Selective distribution of a novel tubulin in the developing and mature rat brain

Developmental Brain Research, 53 (1990) 103-115 Elsevier

103

BRESD 51056

Selective distribution of a novel tubulin in the developing and mature rat brain Jamie M. Gossels* and Vernon M. Ingram Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139 (U.S.A.) (Accepted 7 November 1989) Key words: Tubulin; Selective distribution; Rat brain; Cerebellum; Hippocampus; Purkinje cell

A monoclonal antibody, G8, was isolated which recognizes a form of tubulin (G8-tubulin) with a novel distribution in the rat brain. Immunoblots of rat brain homogenates and immunohistochemicai staining of rat brain sections of various ages with G8 revealed a restricted and developmentally regulated distribution of the G8-tubulin. G8 staining was primarily found in granule cell dendrites in the dentate gyms, in pyramidal cell apical dendrites in the hippocampus, and in Purkinje cell dendrites in the cerebellum. This pattern was much more selective than that observed with three other anti-tubulin antibodies. Relative abundance of G8-tubulin in the brain increases with age between postnatal day 1 (P1) and adulthood. The results suggest that G8 is specific for a novel tubulin form which shows a characteristic distribution in the rat brain. This distribution may indicate that the G8-tubulin possesses functional specificity.

INTRODUCTION Microtubules perform several functions in a variety of cell structures 19. They are particularly important in the brain where they are involved in intracellular organelle transport in neurons as well as in other cell types. In association with microfilaments and intermediate filaments, microtubules are required to establish cell shape and internal structure, especially in neurons. Microtubules are also the major structural elements in cilia and flagella and are involved in chromosome migration during mitosis and meiosis. Tubulin, the major component of microtubules and a major soluble component of the brain 46'51, is a 110 kDa heterodimer composed of one a- and one fl-tubulin. Several a- and fl-tubulin forms exist in the brains of many species t6,22,2s'42. In part, the heterogeneity is due to the expression of multiple tubulin genes H and in part it is generated by post-translational modifications; for example, a-tubulin is subject to cyclical detyrosination and tyrosination of the carboxy-terminus 3'4 and to acetylation of the e-amino group of a lysine residue 38. fl-Tubulin can be phosphorylated in vivo 2°'21'25. The abundance of total tubulin message and protein in rat brain relative to other proteins decreases after postnatal day 10 (P10) 6'5°. Concurrent with this decrease, tubulin

microheterogeneity increases during development15'28"56; changes are complete by P10-P1215. The identification of multiple a- and fl-tubulins suggests that each tubulin might polymerize into distinct microtubules which serve distinct functions associated with microtubules 23. Alternatively, the existence of many tubulin isoforms could be due to a requirement for different sequences for regulation of expression in different environments 49. Some evidence supports and other results refute these hypotheses a2. Support for the theory of distinct functions for different isoforms has come mainly from tubulin forms arising from post-translational modifications 20"25"29'37-39. Tubulin gene products appear to be multifunctional 7"32'33"35'4°. The significance of tubulin heterogeneity remains a major question in tubulin biology. We describe the use of a m0noclonal antibody to characterize a new, developmentally regulated form of tubulin (GS-tubulin). GS-tubulin is distributed differently and more selectively in the rat brain than are other tubulins. These characteristics suggest that the antibody recognizes a previously undescribed form of tubulin which might serve a specific subset of tubulin functions. The GS-tubulin pattern of specific localization also defines a subset of regions of the brain which must share a c o m m o n functional or regulatory characteristic with respect to expression of this form of tubulin.

* Present address: Mailman Research Center, McLean Hospital, 115 Mill Street, Belmont, MA 02178, U.S.A. Correspondence: V. Ingrain, Department of Biology, Massachusetts Institute of Biology, 77 Massachusetts Avenue, 56-50l Cambridge, MA 02139, U.S.A. 0165-3806/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)

104 MATERIALS AND METHODS

Preparation of the G8 antibody To identify growth cone-specific antigens, we prepared growth cone particles to use as an immunogen to generate monoclonal antibodies. G8, an anti-tubulin antibody, was found during the course of screening hybridomas. A crude preparation reported to be enriched in 'growth cone particles '47 was made from 90 embryonic day 17 (El7) rats (Charles River, Wilmington, NY). The pooled fractions from the first peak of the controlled pore glass column were spun in an SS34 rotor in a Sorvall RC2-B centrifuge at 15,000 rpm for 45 min at 4 °C, and the pellet was washed once in calcium, magnesium-free phosphatebuffered solution (PBS). Two BALB/c mice (Jackson Labs, Bar Harbor, ME) were each immunized subcutaneously with 30 /~g protein of adult rat brain homogenate in Freund's complete adjuvant. Two days later, they were immunosuppressed by intraperitoneal cyclophosphamide

