Fractionation and from Mammalian Initial Characterization of the Metaphase Chromosomes Kinetochore

We have partially isolated the kinetochore and associated centromeric structures from mammalian metaphase chromosomes. Human autoantibodies from scleroderma CREST (calcinosis, Raynaud's phenomenon, esophageal dysmotility, sclerodactyly, telangiectasia) patients were used as immunofluorescent probes to monitor fractionation. The procedure includes digestion of total chromosomal DNA with micrococcal nuclease, dehistonization with heparin, and dissociation of the remaining material with detergent and urea. We used a density gradient (metrizamide) to obtain an enriched fraction of stained material (kinetochore). When examined by electron microscopy, the kinetochore fraction is seen to contain numerous small immunoperoxidase-positive masses which are morphologically similar to the centromere/ kinetochore region of intact metaphase chromosomes. The particulate fraction that contains kinetochore components represents <5% of total chromosomal proteins and contains <1% of total DNA. Two polypeptides of 18 and 80 kD were identified as kinetochore antigens by immunoblotting with CREST antiserum. In this paper we discuss the distribution of these kinetochore polypeptides with the associated centromeric chromatin. The genetic information encoded in interphase chromatin is uniformly distributed to the daughter cells in normal mitosis. One specific chromosomal locus, the kinetochore, is believed to be responsible for chromosome attachment to the mitotic spindle and segregation during mitosis and meiosis. Although the cytology and ultrastructure of the kinetochore are well documented (6, 9, 16), relatively little is known of its biochemical composition. As shown by ultrastructural studies, the typical kinetochore of metaphase chromosomes consists of a trilamellar disk composed of a dense outer layer, a lightly stained middle layer, and a dense inner layer juxtaposed with the centromeric chromatin. The kinetochore-associated microtubules attach to the outermost layer. Although very little is known of the molecular composition of the kinetochore plates, cytochemical studies have suggested the presence of RNA (30), DNA (28), and tubulin (26). A major advance in the investigation of kinetochores came with the discovery of human autoantibodies from scleroderma CREST ~ (calcinosis, Raynaud's phenomenon, esophageal dys~ Abbreviations used in this paper: CHO, Chinese hamster ovary; CREST, calcinosis, Raynaud's phenomenon, esophogeal dysmotility, motility, sclerodactyly, telangiectasia) patients that selectively stain centromeres of metaphase chromosomes by indirect immunofluorescence (23). Using immunoperoxidase staining at the electron microscopic level, Brenner et al. (5) discovered that CREST antiserum binds specifically to kinetochore plates. In the present study, we have used CREST antiserum as an immunofluorescent probe to monitor the steps of extraction and fractionation of the kinetochore from metaphase chromosomes and to identify some of its polypeptide components by immunoblott ing experiments. MATERIALS AND METHODS Cultured Cells and Chromosome Isolation: HeLa cells were grown in McCoy's 5a medium supplemented with 8% fetal calf serum. The ceils were synchronized with 2.5 mM thymidine (Sigma Chemical Co., St. Louis, MO) added to the medium for 20 h and then treated with colcemid at 0.06 pg/ml for 16 h. Chinese hamster ovary cells (CHO) were grown in Ham's F-10 medium supplemented with 7% fetal calf serum. In this case, ceils were partially synchronized by decreasing the serum in the media to 2% for 14 h sclerodactyly, telangiectasia; HMG, high mobility group; MNAse, micrococcal nuclease; TEP, 10 mM Tris, pH 7.1, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride. 1124 THE JOURNAL OF CELL BIOLOGY • VOLUME 101 SEPTEMBER 1985 1124 1134 © The Rockefeller University Press 0021-9525[85/09/1124/11 $1.00 and then incubating for 9 h in colcemid at 0.1 pg/ml in media supplemented with 7% fetal calf serum. The mitotic cells in both cases were collected by shake offand centrifugation. A mitotic index of 98% for HeLa and 50-70% for CHO cells was commonly obtained. For a typical preparation, metaphase chromosomes were isolated from l0 s mitotic cells by the hexylene glycol method of Wray and Stubblefleld (37). After isolation, the crude chromosome suspension was purified by a 30-ml 1050% (vol/vol) glycerol discontinuous gradient in hexylene glycol buffer (13). The chromosomes were sedimented through the gradient by centrifugation at 1,000 rpm in a Beckman JS-13 swinging bucket rotor (Beckman Instruments Inc., Palo Alto, CA) for 50 rain at 4"C. 3-ml fraction were collected from the top of the gradient. Those containing purified chromosomes as determined by phase-contrast microscopy (fractions 4-7, typically) were then pooled. Purified chromosome suspensions containing glycerol were centrifuged in a Beckman JA-20 rotor for 15 min at 5,000 rpm at 4"C. The chromosomes were stored in hexylene glycol buffer at 4*(]. Protocol for Kinetochore Fractionation: Purified chromosomes (either from HeLa or CHO mitotic cells) in hexylene glycol buffer were centrifuged at 3,000 rpm for l0 min at 4"C and resuspended in nuclease digestion buffer (10 mM Tris, pH 7.1, 2 mM CaCl2 containing l mM phenylmetbylsulfonyl fluoride). The volume of the chromosome suspension was usually 4-5 ml and the A2~ was 7-10. To this, micrococcal nuclease (MNAse) Worthington Biochemical Corp., Freehold, N J) was added at a final concentra, tion of 200 U/ml, and the solution was incubated at 37*(] for 60 min with occasional gentle swirling. Later, the suspension was centrifuged in a Beckman JA-20 rotor at 5,000 rpm for l0 rain at 4"C, and the supernatant was stored at 4"C. The pellet was resuspended in l0 mM Tris, pH 7.1, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride (TEP buffer). Heparin (Sigma Chemical Co.) at 2 mg/ml was added, and the solution was incubated at 4"C for 120 min with constant shaking. The suspension was centrifuged at 5,000 rpm for 15 min at 4"C, and the supernatant was kept at 4"C. After this spin the pellet was resuspended in TEP buffer. Nonidet P-40 (BDH Chemicals, Poole, U.K.) plus cholate (Sigma Chemical Co.) both at a final concentration of 1% were added, and the suspension was incubated for 60 min at 4"C with constant shaking. The suspension was then centrifuged as above. The superuatant was stored at 4"C and the pellet was resuspended in TEP buffer and incubated for l0 rain at 4"C in the presence of 2 M urea. The suspension was then centrifuged at 10,000 rpm for 15 min at 4"C. The preparation was layered onto a 3 ml 10-50% (wt/ vol) metrizamide (Nyegaard Co., Oslo) gradient in TEP buffer and centrifuged at 4"C for 15 min at 8,000 rpm in a Beckman JS-l 3 swinging bucket rotor. 0.3ml fractions were collected from the top and checked for kinetochore staining by immunofluorescence and electron microscopy. Immunofluorescence and Electron Microscopy: The different steps in the protocol for kinetochore isolation were monitored by centrifuging aliquots onto microscope coverslips coated with poly-L-lysine (Sigma Chemical Co.) at 1 mg/ml and staining first with kinetochore antiserum and then with anti-human IgG labeled with fluorescein isothiocyanate. Sometimes the preparations were also stained with the DNA-binding fluorochrome Hoechst 33258 (American Hoechst Corp., Somerville, NJ) or propidium iodide for visualization of DNA. The immunofluorescence procedure was similar to that described by Pepper and Brinkley (27). Whole chromosomes and aliquots at each step of the kinetocbore fractionation procedure were processed for electron microscopy by the immunoperoxidas¢ technique as described by Brenner et al.