A monoclonal antibody, raised against mammalian centrosomes and screened by recognition of plant microtubule organizing centers, identifies a pericentriolar component in different cell types

VANTARD 0 ANNE-MARIE LAMBERT 0 DIDIER JOB 1 0 Institut de Biologie Moliculaire des Plantes , CNRS, 12 rue du Gtniral Ztmmer, 67084 Strasbourg Cedex , France 1 Dipartement de Biologie Moltculaire et Struclurale, Laboratoire du Cytosquelette, INSERM U244, Centre d'Etudes Nucle'aires , 85 X, 38041 Grenoble Cedex , France A monoclonal antibody, raised against mammalian centrosomes and screened by recognition of plant microtubule organizing centers, identifies a pericentriolar component in different cell types 'Author for correspondence - We have used monoclonal antibodies raised against isolated native calf thymus centrosomes to probe the structure and composition of the pericentriolar material. To distinguish prospective antibodies as specific to conserved elements of this material, we screened clones by their identification of microtubule organizing centers (MTOCs) in different animal and plant cells. Among the clonal antibodies that reacted with MTOCs in both plant and mammalian cells, we describe one (mAb 6C6) that was found to immunostain centrosomes in a variety of bovine and human cells. In cycling cells this signal persisted through the entire cell cycle. Microscopy showed that the mAb 6C6 antigen was a component of the pericentriolar material and this was confirmed by biochemical analysis of centrosomes. Using Immunoblot analysis of protein fractions derived from purified components of centrosomes, we have characterized the mAb 6C6 antigen as a 180 kDa polypeptide. We conclude that we have identified a protein component permanently associated with the pericentriolar material. Microtubules are filamentous structures in the cytoplasm of eucaryotic cells where they perform a remarkable range of functions (for reviews see Dustin, 1984). They play a central role in the organization of the cell interior and in the building of the mitotic apparatus (for reviews see Alberts et al. 1989). Microtubule organizing centers (MTOCs) are required for the assembly, proper organization, and polarity of microtubule networks (Bergen et al. 1980; Mclntosh, 1983; Bornens and Karsenti, 1984; Tucker, 1984; Brinkley, 1985). In animal cells, the major MTOC is the centrosome. Surprisingly, monoclonal antibody 6C6 also stained other mitotic organdies in mammalian cells, in a cellcycle-dependent manner. During prometaphase and metaphase the antibody stained both centrosomes and kinetochores. At the onset of anaphase the klnetochorespecific staining dissociated from chromosomes and was subsequently redistributed onto a newly characterized organelle, the telophase disc while the centrosomal stain remained intact. It is not known if the 180 kDa centrosomal protein itself redistributes during mitosis, or if the pattern observed represents other antigens with shared epitopes. The pericentriolar material is thought to be composed of conserved elements, which appeared very early during the evolution of eukaryotes. Our results strongly suggest that mAb 6C6 identifies one of these elements. Centrosomes have a dual structure. They comprise highly organized and specific elements, namely the centrioles, and a poorly defined pericentriolar material that shows extraordinary flexibility in its organization, location and functions (Mazia, 1984; Karsenti and Maro, 1986; Vorobjev and Nadezhdina, 1987; Bornens, 1991). It shifts from a diffuse intracellular distribution to aggregated forms that appear at appropriate places and possibly induce the formation of centrosomes or of other forms of microtubule organizing centers (Rieder and Borisy, 1982; Maro et al. 1985; Tassin et al. 1985; Karsenti and Maro, 1986; Br6 et al. 1990). Cytoplasmic microtubules are not nucleated directly on centrioles. They originate from the pericentriolar material (Buendia et al. 1992; for reviews see Cande, 1990; Huang, 1990). The explanation of the function of centrosomes in molecular terms relies to a large extent on the biochemical and structural characterization of their components. Several centrosomal proteins have been identified (Frasch et al. 1986; Gosti et al. 1987; Buendia et al. 1990; Moudjou et al. 1991) and for a number of these the cDNA has been cloned and characterized (Snyder and Davis, 1988; Whitfield et al. 1988; Joswig et al. 1991), including ^tubulin (Horio et al. 1991; Stearns et al. 1991; Zheng et al. 1991). However, highly purified centrosomes show complex protein profiles (Bornens, 1991) and the majority of their components remain to be characterized. We have recently described preparative procedures yielding relatively high amounts of purified centrosomes from calf thymus (Komesli et al. 1989). Here, we have used these preparations to generate monoclonal antibodies directed against elements of the pericentriolar material. We report the identification of a protein that in animal cells is specifically and tightly associated with the pericentriolar material and also combines with kinetochores and the midplate during mitosis. In plant cells, a related antigen is found at the expected location of microtubule organizing centers. Materials and methods Materials Ingredients used for the construction of phosphate buffered saline (PBS: 150 mM NaCl, 10 mM sodium phosphate, pH 7.4), bovine serum albumin (BSA), TNM buffer (10 mM TrisHC1, pH 7.2, 10 mM NaCl, 5 mM MgCl2), Pipes buffer (10 mM Pipes, pH 7.2, with KOH), MCM buffer (25 mM Mes, 8 mM CaCl2, 600 mM mannitol, pH 5.5) and Hoechst dye 33258 were from Sigma Chemical Co., St Louis, MO, USA. Urea, 2-mercaptoethanol, uranyl acetate and tannic acid were from Merck, Darmstadt, RFA; osmium tetroxide from Fluka, Mulhouse, France; Epon from TAAB, St Germain-enLaye, France; SDS and Aquamount from BDH, Chassieu, France; Tween 20 (EIA Grade) from Bio-Rad, Ivry, France; ECL Kit from Amersham, les Ulis, France; PVDF Immobilon from Millipore Corporation, Bedford, MA, USA; glutaraldehyde from Polysciences Inc., Warrington, PA, USA. Culture media were from Boehringer Mannheim Diagnostic, Inc, Houston, TX, USA. HeLa cells, SP2O/Agl4 (non-secreting mouse myeloma) and Raji cells (Burkitt lymphoma, human) were kindly provided by Dr R. Berthier. 2017 cells (bovine foetal calf spleen cells) were a generous gift from Dr B. Guillemin. Endosperm cells were isolated from young ovules of Haementhus katherinae bak. and prepared. as previously described (Bajer and Mol6-Bajer, 1986). Balb/c mice were obtained from IFFA CREDO, L'Arbresle, France. Vinblastine sulfate and nocodazole were purchased from Aldrich, Strasbourg, France. Pectolyase Y23 was from Seishin Pharmaceutical Co., Tokyo, Japan; Macerozyme R-10 from Serva, Heidelberg, RFA, and Caylase from Cayla, Toulouse, France. mAb YLl/2, a monoclonal antibody specific to tyrosinated tubulin (Wehland et al. 1983) was a generous gift from Dr J. V. Kilmartin. A polyclonal antibody specific for detyrosinated tubulin was kindly provided by Dr J. C. Bulinski (Gundersen et al. 1984) and CREST serum was a generous gift from Dr B. Rousse (...truncated)