A mitotic role for Mad1 beyond the spindle checkpoint

Abstract

SummaryUnattached kinetochores generate an anaphase inhibitor, through the spindle assembly checkpoint (SAC), that allows cells more time to establish proper kinetochore-microtubule (K-MT) linkages and thus avoid aneuploidy. Mad1 is the receptor for Mad2 at kinetochores, where it catalyzes the formation of Mad2-Cdc20 complexes, an essential part of the anaphase inhibitor, but whether it has any other mitotic function is unknown. We have generated a mad1-null mutation in Drosophila. This mutant is SAC defective and Mad2 is no longer localized to either nuclear envelope or kinetochores, but it displays normal basal mitotic timing. Unlike mad2 mutants, which have relatively normal mitoses, mad1 anaphases show high frequencies of lagging chromatids, at least some of which are caused by persistent merotelic linkages. A transgene expressing GFP-Mad1 rescues both the SAC and the anaphase defects. In an attempt to separate the SAC function from the mitotic function, we made a mad1 transgene with a mutated Mad2-binding domain. Surprisingly, this transgene failed to complement the anaphase phenotype. Thus, Mad1 has activity promoting proper K-MT attachments in addition to its checkpoint function. This activity does not require the presence of Mad2, but it does depend in some unknown way on key residues in the Mad2-binding domain of Mad1.

A maternal effectrough dealmutation suggesting multiple pathways regulating Drosophila RZZ kinetochore recruitment

“…As is the case for Mad1-null mutants (Emre et al, 2011), these lagging chromatids were in most cases correctly segregated, despite their delay in migration (given that overall aneuploidy levels are low). In several cases, the lagging chromatids appeared to be attached to spindle fibers emanating from both poles (merotelic attachment), which is also a feature associated with mitosis in Mad1-null mutants (Emre et al, 2011).…”

“…3F), however, showed that there was an elevated frequency of anaphases in which some kinetochores migrated poleward more slowly than the others. Indeed, the fraction of anaphases with at least one lagging kinetochore was nearly as high in rod Z3 as in rod or Mad1-null mutants (Emre et al, 2011), although only rarely was more than one kinetochore involved (Table 3). As is the case for Mad1-null mutants (Emre et al, 2011), these lagging chromatids were in most cases correctly segregated, despite their delay in migration (given that overall aneuploidy levels are low).…”

“…Indeed, the fraction of anaphases with at least one lagging kinetochore was nearly as high in rod Z3 as in rod or Mad1-null mutants (Emre et al, 2011), although only rarely was more than one kinetochore involved (Table 3). As is the case for Mad1-null mutants (Emre et al, 2011), these lagging chromatids were in most cases correctly segregated, despite their delay in migration (given that overall aneuploidy levels are low). In several cases, the lagging chromatids appeared to be attached to spindle fibers emanating from both poles (merotelic attachment), which is also a feature associated with mitosis in Mad1-null mutants (Emre et al, 2011).…”

The spindle assembly checkpoint promotes chromosome bi-orientation: A novel Mad1 role in chromosome alignment

“…In fly, mad1-null mutant cells show higher rate of lagging chromosomes in anaphase compared to mad2-null mutant cells. 19 This indicates Mad1 has a role in chromosome bi-orientation beyond the SAC. Interestingly, this role depends on Mad2-interacting motif of Mad1, although mad2-null mutant cells undergo normal anaphase.…”

“…Interestingly, this role depends on Mad2-interacting motif of Mad1, although mad2-null mutant cells undergo normal anaphase. 19 Molecular mechanisms how Mad1 promotes chromosome bi-orientation remain elusive. Similarly, in fission yeast, mad1D cells show higher sensitivity to spindle drugs compared to mad2D or mad3D cells.…”