A novel tau transcript in cultured human neuroblastoma cells expressing nuclear tau.

Cell Culture

The cell lines N2A (mouse neuroblastoma) and JC (human neuroblastoma) were cultured in DME supplemented with 10% FBS (Gemini). CG cells (human neuroblastoma) were cultured in DME supplemented with 5% horse serum (GIBCO BRL, Gaithersburg, MD). CHO cells were cultured in McCoy's 5A medium supplemented with 10% CPSR-4 (Controlled Pro- cess Serum Replacement; Sigma, St. Louis, MO). All cell lines were main- tained at 370C in a humidified 5% CO2 atmosphere. Mitotic Cell Preparations Cells were incubated in 0.1/~g/ml of Colcemid for 12 h. Mitotic cells were dislodged from the culture flask by gently shaking the flask. Subsequently, the cells were pelleted by centrifugation (1,000 g for 5 mill), resuspended in PBS, pH 7.4, and washed twice in PBS. The mitotic ceils were swollen in hypotonic (0.075 M KCI) for 5 rain at 370C and then cytospun onto glass coverslipsat 1,500 rpm for 2 rain using a ShandonCytospin (Shandon South- ern Instruments Inc., Sewickley, PA), and then fixed in 3% EM grade form- aldehyde (Tousimis Res. Co., Rockville, MD) in PBS for 30 rain at 24C. Indirect lmmunofluorescence Cells cultured on glass coverslips and fixed with 3% formaldehyde for 30 rain at 24C and mitotic cell preparations described above were pro- cessed for indirectimmunofluoreseencemicroscopy as previously described (Loomis et al., 1990). Briefly, the cells were permeabilized with 0.5% Tri- ton X-100 in PBS for 5 rain, rinsed twice in PBS, reduced in 0.5/~g/ml NaBH4 in PBS for 4 rain, and blocked with 1% BSA in PBS for 10 min. To determine if JC cells contained microtubule-associated tan, the ceils were detergent extracted in microtubule-stabilizingbuffer (to remove solu- ble cytoplasmic proteins) prior to formaldehyde fixation using the protocol of Steuer et al. (1990). Briefly, ceils on coverslipswere rinsed in PBS, placed in PEEM (0.1 M Pipes, pH 6.9, 1 mM MgSO4, 0.1 mM EDTA, and 2 mM EGTA) to stabilize microtubules and then placed in 0.5% Triton X-100 in PEEM containing0.1 mM PMSF and 1mg/ml each of chymostatin, leupep- tin, antipain, and pepstatin. The cells were then stained with the Tan-1 IgG mAb (1:40) followed by fluorescein-conjugated IgG specific sheep anti- mouse and rabbit anti-sheep antibodies respectively (1:20; Boehringer Mannheim Biochemicals, Indianapolis, IN). For tau-microtubule double- label preparations, ceils were further processed using a monoclonal IgM antibody to tubulin (5H1) followedby a goat anti-mouse IgM specificsecon- dary antibody conjugated to Texas red. All antibody incubations were for 30 rain at 37C in a humidified atmosphere. Each incubation was followed by three 5-rain washes in PBS. The preparations were counterstained with 1 /~g/mi Hoechst 33258 (Sigma) in PBS for 5 min to visualize the DNA. The processed coverslips were mounted onto glass slides with a 10:1 (vol/ vol) mixture of glycerol/PBS containing 0.1% p-phenylenediamine(Sigma) to reduce photobleaching, and then sealed with nail polish.

Computer-enhanced VideoMicroscopy and Image Analysis

Cells processed for indirect immunofluorescence were analyzed using a Nikon Optiphot epifluorescence microscope equipped with differential in- The Journal of Cell Biology, Volume 121, 1993 258 terference contrast (DIC) optics and a Dage series 68 SIT video camera (Dage-MTI Inc., Wabash, WI) coupled to an Image-I image analysis system (Universal Imaging, Westchester, PA). Image processing was performed by settingthe gain and black level on beth the computer and camera to provide an optimal signal. Then 128 video frames were summed into the foreground frame buffer, the image was defoeused, and 128 video frames were summed into the background frame buffer which was subsequently subtracted from the foreground buffer. The resultant image was subjected to further contrast enhancement. Overlays of double fluorescence were obtained by aligning processed images in separate frame buffers using discernible fiduciary points within the images and then superimposing the images by adding them together using the "average" logic function of Image-1. The images were printed with a Sony UP-5000 color video printer (Sony, Mont- vale, NJ).