+

G8

"

kD 200 116~.97~ 66~

+

injection 43 to deplete populations of lymphocytes which recognize antigens from adult brain. Three weeks after immunosupression, the spleen cells from these mice were immunized in vitro with the above 'growth cone particles' (5 ~g per spleen43). Immunized spleen cells were fused with P3X63.Ag8.653 ('X63') cells24 at a ratio of 5:1 in 2.5% fetal calf serum (FCS)-RPMI medium. Each dissociated spleen contained 5-7 × 107 cells. Culture supernatants from hybridomas were screened by immunofluorescence on NB2A murine neuroblastoma cells for growth cone staining. Positive wells were cloned by limiting dilution. Fourteen cell lines, G1-G14, were obtained. Culture supernatant from all hybridomas was collected. G8 ascites fluid was also produced in mice which had been primed with 0.5 ml pristane. Hybridoma cells (5 x 105) were injected intraperitoneally. After 2-3 weeks, ascites fluid was tapped 3-4 times.

Other antibodies Primary antibodies tested included G8, 'anti-tubl' (a polyclonal anti-tubulin antibody from ICN Biomedicals, Lisle, IL, catalogue number 65-095), 'anti-tub2' (also known as '429', a polyclonal anti-fl-2-tubulin antibody kindly provided by Wendy Katz and Prof. Frank Solomon, M.I.T.7), 'anti-tub3' (also known as '61D', an IgM monoclonal antibody generated against calf brain tubulin, kindly provided by Dr. Margaret Magendantz and Prof. Frank Solomon, M.I.T.7'41), anti-DNP (an IgM monoclonal antibody against dinitrophenol, kindly provided by Prof. Herman Eisen, M.I.T.), anti-MAP2 (a monoclonal antibody specific for microtubule-associated protein 2, kindly provided by Prof. Ken Kosik, Harvard Medical School), and SMI-31 (an antibody specific for phosphorylated 200 kDa neurofilament protein; Sternberger-Meyer Co., Jarrettsville, MD). Normal rabbit serum was purchased from Organon-Teknika/Cappel Co., Malvern, PA.

anti-tubl

200~

116~ 97~ 66~

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! Fig. 1. Two-dimensional immunoblots of adult rat brain cytoplasmic fraction: comparison of G8 with anti-tubulin reactivity. Protein (25 ktg) was separated by isoelectric focusing in the first dimension and 7.5% SDS-PAGE in the second dimension. Large arrowheads indicate migration positions of most a- and fl-tubulins; in general a-tubulins migrate more slowly than fl-tubulins. G8 ascites fluid diluted 1:20, anti-tubl diluted 1:200. The basic end of each gel is to the right. The arrow indicates the direction of migration in the isoelectric focusing dimension. The pH range of the isoelectric focusing gel is approximately pH 4.5-7.0.

Fig. 2. Immunoblots stained with G8 and anti-tub2 of adult rat brain cytoplasmic fraction and tubulin purified by Phosphocellulose chromatography. SDS-polyacrylamide gels (7.5%); G8 ascites fluid diluted 1:20, anti-tub2 diluted 1:7500. Ad, adult (protein from brain cytoplasmic fraction); T, bovine brain tubulin purified by phosphocellulose chromatography.

105

lmmunofluorescence Cells were plated on 12 × 12 mm coverslips at approximately 1 × 104 cell/coverslip and used 1-4 days later. For initial screening of hybridoma supernatants, NB2A cells were fixed in 3.7% formaldehyde in PBS for 10 min. For G8 and other anti-tubulin antibody staining of Rat-1 fibroblasts cells were fixed in 4% paraformaldehyde in 0.1 M phosphate buffer, pH 7.4 for 10 min at 37 °C followed by permeahilization in 0.1% Triton X-100 in PBS for 4 min at 37 °C. Alternatively, cells were fixed in cold (-20 °C) methanol at -20 °C for 6 min. Following fixation, coverslips were washed twice, 5 min each time in PBS (0.01 M phosphate/0.15 M NaCl, pH 7.2) at room temperature. Coverslips were incubated in 3% normal goat serum (50-100/~l/coverslip) for 30 min at 37 °C followed by two washes in PBS. Primary antibody (50 #l per coverslip) was incubated on the coverslips for 1 h at 37 °C. Coverslips were washed 3 times and incubated for 1 h at 37 °C in a 1:50 or 1:100 dilution of fluorescein isothyocyanate (FITC)- or tetramethylrhodamine isothiocyanate (TRITC)-conjugated goat anti-mouse-IgA+IgM+IgG for the murine monoclonal antibodies or FITC-conjugated goat anti-rabbitIgG for the rabbit polyclonal antibodies (50 ~tl per coverslip; all secondary antibodies obtained from Organon-Teknika/Cappel Co., Malvern, PA). Coverslips were washed 3 times, rinsed in distilled

H20, and mounted in 90% glycerol, 10% Na3PO4.12 Ha0, pH 9.0. Primary and secondary antibodies were diluted in 10% FCS, 3% normal goat serum in PBS. For double label experiments, coverslips were incubated simultaneously with both primary antibodies and then simultaneously with both secondary antibodies.

