Centrosomal Components Immunologically Related to Tektins from Ciliary and flagellar Microtubules
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Journal of Cell Science 107, 2095-2105 (1994) 2095 Printed in Great Britain © The Company of Biologists Limited 1994 Centrosomal components immunologically related to tektins from ciliary and flagellar microtubules Walter Steffen*, Elizabeth A. Fajer† and Richard W. Linck University of Minnesota, Department of Cell Biology and Neuroanatomy, Minneapolis, MN 55455, USA *Present address: University of Vienna, Institute of Biochemistry and Molecular Cell Biology, Dr. Bohrgasse 9, A-1030 Vienna, Austria †Present address: Florida State University, Institute of Molecular Biophysics, Tallahassee, FL 32306-3015, USA SUMMARY Centrosomes are critical for the nucleation and organiz- same protein. Four independently derived monoclonal ation of the microtubule cytoskeleton during both inter- anti-tektins were found to stain centrosomes of S. solidis- phase and cell division. Using antibodies raised against sea sima oocytes and CHO and HeLa cells, by immunofluores- urchin sperm flagellar microtubule proteins, we charac- cence microscopy. In particular, the centrosome staining of terize here the presence and behavior of certain compo- one monoclonal antibody specific for tektin B (tekB3) was nents associated with centrosomes of the surf clam Spisula cell-cycle-dependent for CHO cells, i.e. staining was solidissima and cultured mammalian cells. A Sarkosyl observed only from early prometaphase until late detergent-resistant fraction of axonemal microtubules was anaphase. By immuno-electron microscopy tekB3 specifi- isolated from sea urchin sperm flagella and used to produce cally labeled material surrounding the centrosome, monoclonal antibodies, 16 of which were specific- or cross- whereas a polyclonal anti-tektin B recognized centrioles as specific for the major polypeptides associated with this well as the centrosomal material throughout the cell cycle. microtubule fraction: tektins A, B and C, acetylated α- Finally, by immunoblot analysis tekB3 stained polypep- tubulin, and 77 and 83 kDa polypeptides. By 2-D isoelec- tides of 48-50 kDa in isolated spindles and centrosomes tric focussing/SDS polyacrylamide gel electrophoresis the from CHO cells. tektins separate into several polypeptide spots. Identical spots were recognized by monoclonal and polyclonal anti- bodies against a given tektin, indicating that the different Key words: centriole, intermediate filament, mitosis, Spisula polypeptide spots are isoforms or modified versions of the solidissima, monoclonal antibody, immuno-electron microscopy INTRODUCTION Kimble and Kuriyama, 1992). The PCM can be dissociated from the centrioles in vivo by treatment of cells with the micro- Centrosomes are responsible for nucleating and organizing the tubule drug colchicine (Sellitto and Kuriyama, 1988). From microtubule cytoskeleton of interphase and mitotic cells. Cen- these observations questions arise concerning the structural trosomes are complex structures, which in animal cells are and functional nature of the PCM: what is the framework with usually, but not always, composed of two centrioles and a sur- which the pericentriolar components are associated, and what rounding, amorphous, pericentriolar material (PCM). During organizes the PCM around the centrioles under normal con- the last two decades great effort has gone into studying the ditions? function of centrosomes as microtubule organizing centers, and Recently, a number of components have been found to be it has become clear that the capability for microtubule nucle- associated with the centrosome/PCM. A ~51 kDa polypeptide ation in interphase and mitotic cells is associated with the PCM is associated with the PCM of mitotic spindles in sea urchins rather than with the centrioles (Berns and Richardson, 1977; and appears to be involved in nucleating microtubule assembly Brenner et al., 1977; Gould and Borisy, 1977; Toriyama et al., (Toriyama et al., 1988). In addition, several components have 1988). been identified as being part of the PCM, by immunological Our structural knowledge about centrosomes is predomi- methods (for a complete list, see Kimble and Kuriyama, 1992), nantly restricted to the structure of centrioles with their circular such as centrin (Baron and Salisbury, 1988), centractin, an arrangement of nine triplet microtubules (reviewed by actin homolog (Clark and Meyer, 1992), MAP-1 (Mascardo et Wheatley, 1982). The PCM, on the other hand, appears as a al., 1982; Sato et al., 1983), protein kinase II (Nigg et al., less-defined, osmiophilic material in close association with the 1985), and phosphoproteins (Vandré et al., 1984; Kuriyama, centrioles, and its biochemical composition is just beginning 1989); however, little is known of the importance of these com- to be clarified (for reviews see Bornens and Karsenti, 1986; ponents for centrosomal functions. Certainly, an important 2096 W. Steffen, E. A. Fajer and R. W. Linck finding is the discovery of γ-tubulin (Horio et al., 1991; Oakley precipitating them out of cell culture supernatant with 50% and Oakley, 1989; Oakley