinated axons in the cerebrum and cerebellum. Antibodies to MAP-2 showed similar staining patterns, except that myelinated axons were unstained. These results were confirmed by immunoelectron microscopy of frozen sections through cerebellum using the peroxidase technique. Thereby, the association of MAP-1 with microtubules was also clearly demonstrated. When cultured mouse neuroblastoma N2A cells were examined by immunofluorescence microscopy the antiserum to MAP-1 brightly stained filamentous structures resembling microtubules, whereas relatively weak and diffuse staining of the cytoplasm was observed with the antiserum to MAP-2. In agreement with the immunolocalization, MAP-1, but not MAP-2, was found as a prominent component of microtubules proteins polymerized in vitro by taxol from soluble N2A cell extracts. Together these results indicate that neuronal microtubules are preferentially associated with distinct high mol. wt. polypeptides. Therefore, they support the concept that different complements of associated proteins determine distinct functions of microtubules.