Immunohistochemistry Rats of ages P1, P6, P10, P16, P20 and adult were anesthetized and perfused intracardially with 4% paraformaldehyde, 0.34% lysine, 0.05% sodium periodate (10-150 ml depending on the age of the animal). Tissue was removed soon after perfusion and immersed in the same fixative for 24 h at 4 °C. Fixed tissue was cryoprotected by immersion in 30% sucrose in PBS until it sank completely (at least overnight). The spinal cord was incubated in 30% sucrose for 4 days but did not sink. For retina and dorsal root ganglion experiments, the eye and spinal cord were further cryoprotected for one hour in Tissue-Tek O.C.T. compound (Miles Scientific, Naperville, IL). Cryoprotected tissue was frozen in Tissue-Tek O.C.T. compound on dry ice. Sections (30/lm) were cut on a Bright microtome at -14 °C and were collected floating in PBS. Two brains of each age were sectioned; one was cut parasagittally and the other coronally. Every 10th section was stained with G8 culture supernatant and

Fig. 3. G8 and anti-tub2 double-label immunofluorescent staining of methanol-fixed Rat-1 fibroblasts showing the interphase cytoskeleton. G8: G8 staining visualized with TRITC-conjugated goat anti-mouse-IgA+IgM+IgG. G8 ascites fluid diluted 1:20. anti-tub2:anti-tub2 (diluted 1:500) staining visualized with FITC-conjugated goat anti-rabbit-IgG. Same field as shown for G8. anti-DIVP; anti-DNP (diluted 1:20) staining visualized with TRITC-conjugated goat anti-mouse-IgA+IgM+IgG; pre-s.: Rat-1 cells stained with commercially obtained pre-immune rabbit serum (diluted 1:100) visualized with FITC-conjugated goat anti-rabbit-IgG. Same field as shown for anti-DNP. Bar = 20 ~m.

106 every 40th section was reacted with anti-dinitrophenol (anti-DNP) ascites fluid. Sections were stained using the Vectastain ABC method (Vector Labs, Burlingame, CA). Floating sections were washed for 30 min in Tris-buffered saline (TBS: 0.01 M Tris/0.15 M NaCI, pH 7.5) at room temperature, incubated in 3% normal goat serum for 30 min at room temperature and in primary antibody overnight at room temperature. Dilutions of primary and secondary antibody were in 10% FCS, 1.5% normal goat serum in TBS. Following incubation with primary antibody sections were washed 3 times, 10 min each, in TBS and incubated in 1:200 biotinylated goat anti-mouse-IgM (for the monoclonal antibodies) or goat anti-rabbit-IgG (for the polyclonal antibodies) for 1 h at room temperature. Following 3 washes, 10 min each, in TBS, sections were incubated for 1 h at room temperature in avidin/biotin-peroxidase reagent (Vectastain) diluted in 1.5% normal goat serum in TBS, washed twice, 10 min each, in TBS and once, 10 min, in 0.05 M Tris-HCl, pH 7.5. Color was developed by incubation for 8 min in a freshly prepared mixture of diaminobenzidine (0.5 mg/ml) and 0.01% hydrogen peroxide ir 0.05 M Tris-HCl, pH 7.5. Sections were washed for 10 min in 0.0: M Tris-HCl, pH 7.5 and mounted on subbed slides. Slides wer, allowed to dry, dehydrated through 70-100 t2~ ethanol and xylene, and coverslipped with Permount.

of the isoelectric focusing gels was determined by the method ~I Marotta et al. 4~. After focusing was complete, tube gels were incubated for 3() min in S D S - P A G E sample buffer at room temperature and loaded onto 7 x 8 cm 7.5% SDS-polyacrylamide gels. Two-dimensional gels were stained with 0.1% Coomassie blue or prepared for immunoblotting.