et al., 1990; Stearns et al., 1991; ammonium sulfate, by purifying them over a Protein G column or by Zheng et al., 1991). γ-Tubulin shares significant sequence injecting hybridoma cells into mice to obtain ascites fluid. In this homology with both α- and β-tubulin, it is localized in micro- study, except when indicated otherwise, antibodies obtained only tubule organizing centers (MTOCs) and centrosomes, and from cell culture supernatant were employed. The antibody class of although it appears not to be present along microtubules in monoclonals were determined using the isotyping kit from Boehringer Mannheim (Indianapolis, IN). vivo, it is required for nucleation of microtubules from cen- trosomes. Cell culture In an earlier study we noticed that affinity-purified, poly- Chinese hamster ovary (CHO) cells were cultured in F-10 medium clonal antibodies raised against sea urchin tektins cross-reacted containing 10% fetal bovine serum at 10% CO2 and 37°C. HeLa cells with basal bodies and centrioles from various cell lines (Steffen were cultured in DMEM medium containing 10% fetal calf serum. and Linck, 1988). Tektins are filamentous proteins that form Mouse myeloma (X63-Ag 8.653) and mouse hybridoma cells were polymers in the walls of axonemal doublet microtubules from cultured in DMEM containing 10% fetal calf serum supplemented sea urchin sperm flagella and presumably in triplet micro- with 10 mM hypoxanthine and 1.6 mM thymidine. For immunofluo- tubules of centrioles. By sequence analysis tektins have been rescence microscopy cells were cultured on poly-L-lysine-coated cov- shown to be an independent class of filamentous proteins erslips. (Chen et al., 1993; Norrander et al., 1992), but they share bio- Isolation of centrosomes and spindles chemical and immunological properties with intermediate Centrosomes were isolated from CHO cells according to Mitchison filament proteins (Chang and Piperno, 1987; Linck and and Kirschner (1986). Briefly, confluent cells from 30 cm × 10 cm Stephens, 1987; Steffen and Linck, 1989a), and tektins have a culture dishes of confluent cells were treated with 10 µg/ml nocoda- predicted secondary structural motif similar to that of inter- zole and 5 µg/ml cytochalasin B for 90 minutes to disintegrate the mediate filament (IF) proteins (Norrander et al., 1992). In this cytoskeleton. Cells were harvested in PBS, washed first with diluted study we present the characterization of a set of monoclonal PBS (1/10 the original concentration) containing 8% sucrose and then antibodies raised against axonemal proteins. Using these with 8% sucrose in water. Cells were lysed in 1 mM Tris-HCl, pH 8.0, containing 8 mM β-mercaptoethanol and 0.5% Nonidet P40. The tektin-specific monoclonal and polyclonal antibodies, we β demonstrate the presence of a tektin-like component in cen- lysate was brought to 10 mM PIPES, pH 7.2, 1 mM EGTA, 8 mM - mercaptoethanol, and fractionated on a sucrose gradient. trosomes of molluscan and mammalian cells. Mitotic spindles were isolated from CHO cells according to Kuriyama et al. (1984). Semi-confluent cells were synchronized by MATERIALS AND METHODS arresting them first in S-phase with 3 mM thymidine and then in M- phase with 0.1 µg/ml nocodazole. Harvested cells were freed from the Antibodies culture dishes and washed free of the nocodazole. Mitotic spindles Rabbit polyclonal antibodies were raised against SDS-PAGE-purified were isolated 18 minutes after the removal of the drug by resuspend- tektin A, B and C derived from sperm axonemal doublet microtubules ing the pelleted cells in 2 mM PIPES, pH 6.8, 20 µg/ml taxol, 0.25% of two sea urchin species: Lytechinus pictus (L.p.) and Strongylocen- Triton X-100. trotus purpuratus (S.p.). A detailed characterization of the polyclonal anti-tektins is provided elsewhere (Linck et al., 1987). All polyclonal Isolation of cytoplasmic microtubules and acetylation of anti-tektins employed in this study were affinity purified with tektin tubulin filaments from L. pictus. Eggs from female Strongylocentrotus purpuratus sea urchins were To raise monoclonal antibodies against components of axonemal obtained by shedding them into artificial sea water (423 mM NaCl, 9 microtubules, the antigen was prepared as follows: doublet micro- mM KCl, 23 mM MgCl2, 25 mM MgSO4, 2 mM NaHCO3, 9 mM tubules of S. purpuratus sperm were extracted twice with 0.5% CaCl2, 5 mM Tris-HCl, pH 8.0) at 4°C. They were washed with arti- Sarkosyl in 50 mM Tris, 1 mM EDTA, 1 mM DTT, pH 8.0, on ice, ficial sea water, filtered through cheesecloth and a 95 µm nitex filter resulting in preparations of axonemal ribbons consisting of three to strip off their egg jelly, and then washed three times with PIPES protofilaments each. Protofilament ribbons consisting principally of buffer (100 mM PIPES, 5 mM EGTA, 2 mM MgCl2, 0.1 mM GTP, α- and β-tubulin, tektin A, B and C, and two polypeptides of about pH 6.9). After a final wash the eggs were resuspended in an equal 77 kDa and 83 kDa were dissolved in SDS sample buffer (2% SDS, volume of PIPES buffer containing 1 mM PMSF and homogenized 0.5% β-mercaptoethanol, 2.5 mM Tris, 19.2 mM glycine) and then on ice in a glass Potter homogenizer.