Heterogeneity of microtubule organizing center components as revealed by monoclonal antibodies to mammalian centrosomes and to nucleus-associated bodies from dictyostelium

Abstract

The molecular composition of two morphologically distinct microtubule-organizing centers (MTOCs) was compared by probing with monoclonal antibodies raised against (i) nucleus-associated bodies (NABs) isolated in a complex with nuclei from the cellular slime mold Dictyostelium discoideum and (ii) mammalian mitotic spindles isolated from Chinese hamster ovary (CHO) cells. The staining patterns observed by immunofluorescence microscopy in whole CHO cells and Dictyostelium amoebae showed that the distribution of thirteen MTOC antigens is heterogeneous. Not all antibodies recognized the MTOC in both interphase and mitosis. Most of the anti-MTOC antibodies cross-reacted with other cellular organelles such as nuclei, Golgi apparatus-like aggregates and cytoskeletal elements. Two antibodies, CHO3 and AX3, recognized phosphorylated epitopes present in both mammalian centrosomes and Dictyostelium NABs. On immunoblots, most of the antibodies showed multiple bands, often of high molecular weight, indicating that the antigenic determinants are shared among different molecules. One antibody inhibited the regrowth of microtubules onto centrosomes in vitro after addition of exogenous tubulin to detergent-lysed CHO cells on coverslips; this antibody binds to an antigen(s) that might be essential for the microtubule-nucleating activity of centrosomes. These observations demonstrate that molecular components in different MTOCs exhibit a variety of distinct subcellular localizations and functional properties, and that some antigenic molecules have been conserved among morphologically distinct MTOCs.