Identification of a spindle-associated protein in ciliate micronuclei

Journal of Cell Science Identification of a spindle-associated protein in ciiiate micronuciei GUY KERYER 0 NICOLE GARREAU DE LOUBRESSE 0 NICOLE BORDES 0 MICHEL BORNENS 0 0 'Centre de Ginftique Mole'culaire, Centre National de la Recherche Scientifique , 91198, Cif/Yvette cedex , France 1 Centre de Biologie Cellulaire, Centre National de la Recherche Scientifique , 94201, Ivry/Seine , France Author for correspondence spindle-associated protein; micronucleus; ciliates - Ciliated protozoa display a nuclear dualism, with germinal micronuciei and a somatic macronucleus. During mitosis, which proceeds without disruption of the nuclear envelope, a spindle is organized within the micronucleus from, presumably, intranuclear microtubule-organizing centres (MTOCs). In order to characterize these MTOCs, monoclonal antibodies generated against human centrosomes were screened on several ciliates and particularly on Paramecium tetraurelia. In this ciiiate, the monoclonal antibody CTR 532, which decorates centrosomal and spindle-associated components in mammalian cells, specifically labelled the micronuclei during interphase. At the electron-microscope level, it stained a fibrous material surrounding microtubules localized on the inner face of the nuclear envelope. During mitosis this decoration extended all over the metaphase spindle. At all stages of the cell cycle, the decoration remained specific to the micronucleus and was absent not only from all of the various cytoplasmic and cortical microtubule arrays but also from the macronuclei, even at early stages of their development from the zygotic nucleus. CTR 532 recognizes a single 170xl(pMr polypeptide in the cytoskeletal fraction that contains micronuciei and this polypeptide is absent in the cytoskeletal fraction of amicronucleate cells. Unlike metazoa, protozoa frequently display endomitosis, and the structures that are morphogenetically connected with mitotic spindle formation vary quite considerably. Several types of cytoplasmic or intranuclear microtubule-organizing centres (MTOCs) have been well described in ultrastructural terms (for reviews, see Kubai, 1975; Heath, 1980; Raikov, 1982). However, the biochemical nature of these MTOCs is unknown, as is their relationship to the nuclear envelope. Closed nuclear orthomitoses are characteristic of ciliates. These protozoa display a nuclear dualism with one or more diploid micronuciei and one 'polyploid' macronucleus. During interphase, besides the cytoplasmic and cortical microtubule networks, a layer of microtubules is found under the nuclear envelope in the micronucleus of a number of ciliates and is referred to as a 'residual spindle' (Raikov, 1982). During mitosis or meiosis several microtubule networks are organized into the micro- and the macronuclei (Tucker et al. 1980, 1985; Cohen & Beisson, 1988). Different classes of mitotic microtubules are assembled during successive mitotic 9tages and characterized by their differential stability to cold or anti-mitotic drugs (Eichenlaub-Ritter & Ruthman, 1982) and/or by their protofilament number (Eichenlaub-Ritter & Tucker, 1984). Besides the nuclear spindles, a cortical spindle develops during mitosis (Cohen et al. 1982). It has been suggested that microtubule-associated proteins (MAPs) could contribute to the stability of these different microtubule subclasses in protozoa (EichenlaubRitter & Ruthman, 1982). However, nothing is known about these putative MAPs associated with internal microtubule networks in ciliates. In order to analyse MTOCs and the spindle in ciiiate micronuciei, a library of monoclonal antibodies, generated against purified human centrosomes (Bornens et al. 1987), was screened on Paramecium tetraurelia. One of these monoclonals, CTR 532, which decorates the centrosome and the mitotic spindle in mammalian cells, was observed to label specifically fibres in the micronucleus of interphase paramecia as well as the spindle during mitosis in both Paramecium and Tetrahymena. We describe here the ultrastructural localization and biochemical characterization of the antigen, a 170xl03Mr polypeptide that appears to be similar in both Paramecium and Tetrahymena and is restricted to the micronuclear compartment. Materials and methods Strains and culture conditions Cells of stock d4-2 of Paramecium tetraurelia (Sonneborn, 1975) were grown at 27CC according to the procedure described by Sonneborn (1975), in wheat-germ powder infusion medium, infected the day before use with Aembacter aeivgenes supplemented with ^-sitosterol (0-4/igml~'). The amicronucleate Paramecium cell line (C113 cell line from stock d4-94, a derivative of stock 51 carrying the pw A gene) was a gift from Dr S. Ng (Ng, 1981). Tetrahymena thermophila and Tetrahymena pyriformis strain GL 100 were grown in a medium containing 1% proteose-peptone, 0 ' 5 % yeast extract and 0-87 % dextrose. HeLa and Vero cells were grown in monolayer culture using minimum essential medium (MEM, from GibcoBRL) supplemented with 7% calf serum (Boehringer). Antibodies The monoclonal antibody CTR 532 (IgM class) was obtained from a collection of monoclonal antibodies raised against centrosomes isolated from human lymphoblast (Bornens et al. 1987). It was used as a culture supernatant diluted 1/100 to 1/500 in phosphate-buffered saline (PBS) containing 3 % bovine serum albumin (BSA). The anti-n-tubulin (Blose et al. 1982) from Amersham (France), was used at 1/1000 dilution. The polyclonal antibody raised against Paramecium axonemal tubulin (Cohen et al. 1982) was used at a 1/400 dilution. Immunofluorescence microscopy Immunofluorescence on mammalian cells was performed as follows: the cells were rinsed briefly in PHEM (60mM-Pipes, 25mM-Hepes, 10mM-EGTA, 5mM-MgCl2, p H 6 9 ) buffer as described by Schliwa & Van Blerkom (1981) and permeabilized with 0-5 % Triton X-100 in PHEM buffer for 1 nun; they were fixed with cold methanol for 5 min at 20C. Immunostaining with CTR 532 was done in PBS for 30-45 min, followed by three washes in PBS containing 0-1 % Tween 20. Then the cells were incubated for 30 min with a rhodamine-conjugated goat anti-mouse immunoglobulin (from Cappell), diluted 1/150 in PBS plus 1 % BSA. The cells were washed and mounted as described below for ciliates. Immunofluorescence was performed on paramecia permeabilized in a 1 % Triton X-100-containing PHEM buffer. Two procedures were used for immunostaining. In the first one (Cohen et al. 1982), the permeabilized cells were washed once in the same buffer for antibody incubation and washings. This method provided quite a satisfactory conservation of the cytoskeleton organization and an apparently good labelling specificity as controlled by electron microscopy. However, for stringent antigen-antibody affinity requirement we have used a second procedure: the permeabilized cells were fixed with 2 % paraformaldehyde in PHEM buffer for 1 h at room temperature, rinsed in PBS containing 2mM-MgCl2 and lOmM-EGTA ( (...truncated)