lmmunoblots ('Western blots') SDS-polyacrylamide gels were transferred to nitrocellulose membrane 55. After transfer, unbound sites on the nitrocellulose

a

kD 200

116~" 97~ 66~

Protein preparations All protein preparations were made from brain tissue from rats sacrificed by cervical dislocation or decapitation. For homogenate, membrane, and cytoplasmic, brains were removed and placed in ice-cold PBS. Brain homogenate. Brains were rinsed twice in PBS containing 1 mM phenylmethylsulfonyl fluoride, minced, and homogenized with 10 strokes in a Dounce homogenizer using an A pestle. The homogenate was sonicated on ice 4 times, 10 s each. Aiiquots were stored at -20 *C. Brain membrane preparation. The membrane preparation was made according to the procedure of Hoffman et al. 31. Brain cytoplasmic fraction. Initial steps in the preparation were the same as for preparation of brain homogenate. The brain homogenate was c e n t r i f ~ e d at 15,000 rpm in an SS34 rotor in a Sorvall RC2,B centrifuge for 10 rain at 4 *C. The supernatant was spun at 25,000 rpm ( 1 0 0 , ~ g ) in a SB'283 rotor in an International centrifuge Model IEC/B-60 for 1 h at 4 °C. Aliquots were stored at -20 °C. Triton X-IO0 fractionation. Triton X-100 extraction was according to the procedure of Pruss et al. 4s. Protein concentrations were determined according to the procedure of Bradford 8 (Coomassie blue (Bio-rad Labs reagent)) or Smith et al.52 (Bicinchoninic acid (BCA reagent, Pierce Chemical)).

Gel electrophoresis All protein fractions were prepared for one-dimensional get electrophoresis by mixing with an equal volume of 2 x S D S - P A G E sample buffer (1 x sample buffer: 2% SDS/2% glycerol/0.5% fl-mercaptoethanoi/0.01% bromphenol blue/62.5 mM Tris-HCl, pH 6.8). Samples were boiled for 2 rain. Samples were prepared for two-dimensional gel electrophoresis by boiling for 2 min in 2% SDS and adding a 3-fold volume of Lysis Buffer A 45. SDS, P A G E was according to the method of Laemmli 34. Gels contained 7.5% acrylamide and 0.2% bis-acrylamide. Gels were 1.5 mm thick and 7 x 8 era. Twenty to 30/tg protein was loaded in each of 15 lanes. Gels were stained with 0A% Coomassie blue in 40% methanol/7% acetic acid or prepared for immunoblotting. Two-dimensional gels were run according to the method of O'Farrell45. Tubes for the isoelectric focusing dimension had a radius of 0.7 mm and height of 6 cm and were plugged with 10% acrylamide. The tube gels were composed of 9 M urea, 4% acrylamide, 2% NP-40, 1.6% ampholines pH 5-7 and 0.4% ampholines pH 3-10. After pre-electrophoresis of the gels for 15 min at 200 V, 30 min at 300 V, and 30 min at 400 V, samples (20-30 /~g) were focused for 13 h at 250 V and 1 h at 400 V. The pH range

43~

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6

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Fig. 4. Developmentally regulated expression of tubulins recognized by G8 and by anti-tub2. Immunoblots of 7.5% gels; G8 ascites fluid diluted 1:20, anti-tub2 diluted 1:10,000. a: G8 and anti-tub2 reactivity with brain homogenates. 20 gg protein per lane. Lanes 1-6: G8; Lanes 7-12: anti-tub2. Lanes 1, 7 : E 1 7 brain homogenate: Lanes 2, 8 : E 1 7 brain homogenate, different preparation made according to the same procedure as lanes 1, 7.: Lanes 3, 9:P1 brain homogenate; Lanes 4. 10:P1 brain homogenate, different preparation made according to the same procedure as lanes 3, 9; Lanes 5, 11: adult brain homogenate: Lanes 6,12: adult brain homogenate. different preparation made according to the same procedure as Lanes 5, 11. b: G8 and anti-tub2 reactivity with brain cytoplasmic fractions, 20 /~g protein per lane. Lanes 1-4: G8: Lanes 5-8: anti-tub2. Lanes 1.5: PI: Lanes 2.6: P10; Lanes 3. 7: P22; Lanes 4, 8: adult. Ad, Adult; E17, embryonic day 17; P1, PI0, P22, postnatal days 1, 10, 22.

107

Fig. 5. Developmentally regulated expression of the G8 antigen in the dentate gyrus of the rat hippocampus. G8 labeling shown at P1, P6, P10, P16, and P20. Sections (30~m) were cut parasagittally from brains of rats perfused with 4% paraformaldehyde, 0.34% lysine, 0.5% sodium periodate. G8 culture supernatant undiluted, h, hilus region; g, granule cell layer of the dentate gyrus; m, molecular layer of the dentate gyrus; P1, P6, P10, P16, P20, postnatal days 1, 6, 10, 16, 20. Bar = 30 ~m.

