To study tubulin polymerization and microtubule sliding during spindle elongation in vitro, we developed a method of uncoupling the two processes. When isolated diatom spindles were incubated with biotinylated tubulin (biot-tb) without ATP, biot-tb was incorporated into two regions flanking the zone of microtubule overlap, but the spindles did not elongate. After biot-tb was removed, spindle elongation was initiated by addition of ATP. The incorporated biot-tb was found in the midzone between the original half-spindles. The extent and rate of elongation were increased by preincubation in biot-tb. Serial section reconstruction of spindles elongating in tubulin and ATP showed that the average length of half-spindle microtubules increased due to growth of microtubules from the ends of native microtubules. The characteristic packing pattern between antiparallel microtubules was retained even in the "new" overlap region. Our results suggest that the forces required for spindle elongation are generated by enzymes in the overlap zone that mediate the sliding apart of antiparallel microtubules, and that tubulin polymerization does not contribute to force generation. Changes in the extent of microtubule overlap during spindle elongation were affected by tubulin and ATP concentration in the incubation medium. Spindles continued to elongate even after the overlap zone was composed entirely of newly polymerized microtubules, suggesting that the enzyme responsible for microtubule translocation either is bound to a matrix in the spindle midzone, or else can move on one microtubule toward the spindle midzone and push another microtubule of opposite polarity toward the pole.
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1 August 1988
Article|
August 01 1988
The mechanism of anaphase spindle elongation: uncoupling of tubulin incorporation and microtubule sliding during in vitro spindle reactivation.
H Masuda,
H Masuda
Department of Botany, University of California, Berkeley 94720.
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K L McDonald,
K L McDonald
Department of Botany, University of California, Berkeley 94720.
Search for other works by this author on:
W Z Cande
W Z Cande
Department of Botany, University of California, Berkeley 94720.
Search for other works by this author on:
H Masuda
Department of Botany, University of California, Berkeley 94720.
K L McDonald
Department of Botany, University of California, Berkeley 94720.
W Z Cande
Department of Botany, University of California, Berkeley 94720.
Online ISSN: 1540-8140
Print ISSN: 0021-9525
J Cell Biol (1988) 107 (2): 623–633.
Citation
H Masuda, K L McDonald, W Z Cande; The mechanism of anaphase spindle elongation: uncoupling of tubulin incorporation and microtubule sliding during in vitro spindle reactivation.. J Cell Biol 1 August 1988; 107 (2): 623–633. doi: https://doi.org/10.1083/jcb.107.2.623
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