Incorporation and turnover of labeled exogenous tubulin in the mitotic spindles of Chaetopterus oocytes and HeLa cells.
The incorporation of tubulin into mitotic spindles in situ was studied by incubating permeabilized mitotic cells in solutions containing [3H]GTP-labeled or dichlorotriazinylamino fluorescein (DTAF)-labeled tubulin. Metaphase HeLa cells or spindle-containing "minicells" from Chaetopterus oocytes were lysed in a microtubule-assembly buffer plus 0.5% Nonidet P-40, 1 mg/ml 120,000g supernatant mammalian brain tubulin, and [3H]GTP. After different periods of incubation, mitotic spindles were isolated in 2 M-glycerol-containing assembly buffer and separated from unbound counts by centrifugation through a 4 M-glycerol cushion; 3H counts per mg protein increase linearly for 8-12 min and then reach a plateau or steady state in both Chaetopterus oocytes and HeLa cells. Addition of 4 mM CaCl2 blocks or reverses incorporation. Little or no [3H]GTP is incorporated if exogenous tubulin or lysed cells are omitted from the assembly mixture. To measure the loss rate of [3H]GTP-tubulin from mitotic spindles, cells were incubated in tubulin plus [3H]GTP for 30 min, and a 20-fold excess of cold GTP (2 mM) was added. Samples were removed after incubation for different periods, and spindles were isolated as described above and counted for 3H content. [3H]GTP is lost from spindles at a rate of about 16%/min until a new steady state is reached in about 8 min. These results are consistent with an incorporation and turnover of [3H]GTP-tubulin in spindle microtubules of these lysed-cell models. The location of this newly incorporated tubulin in the spindle was investigated by incorporating fluorescent DTAF-tubulin into mitotic spindles of these lysed cell types.(ABSTRACT TRUNCATED AT 250 WORDS)
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D000818:Animals; D000845:Annelida / Q000648:ultrastructure*; D002463:Cell Membrane Permeability; D003902:Detergents; D005452:Fluoresceins; D005456:Fluorescent Dyes; D006153:Guanosine Diphosphate / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism; D006367:HeLa Cells / Q000648:ultrastructure; D006801:Humans; D007700:Kinetics; D008854:Microscopy, Electron; D008870:Microtubules / Q000378:metabolism; D017830:Octoxynol; D009865:Oocytes / Q000648:ultrastructure*; D011092:Polyethylene Glycols; D008941:Spindle Apparatus / Q000378:metabolism*; D014404:Tubulin / Q000378:metabolism*
D003902:Detergents; D005452:Fluoresceins; D005456:Fluorescent Dyes; D014404:Tubulin; D006153:Guanosine Diphosphate; C039083:5-((4,6-dichloro-1,3,5-triazin-2-yl)amino)fluorescein; D011092:Polyethylene Glycols; D006160:Guanosine Triphosphate; D017830:Octoxynol; C010615:Nonidet P-40