108 were blocked with 5.1% bovine serum albumin (BSA) in 0.02 M Tris/0,15 M NaCI, pH 8.2 for at least 1 h at 37 °C. For staining with the monoelonal antibodies (for which Janssen Life Sciences products Auroprobe BLplus gold-conjugated goat anti-mouse IgG+IgM and IntenSE II silver enhancement were used), the procedure was as described by Janssen Life Sciences, Piscataway, NJ. The nicrocellulose membrane was incubated in primary antibody overnight. For polyelonal antibodies (for which the Vectastain ABC method was used), all washes were for 5 rain in 0.5% Triton X-100/PBS. Following BSA blocking the nitrocellulose was washed 3 times and incubated overnight in primary antibody diluted in 10% FCS in 0.5% Triton X-100/PBS. The membrane was washed 5 times and incubated for one hour in 1:200 biotin-conjugated goat anti-mouse IgM or anti-rabbit IgG (Vector Labs, Burlingame, CA) diluted as above. The nitrocellulose membrane was washed 5 times and incubated for 1 h in avidin/biotin-peroxidase reagent diluted as above. The membrane was washed 5 times and incubated in 30 mg 4-chloro-l-naphthol in 20 ml absolute ethanol, 40 ml 0.05 M Tris/0.15 M NaCI, pH 7.5 with 0.05% HzO2 for 15 min or until stained bands were clearly visible. The nitrocellulose membrane was rinsed in distilled H20. All incubations and washes except the BSA blocking step in both staining procedures were done at room temperature. All incubations were in sealed plastic bags with 0.05 ml of solution per square centimeter of membrane.

PY

sr

RESULTS

G8 recognizes tubulin G8, an I g M m o n o c l o n a l antibody which primarily stains processes in the rat central nervous system, was g e n e r a t e d during a search for antibodies specific for growth cone proteins. G8 recognized a 55 kilodalton ( k D a ) d o u b l e t on two-dimensional i m m u n o b l o t s of adult rat brain cytoplasmic fraction as did the commercial polyclonal anti-tubulin a n t i b o d y ' a n t i - t u b l ' (Fig. 1). This result suggests that G8 recognizes tubulin. O n o n e - d i m e n s i o n a l i m m u n o b l o t s , G8 recognized bovine" brain tubutin purified by cycles of assembly and disassembly and phosphocellulose c h r o m a t o g r a p h y (Fig. 2). The 55 k D a d o u b l e t from adult rat brain was also reactive with G8; this doublet comigrated with tubulin (Fig. 2). A n t i - t u b 2 , a polyclonal anti-tubulin antibody g e n e r a t e d against the fl-2-tubulin isotype, gave similar p a t t e r n s (Fig. 2). A n t i - D N P , an t g M a n t i b o d y specific for the irrelevant hapt¢n dinitrophenol, did not stain the 55 k D a bands recognized by G8 and anti-tub2 above b a c k g r o u n d (data not shown). This a n t i b o d y was used as a control for non-specific IgM staining since G8 is an IgM antibody. The G8 antigen is PBS-soluble, consistent with its identification as tubulin. It was enriched in an adult rat

i~

t i~ i

0

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Fig. 6. Distribution of the G8 antigen in the adult rat hippocampus (CA1). Sections (30/tm) were cut parasagittally from brains of rats perfused with 4% paraformaldehyde, 0.34% lysine. (k05% sodium periodate. G8 culture supernatant undiluted, anti-DNP diluted 1:50. o, oriens layer; py, pyramidal cell layer; sr, stratum radiatum. Bar = 35 #m. brain cytoplasmic fraction c o m p a r e d wtih a m e m b r a n e fraction and in an adult rat brain PBS-soluble fraction c o m p a r e d with PBS-insoluble and Triton X-insoluble fractions (data not shown).

Fig. 7. Developmentally regulated expression of the G8 antigen in the rat cerebellum. G8 labeling shown at P6, P10, P16, P20, and adult. Sections (30 pro) were cut parasagittally from brains of rats perfused with 4% paraformaldehyde, 0.34% lysine, 0.05% sodium periodate. G8 culture supernatant was undiluted. Arrowhead indicates a possible Purkinje cell axon which is labeled with G8. eg, external granule layer; m, molecular layer; p, Purkinje cell layer; ig, internal granule layer; g, granule cell layer; P6, P10, P16, P20, postnatal days 6, 10, 16, 20; Ad, adult. Bar = 10/~m.

