Ortho-vanadate affects both the tyrosination/detyrosination state of spindle microtubules and the organization of XTH-2 spindles.
Mitotic spindles of a cultured endothelial cell line from Xenopus tadpole heart (XTH-2) contain distinct subsets of differentially stabilized microtubules (MTs) enriched in tyrosinated (Tyr) and in nontyrosinated (Glu) tubulin. Generally, the distribution of the more labile Tyr-MTs and of the Glu-MTs indicative for the more stable MT-subclass was remarkably different. During all mitotic stages the Tyr-staining of spindle fibers predominates, whereas the Glu-species was mainly restricted to the peripheral regions of half spindles where the MTs have to sustain a bending stress. Moreover, Glu-tubulin is enriched in prophase centrosomal areas, and, from metaphase on, exclusively also in the centrioles. Glu-positivity does not remain constant, instead, the intensity weakens from metaphase to telophase reflecting the decreasing MT nucleating capacity of the centrosomes. During the spindle cycle, the interzonal spindle at the anaphase-telophase transition exceeds all other stages in Glu-stainability and retains the high amount of detyrosinated MTs during telophase. External application of millimolar vanadate has different effects upon spindle components organization: First, additional assembly of Tyr-MTs together with a Glu-negative reaction of the centrioles was registered and, secondly, a drastic disarrangement of the Tyr-staining spindle fiber component became evident. At the onset of anaphase, an extreme spindle lengthening presumably due to the separation of the Tyr- and Glu-MTs occurred. Obviously, the Glu-spindle fibers were less affected and remained largely in their original spindle position. Redistribution of anti-dynein staining following vanadate incubation suggests a causal relationship between inhibition of dynein motor proteins and disarrangement of different microtubular spindle components. These results suggest that the changes in the spindle framework are at least partly due to malregulation of centrosomal phosphorylation events, respectively to inactivation of special cross-bridging proteins interacting between distinct MT-subsets by a phosphate mimicking effect of vanadate and finally, by a vanadate-induced displacement of polar asters.
Winkelhaus
S
S
Lehrstuhl für Zellmorphologie, Ruhr-Universität, Bochum/Germany.
Hauser
M
M
D000818:Animals; D002455:Cell Division; D002460:Cell Line; D018385:Centrosome / Q000378:metabolism; D004398:Dyneins / Q000378:metabolism; D004730:Endothelium, Vascular; D004791:Enzyme Inhibitors / Q000494:pharmacology; D008870:Microtubules / Q000187:drug effects* / Q000378:metabolism; D008941:Spindle Apparatus / Q000187:drug effects* / Q000378:metabolism; D014404:Tubulin / Q000378:metabolism; D014443:Tyrosine / Q000378:metabolism*; D014638:Vanadates / Q000494:pharmacology*; D014981:Xenopus
D004791:Enzyme Inhibitors; D014404:Tubulin; D014638:Vanadates; D014443:Tyrosine; D004398:Dyneins