Chromosome attachment to the spindle in crane-fly spermatocytes requires actin and is necessary to initiate the anaphase-onset checkpoint

We investigated the possible involvement of actin in the attachment of chromosomes to spindles in crane-fly primary spermatocytes. In a previous study, cytochalasin D,an inhibitor of actin polymerisation, prevented bivalent attachment to microtubules when applied at prophase, but did not cause the detachment of already attached bivalents. We were able to detach the already attached bivalents by first treating prometaphase cells with an anti-tubulin drug, nocodazole, to disrupt spindle microtubules. 2 min after nocodazole addition, we added cytochalasin D, to disrupt actin filaments; then 2 min later nocodazole was removed, and the cells were kept in cytochalasin D until the time of normal anaphase. Double treatment with nocodazole and cytochalasin D blocked reattachment of bivalents to the spindle. Single treatment with nocodazole alone caused chromosome detachment but did not prevent reattachment when nocodazole was washed out. Extended treatment with cytochalasin D alone starting in prometaphase did not cause bivalents to detach from the spindle. These data suggest that actin is needed for attachment of bivalents to spindle microtubules. This protocol is relevant to the anaphase-onset checkpoint. From previous experiments it was argued that the anaphase-onset checkpoint recognises unattached chromosomes only after those chromosomes first interact with (become attached to) the spindle. Our experiments showed that anaphase disjunction occurred at normal times when bivalents were prevented from attaching to the spindle (by adding cytochalasin D in prophase), while anaphase disjunction was greatly delayed when previously attached bivalents were detached (with nocodazole) and then prevented from re-attaching (with cytochalasin D) in the double treated cells. Thus the anaphase-onset checkpoint recognises only those unattached bivalents that previously were attached to the spindle. Other results provided further indication that actin-microtubule interactions are important in spindle organisation. Nocodazole treatment for 4 min caused most microtubules to disappear: bivalents aggregated around remnant microtubules. When cytochalasin D treatment followed nocodazole treatment, remnant spindle microtubules were not seen, suggesting that actin interactions help stabilise those microtubules.