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110

Fig. 8, Comparison of (38 with other anti-tubulin labeling in the adult rat cerebellum. Sections (30 ~m) were cut parasagittally from brains of rats pe,r~sed with 4% paraformaldehyde, 0.34% lysine, 0.05% sodium periodate. G8 culture supernatant undiluted, anti-tub2 diluted 1:5000, anti-tub3 diluted 1:15,000, anti-DNP diluted 1:50. m, molecular layer; p, Purkinje cell layer; g, granule cell layer. Bar = 10 pm. G8 stained the fiber network in interphase Rat-1 fibroblasts, as did anti-tub2 (Fig. 3). Double labelling experiments showed that both antibodies reacted with most but not all of the same fibers. This is additional evidence that G8 recognizes tubulin. Very little staining was observed with anti-DNP or normal rabbit serum.

Developmental expression of G8-tubulin in the rat brain The relative abundance of the G8 antigen increases during development, unlike total tubulin. On immunoblots of equal protein loadings of El7, P1, and adult brain homogenates, G8 staining intensity of both bands increased with age; the increase in the faster band was more dramatic (Fig. 4a). On an immunoblot of equal protein loadings of P1, P10, P22, and adult cytoplasmic fractions, a striking increase in G8 labeling of the faster band was apparent between P1 and P10 (Fig. 4b). Immunoblots of P1, P6, and P10 rat brain homogenates

indicated that this dramatic increase occurs between P1 and P6 (data not shown). In contrast with G8 but consistent with reports of fl-2-tubulin expression 36, anti-tub2 staining of the faster band increased slightly between El7 and P1 and then decreased greatly between P1 and adulthood (Fig. 4a,b). Staining intensity with anti-tubl similarly decreased in the faster band between P1 and adult ages (data not shown).

Distribution of G8-tubulin in the rat brain Immunohistochemistry was performed on cryostat brain sections from perfused rats. The dentate gyrus of the hippocampus undergoes extensive development postnataUy5, paralleled by an increase in G8 reactivity (Fig. 5). At P1, G8 staining in the hippocampus was very weak. Between P6 and P10, granule cell dendrites in the molecular layer of the dentate gyrus became reactive with

111 G8. Staining in the hilus region, in which granule cell axons are found, was not as intense. By P16, dendrite labeling in the molecular layer was more intense than staining in other brain regions. By P20, selective staining of granule cell dendrites was striking. In CA1 and CA3 of the hippocampus, G8 reactivity also increased during development. Pyramidal cell apical dendrites in the stratum radiatum were labeled most intensely (Fig. 6). In the adult, pyramidal cell basal dendrites or axons in the oriens layer stained weakly. Like the dentate gyrus, the cerebellum develops postnatally2; G8 reactivity correlated with Purkinje cell development (Fig. 7). At P6 some short processes, probably Purkine cell dendrites, stained in the molecular layer. Some additional processes also stained; these could be glial cell fibers or those which Airman described as 'unidentified processes with a tubular organization '1. Between P10 and P16, the Purkinje cell dendritic arbor develops, accompanied by extensive G8 labeling of primary and secondary dendrites. The staining intensity of Purkinje cell perikarya also increased with age beginning at P10. At all ages, structures other than Purkinje cell dendrites in the molecular layer stained very weakly. We did observe some staining of unidentifiable structures in the granule layer at P20 and especially in the adult. Anti-tubl, anti-tub2, and anti-tub3 all stained more generally throughout the cerebellum than did G8 (Fig. 8). Purkinje cell somas and short stretches of Purkinje cell dendrites were stained with anti-tubl (not shown) and anti-tub3. Anti-tub2 did not stain Purkinje cell bodies. It could not be determined whether anti-tub2 stained Purkinje cell dendrites. Anti-tub1, anti-tub2, and anti-tub3 all stained beaded fibers and other punctate structures in both the molecular and granule cell layers. These staining patterns were observed even when the antibodies were diluted to different concentrations; even at very low concentrations anti-tubl, anti-tub2, and anti-tub3 staining did not resemble G8 reactivity. In the retina, G8-stained processes in the ganglion cell layer most intensely and faintly labeled processes in the inner and outer plexiform layers (Fig. 9). This staining pattern is similar to that observed with antibodies which stain Muller glial cell fibers 17'18. If G8 staining is of Mfiller glial cell fibers, differences in staining intensity in the ganglion versus plexiform layers cannot be explained. In contrast to the specific staining pattern observed with G8 in the retina, the other 3 anti-tubulin antibodies stained generally and strongly in all layers (data not shown). G8 did not stain the dorsal root ganglion, part of the peripheral nervous system (Fig. 10). Anti-tub2 and anti-tub3 stained both cell bodies and processes in this region (Fig. 10). In the same experiment, G8 did stain

