Checkpoint control in crane-fly spermatocytes: unattached chromosomes induced by cytochalasin D or latrunculin treatment do not prevent or delay the start of anaphase

Abstract

Summary. Variable numbers of bivalents and sex chromosomes do not attach to the spindle when prophase or early prometaphase cranefly spermatocytes (2n = 8) are treated with cytochalasin D or latrunculin. The unattached bivalents lie in the cytoplasm or at the spindle pole, and they do not delay onset of autosomal anaphase; sometimes they disjoin at the same time as the attached bivalents, so they respond to the global signals that initiate anaphase. Unattached sex chromosomes do not delay autosomal anaphase, either. Of various interpretations of these data, we think the best explanation is that the checkpoint system responds to physical rather than chemical cues; we think that the spindle is a "tensegral" structure, that chromosomes need to interact with the spindle in order to be recognised by the anaphase-onset "checkpoint control", and that the physical interaction of chromosomes with spindle acts as a signalling network. Cytochalasin D and latmnculin treatments delay onset of sex chromosome anaphase (which normally occurs about 15 min after autosomal anaphase) and cause altered patterns of sex-chromosome segregation.

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

“…The depolymerising effect of CD on actin was confirmed in live cells by the collapse shortly after CD perfusion of the actin fingers (or "pompons"; La Fountain et al 1992) present near each pole, as described by Forer and Pickett-Heaps (1998a).…”

“…For every treated cell we first followed control cells from the same preparations or from the contralateral testis to ensure that cells proceeded normally through the first meiotic division, from prophase to telophase (60-1.20 rain; Forer and Pickett-Heaps 1998b). Control cells were perfused every 10 rain with IR or with the maximal DMSO concentration (0.2%) used in drug solutions.…”

“…Living crane-fly (Nephrotoma suturalis [Loew]) spermatocytes were prepared as described by Forer and Pickett-Heaps (1998a). In brief, IV-instar larvae were dissected and the testes removed under halocarbon oil; testes were washed in a drop of IR and were pierced in a drop of fibrinogen on previously flamed coverslips.…”

“…Most previous work has considered that "unattached kinetochores.., release an inhibitory signal that delays anaphase until they properly attach to the spindle" (Waters et al 1998(Waters et al : p. 1181). However, experiments on crane-fly spermatocytes suggest that more than "an inhibitory signal" is involved, that chromosomes must first attach to spindle microtubules before they are recognised by the anaphase-onset checkpoint: when chromosomes are prevented from attaching to the spindle (by being treated in prophase with CD or latrunculin B), anaphase disjunction nonetheless occurs at the normal time (Forer and Pickett-Heaps 1998b). This suggests that an initial chromosome-spindle interaction is required to activate the anaphase-onset checkpoint.…”

“…Chromosome attachment to spindle fibres might require actin-microtubule interaction: actin inhibitors cytochalasin D (CD) (S. Brown and Spudich 1979, Cooper 1987, Urbanik and Ware 1989 and latrunculin B (Spector et al 1989) prevent chromosome attachment when applied to crane-fly spermatocytes in prophase (i.e., prior to chromosome attachment) but do not cause chromosome detachment when applied in prometaphase, when the chromosomes already are attached to microtubules (Forer and Pickett-Heaps 1998a). That attachment is blocked indicates that actin might be involved in attachment; that detachment does not occur could be because actin is no longer needed after attachment or could be because actin and kinetochore microtubules bind tightly together, this interaction protecting actin from antiactin drugs.…”