Elastic tethers remain functional during anaphase arrest in partially-lysed crane-fly spermatocytes: a possible approach for studying mitotic tethers

Abstract

Mitotic tethers connect partner telomeres of all segregating anaphase chromosomes in all animal cells that have been tested, as detected by laser-cutting chromosome arms during anaphase and seeing that the arm fragments move rapidly across the equator to their partner chromosome moving to the opposite pole, telomere moving towards telomere. Tethers exert anti-poleward forces on the poleward separating telomeres, but tether elasticity (that produces the backwards forces) diminishes during anaphase: as determined by the behavior of arm fragments; short tethers (early anaphase) are elastic, long tethers (late anaphase) are not elastic, and medium-length tethers transition between the two states. We developed a procedure in which the tethers still functioned after we partially-lysed anaphase crane-fly spermatocytes. The partial lysis consistently arrested chromosome movements, after which the tethers moved the chromosomes backwards, potentially allowing the elastic tethers to be studied biochemically. To ensure that tether function was not altered by the partial cell-lysis procedure, we compared backward chromosome movements in partially-lysed cells with arm fragment movements in control cells. In the partially-lysed cells the backward chromosomal movements had characteristics identical to those of arm fragments in non-lysed (control) cells. In particular, in both control and partially-lysed cells shorter tethers caused backward movements more often than did longer tethers; shorter tethers caused backward movements over greater fractional distances (of the tether) than did longer tethers; and velocities of the backwards movements were the same for tethers of different lengths. We also compared the effects of Calyculin A (an inhibitor of Protein Phosphatase1) in control versus in partially-lysed cells. Calyculin A (CalA) added to control cells in early anaphase blocks dephosphorylation, thereby maintaining tether elasticity throughout anaphase: after the chromosomes reach the poles they move backwards when the usual poleward forces are reduced. Partial lysis preserves this tether functionality: after partial lysis of CalA-treated cells the chromosomes move backward and reach the partner telomeres at even very long tether lengths. We conclude that partial cell-lysis arrests anaphase chromosome poleward movement but does not affect tether function.