processes in the white and gray matter of the spinal cord (data not shown). Throughout the brain most G8 labeling appeared to be of neuronal processes resembling dendrites. In general, G8 staining intensity increased with age from P1 to adult. Labeling was most striking in the cerebellum and hippocampus, particularly after P10. Processes in the cerebral cortex and primary olfactory cortex stained at all ages. Most other areas showed little labeling at any age. In several regions of the adult brain, localization of G8 staining to dendrites was confirmed by co-localization with the dendrite-specific anti-MAP2 antibody (data not shown). Few fiber tracts stained at any age, indicating limited axon staining. Beginning at P16, cell bodies in certain regions were labeled with G8. Anti-tubl, anti-tub2, and anti-tub3 were more generally reactive than G8 throughout the brain, as expected from reports by others T M who observed a general distribution of tubulin in the rat brain. Staining with anti-tubl, anti-tub2, and anti-tub3 was weaker at older ages, as expected from the known decrease in tubulin during development.

f

G8

Fig. 9. G8 staining in the adult rat retina. Sections (30 pm) were cut from the eye of a rat perfused with 4% paraformaldehyde, 0.34% lysine, 0.05% sodium periodate. G8 culture supernatant undiluted. ga, ganglion cell layer; ip, inner plexiform layer; b-h-a, bipolar, horizontal, amacrine cell layer; ph, photoreceptor cell layer, Nomarski optics. Bar = 20 pm.

112

Biochemical nature of the G8 epitope The biochemical nature of the G8 epitope is unknown. G8 did not react with the fusion protein made in E. coli transformed with any of the 4 identified fl-tubulin genes

expressed in mouse brain (data not shown; clones kindly provided by Prof. Nicholas Cowan, N.Y.U. Medical Center35). These are highly homologous to rat fl-tubulin genes, a-Tubulin clones were unavailable for testing. The possibility that G8 is specific for a phosphorylated epitope was investigated by treating an adult rat brain cytoplasmic fraction with E. coli alkaline phosphatase. G8 reactivity with treated and untreated protein was quite similar (data not shown). Phosphatase treatment of a rat brain Triton X-100-insoluble fraction virtually abolished reactivity with an antibody known to react only with a phosphorylated epitope of the largest neurofilament (SMI-3153), indicating that the enzyme was active. DISCUSSION

Fig. 10. Comparison of G8 with other anti-tubulin labeling in the adult rat dorsal root ganglion. Sections (30 pm) were cut horizontally from spinal cord with dorsal root ganglia from rat perfused with 4% paraformaldehyde, 0.34% lysine, 0.05% sodium periodate. G8 culture supernatant undiluted, anti-tub2 diluted 1:7500, anti-tub3 1:15,000. drg, dorsal root ganglion; a, axon region. Bar = 35 pm.

Our immunoblot results provide strong evidence that G8 recognizes a form of tubulin which we call G8tubulin. Our experiments do not allow us to determine whether G8-tubulin is an a- or fl-tubulin. Under similar two-dimensional gel conditions, a-tubulins have been defined as the more slowly migrating, more basic species and fl-tubulins have been designated as the faster migrating, more acidic species 6'4e'54. An accurate identification of a spot as a- or fl-tubulin must rely, however, on amino acid sequence; this has not been obtained for the G8-tubulin. The problem of distinguishing between a- and fl-tubulin is illustrated by the staining pattern of anti-tub2 (Fig. 2). Although anti-tub2 was generated against the carboxy-terminus of chick fl-2-tubulin, the antibody labeled both bands of the tubulin doublet in rat brain. It is not known whether either of these two bands is an a-tubulin. The immunohistochemical staining pattern of G8 was much more restricted than that of the other anti-tubulin antibodies. The fact that anti-tubl, a polyclonal antibody, stained structures and regions throughout the brain confirms other reports 9'44 that the combined distribution of many forms of tubulin is general throughout the rat brain. Anti-tub2, which is specific for fl-2-tubulin, stained more generally than G8 in the rat brain. This suggests that not all tubulin forms are as restricted in distribution as G8-tubulin. The strength of this conclusion is slightly weakened by our finding that anti-tub2 reacted with two bands on immunoblots and might recognize more than one type of tubulin on rat brain sections. The G8-tubulin appeared to be enriched in dendrites rather than axons; however, some G8 labeling of axons was also seen. The staining complexity near cell bodies and the faint labeling of thin processes made definitive identification of axons in the hippocampus and cerebellum difficult. In addition, G8 stained some fibers in the axon-rich spinal cord. The other anti-tubulin antibodies

