Reconstitution of microtubule nucleation potential in centrosomes isolated from Spisula solidissima oocytes.

Treatment of isolated Spisula solidissima centrosomes with KI removes (gamma)-tubulin, 25 nm rings, and their microtubule nucleation potential, revealing the presence of a filamentous lattice, the 'centromatrix'. Treatment of this centromatrix with Spisula oocyte extract results in the binding of (gamma)-tubulin and 25 nm rings, and the recovery of microtubule nucleation potential. Fractionation of this extract resulted in the separation of elements that are required for the recovery of microtubule nucleation potential. We show that some, but not all, of the elements needed cosediment with microtubules. Further, extracts prepared from activated (meiotic) and non-activated (interphase) Spisula oocytes, CHO cells blocked in S phase, Drosophila embryos and Xenopus oocytes all support the recovery of microtubule nucleation potential by the Spisula centromatrix. These results demonstrate that components necessary for centrosome-dependent microtubule nucleation are functionally conserved and abundant in both interphase and meiotic/mitotic cytoplasm.

Autoantibodies from a patient with scleroderma CREST recognized kinetochores of the higher plant Haemanthus.

Human autoantibodies from a patient with scleroderma CREST (calcinosis, Raynaud phenomenon, esophageal dismotility, sclerodactyly, telangiectasia) were used to immunostain kinetochores on chromosomes in endosperm of the seed of the monocot Haemanthus katherinae Bak. Kinetochores of mitotic chromosomes and prekinetochores of interphase cells were specifically stained using conventional indirect immunofluorescence procedures as well as a nonfading immunogold-silver-enhanced technique and analyzed by fluorescence and video microscopy. In interphase, prekinetochores were either single or double structures depending on the stage of the cell cycle but became quadruple (two distinct stained dots on each chromatid) in mid-to-late prophase. In favorable preparations of prometaphase chromosomes, multiple subunits could be resolved within each sister kinetochore suggesting a compound organization. Western blot analysis demonstrated common epitopes in centromeric peptides of HeLa and Haemanthus cell extracts. Although the molecular mass of individual polypeptides differed in the two species, the presence of shared epitopes indicates striking conservation of centromere/kinetochore components throughout evolution.

Synthesis of azidotubulin: a photoaffinity label for tubulin-binding proteins.

A photoaffinity label for the identification of tubulin-binding proteins was synthesized from phosphocellulose-purified bovine brain tubulin and (N-hydroxysuccinimidyl)-4-azidosalicylic acid. The azidotubulin derivative retained the ability to undergo temperature-dependent microtubule assembly and disassembly. When incubated with purified tau protein, the azidotubulin and tau formed cross-linked complexes upon photoactivation. When 125I-labeled azidotubulin was used to photoaffinity label tubulin-binding proteins within the kinetochore of isolated mammalian chromosomes, a 130-kDa band was identified on autoradiographs of SDS-polyacrylamide gels of the 125I-labeled azidotubulin/chromosome preparations. The 130-kDa complex was isolated by antitubulin affinity chromatography and analyzed by immunoblotting using both antitubulin and kinetochore-specific sera obtained from human patients with the autoimmune disease scleroderma CREST. The immunoblots demonstrated that the 130-kDa band that was observed on autoradiographs was a complex of a subunit of the tubulin dimer and an 80-kDa CREST-specific kinetochore protein. The binding of azidotubulin to the 80-kDa kinetochore protein was significantly decreased when chromosomes were treated with a mixture of 9 parts underivatized tubulin to 1 part azidotubulin prior to photolysis. The formation of the 130-kDa azidotubulin/kinetochore protein complex was not inhibited by pretreating the chromosomes with CREST serum prior to incubation with azidotubulin. Azidotubulin should be a useful probe for the identification and characterization of tubulin-binding proteins.

Tubulin interaction with kinetochore proteins: analysis by in vitro assembly and chemical cross-linking.

The sera from patients with the CREST (calcinosis, Raynaud's phenomenon, esophageal dysmotility, sclerodactyly, telangiectasia) variation of the autoimmune disease scleroderma contain autoantibodies that specifically recognize the kinetochore by immunofluorescence. Two major antigens of molecular masses 18 and 80 kD are consistently identified by Western blotting of proteins of isolated chromosomes using CREST sera. In this paper, the possible roles that these two proteins play in the interaction of metaphase chromosomes with tubulin and microtubules are examined using two different procedures. In one set of experiments. Chinese hamster ovary (CHO) chromosomes were extracted with 1-2 M NaCl before incubating with phosphocellulose-purified tubulin under in vitro microtubule assembly conditions. After this treatment, the kinetochores of the residual chromosome scaffolds can still initiate the in vitro assembly of microtubules. Immunoblots of the chromosome scaffold proteins demonstrate that the 18-kD protein has been solubilized by the 1-2 M NaCl extraction, suggesting that this protein is not essential for microtubule assembly at the kinetochore. In a second approach, tubulin was covalently cross-linked to kinetochores of CHO chromosomes using the reversible cross-linking reagent dithiobis (succinimidyl propionate). After DNase I digestion, the chromosomes were solubilized and subjected to anti-tubulin affinity chromatography. Tubulin-kinetochore protein complexes were specifically eluted and analyzed by PAGE and immunoblotting with scleroderma CREST serum. Only a small number of proteins were eluted from the antitubulin affinity column as shown by Coomassie Blue-stained gels. In addition to tubulin, an 80-kD polypeptide, bands at 110 and 24 kD, as well as a faint band at 54 kD, can be resolved. Several minor bands can also be seen in silver-stained gels. The 80-kD protein band from whole metaphase chromosomes reacted with scleroderma CREST serum by immunoblotting and therefore probably represents the major centromere antigen CENP-B. This report provides evidence for a specific protein complex on metaphase chromosomes that is contiguous with kinetochore-bound tubulin and may be involved in microtubule-kinetochore interactions during mitosis.

Arrangements of kinetochores in mouse cells during meiosis and spermiogenesis.

Antibodies from the serum of patients with the autoimmune disease scleroderma CREST were used to investigate the association and distribution of kinetochores in mouse cells during meiosis and spermiogenesis. The pattern of indirect immunofluorescent staining in pachytene nuclei indicated that each autosomal bivalent contains one fluorescent spot. Throughout pachytene, the kinetochores were arranged non-randomly into several clusters and distributed around the periphery of the nucleus. In subsequent stages of meiotic prophase I, distribution was random and the number of fluorescent spots increased from 21 to 40 corresponding to the diploid chromosome number and the number of halfbivalents oriented to the spindle poles at the metaphase I. Twenty pairs of kinetochores were observed at metaphase II. During spermiogenesis, the number of kinetochores correlated with the haploid chromosome number in early spermatids but tandem association of centromeres and clustering into a conspicuous chromocenter corresponded to a significant reduction in the number of fluorescent foci in mid-spermatid nuclei. The number of stained sites per nucleus continued to decrease during sperm maturation and total absence of staining was apparent in mature spermatozoa. Immunoblotting of proteins extracted from mature sperm however, indicated that a kinetochore antigen of Mr 80,000 was still present. Therefore, the absence of kinetochore staining in mature spermatozoa is probably due to the blockage of epitopes during chromatin condensation.