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.

Identification of ubiquitous high-molecular-mass, heat-stable microtubule-associated proteins (MAPs) that are related to the Drosophila 205-kDa MAP but are not related to the mammalian MAP-4.

“…Using a monoclonal antibody (mAb; AX3) raised against microtubule-organizing centers from the cellular slime mold Dictyostelium discoideum, we have identified a highmolecular mass, heat-stable MAP that occurs in species ranging from Dictyostelium to humans (16). We show here that the proteins recognized by mAb AX3 in mouse and human are distinct from the previously identified MAP-2 and MAP-4 proteins.…”

“…The AX3 mAb was raised against an isolated nucleus/nucleus-associated body (the primary microtubuleorganizing center of Dictyostelium) fraction from the Ax-3 strain of Dictyostelium discoideum. The production and preliminary characterization of this mAb are described elsewhere (16).…”

“…Dictyostelium amoebae grown in suspension to a final density of 1-5 x 105 were allowed to attach to polylysine-coated coverslips and fixed with 3.7% (vol/vol) formaldehyde in a cytoskeleton buffer (16), according to the two-step fixation protocol of Roos (21). HeLa and EMF cells were cultured directly on coverslips in Dulbecco's minimal Eagle's medium or F-10 medium, washed two times with phosphate-buffered saline, and then fixed for 10 min with 4% formaldehyde in 100 mM Pipes (pH 6.9)/5 mM EGTA/2 mM MgCl2/0.2% Triton X-100.…”

“…HeLa and EMF cells were cultured directly on coverslips in Dulbecco's minimal Eagle's medium or F-10 medium, washed two times with phosphate-buffered saline, and then fixed for 10 min with 4% formaldehyde in 100 mM Pipes (pH 6.9)/5 mM EGTA/2 mM MgCl2/0.2% Triton X-100. Incubation of cells with antibodies and detection of the fluorescent signal were as described (16,19).…”

“…For immunoblot analysis, protein samples separated on 7 or 10%o acrylamide gels were transferred to nitrocellulose and further incubated with the primary antibodies as before (16,19). Bound antibodies were detected by using alkaline phosphatase-conjugated secondary antibodies, with nitro blue tetrazolium chloride and 5-bromo-4-chloro-3-indoyl phosphate as enzyme substrates (19).…”