113 stained most fibers in the spinal cord. The regional specificity of the G8-tubulin was exemplified by extensive labeling in the hippocampus and in cerebellar Purkinje cells compared to minimal staining in other regions. Characteristics common to only the G8positive brain regions are not obvious. The time at which a particular region develops does not correlate with G8 reactivity. Some G8-positive regions such as the cerebellum and dentate gyrus develop postnatally 2'5 but the G8-immunoreactive hippocampus develops between El7 and E l 9 in the rat 5. The restricted staining pattern of G8 throughout the brain is not likely to have been caused by limited accessibility to its epitope due to the large size of the IgM molecule, since anti-tub3, also an IgM antibody, did not produce a restricted staining pattern. The G8-tubulin has a unique distribution in the rat brain. Other anti-tubulin antibodies have been reported to stain selectively in the rat brain but none produce the same staining pattern as did G8. Acetylated a-tubulin is preferentially localized in granule cell parallel fibers and other axons in the cerebellum 1°. Tyrosinated a-tubulin is present in granule cell parallel fibers at postnatal day 10 but is absent from these axons in the adult 13'14. An antibody which is not specific for the tyrosinated form of tubulin stained throughout all layers of the cerebellum at all stages of development 14. A monoclonal antibody specific for a-tubulin stains rat hippocampus and cortex pyramidal cell apical but not basal dendrites or perikarya 3°. Antibodies specific for 3 synthetic peptides corresponding to the carboxy-terminus of fl-tubulin stained selectively in the rat cerebellum. One of these antibodies selectively stained Purkinje cells and their dendrites and axons; unlike G8, it stained axons in the cerebellar white matter as well. The other two antibodies produced staining patterns very different from that of G826. The specific distribution of G8-tubulin in the rat brain suggest a functional specificity. The function of G8tubulin is unknown. G8-tubulin's appearance later during development suggests that it is unlikely to be involved in cell proliferation or differentiation. This is supported by our observation that G8-tubulin appears to be absent from mitotic spindles 27. Its presence in the interphase cytoskeleton of Rat-1 fibroblasts as well as in some neurons and glia suggests that G8-tubulin might serve a structural purpose in a variety of cell types. The absence of G8-tubulin in the dorsal root ganglion might suggest that G8-tubulin is specific to the central rather REFERENCES 1 Altman, J., Postnatal development of the cerebellar cortex in the rat I. The external germinal layei"and the transitional molecular layer, J. Comp. Neurol., 145 (1972) 353-398.

than peripheral nervous system. However, since the dorsal root ganglion was the only PNS region examined, CNS specificity cannot be confirmed from these data. The biochemical nature of the tubulin epitope recognized by G8 is unknown. G8 did not react on an immunoblot with the fusion protein product made in E. coli from a plasmid encoding any one of the 4 identified i3-tubulin genes expressed in mouse brain. This may indicate that G8 reacts with the protein product of an unidentified/3-tubulin gene or that G8 recognizes only a-tubulins or that G8 recognizes a post-translational modification. It is also possible, though less likely, that the negative result could be explained by an interference of the/3-galactosidase portion of the fusion protein on G8 binding to its antigen. It seems unlikely from our experiments that phosphorylation is the post-translational modification giving rise to the G8 epitope. Most tubulin forms which have been reported to be selectively distributed in the rat brain 1°'13'14 and within the cell 29 are post-translationally modified. In contrast, subcellular sorting of different fl-tubulin gene products was observed not to occur 35'4°. By correlation this evidence suggests that the G8 epitope might be a post-translational modification. If G8 is specific a posttranslational modification, this modification may be present on more than one tubulin form or the modification state may vary since G8 recognizes more than one species on immunoblots. The results described here demonstrate the developmentally regulated, selective distribution of a novel tubulin in the rat brain. It is possible that this distribution is achieved by selective masking of the epitope in other regions of the brain by microtubule associated proteins. This distribution suggests that the G8-tubulin might polymerize into functionally specific microtubules. Further characterization of G8-tubulin might contribute not only to a description of a particular tubulin isoform but also to an understanding of the shared characteristics of the cells in the regions of the brain in which the G8-tubulin is abundant. Acknowlegements. We thank Wendy Katz, Margaret Magendantz, and Prof. Frank Solomon for the anti-tub2 and anti-tub3 antibodies and bovine purified tubulin and Prof. Herman Eisen for the anti-DNP antibody. We also appreciate comments on the manuscript from our colleagues Timothy H. Bestor, Corinne E. Miller, and Nora Perrone-Bizzozero. This work was supported by a grant from the Whitaker Health Sciences Fund to V.M.I.J.M.G. was supported by a National Science Foundation predoctoral fellowship.

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