Nuclear Isolation and Extraction

Nuclear and cytoplasmic fractions from the various cell lines were isolated as described by Mitchison and Kirschner (1985). The isolated nuclei were washed, resuspended in 50 mM Tris, pH 6.8, sonicated for 30 s, extracted with 2.5% formic acid for 60 s and then centrifuged in a microfuge for 2 rain to pellet insoluble material. The supernatant was then desalted into 25 mM Tris-HCl (pH 6.8) and solubilized by boiling in electrophoresissample buffer (62.5 mM Tris, pH 6.8, 2% SDS, 10% glycerol, 5% 2-mercapto- ethanol; Laemmii, 1970). Cytoplasmic fractions were concentrated using an ultrafiltration device (Diaflo, YM10 filter; Amicon Corp., Arlington Heights, IL) prior to solubilization with electrophoresis sample buffer as described above. The purity of nuclearand cytoplasmic fractions was deter- mined by phase microscopy and fluorescence microscopy using the DNA bindingdye Hoechst 33258. Additionally, the purity of the nuclear fractions was confirmed by immunoblotting with mAbs to tubulin as previously de- scribed (Loomis et ai., 1990). SDS-PAGE and WesternBlotting Protein concentrations were determined by a modification of the method of Lowry et al. (1951). 25 ttg of an extract from human frontal cortex, 75 ttg of nuclear extracts from all the cell lines, 50 pg of the cytoplasmic extracts from CHO, CG, and JC and 25 Itg of the cytoplasmic extract from N2A were separated by SDS-PAGE using a 5-12.5% linear polyacrylamide gra- dient gel (Laemmli, 1970). Less total N2A cytoplasmic protein was loaded because N2A cells expressed more tau than the other cell lines assayed. After electrophoresis, proteins were transferred to nitrocellulose (Towbin et al., 1979) and probed with the mouse mAb, Tau 46.1. Bound antibodies were detected using a peroxidase-conjugated secondary antibody followed by reaction with H2Oz and an appropriate chromogen as described previ- ously (Binder et al., 1985). mRNA Isolation and Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR) Poly A+ mRNA was isolated from cultured cells by the method of Badley et al. (1988) and separated by electrophoresis on denaturing 1% low melt agarose gels containing 2.2 M formaldehyde (Sambrook et al., 1989). The 2- and 6-kb regions of the resultant RNA gels were excised, the mRNA ex- tracted withphenol/chloroform, precipitated withethanol, and reverse tran- scribed to eDNA using the 1st Strand Synthesis Kit from Stratagene (La Jolla, CA). PCR was performed with Taq polymerase (Perkin Elmer/Cetus, Norwalk, CT) using the conditions specified by the manufacturer. Briefly, 5 td of eDNA from the RT-eDNA pool was amplified by adding 25 pmol of each primer, 200 ttM of each dNTP, 1x reactionbuffer(10mM Tris HC1, pH 8.3, 50 mM KCl, 1.5 mM MgC12, 1% gelatin), and 2.5 U of Taq poly- merase in a final volume of 100 td. The samples were subjected to 30 cycles of denaturation (I rain at 94"C)1 annealing (2 rain at 55C) and extension (3 min at 72C) using a DNA Thermal Cycler 480 (Perkin Elmer/Cetus). The products of the reaction were analyzed on a I% agarose/ethidium bro- mide gel. PCR was first performed with two primers (nucleotides [-6]-12 and nucleotides 1308-1326) to amplify the whole coding region of human tan. The nucleotide numbers listed here and below refer to the longest hu- man tau isoform (441 amino acids) containing the fourth repeat in the microtubulebindingdomain (exon 10)as well as exons 2 and 3 in the amino end of thetau molecule (Goedert et al., 1989). The resultantproducts could not be seenwith ethidiumbromide stainbut were detected by Southernblots (data not shown). These low abundance cDNAs were reamplified with two primer pairs that amplify from the beginningof exon 1 (nucleotides [-6]-12) to the middle of exon 9 (nucleotides 669-696) as well as from the middle of exon 9 (nucleotides 669-696) to the 3' end of the tan coding region (nucleotides 1308-1326). The primers used within exon 9 provided an over- lap region to assure that the entire coding region of tan was present in the 2-kb message. The PCR fragments generated from the 2-kb mRNA were separated on a 1% agarose gel, stained with ethidium bromide, and the ap- propriate band was excised, purified and ligated into the sequencing vector (TA cloningTM from Invitrngen). DNA sequencing was performed by the dideoxy-chain termination method using the Sequenase DNA sequencing kit (version 1.0; United States Biochemical Corp., Cleveland, OH).

Northern Blot Analysis

Equal amounts of poly A+ mRNA extracted and purified as outlined above were loaded onto denaturing RNA gels and, after electrophoresis, trans- ferred to an Irnmobilon membrane (Mfllipore Corp., Milford, MA). Hy- bridization was performed for 20 h at 42"C in 50% formamide, 5x SSC, 5x Denhardt's, 1% SDS, and 100 ttg/ml salmon sperm DNA using a human three repeat tau eDNA (courtesy of Dr. Gloria Lee, Harvard Medical School, Cambridge, MA) labeled by nick-translation with ot-32P-dCTP. After washing, the blots were dried and then autoradiographed at -70C for 1-2 d. Transient Transfection of Tau cDNA CHO cells which have been shown not to express tan were plated on cover- slips. The pEn123c eukaryotic expression vector (generously provided by Dr. Gloria Lee) containing the whole coding region of three repeat short human tan (lacking exons 2, 3, and 10) was transfected into cultured CHO cells using the LIPOFECTIN reagent (GIBCO BRL). The CHO cells were cultured in a six-well plate until 40-60% confluent. 4 Izgs of DNA was added with the LIPOFECTIN reagent according to the manufacturer's pro- tocol. The cells were incubated for 6 h at 37C and then the DNA- LIPOFECTIN-containing medium was removed. The cells were cultured in fresh serum-containingmedium for an additional42 h before fixation and processing for IF using the Tau-1 mAb.